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https://f1000research.com/articles/5-82/v1
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19 Jan 16
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{
"type": "Review",
"title": "Chikungunya: epidemiology",
"authors": [
"Lyle R. Petersen",
"Ann M. Powers",
"Ann M. Powers"
],
"abstract": "Chikungunya virus is a mosquito-borne alphavirus that causes fever and debilitating joint pains in humans. Joint pains may last months or years. It is vectored primarily by the tropical and sub-tropical mosquito, Aedes aegypti, but is also found to be transmitted by Aedes albopictus, a mosquito species that can also be found in more temperate climates. In recent years, the virus has risen from relative obscurity to become a global public health menace affecting millions of persons throughout the tropical and sub-tropical world and, as such, has also become a frequent cause of travel-associated febrile illness. In this review, we discuss our current understanding of the biological and sociological underpinnings of its emergence and its future global outlook.",
"keywords": [
"Chikungunya",
"mosquito",
"alphavirus"
],
"content": "Introduction\n\nAs with several other mosquito-borne alphaviruses, chikungunya virus causes a fever-rash-arthralgia syndrome in humans. The name chikungunya derives from the debilitating joint pain noted by local populations during an outbreak in 1952–53 in what is now Tanzania. The local word “chikungunya”, meaning “that which bends up” was given as a result of the stooped posture that resulted from the pain of the disease1,2. The arthralgia can persist for months or even years in some affected persons and can progress to frank arthritis in some3–15. Symptoms typically occur in 72–97% of those infected15–19, but one study, in an area where the virus has been long established endemically, showed that only 18% of infections resulted in clinical illness, possibly due to re-exposure events that did not result in clinical illness20. Since 2004, massive urban outbreaks producing considerable morbidity in a widening geographical area have occurred throughout the topical and sub-tropical world. In this review, we will explore the complex interplay of entomological, virological, and sociological factors contributing to its emergence, speculate on future epidemiological trends, and outline the possibilities for control.\n\n\nTransmission cycles and mosquito vectors\n\nThree chikungunya viral genotypes are recognized, which historically have circulated in the distinct geographical regions for which they are named: West African genotype, East Central South African (ECSA) genotype, and Asian genotype21. Phylogenetic evidence suggests that the Asian genotype virus derived from the ECSA virus sometime between 1879 and 192722. In Africa, the virus is maintained in a sylvatic cycle involving non-human primates and forest-dwelling Aedes spp. mosquitoes23. In these rural regions, human outbreaks tend to be small and dependent on environmental conditions (such as increased rainfall) that increase sylvatic mosquito densities, particularly of the Aedes furcifer-tayleri group23,24. A sylvatic transmission cycle has not been identified in Asia, but is likely present due to ongoing, low level, human activity.\n\nLike the arthropod-borne viruses (arboviruses) dengue, yellow fever, o’nyong’nyong, and Zika, humans are not dead-end hosts for chikungunya virus but rather serve as part of the transmission cycle by efficiently infecting Aedes aegypti mosquitoes. This property enables rapid human-mosquito-human transmission cycles in urban areas, which can produce massive outbreaks. Aedes aegypti is a highly efficient urban vector because it preferentially bites humans and often bites multiple humans in the course of acquiring a complete, single blood meal. Furthermore, Aedes aegypti breeds in the ubiquitous small pools of water found around human habitation, often bringing the vectors in close proximity to human hosts facilitating further transmission25,26.\n\nIn addition to Aedes aegypti, other mosquito vectors must be considered. Of particular interest is Aedes albopictus (Asian tiger mosquito), an aggressive, human-biting mosquito that has spread globally from its native Asia largely from international trade in used tires and other commodities in recent years27,28. Unlike Aedes aegypti, which exists in tropical and subtropical areas, Aedes albopictus can also thrive in temperate regions, thus potentially introducing chikungunya virus to new ecological niches29,30. Furthermore, other members of the Stegomyia subgenus may be important under certain circumstances. Aedes henselli was the principal vector of a large outbreak on Yap Island in Micronesia31,32. Other species present in focal areas may be of local importance.\n\n\nEarly outbreaks\n\nAlthough chikungunya virus cannot be definitively linked to outbreaks before its discovery in the mid-1950s, outbreaks of fever and debilitating polyarthralgia affecting a substantial proportion of the affected population were noted in many areas during the 18th and 19th centuries, including Africa, Asia, India, the West Indies, Indonesia, and the southern United States33,34. Interestingly, contemporary physicians called this disease “dengue” and noted its clinical similarities as well as its differences between what is currently known as “dengue” and “breakbone fever”. In the mid-1950s, several authors (including Sabin)35 isolated the “breakbone fever” viruses and called them “dengue viruses”, and the former “dengue” became “chikungunya” following the 1952–53 outbreak that produced the first isolation of chikungunya virus. While some of these previous historical outbreaks could have been due to chikungunya virus, they also could have been caused by other viruses already present in specific locations and causing the same clinical syndrome, such as Mayaro virus in the Americas or o’nyong’nyong virus in Africa.\n\nFollowing the discovery of chikungunya virus, numerous small outbreaks were noted in Africa. However, massive outbreaks were noted in Thailand in the late 1950s and early 1960s36,37, and in India from the early 1960s into the 1970s38. Approximately 31% of the population of Bangkok became infected during the 1962 outbreak36. Antibody prevalence ranged from 10–20% of the 1–2 year olds to 70–85% among adults, suggesting long-standing endemicity in that area. High attack rates were noted during outbreaks in Madras (40%) in 1962–64 and in Barsi (37%) in 197339. For unknown reasons, outbreaks in India abruptly stopped, not to reoccur for the next 32 years40.\n\n\nCurrent epidemic\n\nThe current epidemic, ongoing since 2004, involves many tropical and sub-tropical areas of Africa, Asia, Europe, the Pacific archipelago, and the Americas. Both ECSA and Asian genotype viruses, sometimes together, are responsible for epidemics, depending on location. The fact that both genotypes have nearly simultaneously re-emerged after years of relatively little activity suggests that similar forces may be driving their re-emergence. This hypothesis is reinforced by our recent experience of substantial geographical spread and massive increased disease incidence of the four dengue serotype viruses which, as noted earlier, are vectored also by Aedes aegypti mosquitoes in human-mosquito-human urban transmission cycles41. Factors attributed to dengue emergence include increased human travel, urbanization of human populations, uncontrolled urban growth (leading to increases in Aedes aegypti breeding sites), and lack of adequate control measures42,43. While dengue incidence in the Americas has been increasing for more than three decades, large increases have occurred in the last decade, suggesting an epidemiological turning point highly permissive for the transmission and spread of Aedes aegypti-vectored arboviruses. This point was recently reinforced by the spread throughout the Pacific44,45 and into the Americas (Brazil)46,47 for the first time of Zika virus, a flavivirus also spread from human to human via Aedes aegypti.\n\nAs mentioned previously, chikungunya outbreaks now occurring globally are caused by both ECSA and Asian genotypes. The current ECSA outbreak began on Lamu Island on coastal Kenya in 200411. This outbreak involved an estimated 13,500 persons, which was quite substantial compared to other contemporary African chikungunya outbreaks. Eight months later on Comoros, an island off the coast of Tanzania, an outbreak involved nearly 215,000 of that island’s residents48. Phylogenetic analysis showed that the causative ECSA genotype virus was nearly genetically identical to the Lamu Island outbreak, suggesting that the Comoros outbreak was simply an extension of the Lamu Island outbreak49. A small outbreak on La Reunion Island also began in 2005, an island with very low Aedes aegypti populations. Atypical of recent chikungunya outbreaks, this outbreak smouldered until December 2005 when incidence dramatically increased, eventually involving >244,000 persons50. Viral isolates collected in 2006 contained an envelope protein mutation (E1: A226V) that was found to increase viral fitness in Aedes albopictus mosquitoes51,52, which led to the hypothesis that the lack of abundant Aedes aegypti populations produced the outbreak’s slow onset, but incidence picked up when the viral mutation increased transmission efficiency for the abundant Aedes albopictus mosquito.\n\nThe ECSA virus containing the E1: A226V mutation subsequently spread from La Reunion Island to India by 2006, where over 1 million cases were reported in the first year alone53. Activity in India still continues nearly a decade later. From India, the Indian Ocean lineage strain spread to Southeast Asia and to northern Italy17,54. The Italian outbreak, which involved approximately 300 persons, was significant as this was the first outbreak documented in a subtropical climate in an area where the only vector species was Aedes albopictus.\n\nAutochthonous transmission of an ECSA genotype virus was identified for the first time in the Americas in Brazil in 201455. Subsequently, this virus has spread to multiple areas in that country. The index case was a traveller from Angola and the virus does not contain the E1: A226V mutation, suggesting that it might have limited infectivity for Aedes albopictus.\n\nThe Asian genotype has spread throughout the Pacific in recent years. A small 2011 outbreak in New Caledonia began after two infected travellers returned from Indonesia where the virus had been circulating continuously for at least a decade56. The Asian genotype subsequently was noted during outbreaks in the following locations: Papua New Guinea (2012); Yap Island, Federated States of Micronesia (2013); Tonga, Samoa, American Samoa, Tokelau, French Polynesia (2014); and Kiribati and the Cook Islands (2015)44,47,57.\n\nHowever, the most dramatic expansion occurred in the Americas where autochthonous transmission began on the Island of St. Martin in 201358. Genetic sequencing indicated that the virus was similar to chikungunya viruses recently identified from the Federated States of Micronesia, Philippines, and Indonesia59. Within a year, the virus spread to 26 islands and 14 mainland countries, resulting in more than 1 million reported cases. As of September 2015, 1.7 million cases and 240 deaths were reported from 45 of the 53 countries or territories reporting to the Pan American Health Organization (Figure 1). The true number of affected persons is undoubtedly substantially higher; since laboratory confirmation is completed in only a fraction of cases, infected individuals often do not seek medical care, and reporting may be incomplete. For example, Cuba has not reported any cases, yet infected travellers have returned to the United States from that country. While activity in many island countries in the Caribbean has decreased or stopped, transmission continues in most mainland countries.\n\nIn 2014, a total of 2788 cases were reported among travellers returning to the United States, nearly all from the Caribbean and Latin America. Despite this large influx, only 11 instances of autochthonous transmission have been identified in the contiguous United States, all in Florida60.\n\n\nFuture outlook\n\nThe continuing anthropogenic factors promoting the emergence and spread of chikungunya and other Aedes aegypti-borne diseases suggest that the current global epidemic will continue to spread to previously unaffected areas for some time. However, as the outbreak continues, herd immunity in humans will eventually curtail the scope and frequency of these outbreaks since humans are the virus’s only significant vertebrate host in urban settings and life-long immunity follows infection. The level of herd immunity required to stop outbreaks is unknown and likely varies according to local underlying transmission dynamics, such as human population size and mosquito abundance. Studies demonstrated that upwards of 60% of the population became infected during some outbreaks48, after which outbreaks ceased or were greatly reduced. One or two transmission seasons seem to be sufficient to curtail transmission activity in small, isolated populations such as islands, as has occurred in smaller Pacific Islands recently and now appears to be occurring in some Caribbean islands. At the other extreme, outbreaks in India, with its large and dispersed population, have moved from place to place now for nearly a decade, as they had in the early 1960s and 1970s. However, even in India, the reduction in susceptible populations had been apparently sufficient to prevent another large outbreak for 32 years.\n\nIn the Americas, several critical questions remain. As mentioned previously, the eventual duration and extent of epidemic activity is unknown, but will likely vary geographically. It is also unknown whether a sylvatic cycle will develop, enabling viral persistence without human-to-human transmission. The current outbreak’s substantial geographical reach and incidence make enduring sylvatic transmission a likely possibility if a mechanism exists for it to do so. Another question is to what extent the ECSA genotype introduced in Brazil will spread, which has important implications discussed later.\n\nThe potential for large chikungunya virus outbreaks on the fringes of the Aedes aegypti distribution, such as the southern United States, seems limited given our experiences with dengue61. Hundreds of dengue-infected travellers entering the contiguous United States are reported each year62, but Aedes aegypti-vectored dengue outbreaks have been relatively infrequent, focal and self-limited. One exception was a dengue outbreak spanning two years and involving several hundred persons in Key West, Florida, the most southern point in the contiguous United States63. However, most dengue outbreaks have occurred in Texas when large outbreaks in northern Mexico spilled over into towns along the border. Investigations in Texas showed that sociological conditions that limit contact with the indoor-biting Aedes aegypti mosquito, such as the use of air conditioning, greatly limited transmission despite the presence of abundant Aedes aegypti populations63–66. Dengue outbreaks have not occurred in areas with Aedes albopictus but without Aedes aegypti in the contiguous United States.\n\nSo far, it appears that chikungunya virus is following a similar pattern to dengue in subtropical and temperate areas of the United States. The thousands of infected returning travellers have produced only a handful of identified autochthonous transmission cases and no outbreaks. This dynamic could change, however, if large chikungunya outbreaks in northern Mexico spill over into border towns, as has occurred for dengue.\n\nOf some concern, Aedes albopictus is endemic in much of the southern and eastern United States67 and laboratory experiments show that Aedes albopictus mosquitoes collected throughout the Americas are generally competent to transmit the Asian genotype chikungunya virus68,69. Nevertheless, to date the Italian outbreak of 2007 is the only documented subtropical outbreak vectored solely by Aedes albopictus and this involved the ECSA virus with the E1: A226V mutation54. The ECSA genotype now circulating in Brazil may be less competent for transmission by Aedes albopictus mosquitoes than ECSA strains circulating in the Old World, as it does not possess the E1: A226V mutation, but it could prove problematic if the virus acquires mutations that increase vector competence and is introduced to subtropical and temperate areas endemic for Aedes albopictus.\n\n\nOptions for control\n\nHistorically, control of mosquito-transmitted viruses has relied heavily upon efforts aimed at reducing mosquito populations. These control activities focus on eliminating mosquito larval habitat and adulticiding. However, because Aedes aegypti and Aedes albopictus mosquitoes are container-breeding species that will lay eggs in nearly any water habitat, larval control efforts are a challenging, if not impossible, task70. Control efforts aimed at reducing adult mosquito populations also fail, because truck-based or aerial pesticide applications do not reach many adults, which rest and bite indoors. Indoor residual pesticide spraying might be effective, but is impractical in large urban areas. In addition, the threshold Aedes aegypti population levels required to stop chikungunya transmission are unknown, but are likely quite low. Ominously, Singapore, a country with perhaps the most effective Aedes aegypti control program globally, has experienced large chikungunya outbreaks vectored by both Aedes aegypti and Aedes albopictus71,72. Several new Aedes aegypti control approaches are under development, but none are currently ready for widespread use73–75.\n\nAn alternative control option is the development of a chikungunya vaccine. Several platform options have been explored ranging from virus-like particles, live attenuated variants, virus-vectored products, subunit vaccines, DNA vaccines, or inactivated products76. At least nine distinct options have been examined in pre-clinical research with most demonstrating the potential for protection in animal model systems. Only three have been tested in clinical trials: a live attenuated product developed by scientists at the Walter Reed Army Institute of Research (WRAIR) in the 1980s and 1990s77,78, a virus-like particles product developed recently at the National Institutes of Health79, and a measles-vectored product expressing the chikungunya virus structural genes80. While all appear to be promising candidates, the likelihood of a product reaching a commercial market are slim, due to the unpredictable nature of chikungunya virus outbreaks, the challenges associated with performing efficacy trials, and the uncertainty of revenue generating capacity. However, despite the challenges, a vaccine is one of the best options for preventing further outbreaks.\n\n\nConcluding remarks\n\nUrbanization, human travel, viral adaption, lack of effective control measures, and spread of new vectors likely have contributed to recent re-emergence of chikungunya. The current global outbreak, unprecedented in its size and geographical scope, is comprised of many smaller outbreaks that extend from place to place via human movement, and continue unabated until sufficient herd immunity in local human populations develops, or changes in other conditions, such as weather, inhibit further transmission. In smaller, island populations only one or two transmission seasons seem sufficient to greatly curtail or eliminate transmission; whereas, in larger populations such as India, transmission may extend beyond a decade. Like many other mosquito-borne diseases, it is very difficult to predict if and when a chikungunya outbreak will occur in any given location. Another uncertainty is the risk of epidemics in subtropical and temperate regions of the world where Aedes albopictus is a potential vector. The Italian outbreak demonstrated the possibility of such outbreaks, but to date outbreaks have yet to materialize in the United States, despite thousands of imported cases. While new chikungunya vaccines seem an attractive control possibility, many obstacles exist for their eventual commercialization. The dramatic spread of the dengue, chikungunya, and Zika viruses in recent years highlights the urgent need to identify scalable and cost-effective Aedes aegypti control options.\n\n\nDisclaimer\n\nThe findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention.",
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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLanciotti RS, Valadere AM: Transcontinental movement of Asian genotype chikungunya virus. Emerging Infect Dis. 2014; 20(8): 1400–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKendrick K, Stanek D, Blackmore C, et al.: Notes from the field: Transmission of chikungunya virus in the continental United States--Florida, 2014. MMWR Morb Mortal Wkly Rep. 2014; 63(48): 1137. PubMed Abstract | Faculty Opinions Recommendation\n\nEisen L, Moore CG: Aedes (Stegomyia) aegypti in the continental United States: a vector at the cool margin of its geographic range. J Med Entomol. 2013; 50(3): 467–78. PubMed Abstract | Publisher Full Text\n\nMohammed HP, Ramos MM, Rivera A, et al.: Travel-associated dengue infections in the United States, 1996 to 2005. J Travel Med. 2010; 17(1): 8–14. 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PubMed Abstract | Free Full Text | Faculty Opinions Recommendation\n\nScott TW, Morrison AC: Vector dynamics and transmission of dengue virus: implications for dengue surveillance and prevention strategies: vector dynamics and dengue prevention. Curr Top Microbiol Immunol. 2010; 338: 115–28. PubMed Abstract | Publisher Full Text\n\nLeo YS, Chow AL, Tan LK, et al.: Chikungunya outbreak, Singapore, 2008. Emerging Infect Dis. 2009; 15(5): 836–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOon LL, Ng LC: Chikungunya in Singapore - the Battle Continues. Ann Acad Med Singapore. 2014; 43(6): 325–7. PubMed Abstract | Faculty Opinions Recommendation\n\nCarvalho DO, McKemey AR, Garziera L, et al.: Suppression of a Field Population of Aedes aegypti in Brazil by Sustained Release of Transgenic Male Mosquitoes. PLoS Negl Trop Dis. 2015; 9(7): e0003864. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLambrechts L, Ferguson NM, Harris E, et al.: Assessing the epidemiological effect of wolbachia for dengue control. Lancet Infect Dis. 2015; 15(7): 862–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nIturbe-Ormaetxe I, Walker T, O' Neill SL: Wolbachia and the biological control of mosquito-borne disease. EMBO Rep. 2011; 12(6): 508–18. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAhola T, Courderc T, Ng LF, et al.: Therapeutics and vaccines against chikungunya virus. Vector Borne Zoonotic Dis. 2015; 15(4): 250–7. PubMed Abstract | Publisher Full Text\n\nHoke CH Jr, Pace-Templeton J, Pittman P, et al.: US Military contributions to the global response to pandemic chikungunya. Vaccine. 2012; 30(47): 6713–20. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLevitt NH, Ramsburg HH, Hasty SE, et al.: Development of an attenuated strain of chikungunya virus for use in vaccine production. Vaccine. 1986; 4(3): 157–62. PubMed Abstract | Publisher Full Text\n\nChang LJ, Dowd KA, Mendoza FH, et al.: Safety and tolerability of chikungunya virus-like particle vaccine in healthy adults: a phase 1 dose-escalation trial. Lancet. 2014; 384(9959): 2046–52. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRamsauer K, Schwameis M, Firbas C, et al.: Immunogenicity, safety, and tolerability of a recombinant measles-virus-based chikungunya vaccine: a randomised, double-blind, placebo-controlled, active-comparator, first-in-man trial. Lancet Infect Dis. 2015; 15(5): 519–27. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11987",
"date": "19 Jan 2016",
"name": "Carina G.M. Blackmore",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11988",
"date": "19 Jan 2016",
"name": "Stephen Higgs",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-82
|
https://f1000research.com/articles/5-81/v1
|
19 Jan 16
|
{
"type": "Method Article",
"title": "A clinical test for visual crowding",
"authors": [
"Denis G. Pelli",
"Sarah J. Waugh",
"Marialuisa Martelli",
"Sebastian J. Crutch",
"Silvia Primativo",
"Keir X. Yong",
"Marjorie Rhodes",
"Kathryn Yee",
"Xiuyun Wu",
"Hannes F. Famira",
"Hörmet Yiltiz",
"Sarah J. Waugh",
"Marialuisa Martelli",
"Sebastian J. Crutch",
"Silvia Primativo",
"Keir X. Yong",
"Marjorie Rhodes",
"Kathryn Yee",
"Xiuyun Wu",
"Hannes F. Famira",
"Hörmet Yiltiz"
],
"abstract": "Crowding is a major limitation of visual perception. Because of crowding, a simple object, like a letter, can only be recognized if clutter is a certain critical spacing away. Crowding is only weakly associated with acuity. The critical spacing of crowding is lowest in the normal fovea, and grows with increasing eccentricity in peripheral vision. Foveal crowding is more prominent in certain patient groups, including those with strabismic amblyopia and apperceptive agnosia. Crowding may lessen with age during childhood as reading speed increases. The range of crowding predicts much of the slowness of reading in children with developmental dyslexia. There is tantalizing evidence suggesting that the critical spacing of crowding indicates neural density (participating neurons per square deg) in the visual cortex. Thus, for basic and applied reasons, it would be very interesting to measure foveal crowding clinically in children and adults with normal and impaired vision, and to track the development of crowding during childhood. While many labs routinely measure peripheral crowding as part of their basic research in visual perception, current tests are not well suited to routine clinical testing because they take too much time, require good fixation, and are mostly not applicable to foveal vision. Here we report a new test for clinical measurement of crowding in the fovea. It is quick and accurate, works well with children and adults, and we expect it to work well with dementia patients as well. The task is to identify a numerical digit, 1-9, using a new “Pelli” font that is identifiable at tiny width (0.02 deg, about 1 minarc, in normal adult fovea). This allows quick measurement of the very small (0.05 deg) critical spacing in the normal adult fovea, as well as with other groups that have higher critical spacing. Preliminary results from healthy adults and children are presented.",
"keywords": [
"Crowding",
"critical spacing",
"repeated letters",
"neural density"
],
"content": "Introduction\n\nCrowding is a major limitation of visual perception. Because of crowding, a simple object, like a letter, can only be recognized if clutter is a certain critical spacing away (Bouma, 1970; Levi, 2008; Pelli & Tillman, 2008). That needed spacing grows linearly with eccentricity. In the fovea, we find the critical spacing of crowding to be about 0.05 deg in healthy adults, but it is much higher in certain clinical conditions, such as strabismic amblyopia and apperceptive agnosia (Song et al., 2014; Strappini, Pelli, Di Pace, & Martelli, unpublished report). Even when text is scaled in proportion to eccentricity, peripheral reading is slow and may be a useful model for slow central reading (Latham & Whittaker, 1996; Legge et al., 2001; Pelli et al., 2007). There is some evidence that the critical spacing of crowding drops during childhood as reading speed increases (Kwon et al., 2007; Pelli & Tillman, 2008). In shallow orthography languages, children with developmental dyslexia are doomed to read slowly relative to their peers and have an abnormally large critical spacing (Bouma & Legein, 1977; Martelli et al., 2009; O’Brien et al., 2005).\n\nCrowding is only weakly associated with acuity; some patient groups have greatly worsened crowding with near normal acuity and others have greatly worsened acuity with near normal crowding (Song et al., 2014). There is tantalizing evidence suggesting that the critical spacing of crowding indicates neural density (participating neurons per square deg) in the visual cortex (Strappini, Pelli, Di Pace, & Martelli, unpublished report). Similarly, crowding has been linked to reading speed in children and in patients, so it might be a useful assay of cortical health and development. Thus, for basic and applied reasons, it would be very interesting to measure foveal crowding clinically in children and adults with normal and impaired vision, and to track the development of crowding during childhood.\n\nIn normal vision, letter acuity size A and the critical spacing of crowding S crowding both grow linearly with eccentricity φ (Levi et al., 1985; Toet & Levi, 1992). Based on their measurements in the peripheral visual field, Song et al. (2014) provided these formulas:\n\nA = 0.029 (φ + 2.72 deg), (1)\n\nS crowding = 0.3 (φ + 0.45 deg) (2)\n\nThey showed that a letter is recognized only if it respects three limits: acuity, crowding, and overlap masking. They found no interaction among the three limits. Overlap masking can be completely avoided by using a center-to-center spacing of at least 1.4 letter widths.\n\nTo measure crowding with letters, the letters must be above the acuity limit, yet smaller than the critical spacing. This is easy to achieve in the periphery, where the critical spacing is much larger than the acuity. The ratio of critical spacing to acuity is\n\nS crowding / A = 0.3 (φ + 0.45 deg) / 0.029 (φ + 2.72 deg),\n\n= 10.3 (φ + 0.45 deg) / (φ + 2.72 deg), (3)\n\nAt large eccentricity, beyond 3 deg, this ratio is large and asymptotically approaches a value of about 10:1. Most studies of crowding are done in the periphery with small targets that are above acuity yet much smaller than the critical spacing to be measured. At small eccentricities, in the fovea, this ratio is approximately 1.7:1. The critical spacing 0.14 deg (according to the formula) is less than twice the threshold size of 0.08 deg. In fact, our foveal measurements reveal a smaller critical spacing, less than 0.1 deg, which is impossible to measure with 0.14 deg letters without overlap. The fovea is the hardest place to measure crowding, but that is the site that is most affected by deficits like strabismic amblyopia and is also the site associated with highest neural density, so it seems worth the effort.\n\nDespite this difficulty, there have been a number of reports of foveal crowding (Atkinson et al., 1988; Atkinson et al., 1986; Bedell et al., 2015; Bedell et al., 2013; Danilova & Bondarko, 2007; Hess et al., 2000; Kwon et al., 2007; Liu & Arditi, 2000; Malania et al., 2007; Semenov et al., 2000; Siderov et al., 2013). The thinnest discriminable target, for this purpose, is the Vernier target (Malania et al., 2007). Observers can detect a 0.01 deg misalignment of two thin lines in a Vernier target. However, binary discrimination is not an ideal clinical task because it yields information slowly. With two choices there is a high, 50%, chance of correctly guessing.\n\nFor faster testing, we wanted to use letter identification, with many possible letters, to minimize the guessing rate (Pelli & Robson, 1991). We needed a font that can be identified at a tiny width, a small fraction of the 0.05 deg critical spacing we seek to measure. We evaluated many fonts, and designed several of our own, to achieve a legible width approaching that of a vernier target. We call the new optotypes the “Pelli” font. It has a 5:1 aspect ratio and has a stroke width that is one half the letter width. The Sloan letters, much used in clinical testing, and designated as the standard optotypes for acuity testing in the USA, have a 1:1 aspect ratio and a stroke width of 1/5 the letter size (Sloan, 1959). Both fonts are displayed in Figure 1.\n\nThe new “Pelli” font has been designed to measure the spacing threshold. The Sloan font was designed by Louise Sloan to measure the size threshold, and has become the US standard for acuity testing (Sloan, 1959). (See Software availability.)\n\nFigure 2 allows you to test your own eye. The figure is an acuity test chart, but this test is unusual in focusing exclusively on width. It measures the smallest legible width for three fonts. On each line, the letters of the several fonts have various heights, but they all have the same width. From left to right, the fonts are Pelli, Gotham (Condensed Light), and Sloan. The Gotham font, from Hoefler and Co. (http://www.typography.com), is a commercial font for general text setting, with some attention given to performing well at small sizes, e.g. in tables. It comes in a wide variety of styles including Narrow and Condensed, and, of those we tried, the “Condensed Light” style gave the smallest legible width of 0.04 deg. We also tested two other fonts that have been designed to perform well at small visual angles: Hoefler and Co. Retina Micro font, designed for stock price tables in the Wall Street Journal and Clearview Hwy 1-B, designed for highway signs and adopted as the standard in many US states.\n\nOn each horizontal line of numbers and letters there are three fonts, and all characters in the line have the same width, though their heights vary greatly. The next line down is always smaller by a factor of 2-0.5 = 71%. Thus, going down two lines halves the letter size. The Sloan font, or optotype, is the USA standard for acuity testing. It has a 1:1 aspect ratio. Among the commercially available fonts that we tested, Gotham Condensed Light, with an aspect ratio 2.8:1, has the narrowest legible width. We created several experimental fonts (Arouet and Sticks, not shown) and finally created the “Pelli” font, which has the narrowest legible width. It has a 5:1 aspect ratio. Sloan’s stroke is 1/5 its width; Pelli’s stroke is 1/2 its width. In our sample of normally sighted adults, threshold width is about 0.02 deg for Pelli, 0.04 deg for Gotham (Condensed Light), and 0.05 deg for Sloan. You can use this chart to confirm this for your own eyes. At any viewing distance greater than 2 m, once you reach your limit for Sloan, you’ll be able to read four more lines of the Pelli font.\n\nTo test children as young as 4 years, we considered the use of popular pictograms, such as Lea Symbols and Patti Pics, used for illiterate testing, but they seemed unlikely to yield the tiny threshold width we need (Mercer et al., 2013). Thus, we decided to use numbers, anticipating that most children will have some familiarity. We gave each child a page with the 9 possible numbers so that they can respond by pointing instead of speaking if that seems easier. We’ve had good results from this with the several children we have tested so far.\n\nThe new test uses the digits 1–9, familiar to most children and patients. The 9 categories are sufficiently many to yield a low guessing rate (1/9) for fast threshold estimation. A new font, “Pelli”, with no internal white space, designed for this test with help from Hannes Famira, a professional font designer, is legible down to very small width: 0.02 deg (1.2 minarc) in the normal adult fovea. In the same spirit as David Regan’s repeat-letter acuity chart (Regan et al., 1992), our test alternates two different target digits over the whole display. These two alternating targets crowd each other. As in Regan’s chart, no matter where the observer’s eye lands on the screen, a target will be imaged on the observer’s fovea, so the test can accurately assess foveal function even in observers with poor fixation.\n\nFigure 3 demonstrates the principle. There are two charts, one using the Pelli font, the other using the Sloan font. Each chart has two halves, left and right. These charts measure threshold spacing. Any given row has the same letter or number spacing (center to center), all the way across within each chart and across charts. The two halves of each chart have different character sizes. The ratio of spacing to size is 1.2 on the left and 1.8 on the right, on both charts. When you read down as far as you can go, you might be able to read farther down the left half because it has larger letters. The left-side letters are 1.8/1.2=1.5 times bigger, and the successive rows of the chart are approximately 1.4:1, so, if you are size-limited, you will read one line farther down on the left side. However, if you are spacing-limited, the bigger letters won’t help. This left-right difference is diagnostic. The critical spacing is small, so in order to reach it, letters must be legible at very narrow width. Testing ourselves, we find no left-right difference in our limit of reading on the Pelli chart (left). We do find a one-line difference on the Sloan chart on the right. Thus the Pelli font allows measurement of critical spacing in the healthy fovea, and the Sloan font does not.\n\nThe center-to-center letter or number spacing is fixed for each row, all the way across both charts. See text for details.\n\nIn our new test, the QUEST adaptive procedure adjusts the spacing of each chart to efficiently estimate threshold spacing (Watson & Pelli, 1983). Size is proportional to spacing, usually with a 1.4:1 ratio of spacing to size. Once overlap masking has been avoided, a target letter is identifiable if and only if the target and flankers satisfy both the size limit of acuity and the spacing limit of crowding.\n\nThe observer is asked to identify both targets in each presentation, in any order, and each identification response counts as a trial, so each presentation yields two trials. In 20 trials (i.e. 10 presentations) QUEST achieves an accurate estimate of threshold. We present preliminary results showing that the measured threshold spacing is practically independent of the spacing-to-size ratio used to measure it.\n\nIn normally sighted adults, Regan’s repeat-letter acuity chart yields the same acuity as a single-letter chart. That is perhaps surprising, since one might expect crowding. However, the studies reported by Pelli et al. (2004) included experiments showing that simple targets are not crowded by identical flankers, but that finding was not discussed in the paper. Presumably the flankers contribute the same features as the target, so combining features from both yields the same summary statistics as from the target alone, and thus identification is unaffected.\n\n\nMethods\n\nAll stimuli are presented on a laptop screen. The observer sits at a long viewing distance (2 to 10 m) away from the display. We compute the minimum viewing distance to achieve at least 400 pixel/deg, so that a 0.02 deg letter will be at least 8 pixels wide. The minimum distance depends on the screen resolution of the particular laptop. The MATLAB formula is\n\nminViewingDistanceCm=57*(minPix/letterDeg)/(screenWidthPix/screenWidthCm); (4)\n\nwhere letterDeg=0.02, minPix=8.\n\nOur experiments ran in MATLAB 2015b with the Psychtoolbox 3.0.12 extensions on laptop computers running OS X or Windows (Kleiner et al., 2007). The Psychtoolbox software is available, free (http://psychtoolbox.org). Our testing program is called CriticalSpacing.m (see Data and software availability). We are making it available, and hope this will encourage more investigators to measure the critical spacing of crowding.\n\nWhen testing, we use a wireless keyboard to receive the observer’s responses, since the screen is so far away. Each presentation is a static chart. We ask healthy adults to respond to each chart by typing the character (digit) corresponding to each of the one or two targets presented. Invalid keys are dead and are ignored. When a valid key is typed, it is echoed by computer speech, e.g. “3”. Each correct response is followed by a faint beep. For each presentation, the scoring ignores the order of responses. The observer is informed that the two targets are always different, so that the observer must respond with two digits. Typing the same key again is ignored. After both target responses have been recorded, if testing a child, the computer randomly says, “Good”, “Very good”, or “Nice”.\n\nA green progress bar is always present on the right side of the screen and grows, after each presentation, from the bottom of the screen, reaching the top of the screen at the end of the run (usually 10 presentations). The computer says “Congratulations” at the end of the run. Figure 4 shows screenshots taken during testing.\n\nThe green bar at the right of each screenshot indicates progress through the run of 20 trials (ten presentations of repeated targets or 20 presentations of single targets). Threshold for spacing with repeated targets (left upper) and a single target (left below). Threshold for size with repeated targets (right upper) and with a single target (right below).\n\nThe static presentation can have one of four configurations. Each measures a size or spacing threshold, using single or repeated targets.\n\nIn the REPEATED-TARGETS conditions there are two different targets; in the SINGLE-TARGET conditions there is one target. The observer is asked to report the targets. The chart is displayed until the observer has given a response for each target (one or two).\n\nThe display consists of characters all drawn at the same size from one font and alphabet. We are most interested in our new Pelli font, using “123456789” as possible targets, but we have also tested Sloan, using “DHKNORSVZ”, and Gotham (Condensed Light), using “123456789”. The entire run uses a single ratio of spacing to size, typically 1.4. QUEST (included in the Psychtoolbox) controls the size or spacing; the other parameter tracks it proportionally. QUEST reports horizontal size and spacing. When characters have an aspect ratio that is not 1:1, the spacing is proportional, i.e. vertical spacing is proportional to height and horizontal spacing is proportional to width.\n\nOn a REPEATED-TARGETS presentation, if the targets were repeated out to the edge of the display, the outermost targets would be exposed on one side and would be less crowded. Our instructions try to minimize this by asking observers to concentrate on the middle of the display. And our design prevents escape from crowding by using a non-informative “margin” character around the edge on every REPEATED-TARGETS presentation. When the alphabet is “DHKNORSVZ”, we use “X” on the margin. When the alphabet is “123456789”, we use “$” on the margin. This avoids problems with edge effects.\n\nOne target is at the center of the display, and other characters are added. In the SINGLE conditions, for SIZE, the target remains alone; for SPACING, we add four random flankers (drawn randomly from the alphabet), left and right, up and down.\n\nIn the REPEATED-TARGETS condition, for SIZE, the screen is divided in two, left and right; each half has its own target. The target is repeated left and right and up and down to fill the display, except for the screen margin. For SPACING, the two targets alternate, left and right and up and down, to fill the whole display, except for the screen margins.\n\nThe threshold estimation procedure is like that used by Song et al. (2014). One parameter (horizontal size or spacing) is controlled by QUEST. The other parameter scales proportionally in a fixed ratio of spacing to size (Figure 5), which is usually 1.4:1, but we also tested other ratios QUEST assumes a Weibull function describing probability of threshold versus log size or spacing and estimates the threshold parameter alpha. The steepness parameter beta is set at 3.0. Each run is 20 trials. Presentations with repeated targets have two targets and thus count as two trials. Presentations with single targets yield just one trial. At the end of the run, the QUEST procedure provides an estimate of threshold.\n\nTo create the Pelli font, we made sketches on paper, which we viewed from a great distance and adjusted to enhance recognition. The sketches were then drawn in GraphicConverter and further adjusted. These pixel-based sketches were traced in RoboFont (Version 1.7). The descender was set to 0 units and x-height, ascender and cap height were set to 1000 units. All characters of this fixed pitch or monospaced font were set to a setwidth of 200 units, except that the space and non-breaking space characters were set to 100 units width. The resulting cubic outlines were generated into an OpenType font. See Data and software availability.\n\n\nResults\n\nWe measured threshold width for various fonts that have a reputation for legibility at small angular subtense (Figure 6). ClearviewHwy (www.clearviewhwy.com) is designed for highway signs, has been approved by the US government, and has been adopted by several states, including New York. Retina Micro was designed for typesetting stock price tables in the Wall Street Journal. Gotham (Hoefler & Co.) is an all-round font that comes in a wide range of styles including a very thin Compressed Light. These thresholds were all measured on one experienced healthy adult observer. Standard errors are about 5% of the plotted means. We are surprised by the cluster including Sloan near 0.05 deg. Only the Pelli font escapes to achieve a much smaller threshold size, of 0.02 deg for this observer, XW.\n\nWe measured threshold spacing with repeated targets with the Pelli font on 6 observers (O1–O4 four adults, C1–C2 two 8-year-old children, all healthy) at 3 spacing:size ratios: 1.2, 1.5, 1.8. We collected each threshold once (4 observers, U Rome-Martelli), twice (1 observer, NYU-Pelli-Qiu), or six times (1 observer, NYU-Pelli-Wu). For the latter two observers, we also collected the same number of threshold spacings with a single target.\n\nFigure 7 shows these threshold spacing for the Pelli font. Observers C1 and C2 are 8-year-old children; the rest are adults.\n\nSpacing thresholds were measured with the Pelli font. For each observer, we fit a linear regression line to each kind of threshold (single or repeated target) that was measured at several spacing:size ratio. The slopes are practically zero, showing that threshold spacing of each observer is conserved across this range of spacing-to-size ratio. This is consistent with spacing-limited threshold and inconsistent with the unit slope of a size limit.\n\nA single threshold measured by co-varying size and spacing might be hitting either a size or spacing limit. (Overlap masking is negligible at the large ratios of size to spacing that we used.) The hypothesis that the thresholds are spacing limited predicts that the spacing threshold will be independent of the spacing:size ratio. The hypothesis that the thresholds are size limited predicts that the spacing threshold will be proportional to the spacing:size ratio. Thus the two hypotheses predict that the measured spacing thresholds will have a log-log slope of 0 or 1, if they are spacing- or size-limited, respectively. We did linear regressions to estimate the log-log slope of all the data for each of the 8 observers. Across all the observers the log-log slope mean±se is 0.02±0.17 which is insignificantly different from zero and about 6 standard errors below 1. This confirms that these spacing thresholds are spacing-, not size-, limited.\n\nThreshold spacing mean±se was 0.065±0.006 (repeated target) and 0.049±0.004 (single target). This small difference (0.065 vs. 0.049 deg) is significant, about three standard errors. The slightly stronger crowding in the repeated-target condition is very likely because the repeated target was flanked on all four sides by other digits, whereas the single target was flanked only on left and right (the exception noted above in the SPACING & SIZE table.).\n\nTo better compare the repeated- and single-target estimates of threshold spacing with flankers on all four sides, we measured both 4 times on two observers (Table 1). The repeated-target thresholds are 9% higher.\n\nEach threshold was measured 4 times.\n\n\nDiscussion\n\nStrappini, Pelli, Di Pace, and Martelli (unpublished report) note that the lesions in apperceptive agnosia are accidental and diverse, from which one might expect diverse effects. Instead they find that the known diversity of the apperceptive agnosic population is roughly accounted for by a one-parameter model: the critical spacing of crowding. Crowding limits vision in the periphery, in strabismic amblyopes, and in patients with apperceptive agnosia, making it urgent to know what drives crowding. Acuity does not. Song et al. (2014) report a double dissociation of acuity and crowding: apperceptive agnosia worsens crowding while sparing acuity, and anisometropic amblyopia worsens acuity while sparing crowding. This shows that acuity and crowding are functionally independent. So what drives crowding? Pelli (2008) shows that the critical spacing of crowding is a fixed distance on the cortical surface, 6 mm in V1. Perhaps the extent of crowding reflects the number of cortical neurons per deg2 participating in the recognition task. This neural density may be reduced by lower cortical magnification (in the periphery), take over by the other eye (in strabismic amblyopia), or cell death (in agnosia).\n\nPosterior Cortical Atrophy (PCA) is Alzheimer’s disease occurring in the visual cortex, which is one etiology for apperceptive agnosia (Crutch, 2014). Crutch & Warrington (2007); Crutch & Warrington (2009) showed that the patients’ reading difficulties are well described as crowding. In their study of crowding in 26 PCA patients, Yong et al. (2014) report a correlation between crowding and lower grey matter volume within the right collateral sulcus, between the fusiform and lingual gyri. With regard to neural density, crowding in the central vision of the agnosic patients might reflect limited plasticity of the ventral stream, i.e. insufficient recruitment of other neurons to entirely make up for the loss in neural density.\n\nWe will be using this new test to measure foveal crowding in children and adults, healthy and patients, to develop norms and evaluate the possibility that critical spacing might track neural density.\n\n\nConclusion\n\nThe critical spacing of crowding in the fovea seems to be an important measure of visual function and cortical health. This new test is a good way to measure it.\n\n\nData and software availability\n\nF1000Research: Dataset 1. Several size thresholds (in deg) for each font listed, 10.5256/f1000research.7835.d111930 (Pelli et al., 2016a).\n\nF1000Research: Dataset 2. Threshold spacing of six observers at several values of spacingOverSize, 10.5256/f1000research.7835.d111931 (Pelli et al., 2016b).\n\nF1000Research: Dataset 3. 4 spacing thresholds for each of 2 conditions (single and repeated target) for each of two observers, 10.5256/f1000research.7835.d111932 (Pelli et al., 2016c).\n\nThe “Pelli” and Sloan fonts are available for noncommercial research use from GitHub: https://github.com/denispelli/Eye-Chart-Fonts/\n\nThe Sloan font file was created by Denis Pelli based on Louise Sloan’s specifications and used for the Pelli-Robson contrast sensitivity chart (Pelli et al., 1988). Louise Sloan’s design has been designated the US standard for acuity testing by the National Academy of Sciences, National Research Council, Committee on Vision (NAS-NRC, 1980). The C is a Landolt C. The C and O are particularly hard to discriminate from each other, so Elliott et al. (1990) recommend that most studies omit the C, as we did here.\n\nCriticalSpacing.m is our MATLAB program that uses any font to measure acuity and critical spacing. It allows testing with single or repeated targets. With single targets, it can test at any eccentricity, using brief presentation. We are making it available here, and hope this will encourage more investigators to measure the critical spacing of crowding. If you publish results collected with software based on our program, please cite us (this article). Thanks!\n\nhttps://github.com/denispelli/CriticalSpacing/\n\nWe welcome improvements to the software. Please use GitHub to submit your suggested change.\n\n\nConsent\n\nWritten informed consent for participation was obtained from each adult participant. Minors and their parents gave written consent. Children gave verbal assent and their parents gave written consent. All our human testing was conducted according to the principles expressed in the Declaration of Helsinki. Our protocols were approved by: NYU University Committee on Activities Involving Human Subjects IRB #13-9694 and #10-7375; Anglia Ruskin University Faculty Research Ethics Panel #FST/FREP/151538; Ethics Committee of the IRCCS Fondazione, Santa Lucia Rome (Prot. CE-PROG.480). Work at the Dementia Research Centre was ethically approved by the NRES Research Committee London - Queen Square (05/Q0512/47).",
"appendix": "Author contributions\n\n\n\nDenis conceived the method, designed the “Pelli” font, wrote the testing software CriticalSpacing.m, and reached out to the other authors to help test it. Denis wrote the first draft, and everyone else helped polish it. Sarah, Marialuisa, Sebastian, Silvia, Keir, Marjorie, and Kathryn helped adapt the testing to accommodate children and dementia patients. Sarah, Marialuisa, Silvia, Kathryn, Keir, and Xiuyun recruited and tested observers. Hörmet designed the “$” character in the Pelli font, helped write the MATLAB testing software, and helped analyze the results. Hörmet created an unfamiliar very thin “Sticks” font that was an important step towards the “Pelli” font. Hannes designed a new font Arouet for this project, which performed better than any other font available then; our tests with Arouet led to the design of the new Pelli font, which achieves smaller legible width. Hannes converted Denis’s PNG drawings into the computer-installable Pelli font. All authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by several grants: Evelyn Trust, Cambridge, UK to S.W. NSF BCS-1147543 to M.R. and an Alzheimer’s Research UK Senior Research Fellowship and ESRC/NIHR (ES/L001810/1) and EPSRC (EP/M006093/1) grants to S.C.\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\nThanks to Michelle Qiu for collecting data on effect of the spacing:size ratio. Thanks to Amy Belfi, Aenne Brielmann, Laura Suciu, and Lauren Vale for helpful comments.\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. PubMed Abstract | Publisher Full Text\n\nAtkinson J, Pimm-Smith E, Evans C, et al.: Visual crowding in young children. Doc Ophthalmol Proc Ser. 1986; 45: 201–213. Publisher Full Text\n\nBedell HE, Siderov J, Formankiewicz MA, et al.: Evidence for an eye-movement contribution to normal foveal crowding. Optom Vis Sci. 2015; 92(2): 237–245. PubMed Abstract | Publisher Full Text\n\nBedell HE, Siderov J, Waugh SJ, et al.: Contour interaction for foveal acuity targets at different luminances. Vision Res. 2013; 89: 90–95. PubMed Abstract | Publisher Full Text\n\nBouma H, Legein CP: Foveal and parafoveal recognition of letters and words by dyslexics and by average readers. Neuropsychologia. 1977; 15(1): 69–80. PubMed Abstract | Publisher Full Text\n\nBouma H: Interaction effects in parafoveal letter recognition. Nature. 1970; 226(5241): 177–178. PubMed Abstract | Publisher Full Text\n\nCrutch SJ, Warrington EK: Foveal crowding in posterior cortical atrophy: a specific early-visual-processing deficit affecting word reading. Cogn Neuropsychol. 2007; 24(8): 843–866. PubMed Abstract | Publisher Full Text\n\nCrutch SJ, Warrington EK: The relationship between visual crowding and letter confusability: towards an understanding of dyslexia in posterior cortical atrophy. Cogn Neuropsychol. 2009; 26(5): 471–498. PubMed Abstract | Publisher Full Text\n\nCrutch SJ: Elizabeth Warrington Prize Lecture. Seeing why they cannot see: understanding the syndrome and causes of posterior cortical atrophy. J Neuropsychol. 2014; 8(2): 157–170. PubMed Abstract | Publisher Full Text\n\nDanilova MV, Bondarko VM: Foveal contour interactions and crowding effects at the resolution limit of the visual system. J Vis. 2007; 7(2): 25.1–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElliott DB, Whitaker D, Bonette L: Differences in the legibility of letters at contrast threshold using the Pelli-Robson chart. Ophthalmic Physiol Opt. 1990; 10(4): 323–6. PubMed Abstract | Publisher Full Text\n\nHess RF, Dakin SC, Kapoor N: The foveal ‘crowding’ effect: physics or physiology? Vision Res. 2000; 40(4): 365–370. PubMed Abstract | Publisher Full Text\n\nKleiner M, Brainard D, Pelli D: What’s new in Psychtoolbox-3? Perception ECVP Abstract Supplement. 2007; 36. Reference Source\n\nKwon M, Legge GE, Dubbels BR: Developmental changes in the visual span for reading. Vision Res. 2007; 47(22): 2889–2900. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLatham K, Whitaker D: Relative roles of resolution and spatial interference in foveal and peripheral vision. Ophthalmic Physiol Opt. 1996; 16(1): 49–57. PubMed Abstract | Publisher Full Text\n\nLegge GE, Mansfield JS, Chung ST: Psychophysics of reading. XX. Linking letter recognition to reading speed in central and peripheral vision. Vision Res. 2001; 41(6): 725–743. PubMed Abstract | Publisher Full Text\n\nLevi DM, Klein SA, Aitsebaomo AP: Vernier acuity, crowding and cortical magnification. Vision Res. 1985; 25(7): 963–977. PubMed Abstract | Publisher Full Text\n\nLevi DM: Crowding--an essential bottleneck for object recognition: a mini-review. Vision Res. 2008; 48(5): 635–654. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu L, Arditi A: Apparent string shortening concomitant with letter crowding. Vision Res. 2000; 40(9): 1059–1067. PubMed Abstract | Publisher Full Text\n\nMalania M, Herzog MH, Westheimer G: Grouping of contextual elements that affect vernier thresholds. J Vis. 2007; 7(2): 1.1–7. PubMed Abstract | Publisher Full Text\n\nMartelli M, Di Filippo G, Spinelli D, et al.: Crowding, reading, and developmental dyslexia. J Vis. 2009; 9(4): 14.1–18. PubMed Abstract | Publisher Full Text\n\nMercer ME, Drover JR, Penney KJ, et al.: Comparison of Patti Pics and Lea Symbols optotypes in children and adults. Optom Vis Sci. 2013; 90(3): 236–241. PubMed Abstract | Publisher Full Text\n\nNAS-NRC: Recommended standard procedures for the clinical measurement and specification of visual acuity. Report of working group 39. Committee on vision. Assembly of Behavioral and Social Sciences, National Research Council, National Academy of Sciences, Washington, D.C. Adv Ophthalmol. 1980; 41: 103–48. PubMed Abstract\n\nO'Brien BA, Mansfield JS, Legge GE: The effect of print size on reading speed in dyslexia. J Res Read. 2005; 28(3): 332–349. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPelli D, Waugh S, Martelli M, et al.: Dataset 1 in: A clinical test for visual crowding. F1000Research. 2016a. Data Source\n\nPelli D, Waugh S, Martelli M, et al.: Dataset 2 in: A clinical test for visual crowding. F1000Research. 2016b. Data Source\n\nPelli D, Waugh S, Martelli M, et al.: Dataset 3 in: A clinical test for visual crowding. F1000Research. 2016c. Data Source\n\nPelli DG, Palomares M, Majaj NJ: Crowding is unlike ordinary masking: distinguishing feature integration from detection. J Vis. 2004; 4(12): 1136–1169. PubMed Abstract | Publisher Full Text\n\nPelli DG, Robson JG, Wilkins AJ: The design of a new letter chart for measuring contrast sensitivity. Clin Vision Sci. 1988; 2(3): 187–199. Reference Source\n\nPelli DG, Robson JG: Are letters better than gratings? Clinical Vision Sciences. 1991; 6(5): 409–411. Reference Source\n\nPelli DG, Tillman KA, Freeman J, et al.: Crowding and eccentricity determine reading rate. J Vis. 2007; 7(2): 20, 1–36. PubMed Abstract | Publisher Full Text\n\nPelli DG, Tillman KA: The uncrowded window of object recognition. Nat Neurosci. 2008; 11(10): 1129–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPelli DG: Crowding: a cortical constraint on object recognition. Curr Opin Neurobiol. 2008; 18(4): 445–451. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRegan D, Giaschi DE, Kraft SP, et al.: Method for identifying amblyopes whose reduced line acuity is caused by defective selection and/or control of gaze. Ophthalmic Physiol Opt. 1992; 12(4): 425–432. PubMed Abstract | Publisher Full Text\n\nSemenov LA, Chernova ND, Bondarko VM: Measurement of visual acuity and crowding effect in 3–9-year-old children. Human Physiol. 2000; 26(1): 16–20. Publisher Full Text\n\nSiderov J, Waugh SJ, Bedell HE: Foveal contour interaction for low contrast acuity targets. Vision Res. 2013; 77: 10–13. PubMed Abstract | Publisher Full Text\n\nSloan LL: New test charts for the measurement of visual acuity at far and near distances. Am J Ophthal. 1959; 48(6): 807–813. PubMed Abstract | Publisher Full Text\n\nSong S, Levi DM, Pelli DG: A double dissociation of the acuity and crowding limits to letter identification, and the promise of improved visual screening. J Vis. 2014; 14(5): 3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nToet A, Levi DM: The two-dimensional shape of spatial interaction zones in the parafovea. Vision Res. 1992; 32(7): 1349–1357. PubMed Abstract | Publisher Full Text\n\nWatson AB, Pelli DG: QUEST: a Bayesian adaptive psychometric method. Percept Psychophys. 1983; 33(2): 113–20. PubMed Abstract | Publisher Full Text\n\nYong KX, Shakespeare TJ, Cash D, et al.: Prominent effects and neural correlates of visual crowding in a neurodegenerative disease population. Brain. 2014; 137(Pt 12): 3284–99. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11982",
"date": "22 Jan 2016",
"name": "Arnold Wilkins",
"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\nVisual crowding is a poorly understood phenomenon, the neural origins of which are “hotly debated”1. It can occur at various levels in a perceptual hierarchy, including that of objects 1. Information is not necessarily lost but can become misappropriated, and, perhaps in consequence, critical spacing differs across stimulus categories 2. It is exaggerated in clinical cases to the extent that objects sometimes cannot be discerned unless presented singly in an uncluttered scene 3. The authors of this paper clearly take the view that crowding is one thing rather than several. It may or may not be one thing, but this does not matter here. Pelli and co-authors present some very challenging ideas and data, together with techniques that have exciting potential in the clinic.\n\nThe following are simply thoughts that have occurred to me on reading the report. They are not suggestions for alterations to the draft, with the exception of the list of items at the end. I have some reservations as to the use of highly contrasted spatially periodic material. We showed that single words and words in sentences are read by fluent readers about 10% more slowly when they have a high first peak in the horizontal autocorrelation of the image of the word. When the component strokes of the letters have a spatial periodicity, as in the word mum, the words are slower to read4. Jainta, Jaschinski and I showed that some of this slowing was due to the time taken for the eyes to re-establish vergence with minimum error following a saccade5. Similar considerations may apply to the use of periodic strokes in the Pelli font when the font is observed with both eyes. Some of the difficulty in reading the letters may be due to vergence correction. If so, monocular viewing may reduce the effects of the spatial periodicity relative to binocular viewing. One might expect the usual reduction in acuity with monocular viewing, but on this account the reduction might be less for the Pelli font because it compromises vergence? A second and related consideration concerns pattern glare. This term refers to the perceptual distortions and instability experienced by some observers on viewing spatially periodic arrays, in particular gratings with spatial frequency within one octave of 3 cycles/degree6. Such arrays would include those of similar letters presented across the page, as a result of both the letter strokes and the lines of letters, depending on scale. Perceptual distortions are usually accompanied by discomfort. Did observers report perceptual distortions, particularly instability (apparent movement) of the letters when presented in closely spaced arrays? If so, did they then have a higher threshold and report discomfort? If the Pelli font is to be used with children it would be nice to have data showing how readily (e.g. with what speed) they can name the digits 1-9 when presented in isolation, and how this changes with age. I suspect that digits in which a contour is represented as a filled square might be particularly difficult for young children to recognise. These digits include 4, 6, 8 and 9. Is it necessary to include these digits? Without them, the choice is 1 of 5, which is surely sufficient to be used in rapid clinical assessment. After all, most alphabetic charts use only a subset of the letters of the alphabet and this difference between the perceived set size and actual set size does not appear to present the problems that might be anticipated. As regards the particulars of the report:There is an infelicity somewhere in the sentence that currently reads: “That needed spacing grows linearly with eccentricity”. Please could the unpublished report by Strappini et al be made available for download? Please could we have some numbers to support the statement that “We’ve had good results from this with the several children we have tested so far.” Please could we have a citation or two to support the assertion that “Similarly, crowding has been linked to reading speed in children and in patients, so…” The statement that “As in Regan’s chart, no matter where the observer’s eye lands on the screen, a target will be imaged on the observer’s fovea” seems a little optimistic without a word or two of additional explanation. All in all, this is a fascinating piece of original research, and I hope the promise of clinical use is borne out. I, for one, would like to know whether there is a relationship between pattern glare and crowding. If there is, it might be possible to bring together two disparate aspects of the literature, which would have useful theoretical spin-off.",
"responses": [
{
"c_id": "1814",
"date": "16 Feb 2016",
"name": "Denis Pelli",
"role": "Author Response",
"response": "Professor A J WilkinsDepartment of PsychologyUniversity of Essex Dear Arnold, Thanks very much for your quick and thoughtful review. We appreciate your work on how periodic structure impairs acuity and reading (Wilkins et al. 1989; Wilkins et al. 2007; Jainta et al., 2010). Yes, reducing periodicity might allow a reduction of the size or spacing limit. It's interesting that one of the most revered experts on type design, Gerrit Noordzij (1985/2005), professor of typeface design at the Royal Academy of Fine Arts in The Hague, Netherlands, wrote that the best type has an even alternation of white and black, which seems to advocate optimizing font design by maximizing the same periodicity that your research recommends minimizing. We don’t yet have any reports of discomfort (Wilkins et al. 1984). We'll record such reports to correlate with results. Thanks. Our paper includes testing on two 8-year olds. Their time per trial and critical spacing were similar to those of the adults we tested. We will be collecting a lot more data on children, down to age 4. We'll keep track of which letters they have trouble with (time and accuracy), and, if necessary, we will adjust or drop (as you suggest) troublesome letters. We share your interest in individual differences in reading (Bouldoukian et al., 2002). In our study, Drs. Martelli (U. Rome Sapienza & IRCCS Fondazione Santa Lucia), Waugh (Anglia Ruskin U.), and Rhodes (NYU) are testing school-age children, including many with dyslexia or amblyopia. Regarding your numbered points:1. There is an infelicity somewhere in the sentence that currently reads: “That needed spacing grows linearly with eccentricity”. Yes, this would be better: \"This critical spacing grows linearly with eccentricity” 2. Please could the unpublished report by Strappini et al be made available for download?We can't. It’s under review at another journal. F1000Research journal style designates this as an \"unpublished report\". We have sent you a copy privately. 3. Please could we have some numbers to support the statement that “We’ve had good results from this with the several children we have tested so far.”As noted above, we reported our results on two 8 year olds. We will be testing children in Cambridge (Waugh), New York (Rhodes), and Rome (Martelli). 4. Please could we have a citation or two to support the assertion that “Similarly, crowding has been linked to reading speed in children and in patients, so…”See Fig. 9 and associated discussion in Pelli, D. G., & Tillman, K. A. (2008) The uncrowded window of object recognition. Nature Neuroscience, 11(10):1129 - 1135. doi: 10.1038/nn.2187 http://www.nature.com/neuro/journal/v11/n10/index.html#peThey cite:Kwon, M., Legge, G.E. & Dubbels, B.R. Developmental changes in the visual span for reading. Vision Res. 47, 2889–2900 (2007). 5. The statement that “As in Regan’s chart, no matter where the observer’s eye lands on the screen, a target will be imaged on the observer’s fovea” seems a little optimistic without a word or two of additional explanation.This may help: “Because the screen is covered with letters less than 1 degree apart, no matter where the eye lands, at least one letter will be imaged in the observer’s 1 deg foveola.”Thank you.Denis, on behalf of my coauthors. ReferencesBouldoukian, J., Wilkins, A. J., & Evans, B. J. (2002). Randomised controlled trial of the effect of coloured overlays on the rate of reading of people with specific learning difficulties. Ophthalmic and Physiological Optics, 22(1), 55-60. Jainta S, Jaschinski W, Wilkins AJ: Periodic letter strokes within a word affect fixation disparity during reading. J Vis. 2010; 10 (13): 2 PubMed Abstract | Publisher Full Text Noordzij G (1985/2005)The Stroke: Theory of Writing, Hyphen Press, London (published in Dutch in 1985, translated into English by Peter Enneson in 2005).http://uedata.amazon.com/The-Stroke-Writing-Gerrit-Noordzij/dp/0907259308 Wilkins AJ, Plant G, Huddy A: Neuropsychological principles applied to rehabilitation of a stroke patient. Lancet. 1989; 1(8628): 54 PubMed Abstract Wilkins A, Nimmo-Smith I, Tait A, McManus C, Della Sala S, Tilley A, Arnold K, Barrie M, Scott S: A neurological basis for visual discomfort. Brain. 1984; 107 ( Pt 4): 989-1017 PubMed Abstract Wilkins AJ, Smith J, Willison CK, Beare T, Boyd A, Hardy G, Mell L, Peach C, Harper S: Stripes within words affect reading. Perception. 2007; 36 (12): 1788-803 PubMed Abstract"
}
]
},
{
"id": "11983",
"date": "04 Feb 2016",
"name": "Christopher Tyler",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general, this is an excellent presentation of the capabilities of a new font designed for the measurement of foveal crowding. In relation to terminology, the standard physical and mathematical unit “arcmin” is to be preferred to the ill-formed “minarc”. In the Introduction it should be clarified that eq 3 is derived from eqs 1 & 2. Are the cited studies of foveal crowding compatible with a 1.7:1 ratio of crowding to acuity? The Vernier acuity limit is closer to 0.1 arcmin than 0.01 deg, and its ratio to its crowding spacing would be much greater than 10:1. According to Levi et al. (1985), the maximal crowding is at 3 arcmin for a ~0.1 arcmin foveal Vernier acuity, or a 30:1 ratio. Fig. 7 caption. “ratio” should be plural in “several spacing:size ratio”. The significance of the invariance of threshold with spacing:size ratio is obscure. It might be clearer to say “threshold spacing of each observer is proportional element size rather than conforming to a fixed spacing independent of element size”. The Discussion should specify what is 6 mm in V1. Is this the diameter of the (circular) crowding zone? In suggesting that the crowding is limited by the number of neurons/mm2 in cortex, it should clarify that the number of neurons within a 6 mm diameter crowding zone would be roughly 1.5 million just in V1, and perhaps 5 million throughout the visual hierarchy. Is this the number that are expected to participate in the acuity performance? The Discussion should reflect the studies of the Cavanagh on the attentional window hypothesis of crowding.",
"responses": [
{
"c_id": "1815",
"date": "17 Feb 2016",
"name": "Denis Pelli",
"role": "Author Response",
"response": "Dr. Christopher TylerSmith-Kettlewell Eye Research Institute Dear Christopher, Thank you for your timely and helpful review. In general, this is an excellent presentation of the capabilities of a new font designed for the measurement of foveal crowding. Thank you.In relation to terminology, the standard physical and mathematical unit \"arcmin\" is to be preferred to the ill-formed \"minarc\". Agreed. We’ll change to: arcmin. In the Introduction it should be clarified that eq 3 is derived from eqs 1 & 2. Yes. We’ll do that. Are the cited studies of foveal crowding compatible with a 1.7:1 ratio of crowding to acuity? We agree that our measure should be compared to the prior literature, but we think that it’s more appropriate to forget acuity and just compare crowding directly. The crowding-to-acuity ratio is dimensionless, a ratio of degrees to degrees, but it is not fundamental. Pelli et al. (2006) Fig. 5b and Pelli et al. (2007) Fig. 6, reproduced below, showed that critical spacing (center to center) is independent of the letter size used to measure it. They got the same critical spacing despite varying letter size over a 2:1 range. Pelli and Tillman (2008) Fig. 5 provides demos, reproduced below, showing that critical spacing is independent of target size. Thus we expect the critical spacing of crowding in the healthy fovea to be a fixed constant, independent of the font and letter size used to measure it. Acuity (threshold size) depends strongly on the font, so the variable ratio is less interesting than the critical spacing itself, which should not change.FIGURE: Critical spacing data from Pelli et al. (2007) Figure 6. Reproduced here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624758/figure/F2/The axes indicate position in the visual field, relative to the fixation point (grey “+” in upper left). In the upper right, also gray, we show a triplet: a target letter between two symmetrically arranged flankers. The colored contour lines trace out the center-to-center target-to-flanker spacing the observer required to achieve 80%-correct identification of the target letter. At each eccentricity, the black, red, and green curves represent different letter sizes. We used larger letters at more peripheral locations, but the large letter size (plotted green) was always approximately twice the small letter size (plotted red). The results show that the critical spacing is proportional to radial eccentricity and independent of letter size. FIGURE: Critical spacing demo from Pelli and Tillman (2008) Figure 5. Reproduced here:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624758/figure/F3/Critical spacing is independent of object and size. Fixating on a red minus, you will be unable to identify the middle object in that row unless you isolate the target object by hiding the flanking objects with your fingers (or two pencils). The ± is our estimate of the fixation point where you can just barely identify the target. Note that the task is easy when you fixate to the right of the ± and hard when you fixate to the left. In rows 1-2, 3-4, and 5-6, note that doubling object size has no effect on critical spacing. It is easy to measure the critical spacing of crowding in the periphery, where it has much longer range than most other effects. It is not easy to measure it in the fovea, where a measured spacing threshold may in fact be limited by acuity or overlap masking. Song et al. (2014) found distinct relationships between threshold size and spacing for the three mechanisms. Crowding has a fixed threshold spacing, independent of size. Acuity is a fixed threshold size, independent of spacing. Overlap masking produces a threshold spacing proportional to threshold size, with a ratio of 1.4. We look for these signatures here, taking conservation of threshold spacing across size as evidence for crowding. The conservation of our 0.05 deg estimate across letter size is strong evidence that it’s a crowding limit. Indeed, at the 0.05 deg threshold size of the Sloan font, two letters will be touching at a 0.05 deg center-to-center spacing. Legibility will be impaired by overlap masking, which extends to a spacing of 1.4 times the acuity size (Song et al.2014). Some studies used tiny targets (e.g. Vernier) to obtain crowding estimates consistent with this (e.g. Levi et al., 1985; Malania et al., 2007). Other studies used bigger targets, like the Sloan font, which cannot measure a spacing as small as that, and end up reporting larger threshold spacings that are limited by overlap masking or acuity, not crowding. We’ll add this point to the paper. Thanks! The Vernier acuity limit is closer to 0.1 arcmin than 0.01 deg, Oops. Yes, thanks for catching this. We’ll fix it. and its ratio to its crowding spacing would be much greater than 10:1. According to Levi et al. (1985), the maximal crowding is at 3 arcmin for a ~0.1 arcmin foveal Vernier acuity, or a 30:1 ratio. As noted above, the ratio is not conserved by crowding. Crowding conserves the threshold spacing. 3 arcmin is 0.05 deg, which agrees with our 0.05 deg estimate made with the Pelli font. Fig. 7 caption. “ratio” should be plural in “several spacing:size ratio”. Yes. Thanks. We’ll fix it. The significance of the invariance of threshold with spacing:size ratio is obscure. It might be clearer to say “threshold spacing of each observer is proportional element size rather than conforming to a fixed spacing independent of element size”. Uh oh. This is an important point, which we need to explain better. As noted above, conservation of threshold spacing, independent of size, is evidence of crowding (Pelli et al. 2006; Pelli et al. 2007; Song et al. 2014). Thus Fig. 3 shows that the Pelli font tests crowding, and the Sloan font does not, because the Pelli threshold spacing is conserved across size while the Sloan threshold is not. Thank you very much for insisting that we be clear. The Discussion should specify what is 6 mm in V1. Is this the diameter of the (circular) crowding zone? Good point. It’s radius, not diameter. 6 mm is the critical spacing, which is the radius of the crowding area, centered on the target (Pelli, 2008). At the visual field, the Bouma law tells us that threshold is linearly related to eccentricity. The logarithmic mapping of the cortical magnification factor (cortical position ∝ log eccentricity) results in a critical spacing on the surface of the cortex that is independent of eccentricity. In suggesting that the crowding is limited by the number of neurons/mm2 in cortex, it should clarify that the number of neurons within a 6 mm diameter crowding zone would be roughly 1.5 million just in V1, and perhaps 5 million throughout the visual hierarchy. Is this the number that are expected to participate in the acuity performance? The critical spacing of crowding is 6 mm in V1 and 5 mm in V2 (Pelli, 2008). The density of cortical neurons is conserved, about 370,000/mm2 in V1 and 150,000/mm2 in the rest of cortex, across individuals and mammalian species (Rockel et al. 1980; Braitenberg & Shüz, 1998), so a circle in V1 with 6 mm radius encompasses 41,000,000 neurons. That is an upper bound on number of participating V1 neurons. For V2, the radius is 5 mm, and the neural density is lower, so the radius encompasses 12,000,000 neurons. The Discussion should reflect the studies of the Cavanagh on the attentional window hypothesis of crowding. Yes, will do. Freeman & Pelli (2007) present a long discussion of He et al. (1996) and Intriligator & Cavanagh (2001).Thank you.Denis Pelli & Sarah Waugh on behalf of the authors,Denis & SarahReferences Braitenberg V, Schüz A (1998). Cortex: Statistics and Geometry of Neuronal Connectivity. Edn 2. Heidelberg, Germany: Springer Verlag. Freeman, J., & Pelli, D. G. (2007) An escape from crowding. Journal of Vision, 7(2):22, 1-14, doi:10.1167/7.2.22, http://journalofvision.org/7/2/22/ He, S. Cavanagh, P. Intriligator, J. (1996). Attentional resolution and the locus of visual awareness. Nature, 383, 334–337. http://www.ncbi.nlm.nih.gov/pubmed/8848045 Intriligator, J., & Cavanagh, P. (2001). The spatial resolution of visual attention. Cognitive Psychology, 43(3), 171-216. http://www.ncbi.nlm.nih.gov/pubmed/11689021 Levi, D. M., Klein, S. A., & Aitsebaomo, A. P. (1985). Vernier acuity, crowding and cortical magnification. Vision Research, 25(7), 963-977. http://www.ncbi.nlm.nih.gov/pubmed/4049746 Pelli, D. G., Palomares, M., & Majaj, N. J. (2004). Crowding is unlike ordinary masking: Distinguishing feature integration from detection. Journal of Vision, 4(12), 1136-1169, http://journalofvision.org/4/12/12/ Pelli, D. G., Tillman, K. A., Freeman, J., Su, M., Berger, T. D., & Majaj, N. J. (2007) Crowding and eccentricity determine reading rate. Journal of Vision, 7(2):20, 1-36, doi:10.1167/7.2.20. http://journalofvision.org/7/2/20/ Pelli DG (2008) Crowding: a cortical constraint on object recognition. Curr Opin Neurobiol. 18(4): 445–451.http://www.ncbi.nlm.nih.gov/pubmed/18835355?dopt=Citation Pelli, D. G., & Tillman, K. A. (2008) The uncrowded window of object recognition. Nature Neuroscience, 11(10):1129 - 1135. doi: 10.1038/nn.2187, http://www.nature.com/neuro/journal/v11/n10/index.html#pe Rockel AJ, Hiorns RW, Powell TP (1980) The basic uniformity in structure of the neocortex. Brain, 103:221-244. http://www.ncbi.nlm.nih.gov/pubmed/6772266"
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https://f1000research.com/articles/5-81
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https://f1000research.com/articles/5-78/v1
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19 Jan 16
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{
"type": "Review",
"title": "Recent advances in understanding dengue",
"authors": [
"Sophie Yacoub",
"Juthathip Mongkolsapaya",
"Gavin Screaton",
"Juthathip Mongkolsapaya",
"Gavin Screaton"
],
"abstract": "Dengue is an emerging threat to billions of people worldwide. In the last 20 years, the incidence has increased four-fold and this trend appears to be continuing. Caused by one of four viral serotypes, dengue can present as a wide range of clinical phenotypes with the severe end of the spectrum being defined by a syndrome of capillary leak, coagulopathy, and organ impairment. The pathogenesis of severe disease is thought to be in part immune mediated, but the exact mechanisms remain to be defined. The current treatment of dengue relies on supportive measures with no licensed therapeutics available to date. There have been recent advances in our understanding of a number of areas of dengue research, of which the following will be discussed in this review: the drivers behind the global dengue pandemic, viral structure and epitope binding, risk factors for severe disease and its pathogenesis, as well as the findings of recent clinical trials including therapeutics and vaccines. We conclude with current and future dengue control measures and key areas for future research.",
"keywords": [
"dengue",
"dengue virus",
"Flavivirus"
],
"content": "Introduction\n\nDengue has emerged in the last two decades as the most abundant vector-borne viral infection globally. The dengue virus belongs to the Flavivirus family and has four serotypes (DENV1-4), which are clinically indistinguishable. Latest estimates suggest 390 million infections of dengue occur each year, of which 100 million result in symptomatic disease1. Dengue may present as a spectrum of clinical syndromes from dengue fever, a non-specific febrile illness, through to severe dengue (Box 1) (replacing the original classification of dengue fever/dengue hemorrhagic fever [DHF]2). The 2009 World Health Organization (WHO) dengue guidelines have outlined a number of warning signs (Box 2) to assist triaging the often vast numbers of patients that can present to clinics in endemic areas; however, the ability to predict which patients will progress to severe disease remains challenging.\n\n\n\n• Severe plasma leakage leading to\n\n1) Shock and/or\n\n2) Fluid accumulation with respiratory distress\n\n• Severe bleeding\n\n• Severe organ involvement\n\nLiver: alanine transaminase or aspartate aminotransferase >=1000\n\nCentral nervous system: impaired consciousness\n\nHeart and other organs\n\n\n\n- Abdominal pain or tenderness\n\n- Persistent vomiting\n\n- Clinical fluid accumulation\n\n- Mucosal bleed\n\n- Lethargy/restlessness\n\n- Liver enlargement >2 cm\n\n- Laboratory increase in hematocrit concurrent with rapid decrease in platelet count\n\nOne of the defining features of severe disease is increased capillary permeability causing plasma leakage, which can lead to intravascular volume depletion and, if left untreated, shock and death. The underlying mechanisms for progressing to severe disease have not been fully elucidated, but due to the strong association of severe dengue and secondary infection with a different serotype, an immune-mediated pathogenesis has been postulated. Both T-cell-mediated immunopathogenesis3 and antibody-dependent enhancement (ADE) have been implicated4. Because of the potential for more severe outcome in sequential infections, developing a safe and balanced vaccine for all four serotypes has been challenging5.\n\nThis review will focus on recent advances in understanding the drivers of the dengue pandemic, viral structure and epitope binding, clinical severity and potential risk factors, plus recent studies investigating the pathogenesis and therapeutic options, concluding with strategies for disease control and future directions.\n\n\nGlobal expansion and disease burden\n\nOver the past 30 years, there has been a huge expansion in the transmission of dengue, and currently it is endemic in more than 100 countries2. Over 70% of the global burden lies in South and South-East Asia, but more recently case numbers have exploded in other parts of Asia, Latin America, and the Caribbean. Although harder to quantify, the African continent has also witnessed a significant increase in cases, with outbreaks reported from a number of East and West African countries6,7. It has also become apparent in recent years that developed countries are at risk, with small outbreaks being reported more from Southern Europe, the USA, and northern Australia. In 2012, Europe experienced its first dengue epidemic since the 1920s when over 2000 cases and 120 hospital admissions were reported from the Portuguese island of Madeira8. The origin of this outbreak was most likely from a viremic traveler from Venezuela, taking into account the volume of travel to Madeira from dengue endemic countries, seasonality in these countries, and also genetically similar viruses circulating in Venezuela at the time of the outbreak9. A similar although smaller outbreak occurred in Japan in 2014, again thought to have involved a viremic traveler with ongoing autochthonous spread associated with a large park in Tokyo10.\n\nOverall, the drivers behind the global expansion in disease are thought to include certain vector and host factors, including the urban-adapted Aedes mosquito vector becoming newly established in many areas of the world through distribution on cargo ships, globalization, and increase in breeding sites through rapid and often poorly planned urbanization of cities11. Other suggested factors include climate change and increase in population mobility and air travel12,13. These factors combined with ineffective vector control programs and no licensed therapeutics or vaccines has meant dengue is now a public health threat for two-thirds of the world’s population.\n\n\nViral structure and epitope binding\n\nThe dengue virus is a single-stranded, positive-sense enveloped RNA virus, 50 nm in diameter. The dengue virus genome encodes three structural proteins (capsid [C], precursor membrane [prM], and envelope [E]) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).\n\nStudies using cell culture have shown prM and E insert into the virion membrane to form the glycoprotein shell of the virus. During viral production and assembly, there is a complex series of rearrangements of prM and E. The virus is assembled in the endoplasmic reticulum, where 180 copies of both prM and E associate into trimeric spikes, each containing three prM and three E proteins14. prM acts as a chaperone protecting the hydrophobic fusion loop of E from triggering premature fusion with host cell membranes. As the virion traffics through the Golgi, furin protease cleaves prM, and as the virion is secreted from the cell the cleaved pr polypeptide is released and the E protein rearranges into 90 dimers, giving a smooth mature virus particle15. Following adhesion to poorly characterized cellular receptors, the virus is endocytosed and acidification of the endocytic vesicle then triggers E to reassociate from dimers to trimers, which exposes the fusion loop, allowing the virion to fuse with the endocytic membrane, releasing the viral RNA into the host cell cytoplasm16. One further complication of this is that furin cleavage of prM is often incomplete, leading to the production of virions with varying amounts of cleaved and uncleaved prM17,18.\n\nThe E protein has three domains (DI-III), is required for receptor binding and cell fusion and entry19, and is the major target for neutralizing antibodies, with potent neutralizing mouse monoclonal antibodies binding to epitopes on the DIII region20,21. The most potent human antibodies appear to bind to conformationally sensitive epitopes that are only found on intact virions and not with denatured or monomeric E protein22. It is now clear that the binding of some antibodies is limited by the accessibility of their epitopes, and that breathing of the virion and conformational change in the arrangement of E in the virion lattice may be required for binding23. In addition, broadly neutralizing anti E monoclonal antibodies directed at DII have been found to increase their avidity following secondary infection24. There are a number of serotype-specific human monoclonal antibodies which also recognize quaternary epitopes: HM14C10, 5J7, and 1F4 bind epitopes across three adjacent E monomers, whilst 2D22 binds across the E dimer25–28. Antibodies to prM are produced at high levels following dengue infection, but they are very poor at neutralizing infection, reaching a threshold of activity with none able to fully neutralize infection29. During the process of viral maturation, prM is cleaved, so anti-prM antibodies may fail to neutralize many viral particles because the antibody binding threshold required for neutralization will not be met. As mentioned above, the cleavage of prM is, however, frequently incomplete, which means that many virions contain enough prM to drive ADE but insufficient to promote neutralization. In addition, immature viruses which are usually non-infectious and which have a high density of uncleaved prM can become infectious to cells via ADE17,29.\n\nAn exciting recent development by our group is the discovery of a new class of antibodies directed at a novel epitope: the E dimer epitope (EDE), which is capable of potently neutralizing all four dengue serotypes30. The structure of these broadly neutralizing antibodies was characterized using X-ray crystallography and cryo-electron microscopy, and revealed that they recognized a serotype-invariant site that is located at the E-dimer interface, which includes contacts to the main chain of the E fusion loop31. This is also the binding site of prM during viral maturation, as previously described32. This has major implications for the future development of a subunit vaccine.\n\nOther advances have recently been made in determining the structure and function of the NS1 protein. NS1 is a 50 kDa glycoprotein that is secreted from dengue-infected cells and can be detected in the patient’s serum from early in the disease through to several days after defervescence. NS1 may play a role in the pathogenesis of severe disease, as higher levels have been detected in dengue shock patients33. Further work has identified that NS1 is secreted from infected cells as a hexamer, which creates a barrel shape around a lipid core34, and using cryo-electron microscopy the assembly and antibody binding of NS1 have also been described35,36. Antibodies against NS1 may be a potential therapeutic target, and modified NS1 may provide an alternative vaccine strategy37.\n\n\nClinical severity and risk prediction\n\nThe severe manifestations that develop in a small proportion of dengue-infected patients occur relatively late in the course of the illness, usually day 4–6, at the time of fever clearance. The most common severe manifestation is vascular leakage, which can lead to hemodynamic compromise, shock, and death. In addition, bleeding from mucosal surfaces and organ impairment in the form of hepatitis, myocarditis, and encephalitis can occur. This 48-hour period around defervescence has been classed as the “critical phase” and is the time when patients require closer monitoring. The WHO updated their classification and guidelines in 2009 to incorporate a set of warning signs to identify higher risk patients. These include a set of signs and symptoms and laboratory parameters to guide clinicians as to which patients are at a higher risk for disease progression (Box 2). In addition, several studies have identified certain risk factors that can influence disease severity in dengue; these include specific host and viral factors that likely act in concert to determine the disease phenotype38.\n\nViral factors include both the infecting serotype and the genotype of the virus, with certain genotypes within each serotype considered more virulent than the others, and have been linked to outbreaks of severe disease39,40. Higher viral loads have been associated with disease severity in both primary and secondary dengue and with different serotypes41,42.\n\nThe underlying immune status of the host is one of the most important factors in determining disease outcome, with a primed immune response, under certain conditions, facilitating a higher viral infected cell mass through ADE43 and original antigenic sin44, which will be discussed further in the pathogenesis section below. Other host factors include age of the host, with children more likely to experience plasma leakage and shock, and adults more likely to develop organ impairment and significant bleeding45. Elderly patients and those with co-morbidities, including diabetes and hypertension, have also been found to be at an increased risk of severe dengue46, possibly due to pre-existing endothelial dysfunction in this group. Female sex and age of less than 5 years have also been identified as risk factors for poor outcomes47. Genetic predisposition is also likely to play a role, with a genome-wide association study in Vietnam identifying two loci that were associated with severe disease, MICB and PLCE148, and a further study confirming these loci were also associated with less severe forms of dengue, as well as with dengue in infants49. Other genetic factors that have been shown to affect disease severity include certain HLA alleles, variations in the vitamin D receptor and Fc gamma receptor IIa, and also CD209 (G allele variant of DCSIGN1-336)50–52.\n\n\nPathogenesis of severe disease\n\nThere have been several recent advances in understanding dengue’s pathogenesis; however, the exact mechanisms remain to be fully defined. The observation that severe dengue occurs more frequently in secondary infections may be explained by ADE, where heterotypic non-neutralizing antibodies from a previous dengue infection facilitate viral binding to Fc receptors of monocytes and macrophages, leading to higher viral loads and more marker inflammatory response43. In addition, cross-reactive memory T cells also appear to play an important role in triggering the inflammatory cascade. The exact role of CD8+ T cells in the pathogenesis of severe disease is a rapidly evolving field, with some studies suggesting a pathogenic role with higher frequencies of cross-reactive CD8+ T cells being found in severe disease during secondary infections. Cells of low affinity for the infecting virus but higher affinity for other, presumed previous serotypes may be less effective at clearing the infection, resulting in a higher viremia. However, other studies suggest an HLA-linked protective role of CD8+ cells with a robust multifunctional response being associated with less severe disease53. Further work has demonstrated the T cell response was most marked to NS3 protein, with high cytokine and low CD107a (a marker of cell degranulation) predominating54. The resulting cytokine release, particularly tumor necrosis factor alpha and other vasoactive mediators, may then play a role in the increase in capillary permeability seen in severe dengue55–57.\n\nThe mechanisms linking these immunopathogenesis studies to vascular injury are still lacking. NS1 has been implicated in the pathogenesis of vascular leak. High levels of the soluble NS1 have been identified in patients’ plasma, from early in the disease and for up to 2 weeks later41, and like the viral load, NS1 antigenemia appears to correlate with disease severity33. NS1, along with the viral E protein, are able to bind to heparan sulfate, one of the major glycosaminoglycans (GAGs) in the glycocalyx of the endothelial cell layer58,59. The glycocalyx consists of a negatively charged network of glycoproteins, proteoglycans, and GAGs that covers the luminal surface of the microvascular endothelium. It provides size and charge selectivity to the capillary wall permeability, as well as acting as a transducer of sheer stress60. The adherence of NS1 and of the DENV E protein to the glycocalyx, and the resulting damage, could alter the permeability properties of the microvascular layer, which may contribute to the characteristic vascular leak that is associated with severe dengue58,59,61.\n\nNS1 and anti-NS1 antibodies have also been implicated in the pathogenesis of thrombocytopenia and coagulopathy that is characteristic in dengue62,63. NS1 can also activate complement, which may contribute to the vascular leak through the generation of anaphylatoxins and the terminal complement complex SC5b-959. High plasma levels of NS1 and SC5b-9 in dengue patients correlated with disease severity, and were also detected along with the anaphylatoxin C5a in the pleural fluid of dengue shock patients. In addition, anti-NS1 antibodies have been implicated in complement-mediated cytolysis and endothelial cell damage64,65. Recent in vitro studies have demonstrated that NS1 can alter endothelial monolayer integrity through the activation of Toll-like receptor 4 on peripheral blood mononuclear cells66, and altered endothelial permeability was prevented in mice by blocking NS1 through vaccination and monoclonal antibodies to NS167.\n\nOther immunological parameters that may play a role in the pathogenesis of severe dengue include plasmablast frequency, with high levels correlating with the critical phase68, mast cell activation and mast-cell-derived mediators, particularly vascular endothelial growth factor69,70, and antibody-immune complexes71,72.\n\n\nCurrent and novel therapeutic options\n\nThe current management of dengue relies on supportive treatment in the form of close monitoring for any of the “warning signs” and careful fluid balance for those identified to have capillary leak. Intravenous fluid is usually only required for patients with significant vascular leak and hemodynamic instability, or patients unable to tolerate oral fluids. The current WHO management guidelines recommend the initial use of crystalloid solutions, followed by colloid solutions for patients with profound or unresponsive shock2. Further trials are required to investigate whether earlier intervention with a colloid solution would benefit patients with dengue shock. Also fluid management in adult/elderly patients and those with co-morbidities is required, as evidence from randomized controlled trials in these groups are lacking.\n\nThere have been several disappointing therapeutic trials in dengue investigating both antivirals and adjunctive therapies. Two recent antiviral trials studying balapiravir in Vietnam and celgosivir in Singapore failed to demonstrate any beneficial effect on viremia or clinical outcome73,74.\n\nIn addition, adjunctive therapies have yet to demonstrate any disease-modifying effect. The anti-malarial drug chloroquine, although it showed promising antiviral effects in vitro75, did not translate into a reduction in viremia or NS1 duration in a randomized controlled trial in adult dengue patients76. Immunomodulation with corticosteroids has also failed to alter disease severity both in patients with established dengue shock and also when given early in the disease course77,78. A study using intravenous immunoglobulin did not impact on the development of severe thrombocytopenia79, nor did prophylactic platelet transfusions have any benefit on bleeding manifestations in adult patients with severe thrombocytopenia80.\n\nIn vitro studies have shown lovastatin is able to interrupt the DENV assembly pathway81 and increase survival in animal models82. A human study investigating lovastatin in early dengue has just been published, again showing no benefit in modifying dengue clinical outcomes83. It is anticipated that dengue drug discovery in the next few years will be assisted by improved animal models for dengue84 and also the possibility of a human infection model85.\n\n\nNew strategies for dengue control\n\nEfforts to control the spread of dengue in the last two decades have failed, mainly due to the lack of a licensed vaccine and difficulties in controlling the major global vectors Aedes aegypti mosquitoes and, more recently, Aedes albopictus86. These day-biting, anthropophilic mosquitoes are highly adapted to the urban environment, breeding primarily in man-made water containers. Previously, vector control efforts were aimed at the elimination of the container breeding sites, improved access to piped water supplies, and improved management of water storage. The use of larvicides and insecticides were mainly used during outbreaks and had many limitations, including resistance87. However, new technologies showing some promise for future dengue control are biologic and genetic modification of mosquitoes. The intracellular bacterium Wolbachia, when introduced into Aedes mosquitoes, can influence the ability of the insects to transmit the virus, indirectly by reducing the mosquito’s life span and directly by reducing viral replication in the mosquito88,89. Field trials are underway in Vietnam90 and Australia91 (also Brazil and Indonesia), and have demonstrated successful invasion of Wolbachia-infected mosquitoes into natural mosquito populations at the release sites92. In addition, there are some promising results of genetically manipulated mosquitoes with engineered sterile male Aedes mosquitoes in field trials in the Cayman Islands93.\n\nAlthough currently there is no global dengue vaccine available for public health use, in the last 2 months the first ever dengue vaccine (CYD-TDV, Sanofi-Pasteur) was licensed in Mexico followed by the Philippines, in addition there are several candidate vaccines in different phases of development, e.g. live attenuated, inactivated whole virus, and subunit and recombinant vaccines. Several live attenuated vaccines have progressed to clinical trials, but the concern for ADE with an unbalanced response to all four serotypes has been a major challenge. The lead candidate and recently licensed in 2 countries is a tetravalent live attenuated vaccine (CYD-TDV, Sanofi-Pasteur) has recently completed the first phase III dengue vaccine trial in Asia94 and Latin America95. The overall vaccine efficacy was 56.5% in Asian children and 64.7% in slightly older children in Latin America. However, this varied by serotype, with poor efficacy for DENV-2 of only 35% in the Asian study and 42.3% in Latin America, and also varied depending on background flavivirus immunity, with poor efficacy demonstrated in flavivirus-naive people. A further study has recently been published reporting the results of the first long-term follow up (3 years post vaccination) of the CYD-TDV vaccine and has shown continued benefit in vaccinated children aged 9–16 years. However, in the younger age group (<9 years), there was an increase in hospitalization when compared to unvaccinated subjects96.\n\nThese studies have also highlighted the need to improve our understanding of the immunological correlates of disease, as neutralizing antibodies to all four serotypes were demonstrated among vaccines in an earlier phase of the study but did not translate to equal protection.\n\nOther live attenuated vaccine candidates have reported promising results from phase 1 trials, including NIH Δ30 and DENVax from Takeda97–99. DENV-1, -3, and -4 of NIH Δ30 candidate were attenuated by deleting 30 nucleotides at the 3’ untranslated region of the viral genome, while DENV-2 was generated by replacing the DENV-4 prM and E genes with those from DENV-2. DENVax is a live attenuated DENV-2 backbone with three recombinant vaccine viruses (serotypes 1, 3, and 4) expressing prM and E genes98.\n\nWhole inactivated tetravalent vaccines may offer a safer alternative strategy, and a recent study in macaques demonstrated good immunogenicity when the vaccine was combined with an adjuvant100. Subunit vaccines using the DENV E protein (domain III) as the major immunogen have shown potential in preclinical trials101, and a subunit vaccine (DEN-80E) developed by Merck has now progressed to clinical trials102. With the recent identification of a conserved epitope on the E protein (EDE), this is an area that is likely to develop further in the future30. As with testing novel dengue therapeutics, vaccine efficacy studies should also benefit from potential human infection models in the near future103.\n\n\nConclusion and future direction\n\nDengue is one of the world’s most rapidly emerging diseases, and as incidence continues to rise in endemic areas, and transmission in new regions of the world becomes established, there are major public health challenges ahead. There have been recent advances in our understanding of the epidemiology, risk factors for severe disease, and pathogenesis, plus the identification of therapeutic targets, which may lead to novel treatments. Improved animal and human infection models should lead to better understanding of disease evolution and assist drug development. In addition, the advance in the study of human monoclonal antibodies has opened up a new avenue for vaccine development, which should concentrate on inducing the potent neutralizing anti-EDE antibodies against all four serotypes and avoid anti-prM antibodies, which have low neutralizing activity and high potential to enhance viral infection through ADE. Future randomized controlled trials of novel therapeutics and fluids, including in adults, will be required to guide evidence-based practice in all patient groups. With the possibility that the first ever dengue vaccine may be licensed in more countries in the next couple of years, and the further deployment of Wolbachia bio-control, reversing the spread of dengue may now be a real prospect.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nAll the authors are funded by the Wellcome Trust.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBhatt S, Gething PW, Brady OJ, et al.: The global distribution and burden of dengue. Nature. 2013; 496(7446): 504–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. Geneva: World Health Organization. 2009. PubMed Abstract\n\nMongkolsapaya J, Dejnirattisai W, Xu XN, et al.: Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med. 2003; 9(7): 921–7. PubMed Abstract | Publisher Full Text\n\nHalstead SB: Pathogenesis of dengue: challenges to molecular biology. Science. 1988; 239(4839): 476–81. 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Science. 2010; 328(5979): 745–8. PubMed Abstract | Free Full Text | F1000 Recommendation\n\nDejnirattisai W, Wongwiwat W, Supasa S, et al.: A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus. Nat Immunol. 2015; 16(2): 170–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRouvinski A, Guardado-Calvo P, Barba-Spaeth G, et al.: Recognition determinants of broadly neutralizing human antibodies against dengue viruses. Nature. 2015; 520(7545): 109–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nYu IM, Zhang W, Holdaway HA, et al.: Structure of the immature dengue virus at low pH primes proteolytic maturation. Science. 2008; 319(5871): 1834–7. PubMed Abstract\n\nLibraty DH, Young PR, Pickering D, et al.: High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis. 2002; 186(8): 1165–8. PubMed Abstract | Publisher Full Text\n\nGutsche I, Coulibaly F, Voss JE, et al.: Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci U S A. 2011; 108(19): 8003–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nEdeling MA, Diamond MS, Fremont DH: Structural basis of Flavivirus NS1 assembly and antibody recognition. Proc Natl Acad Sci U S A. 2014; 111(11): 4285–90. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nAkey DL, Brown WC, Dutta S, et al.: Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system. Science. 2014; 343(6173): 881–5. PubMed Abstract | Free Full Text | F1000 Recommendation\n\nWan SW, Lu YT, Huang CH, et al.: Protection against dengue virus infection in mice by administration of antibodies against modified nonstructural protein 1. PLoS One. 2014; 9(3): e92495. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYacoub S, Wills B: Predicting outcome from dengue. BMC Med. 2014; 12: 147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFried JR, Gibbons RV, Kalayanarooj S, et al.: Serotype-specific differences in the risk of dengue hemorrhagic fever: an analysis of data collected in Bangkok, Thailand from 1994 to 2006. PLoS Negl Trop Dis. 2010; 4(3): e617. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTissera HA, Ooi EE, Gubler DJ, et al.: New dengue virus type 1 genotype in Colombo, Sri Lanka. Emerg Infect Dis. 2011; 17(11): 2053–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuyen HT, Ngoc TV, Ha do T, et al.: Kinetics of plasma viremia and soluble nonstructural protein 1 concentrations in dengue: differential effects according to serotype and immune status. J Infect Dis. 2011; 203(9): 1292–300. 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PLoS Negl Trop Dis. 2012; 6(6): e1679. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPang J, Salim A, Lee VJ, et al.: Diabetes with hypertension as risk factors for adult dengue hemorrhagic fever in a predominantly dengue serotype 2 epidemic: a case control study. PLoS Negl Trop Dis. 2012; 6(5): e1641. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nAnders KL, Nguyet NM, Chau NV, et al.: Epidemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Vietnam. Am J Trop Med Hyg. 2011; 84(1): 127–34. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKhor CC, Chau TN, Pang J, et al.: Genome-wide association study identifies susceptibility loci for dengue shock syndrome at MICB and PLCE1. Nat Genet. 2011; 43(11): 1139–41. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nWhitehorn J, Chau TN, Nguyet NM, et al.: Genetic variants of MICB and PLCE1 and associations with non-severe dengue. PLoS One. 2013; 8(3): e59067. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLoke H, Bethell D, Phuong CX, et al.: Susceptibility to dengue hemorrhagic fever in Vietnam: evidence of an association with variation in the vitamin d receptor and Fc gamma receptor IIa genes. Am J Trop Med Hyg. 2002; 67(1): 102–6. PubMed Abstract\n\nSakuntabhai A, Turbpaiboon C, Casadémont I, et al.: A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet. 2005; 37(5): 507–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nStephens HA: HLA and other gene associations with dengue disease severity. Curr Top Microbiol Immunol. 2010; 338: 99–114. PubMed Abstract | Publisher Full Text\n\nWeiskopf D, Angelo MA, de Azeredo EL, et al.: Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells. Proc Natl Acad Sci U S A. 2013; 110(22): E2046–53. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDuangchinda T, Dejnirattisai W, Vasanawathana S, et al.: Immunodominant T-cell responses to dengue virus NS3 are associated with DHF. Proc Natl Acad Sci U S A. 2010; 107(39): 16922–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nButthep P, Chunhakan S, Yoksan S, et al.: Alteration of cytokines and chemokines during febrile episodes associated with endothelial cell damage and plasma leakage in dengue hemorrhagic fever. Pediatr Infect Dis J. 2012; 31(12): e232–8. PubMed Abstract | Publisher Full Text\n\nDong T, Moran E, Vinh Chau N, et al.: High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection. PLoS One. 2007; 2(12): e1192. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSierra B, Pérez AB, Alvarez M, et al.: Variation in inflammatory/regulatory cytokines in secondary, tertiary, and quaternary challenges with dengue virus. Am J Trop Med Hyg. 2012; 87(3): 538–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAvirutnan P, Zhang L, Punyadee N, et al.: Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate E. PLoS Pathog. 2007; 3(11): e183. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAvirutnan P, Punyadee N, Noisakran S, et al.: Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J Infect Dis. 2006; 193(8): 1078–88. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWeinbaum S, Zhang X, Han Y, et al.: Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A. 2003; 100(13): 7988–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Maguire T, Hileman RE, et al.: Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nat Med. 1997; 3(8): 866–71. PubMed Abstract | Publisher Full Text\n\nLin SW, Chuang YC, Lin YS, et al.: Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation. J Infect. 2012; 64(3): 325–34. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCheng HJ, Lei HY, Lin CF, et al.: Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation. Mol Immunol. 2009; 47(2–3): 398–406. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLin CF, Lei HY, Shiau AL, et al.: Endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein 1 via production of nitric oxide. J Immunol. 2002; 169(2): 657–64. PubMed Abstract | Publisher Full Text\n\nLin CF, Lei HY, Shiau AL, et al.: Antibodies from dengue patient sera cross-react with endothelial cells and induce damage. J Med Virol. 2003; 69(1): 82–90. PubMed Abstract | Publisher Full Text\n\nModhiran N, Watterson D, Muller DA, et al.: Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity. Sci Transl Med. 2015; 7(304): 304ra142. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBeatty PR, Puerta-Guardo H, Killingbeck SS, et al.: Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination. Sci Transl Med. 2015; 7(304): 304ra141. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGarcia-Bates TM, Cordeiro MT, Nascimento EJ, et al.: Association between magnitude of the virus-specific plasmablast response and disease severity in dengue patients. J Immunol. 2013; 190(1): 80–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFuruta T, Murao LA, Lan NT, et al.: Association of mast cell-derived VEGF and proteases in Dengue shock syndrome. PLoS Negl Trop Dis. 2012; 6(2): e1505. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSt John AL, Rathore AP, Raghavan B, et al.: Contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage. eLife. 2013; 2: e00481. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSun P, García J, Comach G, et al.: Sequential waves of gene expression in patients with clinically defined dengue illnesses reveal subtle disease phases and predict disease severity. PLoS Negl Trop Dis. 2013; 7(7): e2298. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang WK, Chen HL, Yang CF, et al.: Slower rates of clearance of viral load and virus-containing immune complexes in patients with dengue hemorrhagic fever. Clin Infect Dis. 2006; 43(8): 1023–30. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLow JG, Sung C, Wijaya L, et al.: Efficacy and safety of celgosivir in patients with dengue fever (CELADEN): a phase 1b, randomised, double-blind, placebo-controlled, proof-of-concept trial. Lancet Infect Dis. 2014; 14(8): 706–15. PubMed Abstract | Publisher Full Text\n\nNguyen NM, Tran CN, Phung LK, et al.: A randomized, double-blind placebo controlled trial of balapiravir, a polymerase inhibitor, in adult dengue patients. J Infect Dis. 2013; 207(9): 1442–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarias KJ, Machado PR, de Almeida Junior RF, et al.: Chloroquine interferes with dengue-2 virus replication in U937 cells. Microbiol Immunol. 2014; 58(6): 318–26. PubMed Abstract | Publisher Full Text\n\nTricou V, Minh NN, Van TP, et al.: A randomized controlled trial of chloroquine for the treatment of dengue in Vietnamese adults. PLoS Negl Trop Dis. 2010; 4(8): e785. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nTassniyom S, Vasanawathana S, Chirawatkul A, et al.: Failure of high-dose methylprednisolone in established dengue shock syndrome: a placebo-controlled, double-blind study. Pediatrics. 1993; 92(1): 111–5. PubMed Abstract\n\nTam DT, Ngoc TV, Tien NT, et al.: Effects of short-course oral corticosteroid therapy in early dengue infection in Vietnamese patients: a randomized, placebo-controlled trial. Clin Infect Dis. 2012; 55(9): 1216–24. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDimaano EM, Saito M, Honda S, et al.: Lack of efficacy of high-dose intravenous immunoglobulin treatment of severe thrombocytopenia in patients with secondary dengue virus infection. Am J Trop Med Hyg. 2007; 77(6): 1135–8. PubMed Abstract\n\nLye DC, Lee VJ, Sun Y, et al.: Lack of efficacy of prophylactic platelet transfusion for severe thrombocytopenia in adults with acute uncomplicated dengue infection. Clin Infect Dis. 2009; 48(9): 1262–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMartínez-Gutierrez M, Castellanos JE, Gallego-Gómez JC: Statins reduce dengue virus production via decreased virion assembly. Intervirology. 2011; 54(4): 202–16. PubMed Abstract | Publisher Full Text\n\nMartinez-Gutierrez M, Correa-Londoño LA, Castellanos JE, et al.: Lovastatin delays infection and increases survival rates in AG129 mice infected with dengue virus serotype 2. PLoS One. 2014; 9(2): e87412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhitehorn J, Nguyen CV, Khanh LP, et al.: Lovastatin for the Treatment of Adult Patients With Dengue: A Randomized, Double-Blind, Placebo-Controlled Trial. Clin Infect Dis. 2015; pii: civ949. PubMed Abstract | Publisher Full Text\n\nZompi S, Harris E: Animal models of dengue virus infection. Viruses. 2012; 4(1): 62–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhitehorn J, Van VC, Simmons CP: Dengue human infection models supporting drug development. J Infect Dis. 2014; 209(Suppl 2): S66–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaupy C, Delatte H, Bagny L, et al.: Aedes albopictus, an arbovirus vector: from the darkness to the light. Microbes Infect. 2009; 11(14–15): 1177–85. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLuz PM, Vanni T, Medlock J, et al.: Dengue vector control strategies in an urban setting: an economic modelling assessment. Lancet. 2011; 377(9778): 1673–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcMeniman CJ, Lane RV, Cass BN, et al.: Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedes aegypti. Science. 2009; 323(5910): 141–4. PubMed Abstract | F1000 Recommendation\n\nMoreira LA, Iturbe-Ormaetxe I, Jeffery JA, et al.: A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium. Cell. 2009; 139(7): 1268–78. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHugo LE, Jeffery JA, Trewin BJ, et al.: Adult survivorship of the dengue mosquito Aedes aegypti varies seasonally in central Vietnam. PLoS Negl Trop Dis. 2014; 8(2): e2669. 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}
|
[
{
"id": "11975",
"date": "19 Jan 2016",
"name": "Aravinda M de Silva",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "11976",
"date": "19 Jan 2016",
"name": "Annelies Wilder-Smith",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "11977",
"date": "19 Jan 2016",
"name": "Maria Guzman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-78
|
https://f1000research.com/articles/5-72/v1
|
18 Jan 16
|
{
"type": "Review",
"title": "The cardiac enigma: current conundrums in heart failure research",
"authors": [
"Michael S. Kapiloff",
"Craig A. Emter",
"Craig A. Emter"
],
"abstract": "The prevalence of heart failure is expected to increase almost 50% in the next 15 years because of aging of the general population, an increased frequency of comorbidities, and an improved survival following cardiac events. Conventional treatments for heart failure have remained largely static over the past 20 years, illustrating the pressing need for the discovery of novel therapeutic agents for this patient population. Given the heterogeneous nature of heart failure, it is important to specifically define the cellular mechanisms in the heart that drive the patient’s symptoms, particularly when considering new treatment strategies. This report highlights the latest research efforts, as well as the possible pitfalls, in cardiac disease translational research and discusses future questions and considerations needed to advance the development of new heart failure therapies. In particular, we discuss cardiac remodeling and the translation of animal work to humans and how advancements in our understanding of these concepts relative to disease are central to new discoveries that can improve cardiovascular health.",
"keywords": [
"Heart failure",
"clinical trials",
"angiotensin-converting enzyme"
],
"content": "Introduction\n\nHeart failure, the common end stage of heart disease, is defined clinically by fatigue, shortness of breath, and fluid retention, including pulmonary edema1. Heart failure is a syndrome of major public health significance, impacting 5.7 million worldwide with an incidence of 870,000 adults in the United States alone. The prevalence of heart failure is expected to increase by 46% by 2030, and this is due in large part to aging of the general population but also to the improved survival following events such as myocardial infarction and the increased prevalence of comorbidities such as obesity and diabetes2. The cost to society is consequential. The 5-year mortality for heart failure remains approximately 50%, despite current therapies. Moreover, the financial costs associated with heart failure are expected to balloon to over $70 billion per year by 20301. As a result, the discovery of new drug targets for heart failure prevention or treatment (or both) remains an area of pressing concern.\n\n\nThe current approach to chronic heart failure therapy\n\nCurrent therapies for heart failure are both medicinal and device-driven3. The mainstays of heart failure pharmacotherapy include β-blockers (β-adrenergic receptor antagonists such as carvedilol, metoprolol, and bisoprolol), angiotensin-converting enzyme inhibitors ([ACEI] e.g., enalapril and lisinopril), angiotensin II receptor blockers (e.g., losartan and valsartan), aldosterone antagonists, hydralazine and isosorbide dinitrate, and diuretics4. Ventricular assist devices, such as the implantable cardioverter-defibrillator, left ventricular (LV) assist device, and cardiac resynchronization therapy, are widely used, and cardiac transplant remains a therapy of last resort for some patients. There has been tremendous excitement this year as the first new drugs since 1999 have been approved by the US Food and Drug Administration for chronic heart failure. Ivabradine (Corlanor) is a sinoatrial node If current inhibitor that has been shown to have efficacy in a variety of clinical trials, including SHIFT (Systolic Heart failure treatment with the If inhibitor ivabradine Trial)5–7. LCZ696 (Entresto™) is a combination drug including a neprilysin endopeptidase inhibitor (sacubitril) and angiotensin receptor blocker (valsartan). In the PARADIGM-HF (Prospective Comparison of ARNI With ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure) trial, LCZ696 was shown to reduce mortality in comparison with enalapril8. Finally, ranolazine improved hemodynamic status in the proof-of-concept RALI-DHF (RAnoLazIne for the Treatment of Diastolic Heart Failure) trial9. However, even with these new drugs, heart failure morbidity and mortality are expected to increase substantially in the near future.\n\n\nHeart Failure: HFrEF vs. HFpEF\n\nIn considering the future of heart failure therapies, it is important to remember that heart failure is not a homogenous entity but instead a clinical syndrome related to end-stage heart disease. Heart failure can be divided into two groups: (a) reduced ejection fraction (HFrEF) and (b) preserved ejection fraction (HFpEF) (ejection fraction >45%); the two forms confer a similar prognosis and have similar prevalence10–12. As the names suggest, HFrEF includes prominent systolic cardiac dysfunction, whereas HFpEF is more closely associated with diastolic dysfunction. Although the current therapies for heart failure have been established in clinical trials involving HFrEF patients, none has been therapeutically useful for HFpEF patients10,11,13,14. Instead, exercise may be the only clinically effective, currently available HFpEF treatment15, and several small-scale clinical trials show that exercise improves cardiorespiratory variables such as oxygen consumption (VO2), diastolic dysfunction, and quality of life in HFpEF patients16. The Exercise Training in Diastolic Heart Failure (EX-DHF) (ISRCTN 86879094) study will examine these findings on a larger scale and hopefully provide insight regarding the use of exercise to improve mortality. The lack of drug therapies for HFpEF may be due to the limited efficacy of current drugs to treat diastolic dysfunction. Alternatively, it was recently suggested that many patients with diagnosed HFpEF, typically older women with diabetes and obesity, do not have structural heart disease but instead exhibit fairly general signs that define heart failure due to non-cardiac problems17. For example, although pulmonary edema is usually a reliable indicator of LV dysfunction (and high diastolic pressures), it can also be caused by primary pulmonary disease (e.g., adult respiratory distress syndrome) or low plasma colloid oncotic pressure due to other etiologies18. HFpEF should also be distinguished from high-output heart failure, a form of heart failure that typically is secondary to a non-cardiac disease19. In high-output heart failure, the heart responds normally and reversibly to extra-cardiac stress, often undergoing physiologic hypertrophy that is induced by low afterload and volume overload on the heart. Thus, when novel approaches to the treatment of heart failure are considered, it is important to precisely define the cohort of patients being considered and whether the heart is in fact the relevant target for therapeutic intervention.\n\n\nPathological cardiac remodeling\n\nA long-term goal of heart failure therapies is to reverse or prevent cardiac remodeling, the general term referring to the structural changes in the heart induced by chronic stress. While the heart can respond to acute demands for increased output by changes in chronotropy (heart rate), inotropy (contractility), and lusitropy (relaxation), the adult heart is generally limited to hypertrophy as a compensatory mechanism20. Cardiac hypertrophy at the whole-organ level reflects non-mitotic growth of the cardiac myocytes. The generally cylindrical adult myocyte can grow in either width (diameter) or length, resulting in thickened ventricular walls or chamber dilation, respectively. In theory, concentric myocyte growth increases the width of cardiomyocytes, inducing parallel assembly of sarcomeres and thereby reducing ventricular wall stress (Laplace’s law). In contrast, eccentric myocyte growth increases cardiomyocyte length, inducing serial addition of sarcomeres to accommodate greater ventricular volumes without stretching individual sarcomeres beyond the optimum length for contraction (Frank-Starling law)21. In pressure overload diseases, such as aortic stenosis or hypertension, there is increased systolic wall stress, and concentric hypertrophy initially predominates. In volume overload diseases, such as following a myocardial infarction or dilated cardiomyopathy, eccentric hypertrophy predominates, presumably in response to increased diastolic wall stress. Although sarcomeric assembly is considered initially compensatory, myocyte hypertrophy is eventually concomitant with altered myocyte gene expression, metabolism, excitation-contraction coupling, increased cell death, and myocardial fibrosis. Together, these factors contribute to systolic and diastolic cardiac dysfunction and promote pathological cardiac remodeling and subsequent heart failure.\n\n\nOutstanding questions in heart failure research\n\nThe above description of heart failure is the basis for the underlying paradigm driving most research in the field, primarily the identification of potential drug targets that protect cardiac contractility and inhibit myocyte death and interstitial fibrosis22,23. We propose the following issues as central to advancing the treatment of heart failure, including questions to stimulate discussion about the underlying assumptions concerning disease development:\n\n1. A fundamental question concerns whether any of the features in cardiac remodeling are necessarily compensatory (i.e., can be safely targeted in the face of cardiac stress). For example, as recently discussed in a point-counterpoint editorial series in Circulation24,25, concentric LV hypertrophy is often considered compensatory in diseases of increased afterload. The current first-line therapies for heart failure target the adrenergic and renin-angiotensin systems, having effects both on cardiac myocytes and on the vasculature25. It has been argued that lowering afterload and LV wall stress is essential to the efficacy of these drugs in patients (whether by lowering blood pressure for hypertension or by contemporaneous aortic valve replacement for aortic stenosis) and that attenuating hypertrophy without lowering afterload would not be tolerated in humans26. However, diverse studies in rodents using pharmacological agents such as cyclosporine, and more elegantly with genetically modified mice, have shown that inhibition of hypertrophy not only is tolerated in the face of persistent pressure overload but also can prevent or treat heart failure22.\n\n2. It remains unclear which features in cardiac remodeling are co-regulated by signal pathways that may be targeted and which features may be specifically targeted independently of other aspects of remodeling. Although most studies show that hypertrophy and fibrosis are tightly associated in disease, recent findings by the Backs laboratory showed that mice lacking the γ and δ isoforms of Ca2+/calmodulin-dependent protein kinase II had improved cardiac function and decreased fibrosis but persistent hypertrophy following transverse aortic constriction27. Whereas some aspects of the changes in myocyte gene expression, metabolism, and excitation-contraction coupling may be detrimental (e.g., the decreased sarcoplasmic reticulum Ca2+ ATPase [SERCA2a] activity in heart failure), others may be beneficial (e.g., increased natriuretic peptide expression or concentric hypertrophy that decreases wall stress). These exciting findings suggest that the beneficial aspects of remodeling may be retained while therapeutically combatting the deleterious aspects that lead to heart failure.\n\n3. It remains unclear what causes diastolic dysfunction, especially in HFpEF. Diastolic dysfunction is the result of reduced active relaxation or ventricular compliance. Active relaxation occurs in large part due to ATP-dependent Ca2+ reuptake during diastole primarily through SERCA2a, and reduced SERCA2a activity is associated with heart failure. Accordingly, in animal models, SERCA2a replacement has been effective in improving overall cardiac function28. Decreased compliance and associated atrial hypertrophy have long been associated with interstitial fibrosis29,30. However, the relative extent of fibrosis and altered Ca2+ reuptake can vary in different models for diastolic dysfunction30,31, raising the question of what should be targeted in diastolic dysfunction under different clinical scenarios. In numerous animal models, reversal or reduction of LV hypertrophy has been shown to improve diastolic function independently of hemodynamic alteration, implying that hypertrophy itself plays a role in diastolic dysfunction. However, clinical studies have shown that this relationship is less apparent in humans32. Finally, coronary vascular dysfunction may have a profound impact on myocardial oxidative capacity and diastolic dysfunction in heart failure33. Recent work has shown that swine with diastolic dysfunction have myocardial oxygen supply/demand imbalance34. This suggests that impaired coronary vasculature function may contribute to the inability of HFpEF patients to respond to situations of increasing stress by limiting ATP production and subsequent active relaxation.\n\n4. Humans are not large mice. Little is known or being investigated about cardiac signal transduction in large mammals, despite the significant differences between large and small mammalian hearts. Instead, most of what is known about the regulation of myocyte hypertrophy and cardiac remodeling has been defined in mice and rats25. Differences between large and small mammals include life span, heart rate, excitation-contraction coupling and Ca2+ handling, α:β-myosin heavy chain ratio, tolerance for myocardial injury, and rate of progression of cardiac remodeling25. The successful development of therapeutics for human patients is dependent upon the identification of mechanisms that are in fact relevant to the large mammalian heart. For example, many question whether large animals subject to pressure overload can tolerate diminished hypertrophy similar to mice24. A review by Dixon and Spinale discusses the importance of large animal models in translating basic science findings to the clinic and addresses the lack of studies using large animals to address pressure overload LV hypertrophy and its role in the development of heart failure35. An advantage of large animal models is that key determinants of myocardial work and energy consumption, including LV wall tension, heart rate, and vascular wall-to-lumen ratios36, are similar to those in humans. Thus, large animal models could provide an essential link to implement discoveries made in murines into models exhibiting functional and anatomical similarities more analogous to humans as a means to assess therapeutic potential for treating heart failure clinically. Significant financial challenges exist in generating, sustaining, and implementing large animal models of heart failure into research programs. Specific programs aimed at soliciting and supporting large animal cardiovascular research from both federal and private sources would help stimulate more large animal studies in the future and aid in bridging the gap between small animals, large animals, and humans.\n\n\nHope for the future\n\nIt is an exciting time to be involved in heart failure research. There are ample animal models, both small and large, addressing diverse types of heart diseases, as well as protocols to study cardiac cell types in vitro. Our knowledge of cardiac cell regulation continues to rapidly increase, and new tools, including novel methods for visualizing signaling in real time, are being developed37. In addition, adeno-associated viruses are emerging as a viable therapeutic approach to deliver both small interfering RNA (siRNA) and proteins in vivo for both scientific and clinical purposes38. These advances portend the discovery of additional cardiac therapeutics in the coming years.",
"appendix": "Competing interests\n\n\n\nMSK is a co-inventor of patented intellectual property concerning the use of RSK3 inhibitors for the treatment of heart failure, by which he and the University of Miami may gain royalties from future commercialization. He is the manager of Anchored RSK3 Inhibitors, LLC (Miami Beach, FL, USA) and is on the board of directors of Cardiac RSK3 Inhibitors, LLC (Miami Beach, FL, USA), companies interested in developing RSK3-targeted therapies and in which he holds equity. CAE declares that he has no competing interests.\n\n\nGrant information\n\nThis work was funded in part by National Institutes of Health grants R01HL075398 (MSK) and R01HL112998 (CAE).\n\n\nReferences\n\nMozaffarian D, Benjamin EJ, Go AS, et al.: Heart disease and stroke statistics--2015 update: a report from the American Heart Association. Circulation. 2015; 131(4): e29–322. PubMed Abstract | Publisher Full Text\n\nHeidenreich PA, Albert NM, Allen LA, et al.: Forecasting the impact of heart failure in the United States: a policy statement from the American Heart Association. Circ Heart Fail. 2013; 6(3): 606–19. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nYancy CW, Jessup M, Bozkurt B, et al.: 2013 ACCF/AHA guideline for the management of heart failure: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2013; 128(16): 1810–52. PubMed Abstract | Publisher Full Text\n\nXie M, Burchfield JS, Hill JA: Pathological ventricular remodeling: therapies: part 2 of 2. Circulation. 2013; 128(9): 1021–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKomajda M, Tavazzi L, Francq BG, et al.: Efficacy and safety of ivabradine in patients with chronic systolic heart failure and diabetes: an analysis from the SHIFT trial. Eur J Heart Fail. 2015; 17(12): 1294–301. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRogers JK, Kielhorn A, Borer JS, et al.: Effect of ivabradine on numbers needed to treat for the prevention of recurrent hospitalizations in heart failure patients. Curr Med Res Opin. 2015; 31(10): 1903–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBorer JS, Böhm M, Ford I, et al.: Efficacy and safety of ivabradine in patients with severe chronic systolic heart failure (from the SHIFT study). Am J Cardiol. 2014; 113(3): 497–503. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMcMurray JJ, Packer M, Desai AS, et al.: Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med. 2014; 371(11): 993–1004. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMaier LS, Layug B, Karwatowska-Prokopczuk E, et al.: RAnoLazIne for the treatment of diastolic heart failure in patients with preserved ejection fraction: the RALI-DHF proof-of-concept study. JACC Heart Fail. 2013; 1(2): 115–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBorlaug BA, Paulus WJ: Heart failure with preserved ejection fraction: pathophysiology, diagnosis, and treatment. Eur Heart J. 2011; 32(6): 670–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaeder MT, Kaye DM: Heart failure with normal left ventricular ejection fraction. J Am Coll Cardiol. 2009; 53(11): 905–18. PubMed Abstract | Publisher Full Text\n\nHunt SA, Abraham WT, Chin MH, et al.: ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation. 2005; 112(12): e154–235. PubMed Abstract | Publisher Full Text\n\nPaulus WJ, van Ballegoij JJ: Treatment of heart failure with normal ejection fraction: an inconvenient truth! J Am Coll Cardiol. 2010; 55(6): 526–37. PubMed Abstract | Publisher Full Text\n\nDesai AS: Heart failure with preserved ejection fraction: time for a new approach? J Am Coll Cardiol. 2013; 62(4): 272–4. PubMed Abstract | Publisher Full Text\n\nMcDonald KS, Emter CA: Exploring new concepts in the management of heart failure with preserved ejection fraction: is exercise the key for improving treatment? J Appl Physiol (1985). 2015; 119(6): 724–5. PubMed Abstract | Publisher Full Text\n\nDieberg G, Ismail H, Giallauria F, et al.: Clinical outcomes and cardiovascular responses to exercise training in heart failure patients with preserved ejection fraction: a systematic review and meta-analysis. J Appl Physiol (1985). 2015; 119(6): 726–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSharma K, Kass DA: Heart failure with preserved ejection fraction: mechanisms, clinical features, and therapies. Circ Res. 2014; 115(1): 79–96. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nStein L, Beraud JJ, Morissette M, et al.: Pulmonary edema during volume infusion. Circulation. 1975; 52(3): 483–9. PubMed Abstract | Publisher Full Text\n\nAnand IS, Florea VG: High Output Cardiac Failure. Curr Treat Options Cardiovasc Med. 2001; 3(2): 151–9. PubMed Abstract\n\nHill JA, Olson EN: Cardiac plasticity. N Engl J Med. 2008; 358(13): 1370–80. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGrossman W, Jones D, McLaurin LP: Wall stress and patterns of hypertrophy in the human left ventricle. J Clin Invest. 1975; 56(1): 56–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Berlo JH, Maillet M, Molkentin JD: Signaling effectors underlying pathologic growth and remodeling of the heart. J Clin Invest. 2013; 123(1): 37–45. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBurchfield JS, Xie M, Hill JA: Pathological ventricular remodeling: mechanisms: part 1 of 2. Circulation. 2013; 128(4): 388–400. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCrozatier B, Ventura-Clapier R: Inhibition of hypertrophy, per se, may not be a good therapeutic strategy in ventricular pressure overload: other approaches could be more beneficial. Circulation. 2015; 131(16): 1448–57. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSchiattarella GG, Hill JA: Inhibition of hypertrophy is a good therapeutic strategy in ventricular pressure overload. Circulation. 2015; 131(16): 1435–47. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDrazner MH: The progression of hypertensive heart disease. Circulation. 2011; 123(3): 327–34. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKreusser MM, Lehmann LH, Keranov S, et al.: Cardiac CaM Kinase II genes δ and γ contribute to adverse remodeling but redundantly inhibit calcineurin-induced myocardial hypertrophy. Circulation. 2014; 130(15): 1262–73. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKho C, Lee A, Hajjar RJ: Altered sarcoplasmic reticulum calcium cycling--targets for heart failure therapy. Nat Rev Cardiol. 2012; 9(12): 717–33. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nVillari B, Campbell SE, Hess OM, et al.: Influence of collagen network on left ventricular systolic and diastolic function in aortic valve disease. J Am Coll Cardiol. 1993; 22(5): 1477–84. PubMed Abstract | Publisher Full Text\n\nIshikawa K, Aguero J, Oh JG, et al.: Increased stiffness is the major early abnormality in a pig model of severe aortic stenosis and predisposes to congestive heart failure in the absence of systolic dysfunction. J Am Heart Assoc. 2015; 4(5): pii: e001925. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHiemstra JA, Gutiérrez-Aguilar M, Marshall KD, et al.: A new twist on an old idea part 2: cyclosporine preserves normal mitochondrial but not cardiomyocyte function in mini-swine with compensated heart failure. Physiol Rep. 2014; 2(6): pii: e12050. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeinzel FR, Hohendanner F, Jin G, et al.: Myocardial hypertrophy and its role in heart failure with preserved ejection fraction. J Appl Physiol (1985). 2015; 119(10): 1233–42. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHeinonen I, Sorop O, de Beer VJ, et al.: What can we learn about treating heart failure from the heart's response to acute exercise? Focus on the coronary microcirculation. J Appl Physiol (1985). 2015; 119(8): 934–43. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMarshall KD, Muller BN, Krenz M, et al.: Heart failure with preserved ejection fraction: chronic low-intensity interval exercise training preserves myocardial O2 balance and diastolic function. J Appl Physiol (1985). 2013; 114(1): 131–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDixon JA, Spinale FG: Large animal models of heart failure: a critical link in the translation of basic science to clinical practice. Circ Heart Fail. 2009; 2(3): 262–71. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDouglas WR: Of pigs and men and research: a review of applications and analogies of the pig, sus scrofa, in human medical research. Space Life Sci. 1972; 3(3): 226–34. PubMed Abstract | Publisher Full Text\n\nSprenger JU, Nikolaev VO: Biophysical techniques for detection of cAMP and cGMP in living cells. Int J Mol Sci. 2013; 14(4): 8025–46. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nZacchigna S, Zentilin L, Giacca M: Adeno-associated virus vectors as therapeutic and investigational tools in the cardiovascular system. Circ Res. 2014; 114(11): 1827–46. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11949",
"date": "18 Jan 2016",
"name": "Maria Kontaridis",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11950",
"date": "18 Jan 2016",
"name": "Sakthi Sadayappan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11951",
"date": "18 Jan 2016",
"name": "Timothy McKinsey",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-72
|
https://f1000research.com/articles/5-65/v1
|
14 Jan 16
|
{
"type": "Review",
"title": "Prevention of Device-Related Healthcare-Associated Infections",
"authors": [
"Edward J. Septimus",
"Julia Moody",
"Julia Moody"
],
"abstract": "Healthcare-associated infections (HAIs) are a leading cause of morbidity and mortality in hospitalized patients. Up to 15% of patients develop an infection while hospitalized in the United States, which accounts for approximately 1.7 million HAIs, 99,000 deaths annually and over 10 billion dollars in costs per year. A significant percentage of HAIs are preventable using evidenced-based strategies. In terms of device-related HAIs it is estimated that 65-70% of catheter-line associated bloodstream infections (CLABSIs) and catheter-associated urinary tract infections (CAUTIs) are preventable. To prevent CLABSIs a bundle which includes hand hygiene prior to insertion and catheter manipulation, use of chlorhexidene alcohol for site preparation and maintenance, use of maximum barrier for catheter insertion, site selection, removing nonessential lines, disinfect catheter hubs before assessing line, and dressing changes are essential elements of basic practices. To prevent CAUTIs a bundle that includes hand hygiene for insertion and catheter or bag manipulation, inserting catheters for appropriate indications, insert using aseptic technique, remove catheters when no longer needed, maintain a close system keeping bag and tubing below the bladder are the key components of basic practices.",
"keywords": [
"CLABSI",
"central line–associated bloodstream infections",
"healthcare-associated infections",
"CAUTI",
"catheter-associated urinary tract infections"
],
"content": "Introduction\n\nHealth care-associated infections (HAIs) are a leading cause of morbidity and mortality in hospitalized patients. Up to 15% of patients develop an infection while hospitalized. In the US, this accounts for approximately 1.7 million HAIs and 99,000 deaths annually. HAIs are now the fifth leading cause of death in US acute care hospitals1. A recent report estimated US health care system costs attributable to the five most common HAIs—central line-associated bloodstream infections (CLABSIs), catheter-associated urinary tract infections (CAUTIs), ventilator-associated pneumonia, surgical site infection, and Clostridium difficile infection—to be $9.8 billion, even without considering the sizable societal costs2. We now know a significant percentage of HAIs are preventable by using evidence-based strategies3. In terms of device-related HAIs, it is estimated that 65% to 70% of CLABSIs and CAUTIs are now preventable. There is now coordination of federal efforts aimed at HAI prevention, including public reporting of hospital-specific HAI rates and linking hospital-specific HAI performance to financial reimbursement as a strategy to motivate hospitals’ HAI prevention efforts. Since 2011, hospitals have been required to report CLABSIs among patients in intensive care units (ICUs) to the Centers for Disease Control and Prevention’s (CDC) National Healthcare Safety Network (NHSN) in order to qualify for annual payment updates. CAUTI reporting in ICUs was added in 2012. In addition, starting in 2015, CLABSIs and CAUTIs are reported in general medical surgical wards. Along with other quality metrics, these HAI data will be used to determine hospital-specific Centers for Medicare & Medicaid Services reimbursement levels as part of value-based purchasing, thereby shifting reimbursement from volume-driven to performance-driven.\n\nEvidence-based recommendations provided through guidelines or guidance documents form the foundation for HAI prevention efforts. Translating knowledge into practice requires an integrated approach to address both technical and adaptive work, including a deep understanding of the health care delivery system and human behavior, fostering engagement and ownership of the improvement process by local interdisciplinary teams, creating centralized support for the technical work, encouraging local adaptation of the intervention bundle, and ensuring a collaborative culture within the facility4. The use of prevention bundles has been shown to reduce HAI rates. A bundle is best defined as a grouping of evidence-based practices that individually improve care. A number of studies have demonstrated the impact of catheter insertion and maintenance bundles on CLABSI rates and have shown that CLABSI prevention bundles are effective, sustainable, and cost-effective for both adults and children5,6. Bundles have also been used in successful multifaceted efforts to reduce CAUTI7. The purpose of this article is to review the strategies to prevent device-related infections, CLABSIs and CAUTIs.\n\n\nCatheter line-associated bloodstream infection\n\nAn estimated 41,000 CLABSIs occurred in the US in 20098. Although the primary focus over the last two decades has been the ICU, the majority of CLABSIs occur outside the ICU9. In 2013, Zimlichman et al. published a meta-analysis of costs and financial impact of HAIs on the US health care system2. On a per-case basis, CLABSIs were found to be the most costly HAIs at $45,814.\n\nThere are two major sources for contaminated catheters and subsequent CLABSI: (1) colonization at the insertion site with migration of organism(s) along the external surface of the catheter is the most common source of CLABSI in catheters within the first week of catheterization, and (2) direct contamination of connectors/hubs resulting in internal colonization and subsequent CLABSI is the major source in catheters in place for at least 1 week. Less commonly, catheters can be seeded hematogenously from another site of infection and rarely from contaminated intravenous (IV) fluids10.\n\nCLABSI is a term used for surveillance purposes by the CDC’s NHSN to identify bloodstream infections (BSIs) that occur in patients with a central venous line. A central line is an intravascular catheter that terminates at or close to the heart or in one of the great vessels which is used for infusion, withdrawal of blood, or hemodynamic monitoring. A laboratory-confirmed CLABSI (LCBI) is where a central line was in place for more than 2 calendar days on the date of event, with day of device placement being day 1, and a central line was in place on the date of event or the day before. If a central line was in place for more than 2 calendar days and then removed, the date of event of the LCBI must be the day of discontinuation or the next day. There are two criteria: (1) the patient has a recognized pathogen cultured from one or more blood cultures, and the organism cultured from blood is not related to an infection at another site, or (2) the patient has at least one of the following signs or symptoms: fever (>38.0°C), chills, or hypotension and the organism cultured from blood is not related to an infection at another site, and the same common commensal—i.e. diphtheroids (Corynebacterium spp. not C. diphtheriae), Bacillus spp. (not B. anthracis), Propionibacterium spp., coagulase-negative staphylococci (including S. epidermidis), viridans group streptococci, Aerococcus spp., and Micrococcus spp.—is cultured from two or more blood cultures drawn on separate occasions11.\n\nPrevention strategies are divided into insertion and maintenance, as well as basic practices and special approaches. Basic practices should be implemented in all acute care hospitals, whereas special approaches should be considered only when CLABSIs are not controlled by use of basic practices12 (Table 1).\n\n1. Have a process, such as a checklist, to ensure compliance with evidence-based infection prevention strategies to prevent CLABSIs.\n\n2. Perform hand hygiene before and after insertion, dressing change, or hub access. Multiple studies have documented reduction of HAIs, including CLABSIs, with strict adherence to hand hygiene. A recent publication reported a decrease in CLABSI rates from 4.08 per 1000 catheter line days to 0.42 per 1000 catheter line days after a multifactorial action plan to improve hand hygiene compliance13.\n\n3. Use a chlorhexidine gluconate (CHG)-alcohol antiseptic for skin preparation. Multiple studies show a greater reduction in both colonization and infection by using CHG-alcohol compared with a povidone-iodine (PI) preparation14. A recent study compared CHG-alcohol with PI-alcohol for prevention of CLABSIs. CHG-alcohol was associated with a significantly lower incidence of CLABSI compared with PI-alcohol (0.28 versus 1.77 per 1000 catheter days; hazard ratio 0.15, 95% confidence interval [CI] 0.05 to 0.41; P = 0.0002)15.\n\n4. Use maximum sterile barrier (MSB) precautions during central line insertion. MSB precautions include mask, cap, sterile gown, and sterile gloves by all health care workers involved in the catheter insertion. In addition, the patient should be covered with a sterile full-body drape during insertion. Most studies have shown a reduction in CLABSIs when maximum barrier precautions are enforced. In a randomized control trial, MSB precautions during insertion of central venous catheter (CVC) were compared with sterile gloves and a small drape. The MSB group had fewer episodes of both catheter colonization (relative risk [RR] = 0.32, 95% CI 0.10 to 0.96, P = 0.04) and CLABSIs (RR = 0.16, 95% CI 0.02 to 1.30, P = 0.06)16. However, a recent prospective randomized trial in surgical patients failed to show a benefit of maximum barrier precautions17. Nonetheless, the majority of evidence suggests a reduction in CLABSI rates with maximum barrier precautions.\n\n5. Avoid using the femoral site for central venous pressure (CVP) access in adult obese patients when placed under elective and controlled situations12,18. In pediatrics, the risk of infection is equal in femoral site insertion versus non-femoral sites19.\n\n6. Use ultrasound guidance for internal jugular catheter insertion. Ultrasound use has been shown to reduce the risk of CLABSI and non-infectious complications during insertion20. In a recent systemic review and meta-analysis, ultrasound-guided subclavian insertion also reduced the risk of adverse events21.\n\n1. Disinfect catheter hubs and connectors before accessing the catheter. Recent studies suggest that friction for at least 5 seconds is needed for reducing contamination of split-septum needless connectors22. Monitoring scrubbing of the hub is important to ensure compliance.\n\n2. Promptly remove catheters when no longer needed, since prolonged catheter use increases the risk of CLABSI23. Multiple concurrent lines are also associated with increased risk for CLABSIs24. In addition, central lines placed under conditions that are not compliant with asepsis (emergently placed) should be replaced or removed whenever feasible. Therefore, facilities should monitor all lines for indications on a daily basis.\n\n3. Minimize unnecessary manipulation of lines, such as drawing blood through lines for convenience.\n\n4. Bathe ICU patients over 2 months of age with CHG on a daily basis. In the last few years, there has been growing evidence that daily CHG bathing in the ICU reduces CLABSIs in both adult ICUs and pediatric ICUs. Milstone et al. found that 2% CHG cloth bathing was significantly associated with a significant decline in BSIs compared with standard bathing25. In another trial, called the REDUCE MRSA (Randomized Evaluation of Decolonization versus Universal Clearance to Eliminate MRSA), universal decolonization with daily CHG with 2% CHG cloths along with 5 days of mupirocin was compared with targeted decolonization or screening and isolation alone. The trial demonstrated universal decolonization was more effective than targeted decolonization or screening and isolation in significantly reducing all cause bloodstream infection by 44% (P<0.001)26. The role of CHG bathing outside the ICU remains to be determined.\n\n5. Change transparent dressing and perform site care with a CHG-based product every 5 to 7 days or every 2 days for a gauze dressing12. Replace dressing immediately if dressing becomes damp, loose, or visibly soiled. Recently, Timsit et al. found that the number of dressing disruptions was related to increased risk of colonization around the insertion site and that the risk of CLABSI increased threefold after the second dressing disruption27.\n\n6. Replace administration sets not used for blood, blood products, total parenteral nutrition (TPN), or lipids at intervals of no longer than 96 hours. Administration sets used for blood, blood products, TPN, or lipids should be changed every 24 hours12.\n\n1. Antimicrobial-/antiseptic-impregnated catheters: Use of a chlorhexidine/silver sulfadiazine- or minocycline/rifampin-impregnated CVC in adults whose catheter is expected to remain in place for more than 5 days has been shown to reduce the risk of CLABSIs28,29.\n\n2. Use a CHG-containing dressing for CVPs in patients over 2 months of age: in a large multicenter randomized controlled trial, the investigators compared chlorhexidine-impregnated sponge dressing versus standard dressings in ICU patients. They found a significant reduction in CLABSIs (6/1953 catheters, 0.40 versus 17/1825 catheters, 1.3 per 1000 catheter-days; hazard ratio 0.24, 95% CI 0.09 to 0.65)30. In a follow-up study, the authors published a large randomized trial using a CHG-gel-impregnated transparent dressing and also found a significant decrease in CLABSIs31.\n\n3. Use an antiseptic-containing cap to cover connectors: in an observational trial in a tertiary care unit, the practice of central line hub care was changed from cleaning with alcohol wipes to using alcohol-impregnated protectors. The implementation of alcohol-impregnated protectors significantly reduced the rate of CLABSIs. The rate of CLABSIs decreased from 2.3/1000 central line-days in the preintervention period to 0.3/1000 central line-days in the intervention period (RR 0.14; 95% CI 0.02 to 1.07; P = 0.03)32. Wright et al. published a multifacility, quasi-experimental study of adult patients with central lines divided into P1 (baseline), when the standard scrub was used; P2, when an alcohol cap was used on all central lines; and P3, when standard disinfection was reinstituted. CLABSI rates declined from 1.43 per 1000 line-days (16/11,154) to 0.69 (13/18,972) in P2 (P = 0.04), and increased to 1.31 (7/5354) in P333. Some of the studies had blood cultures drawn from lines; therefore, some reduction may have been reduction of colonization rather than true infection.\n\n4. Antimicrobial lock solutions: to use lock solutions, supratherapeutic concentrations of an antimicrobial solution are allowed to dwell rather than simply flush through the catheter. Owing to concerns regarding potential for emergence of resistance, antimicrobial locks have been reserved for patients with long-term hemodialysis catheters, patients with limited IV access and a history of recurrent CLABSIs, and patients at risk for severe sequelae, such as those with recently implanted intravascular devices (e.g. prosthetic heart valve or intravascular graft)12. In a recent meta-analysis, Zacharioudakis et al. reported on 23 studies involving adult patients undergoing hemodialysis, adult and pediatric oncology patients, neonates, and patients receiving parenteral nutrition34. The authors found that the use of antimicrobial lock solutions led to a 69% reduction in CLABSI rate (RR 0.31, 95% CI 0.24 to 0.40) and a 32% reduction in the rate of exit site infections (RR 0.68, 95% CI 0.49 to 0.95) compared with heparin. They concluded that antimicrobial lock solutions are effective at reducing CLABSIs in select populations and are additive to basic practices34. Owing to concern over the development of antimicrobial resistance, there has been recent interest in non-antibiotic antimicrobial solutions such as ethanol35. The effectiveness of ethanol has been studied almost exclusively in hemodialysis patients, demonstrating a reduction of CLABSIs with long dwell times.\n\n\nCatheter-associated urinary tract infection\n\nUrinary tract infections (UTIs) are common hospital-acquired infections accounting for 15% of HAIs, and approximately 70% are associated with an indwelling urethral catheter. Up to 16% of inpatients have a urinary catheter at some point during their admission1. Although the primary focus over the last two decades has been the ICU, the majority of CAUTIs occur outside the ICU36. In 2013, Zimlichman et al. published a meta-analysis of costs and financial impact of HAIs on the US health care system2. On a per-case basis, CAUTIs cost at least at $896. The incidence rate was third highest among US adult inpatients. In addition, there are many non-infectious complications that are at least as common as urinary tract infections (UTIs). Urinary catheters can operate as physical restraints by reducing mobility, leading to increased falls, risk of venous thromboembolism, and pressure ulcers37. Leakage, urethral strictures, gross hematuria, and blockage have also been reported38.\n\nThe major contributing risk for developing CAUTI is the duration the urinary device is present. Other risk factors include meatal colonization with uropathogens, microbiological colonization of the draining bag, and gaps in catheter insertion and care.\n\nCAUTI is a term used for surveillance purposes by the CDC’s NHSN to identify UTIs that occur in patients with an indwelling Foley urinary catheter. A laboratory-confirmed CAUTI is where the indwelling urinary catheter is in place for more than 2 calendar days on the date of event, with day of device placement being day 1, and an indwelling urinary catheter was in place on the date of event or the day before. If an indwelling urinary catheter was in place for more than 2 calendar days and then removed, the date of event of the CAUTI must be the day of discontinuation or the next day. In 2015, two significant changes occurred in the surveillance definitions: (1) retirement of cultures with less than 100,000 colony-forming units (CFU)/ml and positive urinalysis diagnostic tests, and (2) removal of Candida species and yeast in the qualifying urinary pathogen list.\n\nThe 2015 surveillance criteria for symptomatic UTI in adults are the patient has a recognized pathogen cultured from urine of no more than two organism species, where at least one organism is a bacteria of more than 100,000 CFU/ml and the patient has at least one of the following signs or symptoms: fever (>38.0°C), suprapubic tenderness, costovertebral angle pain or tenderness, urinary urgency, urinary frequency or dysuria, where symptoms except for fever have no other recognized cause within the infection window period39. Prevention strategies are divided into basic practices of insertion, maintenance, and removal and should be implemented in all acute care hospitals40 (Table 2).\n\n1. Establish processes to assess for medical necessity for patient care and appropriateness of device in care areas such as emergency room, ICU, non-ICU ward, perioperative suites, and inserting urinary catheters. Table 3 includes suggested indication for urinary catheter insertion40. Consider alternative devices or intermittent catheterization when applicable. One statewide effort reduced catheter device utilization through education and monitoring compliance with appropriate indications41.\n\n2. Perform hand hygiene before and after insertion.\n\n3. Cleanse or perform pericare to remove gross material prior to applying antiseptic solution to maximize antiseptic bioactivity and minimize introduction of bacteria, fluids, and secretions into the bladder.\n\n4. Use aseptic insertion technique and sterile supplies. All-inclusive prepackaged kits or having necessary supplies conveniently located support aseptic insertion and evidence-based practice.\n\n5. Apply securement device to prevent movement and traction.\n\n1. Perform hand hygiene before and after catheter or bag manipulation.\n\n2. Perform regular pericare and incontinence care to keep catheter clean.\n\n3. Practice bundles to optimize unobstructed urine flow include the following:\n\n○ Maintaining a sterile, continuous closed system.\n\n○ Minimizing dependent loops and kinks.\n\n○ Keeping bag below bladder, including during transport.\n\n○ Emptying urine from the collecting bag regularly by using a patient-dedicated collection container.\n\n4. Collect urine samples from the port, not drain tubing.\n\n5. Replace devices when breaks or leaks occur with the catheter and collection system.\n\n6. Assess daily for medical necessity for patient care. Consider other methods such as intermittent catheterization where appropriate.\n\n1. Remove when no longer medically necessary. Reminders, stop orders, and protocolizing removal for patients meeting appropriate indications have been shown to be successful in timely removal of urinary catheters when no longer necessary for patient care42,43. Successful outcomes have occurred when partnering with clinicians and nurses to adopt removal protocols in qualifying patients upon change in level of care from ICU to non-ICU wards, or post-anesthesia care unit to inpatient units. Directives that allow nurses to remove urinary catheters on the basis of specified criteria have been shown to reduce catheter days and CAUTIs44.\n\n2. Support toileting and consider alternative urinary devices such as intermittent catheterization, external male condom catheters, and urinals.\n\n3. Use bladder scanners to assess urinary retention.\n\n1. Use of antimicrobial-/antiseptic-impregnated catheters is unproven for routine use. Pickard et al. published a prospective randomized three-arm trial comparing a standard latex catheter, latex silver alloy-coated catheter and a nitrofurazone silicone-impregnated catheter45. They found no differences in symptomatic culture-confirmed urinary infection at 6 weeks with use of the two latex catheters and only a small decrease with the nitrofurazone silicone catheter (odds ratio 0.68, 97.5% CI 0.48 to 0.99; P = 0.017). However, the nitrofurazone catheter was associated with greater patient discomfort and increased catheter removal. They concluded that routine use of antimicrobial-impregnated catheters was not supported45.\n\n2. Avoid screening for asymptomatic bacteriuria (ABU) in catheterized patients. Studies have confirmed that overtreatment of ABU with urinary catheters remains high and can lead to increased antimicrobial resistance and C. difficile infections. Recently, Trautner et al. demonstrated that an intervention to decrease inappropriate screening for ABU significantly decreased overtreatment of ABU46.\n\n3. Do not change catheters routinely.\n\n\nSummary\n\nEffective sustainable reduction of CLABSIs and CAUTIs requires a multifaceted approach implementing evidence-based strategies plus an adaptive approach which aligns health care professional behaviors. We have made significant progress in the last 10 years, but more can be done in eliminating preventable CLABSIs and CAUTIs.",
"appendix": "Competing interests\n\n\n\nES and JM are currently conducting a trial in which participating hospitals are receiving products contributed by Mölnlycke Health Care (Gothenburg, Sweden) and Sage Products (Cary, IL, USA). However, the manufacturers had no part in the design or implementation of the trials.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMagill SS, Edwards JR, Bamberg W, et al.: Multistate point-prevalence survey of health care-associated infections. N Engl J Med. 2014; 370(13): 1198–208. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nZimlichman E, Henderson D, Tamir O, et al.: Health care-associated infections: a meta-analysis of costs and financial impact on the US health care system. JAMA Intern Med. 2013; 173(22): 2039–46. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nUmscheid CA, Mitchell MD, Doshi JA, et al.: Estimating the proportion of healthcare-associated infections that are reasonably preventable and the related mortality and costs. Infect Control Hosp Epidemiol. 2011; 32(2): 101–14. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSeptimus E, Yokoe DS, Weinstein RA, et al.: Maintaining the momentum of change: the role of the 2014 updates to the compendium in preventing healthcare-associated infections. Infect Control Hosp Epidemiol. 2014; 35(5): 460–3. PubMed Abstract | Publisher Full Text\n\nPronovost P, Needham D, Berenholtz S, et al.: An intervention to decrease catheter-related bloodstream infections in the ICU. N Engl J Med. 2006; 355(26): 2725–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMiller MR, Griswold M, Harris JM 2nd, et al.: Decreasing PICU catheter-associated bloodstream infections: NACHRI's quality transformation efforts. Pediatrics. 2010; 125(2): 206–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSaint S, Olmsted RN, Fakih MG, et al.: Translating health care-associated urinary tract infection prevention research into practice via the bladder bundle. Jt Comm J Qual Patient Saf. 2009; 35(9): 449–55. PubMed Abstract | Free Full Text\n\nCenters for Disease Control and Prevention: Vital signs: central line-associated blood stream infections--United States, 2001, 2008, and 2009. MMWR Morb Mortal Wkly Rep. 2011; 60(8): 243–8. PubMed Abstract\n\nVonberg R, Behnke M, Geffers C, et al.: Device-associated infection rates for non-intensive care unit patients. Infect Control Hosp Epidemiol. 2006; 27(4): 357–61. PubMed Abstract | Publisher Full Text\n\nCrnich CJ, Maki DG: The promise of novel technology for the prevention of intravascular device-related bloodstream infection. I. Pathogenesis and short-term devices. Clin Infect Dis. 2002; 34(9): 1232–42. PubMed Abstract | Publisher Full Text\n\nCenters for Disease Control and Prevention: Bloodstream Infection Event (Central Line-Associated Bloodstream Infection and Non-central line-associated Bloodstream Infection). 2016; Accessed October 2, 2015. Reference Source\n\nMarschall J, Mermel LA, Fakih M, et al.: Strategies to prevent central line-associated bloodstream infections in acute care hospitals: 2014 update. Infect Control Hosp Epidemiol. 2014; 35(7): 753–71. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJohnson L, Grueber S, Schlotzhauer C, et al.: A multifactorial action plan improves hand hygiene adherence and significantly reduces central line-associated bloodstream infections. Am J Infect Control. 2014; 42(11): 1146–51. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChaiyakunapruk N, Veenstra DL, Lipsky BA, et al.: Chlorhexidine compared with povidone-iodine solution for vascular catheter-site care: a meta-analysis. Ann Intern Med. 2002; 136(11): 792–801. PubMed Abstract | Publisher Full Text\n\nMimoz O, Lucet J, Kerforne T, et al.: Skin antisepsis with chlorhexidine-alcohol versus povidone iodine-alcohol, with and without skin scrubbing, for prevention of intravascular-catheter-related infection (CLEAN): an open-label, multicentre, randomised, controlled, two-by-two factorial trial. Lancet. 2015; 386(10008): 2069–77. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRaad II, Hohn DC, Gilbreath BJ, et al.: Prevention of central venous catheter-related infections by using maximal sterile barrier precautions during insertion. Infect Control Hosp Epidemiol. 1994; 15(4 Pt 1): 231–8. PubMed Abstract\n\nIshikawa Y, Kiyama T, Haga Y, et al.: Maximal sterile barrier precautions do not reduce catheter-related bloodstream infections in general surgery units: a multi-institutional randomized controlled trial. Ann Surg. 2010; 251(4): 620–3. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMarik PE, Flemmer M, Harrison W: The risk of catheter-related bloodstream infection with femoral venous catheters as compared to subclavian and internal jugular venous catheters: a systematic review of the literature and meta-analysis. Crit Care Med. 2012; 40(8): 2479–85. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nde Jonge RC, Polderman KH, Gemke RJ: Central venous catheter use in the pediatric patient: mechanical and infectious complications. Pediatr Crit Care Med. 2005; 6(3): 329–39. PubMed Abstract | Publisher Full Text\n\nHind D, Calvert N, McWilliams R, et al.: Ultrasonic locating devices for central venous cannulation: meta-analysis. BMJ. 2003; 327(7411): 361. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLalu MM, Fayad A, Ahmed O, et al.: Ultrasound-Guided Subclavian Vein Catheterization: A Systematic Review and Meta-Analysis. Crit Care Med. 2015; 43(7): 1498–507. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRupp ME, Yu S, Huerta T, et al.: Adequate disinfection of a split-septum needleless intravascular connector with a 5-second alcohol scrub. Infect Control Hosp Epidemiol. 2012; 33(7): 661–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMcLaws M, Berry G: Nonuniform risk of bloodstream infection with increasing central venous catheter-days. Infect Control Hosp Epidemiol. 2005; 26(8): 715–9. PubMed Abstract | Publisher Full Text\n\nConcannon C, van Wijngaarden E, Stevens V, et al.: The effect of multiple concurrent central venous catheters on central line-associated bloodstream infections. Infect Control Hosp Epidemiol. 2014; 35(9): 1140–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMilstone AM, Elward A, Song X, et al.: Daily chlorhexidine bathing to reduce bacteraemia in critically ill children: a multicentre, cluster-randomised, crossover trial. Lancet. 2013; 381(9872): 1099–106. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHuang SS, Septimus E, Kleinman K, et al.: Targeted versus universal decolonization to prevent ICU infection. N Engl J Med. 2013; 368(24): 2255–65. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTimsit JF, Bouadma L, Ruckly S, et al.: Dressing disruption is a major risk factor for catheter-related infections. Crit Care Med. 2012; 40(6): 1707–14. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDarouiche RO, Raad II, Heard SO, et al.: A comparison of two antimicrobial-impregnated central venous catheters. Catheter Study Group. N Engl J Med. 1999; 340(1): 1–8. PubMed Abstract | Publisher Full Text\n\nRupp ME, Lisco SJ, Lipsett PA, et al.: Effect of a second-generation venous catheter impregnated with chlorhexidine and silver sulfadiazine on central catheter-related infections: a randomized, controlled trial. Ann Intern Med. 2005; 143(8): 570–80. PubMed Abstract | Publisher Full Text\n\nTimsit JF, Schwebel C, Bouadma L, et al.: Chlorhexidine-impregnated sponges and less frequent dressing changes for prevention of catheter-related infections in critically ill adults: a randomized controlled trial. JAMA. 2009; 301(12): 1231–41. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTimsit JF, Mimoz O, Mourvillier B, et al.: Randomized controlled trial of chlorhexidine dressing and highly adhesive dressing for preventing catheter-related infections in critically ill adults. Am J Respir Crit Care Med. 2012; 186(12): 1272–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSweet MA, Cumpston A, Briggs F, et al.: Impact of alcohol-impregnated port protectors and needleless neutral pressure connectors on central line-associated bloodstream infections and contamination of blood cultures in an inpatient oncology unit. Am J Infect Control. 2012; 40(10): 931–4. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWright MO, Tropp J, Schora DM, et al.: Continuous passive disinfection of catheter hubs prevents contamination and bloodstream infection. Am J Infect Control. 2013; 41(1): 33–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZacharioudakis IM, Zervou FN, Arvanitis M, et al.: Antimicrobial lock solutions as a method to prevent central line-associated bloodstream infections: a meta-analysis of randomized controlled trials. Clin Infect Dis. 2014; 59(12): 1741–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBroom JK, Krishnasamy R, Hawley CM, et al.: A randomised controlled trial of Heparin versus EthAnol Lock THerapY for the prevention of Catheter Associated infecTion in Haemodialysis patients--the HEALTHY-CATH trial. BMC Nephrol. 2012; 13: 146. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDudeck MA, Edwards JR, Allen-Bridson K, et al.: National Healthcare Safety Network report, data summary for 2013, Device-associated Module. Am J Infect Control. 2015; 43(3): 206–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaint S, Lipsky BA, Goold SD: Indwelling urinary catheters: a one-point restraint? Ann Intern Med. 2002; 137(2): 125–7. PubMed Abstract | Publisher Full Text\n\nHollingsworth JM, Rogers MA, Krein SL, et al.: Determining the noninfectious complications of indwelling urethral catheters: a systematic review and meta-analysis. Ann Intern Med. 2013; 159(6): 401–10. PubMed Abstract | Publisher Full Text\n\nCenters for Disease Control and Prevention: Urinary Tract Infection (Catheter-Associated Urinary Tract Infection [CAUTI] and Non-Catheter-Associated Urinary Tract Infection [UTI]) and Other Urinary System Infection [USI]) Events. 2016. Accessed 10-02-2015. Reference Source\n\nLo E, Nicolle LE, Coffin SE, et al.: Strategies to prevent catheter-associated urinary tract infections in acute care hospitals: 2014 update. Infect Control Hosp Epidemiol. 2014; 35(5): 464–79. PubMed Abstract | Publisher Full Text\n\nFakih MG, Watson SR, Greene MT, et al.: Reducing inappropriate urinary catheter use: a statewide effort. Arch Intern Med. 2012; 172(3): 255–60. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSaint S, Greene MT, Kowalski CP, et al.: Preventing catheter-associated urinary tract infection in the United States: a national comparative study. JAMA Intern Med. 2013; 173(10): 874–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSaint S, Kowalski CP, Forman J, et al.: A multicenter qualitative study on preventing hospital-acquired urinary tract infection in US hospitals. Infect Control Hosp Epidemiol. 2008; 29(4): 333–41. PubMed Abstract | Publisher Full Text\n\nParry MF, Grant B, Sestovic M: Successful reduction in catheter-associated urinary tract infections: focus on nurse-directed catheter removal. Am J Infect Control. 2013; 41(12): 1178–81. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPickard R, Lam T, MacLennan G, et al.: Antimicrobial catheters for reduction of symptomatic urinary tract infection in adults requiring short-term catheterisation in hospital: a multicentre randomised controlled trial. Lancet. 2012; 380(9857): 1927–35. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTrautner BW, Grigoryan L, Petersen NJ, et al.: Effectiveness of an Antimicrobial Stewardship Approach for Urinary Catheter-Associated Asymptomatic Bacteriuria. JAMA Intern Med. 2015; 175(7): 1120–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11940",
"date": "14 Jan 2016",
"name": "Mohamad Fakih",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11941",
"date": "14 Jan 2016",
"name": "Keith S. Kaye",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-65
|
https://f1000research.com/articles/5-59/v1
|
13 Jan 16
|
{
"type": "Review",
"title": "Implications of circadian rhythm and stress in addiction vulnerability",
"authors": [
"Darius Becker-Krail",
"Colleen McClung",
"Darius Becker-Krail"
],
"abstract": "In the face of chronic stress, some individuals can maintain normal function while others go on to develop mental illness. Addiction, affecting one in every twelve people in America, is a substance use disorder long associated with stressful life events and disruptions in the sleep/wake cycle. The circadian and stress response systems have evolved to afford adaptability to environmental changes and allow for maintenance of functional stability, or homeostasis. This mini-review will discuss how circadian rhythms and stress individually affect drug response, affect each other, and how their interactions may regulate reward-related behavior. In particular, we will focus on the interactions between the circadian clock and the regulation of glucocorticoids by the hypothalamic-pituitary-adrenal (HPA) axis. Determining how these two systems act on dopaminergic reward circuitry may not only reveal the basis for vulnerability to addiction, but may also illuminate potential therapeutic targets for future investigation.",
"keywords": [
"Addiction",
"Circadian",
"Clock",
"Glucocorticoids",
"HPA",
"Reward",
"Stress",
"Vulnerability"
],
"content": "Introduction\n\nWithin any given environment, an organism must learn to anticipate and adapt to external changes/stressors in order to survive. Two systems, the circadian and stress response systems, have evolved to afford adaptability to both recurring and spontaneous environmental changes. Through the active process of allostasis (McEwen, 1998; Sterling & Eyer, 1988), these systems and many others work to maintain an internal state of stability, or homeostasis, in the presence of these changes. In the face of acute and/or chronic stressors, the ability for an organism to adapt and avoid negative biological consequences is known as resilience. Within the context of psychopathology, resilience can be seen as a person’s ability to maintain physiological and psychobiological homeostasis, in spite of adversity (Charney, 2004; Feder et al., 2009). While resilient individuals uphold “normal” mental health and homeostatic function when exposed to stress, other individuals may go on to develop neuropsychiatric or behavioral disorders. According to the vulnerability-stress model (Ingram & Luxton, 2005; Zubin & Spring, 1977), some individuals may be more biologically vulnerable to developing neuropsychiatric disorders when faced with acute and/or chronic stressors (Pihl & Nantel-Vivier, 2005). To fully understand this phenomenon, current research seeks to understand the effects of stress in the brain (reviewed in McEwen et al., 2015), and elucidate potential structural and/or molecular bases resulting in the vulnerability to develop mental illnesses.\n\nMany neuropsychiatric disorders have long been associated with altered circadian rhythm (Mills et al., 1977; Van Cauter & Turek, 1986; Wehr et al., 1983) and stress response (Albus et al., 1982; Amsterdam et al., 1983; Roy et al., 1988); highlighted here, disruptions in both systems are also known to have implications in substance use disorder/addiction and reward-related behaviors (Briand & Blendy, 2010; Falcón & McClung, 2009; Koob, 2008; Logan et al., 2014). While abnormal circadian and stress response function can independently affect reward-related behavior, understanding the interface of these two systems, during both typical and atypical functioning, may provide greater insight into the complexities of disorder etiology. This mini-review examines the interplay of the circadian and stress response systems, and how this interaction may affect addiction vulnerability.\n\n\nCircadian rhythm and the molecular clock\n\nHighly conserved across most living organisms, circadian rhythms facilitate the anticipation and adaptation of behavior to daily changes in environmental stimuli. In mammalian organisms, system and cellular level rhythms are maintained by a rhythm-generating nucleus in the hypothalamus, the suprachiasmatic nucleus (SCN). The SCN can be entrained by both photic and non-photic cues called zeitgebers, or “time-keepers”, but can ultimately generate a ~24 hour rhythm independent of these cues. At the molecular level, across all cell types, circadian rhythms are upheld by a “molecular clock” consisting of auto-regulatory transcription-translation feedback loops in the nucleus (see Figure 1A). The key proteins that make up the molecular clock are transcription factors: circadian locomotor output cycles kaput (CLOCK), or neuronal PAS domain protein 2 (NPAS2), and brain and muscle Arnt-like protein 1 (BMAL1). Throughout the day, CLOCK/BMAL1 (or NPAS2/BMAL1) heterodimerize to promote the transcription of Period (PER1,2,3), Cryptochrome (CRY1,2), and many other clock controlled genes (CCGs). The feedback loop is established when PER and CRY proteins accumulate in the cytoplasm, form hetero- and homodimers, and eventually shuttle back into the nucleus to inhibit their own expression. Additionally, clock regulated RAR-related orphan receptor alpha (RORα) and reverse-ErbA alpha (REV-ERBα) nuclear receptors act in an auxiliary oscillatory feedback loop to regulate expression of Bmal1, stabilizing the core feedback loop (Guillaumond et al., 2005). The circadian molecular clock cycles on a timescale of ~24 hours and regulates the expression of many genes controlling neuronal, metabolic, endocrine, and immune function (Jin et al., 1999; Lowrey & Takahashi, 2000).\n\nA. The mammalian circadian molecular clock consists of multiple transcription and translation feedback (TTF) loops. Central to the TTF loops, the transcription factors CLOCK (or NPAS2) and BMAL1 heterodimerize and bind to the enhancer box (E-Box) sequence to promote transcription of many target and clock controlled genes (CCGs). The main TTF loop is achieved when PERIOD (PER1,2,3) and CRYPTOCHROME (CRY1,2) proteins accumulate, dimerize, undergo phosphorylation, and shuttle back into the nucleus to inhibit both CLOCK/BMAL1 and, as a result, their own transcription. This negative feedback loop cycles every ~24 hours and is crucial for regulation of circadian rhythm. Among its target genes, CLOCK/BMAL1 also regulates the expression of the nuclear receptors RORα and REV-ERBα, both of which can regulate BMAL1 activity via binding at a response element in its promoter. A recently discovered circadian protein, CHRONO, is clock-regulated and can also inhibit CLOCK/BMAL1 activity via interactions at the E-Box. Taken together, these proteins make up auxiliary TTF loops that work to both stabilize and reinforce rhythm. B. The circadian molecular clock can interact with the stress axis through regulating activity of both glucocorticoid (GCC) and its receptor (GCR). GC is known to be released under tight circadian regulation, with peak levels in the animal’s active phase. Additionally, several circadian proteins are known to rhythmically regulate GCR-dependent transcription activity. CLOCK/BMAL1 can directly attenuate GCR activity via acetylation (A), thereby reducing its binding ability at the Glucocorticoid Response Element (GRE). Simultaneously, CHRONO and CRY1,2 proteins can repress GCR activity via direct interaction in ligand-fashion. CRY proteins can also regulate GCR-dependent transcription through association at the GRE. (+), promote/activate; (-), repress/inhibit.\n\n\nCircadian genes and reward\n\nSeveral studies in the past two decades have shown core circadian genes to be important regulators of reward-related behavior in response to common substances of abuse, as reviewed by Parekh et al. (2015). One of the first studies investigating this link demonstrated that fruit flies with mutations in the Period, Clock, and/or Cycle (similar to Bmal1 in mammals) genes fail to show behavioral sensitization to cocaine (Andretic et al., 1999). In mice, mutations in the mPer1 and mPer2 genes seem to produce a similar but unique effect on cocaine response; mutation in mPer2 increased behavioral sensitization to cocaine but mutation in mPer1 abolished sensitization (Abarca et al., 2002). Recently, our lab has been studying a similar differential regulation of reward, but with CLOCK and its functional homologue, NPAS2. While CLOCK is known to be expressed almost ubiquitously, NPAS2 is primarily expressed in the liver and forebrain (Bertolucci et al., 2008; Reick et al., 2001) and can regulate circadian transcription when CLOCK expression is absent or low. With notable implications for reward, NPAS2 has been shown to have a unique expression pattern in the mesolimbic pathway; NPAS2 is highly enriched in the nucleus accumbens (NAc), but has little to no expression in the ventral tegmental area (VTA) (Garcia et al., 2000).\n\nThe observation that NPAS2 is specifically enriched in the NAc motivated our most recent studies investigating the potential for CLOCK and NPAS2 to differentially regulate gene expression and reward-related behavior, following cocaine exposure. Interestingly, in response to chronic cocaine, only NPAS2 is upregulated in the NAc and caudate putamen (normal rhythm abolished) and has increased binding activity at Period gene promoters; CLOCK did not show these changes in the NAc or caudate putamen (Falcón et al., 2013). Knock-down of CLOCK or NPAS2 in these regions also has different effects on the regulation of reward-related behavior following cocaine administration. Mice containing a globally expressed, dominant negative, single-point mutation in the CLOCK protein (Clock∆19) exhibit a mania-like phenotype with increased baseline activity, decreased anxiety- and depression-like behavior, and increased sensitivity to rewarding stimuli; in response to cocaine, Clock∆19 mice show increased conditioned place preference (CPP) and increased cocaine self-administration, relative to wild-type mice (McClung et al., 2005; Ozburn et al., 2012; Roybal et al., 2007). However, using AAV-shRNA to selectively knock-down CLOCK or NPAS2 in the NAc, near entire reduction of CLOCK in the NAc does not recapitulate increased preference seen in Clock∆19; while mice with either a mutated form of NPAS2 or a NAc specific knock-down of NPAS2 showed decreased cocaine CPP and self-administration (Ozburn et al., 2015).\n\nThe genes central to the circadian molecular clock may have differential roles in the regulation of reward-related behavior depending on the time of day, brain region, and/or the drug’s effect on the specific protein in a particular region. Given the involvement of these core circadian genes in the regulation of reward and behavioral response, it is likely that disruption to the normal functioning/activity of these circadian proteins can contribute to the vulnerability of developing an addiction.\n\n\nHPA axis and reward\n\nWhen encountering physical and/or psychological stressors, activation of the hypothalamic-pituitary-adrenal (HPA) axis is a fundamental, evolutionarily conserved response allowing the organism to adapt both physiologically and behaviorally. Regulated by the HPA axis, glucocorticoids (cortisol in humans and corticosterone in rodents) are a class of steroid hormones responsible for driving the changes seen in stress response. Upon activation by limbic structures, neurons in the hypothalamic paraventricular nucleus (PVN) release corticotropin-releasing hormone (CRH) at the median eminence into the hypophyseal portal system, connecting the hypothalamus with the anterior pituitary. Once stimulated by CRH, the anterior pituitary increases secretion of adrenocorticotropic hormone (ACTH) into the bloodstream, where it will then travel to the adrenal gland. At the adrenal cortex, ACTH causes an increase in synthesis and release of glucocorticoids into the circulatory system to then act on energy metabolism, protein synthesis, immune function, cardiovascular function, attention/arousal/vigilance, and even memory function. Much like the circadian molecular clock, the HPA axis is regulated via a negative feedback loop in which accumulation of glucocorticoids in the hypothalamus (PVN) and anterior pituitary can block the production of CRH and ACTH. (de Kloet et al., 2005; Johnson et al., 1992; Uchoa et al., 2014).\n\nWhile the effects of the HPA axis are transient in response to acute stress, chronic stress and prolonged presence of glucocorticoids can have deleterious outcomes, ranging from neurotoxicity and cell death to impaired metabolic/mitochondrial function and immunosuppression (de Kloet et al., 2005; Dhabhar & McEwen, 1997; McEwen et al., 1999; Picard et al., 2014). Considering the potential detrimental effects of chronic stress, recent studies have investigated the implications of the HPA axis and glucocorticoids in neuropsychiatric disorder etiology (Herane Vives et al., 2015; Jacobson, 2014; Sapolsky, 2000; Yehuda et al., 2015). In the context of addiction, animal model studies have long demonstrated a strong association between chronic stress and increased behavioral response to drugs of abuse (Antelman et al., 1980; Deroche et al., 1995; Herman et al., 1984; MacLennan & Maier, 1983). This increased response can be seen through augmented locomotor effects and increased reward/preference – both of which are associated with elevated glucocorticoid activity (Deroche et al., 1995; Haile et al., 2001; Lepsch et al., 2005). Further confirming the role of glucocorticoids in this phenomenon, if normal stress-induced increases in corticosterone (in rats) are blocked (via adrenalectomy), the stress-related increase in drug behavioral response is abolished; notably, the response remains intact if normal elevation of corticosterone is artificially maintained (Deroche et al., 1995; Prasad et al., 1998; Rougé-Pont et al., 1995). Corticosterone has even been shown to be necessary for drug-induced sensitization in general, suggesting its importance in overall behavioral response and drug-seeking behavior, with or without stress as a factor (de Jong et al., 2009; Przegaliński et al., 2000).\n\nThough it is clear that chronic stress and the biological mediators of stress response may alter one’s sensitivity to the behavioral and/or rewarding effects of drugs of abuse, further investigation into the cellular and molecular bases of this relationship is necessary. Given that the circadian rhythm and stress response systems are both known to be involved in mediating behavioral responses to rewarding-substances, perhaps their interactions may be the key to understanding what drives vulnerability to addiction.\n\n\nCircadian regulation of the HPA axis\n\nLike many other processes in the body, the HPA axis and its hormonal components are under direct circadian regulation by both the SCN and a peripheral clock in the adrenal cortex (Ishida et al., 2005; Nader et al., 2010; Oster et al., 2006; Son et al., 2008). Glucocorticoids, along with the other hormones in the axis, display a prominent diurnal variation in which peak levels correspond with the organism’s active phase (reviewed in: Kalsbeek et al., 2012; Spiga et al., 2014). Rhythms in glucocorticoids are not only mediated by both direct and indirect projections to the CRH producing PVN neurons at the fore-end of the HPA axis (Engeland & Arhnhold, 2005; Kalsbeek et al., 2006), but also through modulation of receptors by core circadian proteins (See Figure 1B). Oster et al. (2006) demonstrated the importance of a functional adrenal molecular clock in “gating” sensitivity to ACTH (via ACTH receptors) in the adrenal cortex, as means of regulating glucocorticoid synthesis. Moreover, the actual glucocorticoid’s effect across the body can also be regulated through acting on glucocorticoid receptor (GCR) function; multiple studies have demonstrated the ability for CLOCK/BMAL1 to modulate GCRs and their sensitivity via circadian mediated acetylation (Charmandari et al., 2011; Kino & Chrousos, 2011; Nader et al., 2009). Alongside CLOCK/BMAL1, CRY1,2 have also been shown to repress GCR-dependent transcription activity via association with GCRs and/or at the glucocorticoid responsive element (GRE) (Lamia et al., 2011). In recent literature, a novel circadian protein named CHRONO, or computationally-highlighted/ChIP-derived repressor of network oscillator, acts as a negative regulator of the molecular clock and may even interact with GCRs to rhythmically repress their function (Anafi et al., 2014; Goriki et al., 2014). While investigation into CHRONO’s function is still in its early stages, there is potential for this novel protein to play an even greater role in connecting the circadian and stress response systems.\n\nSeveral circadian-gene mutant mouse studies have further verified the role of core molecular clock proteins in regulating the HPA axis. In Bmal1 null mutant mice, deficiency in BMAL1 causes a significant reduction in glucocorticoid levels, sensitivity, and altered rhythmicity (Leliavski et al., 2014); similarly, Clock mutant mice lacking functional CLOCK protein also show altered glucocorticoid rhythmicity and decreased total levels (Oishi et al., 2006; Takita et al., 2013). Along with the core CLOCK/BMAL1 complex, null mutations in the Cryptochrome (CRY1,2) or Period (PER1) genes also yield altered rhythmicity and, unlike Clock and Bmal1 mutants, increased total glucocorticoid levels (Dallmann et al., 2006; Destici et al., 2013; Lamia et al., 2011). Even more interesting, cultured BMAL1 and/or PER1,2 deficient mouse embryonic fibroblasts (MEFs) show altered GCR transactivation resulting in hypersensitivity to glucocorticoids (Han et al., 2014). Taken together, these studies demonstrate the importance of the molecular clock in regulating both the output of the stress axis and its efficacy.\n\n\nStress effects on circadian rhythm\n\nOccurring simultaneously, the same components of the molecular clock that regulate HPA axis function can also be reciprocally affected by the stress itself. Most notably, chronic stress is known to affect the rhythmic expression of the core circadian genes. A key example can be found through glucocorticoids mediating the expression of Period genes. With either acute or chronic stress, levels of mPer1 and PER1 are elevated in some neural and peripheral tissues (Al-Safadi et al., 2014; Al-Safadi et al., 2015; Takahashi et al., 2013). PER2 has a similar response to stress (Segall & Amir, 2010; So et al., 2009) and even loses rhythmic expression in the bed nucleus of the stria terminalis (BNST) and in the amygdala, following adrenalectomy (Lamont et al., 2005). Additionally, it was later revealed that PER2 actually requires functional GCRs for its rhythmic expression (Segall et al., 2009). Both Period genes are afforded this unique relationship with glucocorticoids due to the presence of GREs in their promotor regions (So et al., 2009; Yamamoto et al., 2005). Glucocorticoids can target genes that have these GREs, bind, and thus promote their expression. This stress-induced change in gene expression, if occurring chronically, can even result in clock entrainment (Tahara et al., 2015).\n\nPrevious work in our lab has shown that mice exposed to chronic social defeat stress show increased anxiety that correlates with decreased mPer1/2 expression in the NAc (Spencer et al., 2012). While this may appear to conflict with aforementioned stress effects on Period genes, it is likely that region specific changes occur depending on the type of stressor, GCR distribution, and region/network involvement. Illustrating this, we recently showed mice subjected to the unpredictable chronic mild stress (UCMS) paradigm (used to model depression-like behavior) have increased Per2 rhythm amplitude in the NAc, but decreased rhythm amplitude in the SCN (Logan et al., 2015). This is likely due to GCR concentration being high in the NAc (Barik et al., 2010; Der-Avakian et al., 2006) but low/absent in the SCN (Balsalobre et al., 2000; Pezuk et al., 2012; Rosenfeld et al., 1988). The interaction between stress response and the circadian molecular clock in a region specific manner may perhaps be the basis that allows for manifestation of disorder specific vulnerability.\n\n\nCircadian rhythm and stress interactions in dopaminergic transmission\n\nAs described above, a lot can be understood by just examining how each system not only affects each other, but also how they individually affect behavioral response to drugs of abuse. However, in the framework of addiction and reward, the effects on vulnerability may more likely arise from the two systems’ interactions while simultaneously regulating the same reward-related processes. Both systems have been show to directly regulate/affect the dopaminergic reward circuitry and its functions involved in addiction (reviewed in: Marinelli & Piazza, 2003; Parekh et al., 2014). Implemented in addiction, the mesolimbic pathway is a system of dopaminergic neurons connecting the VTA to the NAc, and is important for mediating reward-related behavior. This pathway’s activity is known to be directly affected by both the circadian system and the stress response system (McClung et al., 2007; Trainor, 2011). In controlling dopamine synthesis, circadian molecular clock proteins and glucocorticoids may be working in opposing fashion to regulate expression of tyrosine hydroxylase (TH), a key enzyme in synthesis of dopamine from tyrosine (see Figure 2).\n\nImportant for reward-related behavior, tyrosine hydroxylase (TH) is an enzyme involved in the synthesis of dopamine from the amino acid L-Tyrosine. Transcription of TH is mediated by the binding of cAMP response element-binding protein (CREB) at its response element (CRE) in the TH promoter. Dopamine synthesis is known to be directly regulated by both circadian and stress-related proteins/hormones. At the TH promoter, the core circadian CLOCK/BMAL1 complex binds to the enhancer-box (E-Box) sequence in antiphase with CREB:CRE binding, and negatively regulates the transcription of TH in a time dependent manner. Additionally, the circadian nuclear receptor REV-ERBα and nuclear receptor-related 1 (NURR1) protein regulate TH expression via competitive binding at the REV-ERB/ROR response element (RRE)/NGF1B-response element (NBRE); while NURR1 promotes the expression of TH, REV-ERBα represses expression. Glucocorticoids (GCs) and its receptor (GCR) can also promote expression of TH by binding at the glucocorticoid response element (GRE) in the TH promoter. The above response element spacing is not shown to scale. (+), promote/activate; (-), repress/inhibit.\n\nUnder circadian control, recent work in our lab has demonstrated that CLOCK/BMAL1 acts as a time-dependent, negative regulator of TH transcription via binding at the TH promotor in anti-phase with CREB (Sidor et al., 2015). Opposite of CLOCK/BMAL1, glucocorticoids may positively regulate the expression and activity of TH (Fossom et al., 1992; Kalinina et al., 2012; Núñez et al., 2009; Tank et al., 1986). While the exact mechanism by which glucocorticoids increase TH expression is still being investigated, it has been shown that the TH promotor contains a GRE to which glucocorticoids can bind and promote expression independent of CRE (Hagerty et al., 2001a; Hagerty et al., 2001b). An additional explanation may arise through glucocorticoid regulation of the circadian nuclear receptor, REV-ERBα. In a recent paper by Chung et al. (2014), REV-ERBα has been shown to repress TH transcription through competitive binding with nuclear receptor-related 1 protein (NURR1) at the TH promotor. Given that glucocorticoids have been shown to control REV-ERBα expression in the liver (Torra et al., 2000), it may be possible for glucocorticoids to increase TH through a disinhibition type mechanism.\n\nIn addition to regulation of TH, the two systems can simultaneously act on dopamine receptor (DR) expression/function (Biron et al., 1992; Iasevoli et al., 2013; Ikeda et al., 2013; Spencer et al., 2012) and monoamine oxidase-A (MAO-A) expression/function, an enzyme responsible for degradation of dopamine (Chevillard et al., 1981; Cvijić et al., 1995; Hampp et al., 2008; Soliman et al., 2012). Taken together, these studies demonstrate the interactions of both the circadian system and stress response system in regulating the same aspects of reward related function/behavior. With slight disruption in either of the systems, the negative effects of one can alter the other and result in a self-perpetuating consequence. It is at this level that an understanding of vulnerability to addiction can be more readily obtained.\n\n\nConclusion\n\nReward-related behavior and sensitivity to drug response have both been shown to be regulated by the circadian and stress response systems. Described above, disruption in either of the systems individually can have pronounced effects on the rewarding effects of substances of abuse. Understanding both the tight circadian regulation of the stress axis and how stress/response can affect the molecular clock, the possibility for their interaction to drive vulnerability to addiction is entirely plausible. Both systems act in parallel regulating many aspects of reward related behavior and function. Slight mutations in either system causing minor functional disruption may be innocuous alone, but given the interface of the two systems, a minor change has the potential to be amplified in a reverberating fashion. The once subtle change may even become deleterious with time. It is in this logic that vulnerability to addiction is rooted; as a biological predisposition that becomes exploited in the face of chronic stressors. As an outlook, future studies can begin to consider one or both systems as potential therapeutic targets to mediate drug response in addiction and combat vulnerability.",
"appendix": "Author contributions\n\n\n\nDDBK prepared the manuscript. DDBK and CAM contributed equally to the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbarca C, Albrecht U, Spanagel R: Cocaine sensitization and reward are under the influence of circadian genes and rhythm. Proc Natl Acad Sci U S A. 2002; 99(13): 9026–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlbus M, Ackenheil M, Engel RR, et al.: Situational reactivity of autonomic functions in schizophrenic patients. Psychiatry Res. 1982; 6(3): 361–70. 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PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "12056",
"date": "01 Feb 2016",
"name": "Harry Pantazopoulos",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written and timely summary of interactions between the circadian system and the stress response system set in the framework of drug addiction/reward. The article provides an accurate summary of the current literature and a helpful introduction of these biological systems to readers who are unfamiliar with the field. The figures are clear, well-described, and aid the reader in understanding the molecular mechanisms described in the text. In addition, the evolution/environmental interaction perspective in the introduction provides an excellent framework for the following discussion of how interactions between the circadian and stress systems can go awry. This mini-review is a useful reference to researchers studying basic neuroscience questions on these systems and how these systems are disrupted in psychiatric disorders. The review highlights the importance of recognizing the potential interactions between these systems in both normal and pathological conditions.",
"responses": []
},
{
"id": "11935",
"date": "02 Feb 2016",
"name": "Randy Nelson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide an excellent and timely mini-review of the interactions between the circadian and stress response systems, and their actions on dopaminergic reward circuitry underlying addiction and diseases of drug abuse. They highlight the differential expression levels of BMAL1 binding partners CLOCK and NPAS2 throughout the brain, indicating that NPAS2 is specifically enriched in the nucleus accumbens. This sets the stage for the idea that selective manipulation of the circadian clock through NPAS2 in the nucleus accumbens could influence reward-seeking behavior. Additional evidence is discussed regarding the binding of CLOCK/BMAL1 at the tyrosine hydroxylase promoter to repress transcription and therefore dopamine synthesis.The authors further discuss the fundamental link between the HPA axis and the circadian clock through glucocorticoid-response elements in clock gene and tyrosine hydroxylase promoters, acetylation of the glucocorticoid receptor by CLOCK/BMAL1, and other potential interactions with the newly discovered clock component CHRONO. The review is concise and provides an excellent starting point for anyone interested in the etiology, treatment, or molecular biology of addiction or drug abuse.It may be beyond the scope of this review, but future discussion of the effects drugs of abuse on the circadian clock may be warranted.Is there any evidence for circadian control of opioid reward signaling? It may be worth discussing stress effects on opioid or opioid-receptor expression in the VTA or NAc on a circadian basis. (e.g., Polter et al, 2014, Biological Psychiatry Vol 76: 785-793).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-59
|
https://f1000research.com/articles/5-48/v1
|
11 Jan 16
|
{
"type": "Review",
"title": "Perioperative sleep apnea: a real problem or did we invent a new disease?",
"authors": [
"Sebastian Zaremba",
"James E. Mojica",
"Matthias Eikermann",
"Sebastian Zaremba",
"Matthias Eikermann"
],
"abstract": "Depending on the subpopulation, obstructive sleep apnea (OSA) can affect more than 75% of surgical patients. An increasing body of evidence supports the association between OSA and perioperative complications, but some data indicate important perioperative outcomes do not differ between patients with and without OSA. In this review we will provide an overview of the pathophysiology of sleep apnea and the risk factors for perioperative complications related to sleep apnea. We also discuss a clinical algorithm for the identification and management of OSA patients facing surgery.",
"keywords": [
"Obstructive sleep apnea",
"Perioperative sleep apnea",
"Bariatric surgery",
"Upper airway",
"Anaesthesiology"
],
"content": "Objectives\n\nPerioperative sleep apnea is becoming a major concern for anesthesiologists and intensivists1. However, similar to another problem in the perioperative field (i.e. residual paralysis)2, the negative impact of sleep apnea on postsurgical management and patient outcomes remains unclear. In this article, we will (1) summarize currently available data on the prevalence and pathophysiology of sleep apnea in the perioperative context, (2) discuss the bidirectional effect of anesthesia and surgery on sleep apnea, and (3) suggest a clinical pathway for the perioperative identification and management of sleep apnea patients that is being used by many physicians at the Massachusetts General Hospital in Boston, MA, USA based on in-house discussions and input we received from Dr Shiroh Isono, Chiba, Japan during his visits in our institution.\n\n\nObstructive sleep apnea and why it is important in the perioperative setting\n\nObstructive sleep apnea (OSA) is characterized by recurrent episodes of reduction or cessation of airflow despite continued or increased respiratory effort. Hypopneas are shallow breaths resulting from partial obstruction and reduced intraluminal diameter of the upper airway (UA). Apneas are characterized by the absence of airflow due to complete airway collapse. These respiratory events are associated with oxyhemoglobin desaturations, neuronal arousal, disrupted sleep, and impaired daytime functioning3.\n\nBased on daytime symptoms, the incidence of OSA in the general population ranges from 0.3% to 5%4–6. However, several studies investigating the prevalence of OSA without daytime symptoms using polysomnography (PSG) found much higher rates7–9 (Table 1A) with undiagnosed OSA in up to 80% of patients10. Furthermore, with obesity representing a major risk factor for OSA, one can expect a higher prevalence of sleep apnea as rates of obesity continue to climb11–13.\n\n* = retrospective analysis; OSA = obstructive sleep apnea; AHI = apnea hypopnea index; w = women; m = men; NHANES = National Health and Nutrition Examination Surveys; PSG = polysomnography; CABG = coronary artery bypass graft\n\nIn the perioperative population, the prevalence of OSA varies widely among different subgroups (Table 1B). Bariatric surgery patients, the subpopulation most extensively studied, have been shown to have rates of up to 77.5%14. Many of these patients are asymptomatic despite severe sleep apnea15. Other surgical populations, such as orthopedic surgery patients, have been shown to have rates of OSA that are only slightly higher than the general population16. This broad range of prevalence rates may be related to the diverse distribution of risk factors for OSA, such as obesity based on a high body mass index (BMI), age, and/or comorbidities (e.g. stroke and myocardial infarction). Further research is needed to evaluate if the surgical population has a higher risk of OSA independent of these factors.\n\nIn the general population, OSA is a risk factor for diabetes17 and cardiovascular diseases including cardiac arrhythmias18,19, myocardial infarction20, pulmonary hypertension21, systemic hypertension19,22, heart failure19, renal disease23, stroke, and death24. Proposed mechanisms include hypoxemia, sympathetic activation, metabolic dysregulation, left atrial enlargement, endothelial dysfunction, systemic inflammation, and hypercoagulability25.\n\nPerioperative complications occur more often in patients with OSA compared to controls26–29. These include delirium30,31, reintubation, pneumonia32–35, atrial arrhythmias, myocardial infarction, and pulmonary embolism36 (Table 2). Delirium in the postoperative period is associated with increased morbidity and mortality37,38, as well as long-term cognitive and functional decline39,40. These complications increase utilization of intensive care and length of stay as described in a recent retrospective study in 1,058,710 patients undergoing elective surgeries35. Our group also found that, independent of OSA, reintubation and unplanned intensive care unit (ICU) admission may result in a substantial increase in in-hospital mortality41,42.\n\nOR = odds ratio; 95% CI = 95% confidence interval; ICU = intensive care unit; AHI = apnea hypopnea index; LOS = length of hospital stay; # = not controlled for obesity; OSA = obstructive sleep apnea\n\n(Systematic review of 622 references published later than 2009 retrieved using MedLine search term “sleep apnea postoperative complications” – www.pubmed.org)\n\nYet some studies suggest a decreased risk of postoperative mortality in patients with a known diagnosis of sleep apnea34,35,43. Ischemic preconditioning was hypothesized to be involved in this protective effect of OSA, despite higher rates of cardiovascular comorbidities44,45. Ischemic preconditioning is an experimental strategy during which exposure to short, non-lethal episodes of ischemia results in attenuated tissue injury from ischemia and reperfusion46. The underlying mechanisms may include increased blood vessel collaterality47 and reduced oxidative stress48. Recent studies found patients with OSA to have less severe cardiac injury after acute non-fatal myocardial infarction49. Protective preconditioning from OSA may not be limited to the heart muscle, but may also have beneficial effects in the kidney and the brain50–52.\n\nIt is important to note that published studies investigating the effect of OSA on postoperative mortality are based on retrospective chart review. These retrospective analyses used diagnostic coding of OSA as an independent variable. These studies did not control for intraoperative predictors of postoperative complications, such as blood loss, anesthetics used, and mode of mechanical ventilation53,54. Therefore, one can assume that the true impact of OSA on postoperative outcomes remains unclear.\n\nAdditionally, one could argue that patients already diagnosed with OSA might receive more careful postoperative management. Longer time to extubation55,56 and increased utilization of non-invasive ventilation have been reported in OSA patients33. Furthermore, it is a challenge to isolate the effect of OSA from the known adverse perioperative outcome of other comorbidities seen in patients with OSA, such as hypertension, diabetes, dyslipidemia, and obesity57–59. Although randomized controlled trials are important, such trials are unlikely to be feasible. Given that postoperative pulmonary complications are rare and multifactorial, and that the phenotypes of OSA differ by patient, it is difficult to undertake a trial that can capture all the nuances of this question. Observational studies reflect the real world practice of anesthesiologists and allow for the large sample size that is needed to be able to make inferences on the ideal settings for specific patient populations. Despite the limited data on OSA as an independent perioperative risk factor60, it is intuitive to conclude that OSA patients are at risk of developing severe perioperative complications. Therefore, identification and optimal perioperative management of OSA patients is mandatory.\n\n\nWhy does OSA occur and how can the perioperative setting affect OSA?\n\nThe respiratory pump consists of an anatomically diverse group of muscles including thoracic wall muscles, the diaphragm, and other muscles of the trunk61. The contraction of these muscles increases the thoracic volume, and the lung generates negative intra-thoracic pressure. That negative pressure translates to a negative intraluminal UA pressure and thereby results in inspiratory airflow. If the negative UA pressure drops below a critical value (Pcrit), the UA collapses62,63. In contrast to healthy controls, Pcrit is positive (>0 cmH2O) in OSA patients when paralyzed64 or sedated, and UA dilator muscle activity is required to maintain airway patency65.\n\nThe activity of UA dilating muscles depends on neuronal innervation from subcortical and cortical brain regions that are modulated by physiological feedback and feed forward mechanisms. Subcortical regions of the brainstem and midbrain receive inputs for peripheral and central chemoreceptors that are sensitive to partial pressures of oxygen and carbon dioxide levels. Breathing and UA patency therefore respond to changes in gas exchange66. Excitatory inputs from the UA, in response to a negative pressure, stimulate UA motor neurons to compensate for pressure changes across the respiratory cycle67,68. Respiratory pattern generators in the brainstem provide further excitatory input to the UA motor neurons and increase UA stability just prior to inspiration. While these subcortical regulatory circuits are effective, cortical inputs to the UA motor neurons are strongly connected to wakefulness69. During wakefulness, serotonergic and noradrenergic neurons send excitatory inputs to the UA motor neurons70–73, resulting in increased UA dilator muscle activity. Yet, upon sleep onset, this neuronal input disappears and puts the UA at risk for collapse69,75,76. UA collapse results in desaturations and an increase in the work of breathing that triggers cerebral arousal from sleep. The increased excitatory input to the UA motor neurons resulting from the sleep arousal reestablishes UA patency77 (Figure 1).\n\nRespiratory arousal (grey hexagon) consisting of cortically and subcortically generated excitatory activity increases airway dilator muscle activity, thereby increasing upper airway dilating forces (green arrows). This counteracts the upper airway constricting forces (red arrows) generated by surrounding tissue pressure and negative intraluminal pressure during inspiration. (UA=upper airway yellow).\n\nWhile the main source of airway collapsing forces is the negative intraluminal pressure during inspiration, additional predisposing factors increase the risk for airway collapse in OSA. UA anatomy78 (e.g. hereditary reduction in the size of the retropalatal and retroglossal airway)79,80 and age81,82 are predisposing factors that cannot be altered by intervention. In contrast, increased extraluminal volume due to obesity (e.g. neck circumference) increases the risk of UA collapse81 and can be improved by weight loss83. Yet sleep apnea is not restricted to obese patients84 and other factors such as reduced UA muscle activity (e.g. due to sedatives or alcohol)85 may lead to decreased UA patency and apneas during sleep86,87.\n\nDuring the perioperative period, multiple factors affect UA patency and increase the risk for UA obstruction, especially in patients with OSA88. Mechanical loads to the collapsible segments of the retropalatal and retropharyngeal UA (e.g. postoperative hematoma89,90, peripharyngeal inflammation, and edema, e.g. due to fluid overload and rostral fluid shift91–94) lead to physical compression of the airway. Airway patency may also be affected by intubation and extubation. Supine positioning during surgery and the immediate postoperative period reduces lung volume and oxygen saturation. Reduced lung inflation due to pain-induced splinting and pharmacologic agents can limit tracheal traction on the UA and promote collapse94–99. Furthermore, decreased respiratory muscle function (i.e. diaphragm and intercostal muscles) results in impaired expansion of the lung and often occurs after surgery100. Studies in the ICU have shown that systemic inflammation and mechanical ventilation can both dramatically reduce diaphragmatic function101,102.\n\nBeyond these physical factors, pharmacological agents that are routinely applied during and early after anesthesia also affect breathing and UA patency in a dose-, muscle type-103 and agent-104–106 dependent manner. Negative effects on breathing and UA patency were observed across three classes of gamma-aminobutyric acid (GABA)-ergic narcotics (volatile105,107, propofol104,106,108,109, and benzodiazepine110,111) and have impairing effects on UA dilator muscles. In contrast, ketamine112 might have protective effects. In specific groups of patients (those with high loop gain), barbiturates may have protective effects on UA patency113,114. However, it is unclear if the latter is clinically meaningful in a perioperative scenario where decrease in ventilatory drive as a consequence of respiratory depressant drugs such as opioids may be more relevant in the pathophysiology of postoperative sleep apnea.\n\nThe underlying mechanisms of pharmacological agents on breathing are diverse106. Dose-dependent increases in collapsibility of the UA through depressed respiratory drive, direct inhibition of UA dilator muscle activity (e.g. propofol)104, and reduced responsiveness of UA dilator muscles to negative pressure (e.g. isoflurane)105 have been shown for all GABAergic drugs, but N-methyl-D-aspartate (NMDA) antagonistic drugs such as ketamine and nitrous oxide may have respiratory protective effects, at least in the low-dose range. Nishino and colleagues investigated the differential effects of anesthetics and found greater dampening of hypoglossal nerve input relative to the phrenic nerve115. Since this results in greater impairment of UA dilator muscles compared to respiratory pump muscles, the effects can lead to increased risk for UA collapse. In contrast, ketamine has been found to reduce neural input to the UA dilator muscles and equally to respiratory pump muscles. Ketamine’s effect on the UA dilator muscles was less when compared to GABAergic anesthetics115. Studies in rats have demonstrated dissociation between loss of consciousness and UA dilator muscle function under ketamine anesthesia112. Taken together, these studies suggest that patients with OSA, who have preoperative UA instability, may be at a heightened risk of UA collapse when under the influence of some, but not all, anesthetics. The unique effects associated with ketamine suggest that some anesthetic agents may be a safer choice for patients with OSA. However, prospective clinical studies in surgical patients with OSA are still required to confirm this preclinical finding and investigate the resulting effects on postoperative outcome.\n\nIn addition to reducing arousal and inducing loss of consciousness during surgery with medication, the anesthetist needs to apply neuromuscular blocking agents (NMBAs) carefully to cause muscle relaxation and optimize surgical conditions. The effects of NMBAs may outlast the duration of the surgical procedure. Postoperative residual neuromuscular blockade (rNMB) can affect postoperative respiratory outcome116,117 and has been reported to occur frequently after surgery118–120. rNMB as well as neostigmine reversal may also be associated with an even higher risk of complications in OSA patients. UA and respiratory pump muscles differ in their sensitivity to the effects of NMBAs121,122. These differences may lead to imbalanced activation of pump and dilator muscles, thereby generating excessive negative intraluminal pressure. Weakened UA dilator muscles would be unable to compensate for the excessive negative pressure. Even at levels that produce minimal blockade (as measured by train-of-four ratio 0.5 to 1), NMBAs increased UA collapsibility and impaired the compensatory genioglossus response to negative pharyngeal pressure challenges117. Studies in surgical patients have demonstrated the dose-dependent association between intermediate-acting NMBAs and postoperative respiratory complications. That effect was shown to persist despite neostigmine-based reversal of neuromuscular blockade at the end of surgery42,54,123. Although OSA patients should be more vulnerable to the effects of NMBAs and reversal agents117,121,124, population-based studies do not currently exist.\n\nFollowing surgery, opioids are commonly used for the management of postoperative pain. The use of opioids has been increasingly identified as a contributor to postoperative exacerbation of sleep-disordered breathing94,125,126. OSA patients show a lower pain threshold127–130 and increased sensitivity to the respiratory depressant effects of opioids130, both of which are of particular relevance in the postoperative setting. Increased UA resistance has been described in cats after opioid application131 and may be mediated by direct inhibition of hypoglossal motor neurons with suppressed genioglossus activity132. Therefore, the use of opioids during and immediately after surgery can be an important perioperative factor to consider in patients with OSA. Some data suggest that some interventions such as elevated upper body position94 or continuous positive airway pressure (CPAP)133 can ameliorate the respiratory depressant effects of opioids.\n\nFinally, following surgery, patients commonly experience disrupted, reduced, and poor-quality sleep. Sleep fragmentation can reduce the rapid eye movement (REM) sleep stage134,135. Following sleep deprivation and fragmentation, a rebound effect with increased amount of REM sleep can be seen a few nights after surgery134,136. REM sleep is primetime for sleep apnea, since it is associated with muscle atonia and impaired respiratory arousal137. Sleep deprivation may also contribute to the development of delirium, further disrupting sleep patterns and cortical arousals138.\n\n\nHow to manage patients with OSA perioperatively\n\nDespite the high prevalence of OSA in surgical populations, standardized guidelines for the safe handling of this patient population are limited1. The imperative is to provide the highest level of quality care while scheduling surgery in a timely manner and minding the expanding cost of providing care. Healthcare resources need to be optimally allocated to improve patients’ safety without undue economic impact. Given these restrictions, it is probably not feasible to conduct a sleep study on each patient scheduled for surgery, and there is so far no data indicating that a preoperative sleep medicine consultation improves patient safety. However, a stepwise approach for the detection of patients at risk of sleep apnea may help guide the need for diagnostics and treatment.\n\nPatients should be tested for the risk of sleep apnea, and there are several validated scores available for preoperative testing such as the STOP BANG questionnaire139,140. We have recently developed an OSA screening instrument that is supposed to be applied to patients who have not been scheduled to see an anesthesiologist prior to the day of surgery. The Score for Preoperative prediction of OSA (SPOSA) can be used based on data available preoperatively in the electronic medical record without the need to take a physical exam141. Once patients are identified to be at a high risk for sleep apnea in the perioperative setting, they demand special attention and care during the perioperative period and anesthesia. To some extent, the need for additional testing may depend on the perioperative risk of the scheduled surgical intervention. Data on the optimal intraoperative and perioperative management of sleep apnea is still limited, but OSA patients undergoing surgery and anesthesia with high risk of morbidity should receive specialized treatment during the perioperative period based on the best available local evidence and experience level.\n\nThe use of a standardized algorithm, such as the one developed by physicians at the Massachusetts General Hospital in Boston (Figure 2), may help identify and manage OSA patients facing surgery. In patients with known OSA who have been prescribed CPAP, the use of CPAP is continued during the postoperative period (e.g. in the recovery room). A respiratory therapist may visit the patient preoperatively or postoperatively in the recovery room to make sure the device and its interface function properly. During the postoperative period, when the patient is under lingering effects of anesthetics, CPAP treatment under close guidance of respiratory therapists reduces the number of respiratory events142 and improves breathing early after surgery126. Furthermore, CPAP may even improve postoperative hemodynamics (i.e. blood pressure variance) in patients who are not hypovolemic142. CPAP treatment under close guidance of respiratory therapists likely reduces postoperative complications in OSA patients143. However, successful perioperative CPAP treatment needs a close collaboration among patients, surgeons, anesthesiologists, and respiratory therapists, and sleep physicians may need to be consulted in selected patients.\n\n(modified from Zaremba S, Chamberlin NL, Eikermann M in Miller's Anesthesia 8th Edition, by Miller RD. Eriksson LI, Fleisher LA, Wiener-Kronish JP, Cohen NH, Young WL).\n\nFor patients not previously diagnosed with OSA, clinical management should be performed based on risk stratification. Patients undergoing surgical procedures with moderate to high risk of perioperative complications should be stratified based on OSA risk. Risk for occult or undiagnosed OSA is based on the clinical assessment (e.g. symptoms and/or comorbidities of sleep apnea; see Table 3) and the use of standardized validated questionnaires (e.g. Berlin questionnaire144, STOP questionnaire140, P-SAP score139, or the SPOSA141). Note that these questionnaires have been validated for the identification of OSA, but not other forms of sleep-disordered breathing (e.g. obesity hypoventilation or central sleep apnea)145. Additional testing, such as arterial blood sampling, is required to detect hypercapnia (increased blood carbon dioxide). However, blood gas analysis is not typically included in the standard preoperative workup for surgical patients and might not be available in some settings. In these cases, venous serum bicarbonate concentration, as available on most biochemistry profiles, might be a helpful screening tool for an occult, chronic, respiratory acidosis. A serum bicarbonate level greater than 27 mmol/l has been shown to be highly sensitive (92%) for an elevated arterial partial pressure of carbon dioxide. An elevated bicarbonate level accompanied by mild hypoxemia (peripheral oxygen saturation of 94%) may also indicate high risk of obesity-related alveolar hypoventilation145,146.\n\nPatients deemed at high risk for OSA and/or obesity hypoventilation syndrome based on these screening tools may require sleep medicine consultation prior to or following anesthesia. The sleep specialist can help determine the role of a sleep study (home vs. laboratory based), start therapy with positive airway pressure therapy (e.g. auto-titrating CPAP), and develop strategies to “desensitize” patients to the mask and pressures prior to or following an elective surgical procedure.\n\nThroughout the perioperative period, special attention should be paid to patients with confirmed OSA or high-risk patients under special circumstances. In these patients, specific methods should be used during intubation, intraoperatively, during and early after extubation, and during post-anesthesia care unit (PACU) treatment including fluid management, patient positioning, neuromuscular blockade, protective ventilation53,147, pain management, and choice of anesthesia type and anesthetic (Table 4). However, the currently available data on the choice of anesthetic for the OSA patient facing surgery are limited, but the choice of a sedative agent to be applied intraoperatively can affect OSA patients and increase their risk for postoperative complication, as the majority of currently available anesthetics have been found to impair UA patency (see above). However, the supplementation of anesthesia with NMDA receptor antagonists might be a superior alternative during some surgical and interventional procedures in OSA patients. Ketamine can be particularly useful for pain control during short procedures such as dressing changes in burn patients, endoscopic procedures, and small surgical interventions. Compared to the majority of anesthetics, low-dose ketamine preserves airway reflexes and maintains respiratory drive. Ketamine might even stimulate breathing when applied in low to moderate doses in combination with other anesthetics. Data from animal studies and clinical studies in non-OSA patients showed an increase in respiratory rate112,148,149, tidal volume112, and minute ventilation112 compared to other anesthetic agents, to sleep, and even to wakefulness149. In clinical studies, combined anesthesia with ketamine was associated with improved oxygen saturation150 and reduced time with supplemental oxygen151 compared to regimes not including ketamine. In addition, ketamine’s positive effect on UA patency due to increased activity of UA dilating muscle activity112 might be of special benefit in patients with OSA. Other beneficial effects making ketamine a favorable anesthetic agent for OSA patients include ketamine’s analgesic effects, as ketamine infusions have significantly reduced the requirements of opioids in patients with cancer and non-cancer pain152. However, currently, no studies investigating the effect of ketamine on breathing in OSA patients have been reported. Randomized controlled trials are required to investigate if ketamine and other NMDA receptor antagonists have similar beneficial respiratory effects in patients with OSA and if these effects translate to improved postoperative outcome.\n\nOther considerations for patients with high risk of OSA include the application of neuromuscular blockade. Thus, high doses of NMBAs should be avoided intraoperatively, as these may facilitate rNMB and increase risk of postoperative respiratory complications42,121,153,154. If used, quantitative neuromuscular transmission monitoring is highly recommended to reduce the risk of residual neuromuscular blockade and detect neuromuscular blockade persisting beyond surgery. Reversal of rNMB by administration of a cholinesterase inhibitor (e.g. neostigmine) should be considered when rNMB is present, but the reversal agent has to be titrated carefully, since inappropriate (high-dose) neostigmine reversal has been shown to impair UA function in animals and humans122,155.\n\nPatients undergoing surgical and anesthetic procedure with low risk of postoperative complications may receive standard postoperative care. However, in cases where UA obstruction occurs during intubation, extubation, or the early postoperative period88, the postoperative application of CPAP with monitoring by a health care professional should be considered. Autotitrating CPAP (APAP) devices, which provide variable levels of pressure based on flow limitation, peak flow, vibration (snoring), and airway impedance156, are a reasonable option for the CPAP-naïve patient157. However, some studies indicate APAP without supervision by respiratory therapists does not improve oxygen saturation or outcome in all cases158,159, and difficulties with use can result in a low adherence to the therapy160.\n\nFollowing surgery, specialized attention should continue for patients diagnosed with, or at high risk for, OSA. If adherence to CPAP is limited in CPAP-naïve patients, the use of positional therapy may be a feasible option. Positional therapy, however, has been shown to be less effective compared to CPAP161. Yet UA patency can easily be improved by elevation of the upper body, especially in the recovery room and during sleep94,97. Alternatively, a lateral body position can be used to minimize gravitational impact on UA whenever acceptable98.\n\nOpioids are commonly used for the control of surgical pain during and after anesthesia. This is of special importance in OSA patients who have been found to require higher doses of opioids for adequate pain control162. While pain management may improve the use of the respiratory pump, these analgesics induce a dose-dependent impairment of UA dilator muscle activity94,132,163. Recent data from our group and others indicate that treatment of OSA patients with CPAP early after surgery improves sleep apnea and mitigates negative effects of opioid application. An alternative, such as non-steroidal anti-inflammatory drugs (NSAIDs) or regional anesthesia with local anesthetics, should also be considered.\n\nGiven these perioperative factors, the transfer of an OSA patient from the recovery room or ICU to an unmonitored floor should be carefully considered. Patients should not be moved until their vital signs have recovered to values similar to pre-anesthesia baseline and after passing a room air challenge test. Furthermore, adequate treatment of nausea and pain should be accomplished, preferably by NSAIDs, prior to transfer.\n\n\nConclusion\n\nWhen caring for OSA patients facing surgery, the therapeutic team needs to be aware of the increased risk for post-anesthesia respiratory complications. While these complications are not associated with increased mortality risk, the morbidity of preventable complications may lead to undesired expenses, jeopardize available resources, and may lead to an increased hospital readmission rate. Since the currently available literature on perioperative management of OSA patients is still limited, additional clinical and basic research in this area is needed to improve the safety of OSA patients undergoing anesthesia.\n\nWe hypothesize that early recognition and treatment of sleep apnea reduces perioperative complications. Further research is needed to confirm this clinical suspicion and support the use of diagnostic or therapeutic algorithms for these patients.\n\nPending that research, an institution-specific plan (based on setting, personnel, equipment, medications, and resources) needs to be established for the identification, testing, monitoring, and care of the surgical population. The plan should include (1) stepwise preoperative screening procedures for OSA, (2) an optimized anesthesia regimen and sedation protocol for this high-risk group which eliminates drug-induced respiratory depressant effects at the end of the case, (3) intraoperative neuromuscular monitoring with goal-directed reversal of rNMB, (4) a protocol for the use of CPAP therapy in the recovery room, (5) optimal opioid titration for postoperative pain control, and (6) specific discharge criteria for transfer to the unmonitored ward.\n\n\nAbbreviations\n\nCPAP - continuous positive airway pressure\n\nGABA - gamma-aminobutyric acid\n\nNMBA - neuromuscular blocking agents\n\nNMDA - N-methyl-D-aspartate\n\nNSAID - non-steroidal anti-inflammatory drugs\n\nOSA - obstructive sleep apnea\n\nREM sleep - rapid eye movement sleep\n\nrNMB - residual neuromuscular blockade\n\nUA - upper airway",
"appendix": "Competing interests\n\n\n\nSebastian Zaremba declares that he has no disclosures.\n\nJames E. Mojica declares that he has no disclosures.\n\n\nGrant information\n\nMatthias Eikermann has received research funding from MERCK and ResMed Foundation, and received grants from Jeff and Judy Buzen.\n\n\nReferences\n\nMemtsoudis SG, Besculides MC, Mazumdar M: A rude awakening--the perioperative sleep apnea epidemic. N Engl J Med. 2013; 368(25): 2352–3. PubMed Abstract | Publisher Full Text\n\nDonati F: Residual paralysis: a real problem or did we invent a new disease? Can J Anaesth. 2013; 60(7): 714–29. PubMed Abstract | Publisher Full Text\n\nPatil SP, Schneider H, Schwartz AR, et al.: Adult obstructive sleep apnea: pathophysiology and diagnosis. Chest. 2007; 132(1): 325–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavies RJ, Stradling JR: The epidemiology of sleep apnoea. Thorax. 1996; 51(Suppl 2): S65–70. 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}
|
[
{
"id": "11693",
"date": "11 Jan 2016",
"name": "Sergio Tufik",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11694",
"date": "11 Jan 2016",
"name": "Nico de Vries",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-48
|
https://f1000research.com/articles/4-1080/v1
|
16 Oct 15
|
{
"type": "Software Tool Article",
"title": "From reads to regions: a Bioconductor workflow to detect differential binding in ChIP-seq data",
"authors": [
"Aaron T. L. Lun",
"Gordon K. Smyth"
],
"abstract": "Chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) is widely used to identify the genomic binding sites for protein of interest. Most conventional approaches to ChIP-seq data analysis involve the detection of the absolute presence (or absence) of a binding site. However, an alternative strategy is to identify changes in the binding intensity between two biological conditions, i.e., differential binding (DB). This may yield more relevant results than conventional analyses, as changes in binding can be associated with the biological difference being investigated. The aim of this article is to facilitate the implementation of DB analyses, by comprehensively describing a computational workflow for the detection of DB regions from ChIP-seq data. The workflow is based primarily on R software packages from the open-source Bioconductor project and covers all steps of the analysis pipeline, from alignment of read sequences to interpretation and visualization of putative DB regions. In particular, detection of DB regions will be conducted using the counts for sliding windows from the csaw package, with statistical modelling performed using methods in the edgeR package. Analyses will be demonstrated on real histone mark and transcription factor data sets. This will provide readers with practical usage examples that can be applied in their own studies.",
"keywords": [
"ChIP-seq",
"Differential binding",
"genomics",
"bioinformatics",
"read alignment",
"visualization"
],
"content": "Introduction\n\nChromatin immunoprecipitation with sequencing (ChIP-seq) is a popular technique for identifying the genomic binding sites of a target protein. Conventional analyses of ChIP-seq data aim to detect absolute binding (i.e., the presence or absence of a binding sites) based on peaks in the read coverage. However, a number of recent studies have focused on the detection of changes in the binding profile between conditions (Pal et al., 2013; Ross-Innes et al., 2012). These differential binding (DB) analyses involve counting reads into genomic intervals, and then testing those counts for significant differences between conditions. This defines a set of putative DB regions for further examination. DB analyses are easier to perform than their conventional counterparts, as the effect of genomic biases is largely mitigated when counts for different libraries are compared at the same genomic region. DB regions may also be more relevant as the change in binding can be associated with the biological difference between conditions.\n\nThe key step in the DB analysis is the manner in which reads are counted. The most obvious strategy is to count reads into pre-defined regions of interest, like promoters or gene bodies (Pal et al., 2013). This is simple but will not capture changes outside of those regions. In contrast, de novo analyses do not depend on pre-specified regions, instead using empirically defined peaks or sliding windows for read counting. Peak-based methods are implemented in the DiffBind and DBChIP software packages (Liang & Keles, 2012; Ross-Innes et al., 2012), which count reads into peak intervals that have been identified with software like MACS (Zhang et al., 2008). This requires some care to maintain statistical rigour, as peaks are called with the same data used to test for DB. Alternatively, window-based approaches count reads into sliding windows across the genome. This is a more direct strategy that avoids problems with data re-use and can provide increased DB detection power (Lun & Smyth, 2014). However, its correct implementation is not straightforward due to the subtleties with interpretation of the false discovery rate (FDR).\n\nThis article describes a computational workflow for performing a DB analysis with sliding windows. The aim is to facilitate the practical implementation of window-based DB analyses by providing detailed code and expected output. The workflow described here applies to any ChIP-seq experiment with multiple experimental conditions and with multiple biological samples within one or more of the conditions. It detects and summarizes DB regions between conditions in a de novo manner, i.e., without making any prior assumptions about the location or width of bound regions. Detected regions are then annotated according to their proximity to annotated genes. In addition, the code can be easily adapted to accommodate batch effects, covariates and multiple experimental factors.\n\nThe workflow is based primarily on software packages from the open-source Bioconductor project (Huber et al., 2015). It contains all steps that are necessary for detecting DB regions, starting from the raw read sequences. Reads are first aligned to the genome using the Rsubread package (Liao et al., 2013). These are counted into sliding windows with csaw, to quantify binding intensity across the genome (Lun & Smyth, 2014). Statistical modelling is based on the negative binomial (NB) distribution with generalized linear models (GLMs) in the edgeR package (McCarthy et al., 2012; Robinson et al., 2010), with additional sophistication provided by quasi-likelihood (QL) methods (Lund et al., 2012). Code is also provided for filtering, normalization and region-level control of the FDR. Finally, annotation and visualization of the DB regions is described using Gviz and other packages.\n\nThe application of the methods in this article will be demonstrated on two publicly available ChIP-seq data sets. The first data set studies changes in H3K9ac marking between pro-B and mature B cells (Revilla-I-Domingo et al., 2012). The second data set studies changes in CREB-binding protein (CBP) binding between wild-type and CBP knock-out cells (Kasper et al., 2014). A separate workflow is described for the analysis of each data set, using the sliding window approach in both cases but with different parameter settings. The intention is to provide readers with a variety of usage examples from which they can construct DB analyses of their own data.\n\n\nAligning reads in the H3K9ac libraries\n\nThe first task is to download the relevant ChIP-seq libraries from the NCBI Gene Expression Omnibus (GEO) (Edgar et al., 2002). These are obtained from the data series GSE38046, using the Sequence Read Accession (SRA) numbers listed below. The experiment contains two biological replicates in total for each of the two cell types, i.e., pro-B and mature B. Multiple technical replicates exist for some of the biological replicates, and are indicated as those files with the same grouping.\n\n\n\n\n\nThese files are downloaded in the SRA format, and need to be unpacked to the FASTQ format prior to alignment. This can be done using the fastq-dump utility from the SRA Toolkit.\n\n\n\nTechnical replicates are merged together prior to further processing. This reflects the fact that they originate from a single library of DNA fragments.\n\n\n\nReads in each library are aligned to the mm10 build of the mouse genome, using the Rsubread package (Liao et al., 2013). This assumes that an index has already been constructed with the prefix index/mm10. Here, a consensus threshold of 2 is used instead of the default of 3, to accommodate the shorter length of the reads (32 bp).\n\n\n\nIn each of the resulting BAM files, alignments are re-sorted by their mapping location. This is required for input into csaw, but is also useful for other programs like genome browsers that depend on sorting and indexing for rapid retrieval of reads.\n\n\n\nPotential PCR duplicates are marked using the MarkDuplicates tool from the Picard software suite. These are identified as alignments at the same genomic location, such that they may have originated from PCR-amplified copies of the same DNA fragment.\n\n\n\nThe behaviour of the alignment pipeline for this data set can be easily summarized with some statistics. Ideally, the proportion of mapped reads should be high, while the proportion of marked reads should be low.\n\n\n\n\n\nFinally, the libraries are indexed for rapid retrieval by genomic location. This generates a number of index files at the same location as the BAM files.\n\n\n\n\nObtaining the ENCODE blacklist for mm10\n\nA number of genomic regions contain high artifactual signal in ChIP-seq experiments. These often correspond to genomic features like telomeres or microsatellite repeats. For example, multiple tandem repeats in the real genome are reported as a single unit in the genome build. Alignment of all (non-specifically immunoprecipitated) reads from the former will result in artificially high coverage of the latter. Moreover, differences in repeat copy numbers between conditions can lead to detection of spurious DB.\n\nAs such, these regions must be removed prior to further analysis. This can be done with an annotated blacklist of problematic regions in the mm9 build of the mouse genome. All reads in the blacklist will be ignored during processing in csaw. The blacklist itself was constructed by identifying consistently problematic regions in the ENCODE and modENCODE data sets (ENCODE Project Consortium, 2012).\n\nRecall that the alignments have been performed to the mm10 build, so the mm9 blacklist coordinates must be transferred to their mm10 equivalents. This is done using the liftOver function in the rtracklayer package (Lawrence et al., 2009). The chain file specifies the corresponding coordinates between the two builds and can be obtained here. The new blacklist coordinates are then saved to file for future use.\n\n\n\nAn alternative approach is to use predicted repeat regions from the UCSC genome annotation (Rosenbloom et al., 2015). This tends to remove a greater number of problematic regions (especially microsatellites) compared to the ENCODE blacklist. However, the size of the UCSC list means that genuine DB sites may also be removed. Thus, the ENCODE blacklist is preferred for most applications.\n\n\nTesting for DB between pro-B and mature B cells\n\nHere, the settings for the DB analysis are specified. Recall that the paths to the BAM files are stored in the bam.files vector after alignment. The cell type for each file can be conveniently extracted from the file name.\n\n\n\n\n\nIn the csaw package, the readParam object determines which reads are extracted from the BAM files. The idea is to set this up once and to re-use it in all relevant functions. For this analysis, reads are only used if they have a mapping quality score above 50. This avoids spurious results due to weak or non-unique alignments. Reads are also ignored if they map within blacklist regions or if they do not map to the standard set of mouse nuclear chromosomes.\n\n\n\nStrand bimodality is often observed in ChIP-seq experiments involving sharp binding events like H3K9ac marking. This refers to the presence of distinct subpeaks on each strand and can be quantified with cross-correlation plots (Kharchenko et al., 2008). A strong peak in the cross-correlations should be observed if immunoprecipitation was successful. The delay distance at the peak corresponds to the distance between forward-/reverse-strand subpeaks. This is identified from Figure 1 and is used as the average fragment length for this analysis.\n\n\n\n\n\n\n\nThe delay with the maximum correlation is shown as the red line.\n\nOnly unmarked reads (i.e., not potential PCR duplicates) are used here. This tends to give better signal by reducing the size of the “phantom” peak at the read length (Landt et al., 2012). However, removal of marked reads is risky as it caps the signal in high-coverage regions of the genome. This can result in loss of power to detect DB, or introduction of spurious DB when the same cap is applied to libraries of different sizes. Thus, the marking status of each read will be ignored in the rest of the analysis, i.e., no duplicates will be removed in downstream steps.\n\ncsaw uses a sliding window strategy to quantify binding intensity across the genome. Each read is directionally extended to the average fragment length, to represent the DNA fragment from which that read was sequenced. The number of extended reads overlapping a window is counted. The window is then moved to its next position on the genome, and counting is repeated. This is done for all libraries such that a count is obtained for each window in each library. The windowCounts function produces a RangedSummarizedExperiment object containing these counts in matrix form, where each row corresponds to a window and each column represents a library.\n\n\n\n\n\nTo analyze H3K9ac data, a window size of 150 bp is used here. This corresponds roughly to the length of the DNA in a nucleosome (Humburg et al., 2011), which is the smallest relevant unit for studying histone mark enrichment. The spacing between windows is set to the default of 50 bp, i.e., adjacent window starts are 50 bp apart. By default, windows with very low counts are removed to reduce memory use.\n\nLow-abundance windows contain no binding sites and need to be filtered out. This improves power by removing irrelevant tests prior to the multiple testing correction; avoids problems with discreteness in downstream statistical methods; and reduces computational work for further analyses. Here, filtering is performed using the average abundance of each window (McCarthy et al., 2012). This performs well as an independent filter statistic for NB-distributed count data (Lun & Smyth, 2014).\n\nThe filter threshold is defined based on the assumption that most regions in the genome are not marked by H3K9ac. Reads are counted into large bins and the median coverage across those bins is used as an estimate of the background abundance. Windows are only retained if they have abundances 3-fold higher than the background. This removes a large number of windows that are weakly or not marked and are likely to be irrelevant.\n\n\n\n\n\nThe effect of the fold-change threshold can be examined visually in Figure 2. The chosen threshold is greater than the abundances of most bins in the genome – presumably, those that contain background regions. This suggests that the filter will remove most windows lying within background regions.\n\nThe filter threshold is shown as the red line.\n\n\n\nThe actual filtering itself is done by simply subsetting the RangedSummarizedExperiment object.\n\n\n\nNormalization is required prior to any comparisons between libraries, to eliminate confounding library-specific biases. In particular, a trended bias is often observed between libraries in Figure 3. This refers to a systematic fold-difference in window coverage between libraries that changes according to the average abundance of the window.\n\n\n\nTrended biases cannot be removed by scaling methods like TMM normalization (Robinson & Oshlack, 2010), as the amount of scaling required varies with the abundance of the window. Rather, non-linear normalization methods must be used. csaw implements a version of the fast loess method (Ballman et al., 2004) that is adapted to count data. This produces a matrix of offsets that can be used during GLM fitting.\n\n\n\n\n\nThe effect of non-linear normalization can be visualized with a mean-difference plot comparing the first and last libraries. Once the offsets are applied to adjust the log-fold changes, the trend is eliminated from the plot (Figure 4). The cloud of points is also centred at a log-fold change of zero. This indicates that normalization was successful in removing the differences between libraries.\n\n\n\nLog-fold changes for all windows are shown after normalization.\n\nThe implicit assumption of non-linear methods is that most windows at each abundance are not DB. Any systematic difference between libraries is attributed to bias and is removed. This is not appropriate in situations where large-scale DB is expected, as removal of the difference would result in loss of genuine DB. However, there is no indication that such changes are present in this data set, so non-linear methods can be applied without too much concern.\n\nIntroduction. Counts are modelled using NB GLMs in the edgeR package (McCarthy et al., 2012; Robinson et al., 2010). The NB distribution is useful as it can handle low, discrete counts for each window. The NB dispersion parameter allows modelling of biological variability between replicate libraries. GLMs can also accommodate complex experimental designs, though a simple design is sufficient for this study.\n\n\n\n\n\nEstimating the NB dispersion. The RangedSummarizedExperiment object is coerced into a DGEList object (plus offsets) prior to entry into edgeR. Estimation of the NB dispersion is then performed. Specifically, a NB dispersion trend is fitted to all windows against the average abundance. This means that empirical mean-dispersion trends can be flexibly modelled.\n\n\n\n\n\nThe NB dispersion trend is visualized in Figure 5 as the biological coefficient of variation (BCV), i.e., the square root of the NB dispersion. A trend that decreases to a plateau with increasing abundance is typical of many analyses, including those of RNA-seq and ChIP-seq data. Note that only the trended dispersion will be used here – the common and tagwise values are only shown for diagnostic purposes.\n\nCommon and tagwise estimates are also shown.\n\n\n\nEstimating the QL dispersion. Additional modelling is provided with the QL methods in edgeR (Lund et al., 2012). This introduces a QL dispersion parameter for each window, which captures variability in the NB dispersion around the fitted trend for each window. Thus, the QL dispersion can model window-specific variability, whereas the NB dispersion trend is averaged across many windows. However, with limited replicates, there is not enough information for each window to stably estimate the QL dispersion. This is overcome by sharing information between windows with empirical Bayes (EB) shrinkage. The instability of the QL dispersion estimates is reduced by squeezing the estimates towards an abundance-dependent trend (Figure 6).\n\n\n\nThe extent of shrinkage is determined by the prior degrees of freedom (d.f.). Large prior d.f. indicates that the dispersions were similar across windows, such that strong shrinkage to the trend could be performed to increase stability and power. Small prior d.f. indicates that the dispersions were more variable. In such cases, less squeezing is performed as strong shrinkage would be inappropriate. Also note the use of robust=TRUE, which reduces the sensitivity of the EB procedures to outlier windows.\n\n\n\n\n\nExamining the data with MDS plots. Multi-dimensional scaling (MDS) plots can be used to examine the similarities between libraries. The distance between a pair of libraries on this plot represents the overall log-fold change between those libraries. Ideally, replicates should cluster together while samples from different conditions should be separate. In Figure 7, strong separation in the first dimension is observed between libraries from different cell types. This indicates that significant differences are likely to be present between cell types in this data set.\n\n\n\nLibraries are labelled and coloured according to the cell type.\n\nTesting for DB with QL F-tests. Each window is tested for significant differences between cell types using the QL F-test (Lund et al., 2012). This is superior to the likelihood ratio test that is typically used for GLMs, as the QL F-test accounts for the uncertainty in dispersion estimation. One p-value is produced for each window, representing the evidence against the null hypothesis (i.e., no DB). For this analysis, the comparison is parametrized such that the reported log-fold changes represent that of pro-B cells over mature B counterparts.\n\n\n\n\n\nControlling the FDR across regions. One might attempt to control the FDR by applying the Benjamini-Hochberg (BH) method to the window-level p-values (Benjamini & Hochberg, 1995). However, the features of interest are not windows, but the genomic regions that they represent. Control of the FDR across windows does not guarantee control of the FDR across regions (Lun & Smyth, 2014). The latter is arguably more relevant for the final interpretation of the results.\n\nControl of the region-level FDR can be provided by aggregating windows into regions and combining the p-values. Here, adjacent windows less than 100 bp apart are aggregated into clusters. Each cluster represents a genomic region. Smaller values of tol allow distinct marking events to kept separate, while larger values provide a broader perspective, e.g., by considering adjacent co-regulated sites as a single entity. Chaining effects are mitigated by setting a maximum cluster width of 5 kbp.\n\n\n\nA combined p-value is computed for each cluster using the method of Simes (1986), based on the p-values of the constituent windows. This represents the evidence against the global null hypothesis for each cluster, i.e., that no DB exists in any of its windows. Rejection of this global null indicates that the cluster (and the region that it represents) contains DB. Applying the BH method to the combined p-values allows the region-level FDR to be controlled.\n\n\n\n\n\nEach row of the output table contains the statistics for a single cluster, including the combined p-value before and after the BH correction. The nWindows field describes the total number of windows in the cluster. The logFC.up and logFC.down fields describe the number of windows with a log-fold change above 0.5 or below -0.5 in each cluster, respectively. This can be used to determine the direction of DB in each cluster.\n\nExamining the scope and direction of DB. The total number of DB regions at a FDR of 5% can be easily calculated.\n\n\n\n\n\nDetermining the direction of DB is more complicated, as clusters could potentially contain windows that are changing in opposite directions. One approach is to define the direction based on the number of windows changing in each direction, as described above. Another approach is to use the log-fold change of the most significant window as a proxy for the log-fold change of the cluster. This is generally satisfactory, though it will not capture multiple changes in opposite directions. It also tends to overstate the change in each cluster.\n\n\n\n\n\nIn the above table, each row contains the statistics for each cluster. Of interest are the best and logFC fields. The former is the index of the window that is the most significant in each cluster, while the latter is the log-fold change of that window. This can be used to obtain a summary of the direction of DB across all clusters/regions.\n\n\n\n\n\nResults can be saved to file prior to further manipulation. One approach is to store all statistics in the metadata of a GRanges object. This is useful as it keeps the statistics and coordinates together for each cluster, avoiding problems with synchronization in downstream steps. The midpoint and log-fold change of the best window are also stored.\n\n\n\nFor input into other programs like genome browsers, results can be saved in a more conventional format. Here, coordinates of DB regions are saved in BED format via rtracklayer, using a log-transformed FDR as the score.\n\n\n\nSaving the RangedSummarizedExperiment objects is also recommended. This avoids the need to re-run the time-consuming read counting steps if parts of the analysis need to be repeated. Similarly, the DGEList object is saved so that the edgeR statistics can be easily recovered.\n\n\n\n\nInterpreting the DB results\n\nUsing the detailRanges function. csaw provides its own annotation function, detailRanges. This identifies all genic features overlapping each region and reports them in a compact string form. Briefly, features are reported as SYMBOL|EXONS|STRAND where SYMBOL represents the gene symbol, EXONS lists the overlapping exons (0 for promoters, I for introns), and STRAND reports the strand. Multiple overlapping features for different genes are separated by commas within the string for each region.\n\n\n\n\n\nAnnotated features that flank the region of interest are also reported. The description for each feature is formatted as described above but with an extra [DISTANCE] field, representing the distance (in base pairs) between that feature and the region. By default, only flanking features within 5 kbp of each region are considered.\n\n\n\n\n\n\n\n\n\nThe annotation for each region can then be stored in metadata of the GRanges object. The compact string form is useful for human interpretation, as it allows rapid examination of all genic features neighbouring each region.\n\n\n\nUsing the ChIPpeakAnno package. As its name suggests, the ChIPpeakAnno package is designed to annotate peaks from ChIP-seq experiments (Zhu et al., 2010). A GRanges object containing all regions of interest is supplied to the relevant function, after removing all previous metadata fields to reduce clutter. The gene closest to each region is then reported. Gene coordinates are taken from the NCBI mouse 38 annotation, which is roughly equivalent to the annotation in the mm10 genome build.\n\n\n\n\n\nThe behaviour of ChIPpeakAnno complements that of detailRanges. The latter reports all overlapping and flanking genes, while the former reports only the closest gene (but in greater detail). Which is preferable depends on the proclivities of the user and the purpose of the annotation.\n\nReporting gene-based results. Another approach to annotation is to flip the problem around, such that DB statistics are reported directly for features of interest like genes. This is more convenient when the DB analysis needs to be integrated with, e.g., DE analyses of matching RNA-seq data. In the code below, promoter coordinates are obtained by running detailRanges without specifying any regions. All windows overlapping each promoter are defined as a cluster, and DB statistics are computed as previously described for each cluster/promoter. This directly yields results for annotated features (with some NA values, representing those promoters that have no overlapping windows).\n\n\n\n\n\nNote that this is distinct from counting reads across promoters. Using promoter-level counts would not provide enough spatial resolution to detect sharp binding events that only occur in a subinterval of the promoter. In particular, detection may be compromised by non-specific background or the presence of multiple opposing DB events in the same promoter. Combining window-level statistics is preferable as resolution is maintained for optimal performance.\n\nOverview. Here, the Gviz package is used to visualize read coverage across the data set at regions of interest. Coverage in each BAM file will be represented by a single track. Several additional tracks will also be included in each plot. One is the genome axis track, to display the genomic coordinates across the plotted region. The other is the annotation track containing gene models, with gene IDs replaced by symbols (where possible) for easier reading.\n\n\n\nSimple DB across a broad region. To begin with, the top-ranking DB region will be visualized. This represents a simple DB event where the entire region changes in one direction (Figure 8). Specifically, it represents an increase in H3K9ac marking at the H2-Aa locus. This is consistent with the expected biology – H3K9ac is a mark of active gene expression (Karmodiya et al., 2012) and MHCII components are upregulated in mature B cells (Hoffmann et al., 2002).\n\nRead coverage for each library is shown as a per-million value at each base.\n\n\n\n\n\nOne track is plotted for each library, in addition to the coordinate and annotation tracks. Coverage is plotted in terms of sequencing depth-per-million at each base. This corrects for differences in library sizes between tracks.\n\n\n\nComplex DB across a broad region. Complex DB refers to situations where multiple DB events are occurring within the same enriched region. These are identified as those clusters that contain windows changing in both directions. Here, the second-ranking complex cluster is selected for visualization (the top-ranking complex cluster is adjacent to the region used in the previous example, so another region is chosen for some variety).\n\n\n\n\n\nThis region contains a bidirectional promoter where different genes are marked in the different cell types (Figure 9). Upon differentiation to mature B cells, loss of marking in one part of the region is balanced by a gain in marking in another part of the region. This represents a complex DB event that would not be detected if reads were counted across the entire region.\n\n\n\nSimple DB across a small region. Both of the examples above involve differential marking within broad regions spanning several kilobases. This is consistent with changes in the marking profile across a large number of nucleosomes. However, H3K9ac marking can also be concentrated into small regions, involving only a few nucleosomes. csaw is equally capable of detecting “sharp” DB within these small regions. This can be demonstrated by examining those clusters that contain a smaller number of windows.\n\n\n\n\n\nMarking is increased for mature B cells within a 500 bp region (Figure 10), which is sharper than the changes in the previous two examples. This also coincides with the promoter of the Cd86 gene. Again, this makes biological sense as CD86 is involved in regulating immunoglobulin production in activated B-cells (Podojil & Sanders, 2003).\n\n\n\nNote that the window size will determine whether sharp or broad events are preferentially detected. Larger windows provide more power to detect broad events (as the counts are higher), while smaller windows provide more resolution to detect sharp events. Optimal detection of all features can be obtained by performing analyses with multiple window sizes and consolidating the results, though – for brevity – this will not be described here. In general, smaller window sizes are preferred as strong DB events with sufficient coverage will always be detected. For larger windows, detection may be confounded by other events within the window that distort the log-fold change in the counts between conditions.\n\n\nRepeating the analysis for the CBP data\n\nA window-based DB analysis will be shown for transcription factor (TF) data, to complement the histone mark analysis above. This data set compares CBP binding between wild-type (WT) and CBP knock-out (KO) animals (Kasper et al., 2014). The aim is to use csaw and other Bioconductor packages to identify DB sites between genotypes. Most, if not all, of these sites should be increased in the WT, given that protein function should be compromised in the KO.\n\nLibraries are downloaded from the NCBI GEO data series GSE54453, using the SRA accessions listed below. The data set contains two biological replicates for each of the two genotypes. One file is available for each library, i.e., no technical replicates.\n\n\n\n\n\nSRA files are unpacked to yield FASTQ files with the raw read sequences.\n\n\n\nReads are aligned to the mm10 genome using Rsubread. Here, the default consensus threshold is used as the reads are longer (75 bp). A Phred offset of +64 is also used, instead of the default +33 used in the previous analysis.\n\n\n\nAlignments in each BAM file are sorted by coordinate. Duplicate reads are marked, and the resulting files are indexed.\n\n\n\nSome mapping statistics can be reported as previously described. For brevity, the code will not be shown here, as it is identical to that used for the H3K9ac analysis.\n\n\n\nCounting reads into windows. First, the average fragment length is estimated by maximizing the cross-correlation function.\n\n\n\n\n\nReads are then counted into sliding windows. For TF data analyses, smaller windows are necessary to capture sharp binding sites. A large window size will be suboptimal as the count for a particular site will be “contaminated” by non-specific background in the neighbouring regions. In this case, a window size of 10 bp is used.\n\n\n\n\n\nNormalization for composition biases. Composition biases are introduced when the amount of DB in each condition is unbalanced (Lun & Smyth, 2014; Robinson & Oshlack, 2010). More binding in one condition means that more reads are sequenced at the binding sites, leaving fewer reads for the rest of the genome. This suppresses the genomic coverage at non-DB sites, resulting in spurious differences between libraries. To remove this bias, reads are counted into large genomic bins. Most bins are assumed to represent non-DB background regions. Any systematic differences in the coverage of those bins is attributed to composition bias and is normalized out. Specifically, the TMM method (Robinson & Oshlack, 2010) is applied to compute normalization factors from the bin counts. These factors can then be applied to the DB analysis with the window counts.\n\n\n\n\n\nThe effect of normalization can be visualized with some mean-difference plots between pairs of libraries (Figure 11). The dense cloud in each plot represents the majority of bins in the genome. These are assumed to mostly contain background regions. A non-zero log-fold change for these bins indicates that composition bias is present between libraries. The red line represents the log-ratio of normalization factors and passes through the centre of the cloud in each plot, indicating that the bias has been successfully identified and removed.\n\n\n\nThe red line represents the log-ratio of the normalization factors between libraries.\n\nNote that this normalization strategy is quite different from that in the H3K9ac analysis. Here, systematic DB in one direction is expected between conditions, given that CBP function is lost in the KO genotype. This means that the assumption of a non-DB majority (required for non-linear normalization of the H3K9ac data) is not valid. No such assumption is made by the binned-TMM approach described above, which makes it more appropriate for use in the CBP analysis.\n\nFiltering of low-abundance windows. Removal of low-abundance windows is performed as previously described. The majority of windows in background regions are filtered out upon applying a modest fold-change threshold. This leaves a small set of relevant windows for further analysis.\n\n\n\n\n\n\n\nStatistical modelling of biological variability. Counts for each window are modelled using edgeR as previously described. First, a design matrix needs to be constructed.\n\n\n\n\n\nEstimation of the NB and QL dispersions is then performed. The estimated NB dispersions are substantially larger than those observed in the H3K9ac data set. In addition, the estimated prior d.f. is infinite. This is consistent with a batch effect between replicates. The dispersions for all windows are inflated to a similarly large value by the batch effect, resulting in low variability in the dispersions across windows.\n\n\n\n\n\n\n\n\n\nThe presence of a large batch effect between replicates is not ideal. Nonetheless, the DB analysis can proceed, albeit with some loss of power due to the inflated NB dispersions.\n\nTesting for DB. DB windows are identified using the QL F-test. Windows are clustered into regions, and the region-level FDR is controlled using Simes’ method. All significant regions have increased CBP binding in the WT genotype. This is expected, given that protein function should be lost in the KO genotype.\n\n\n\n\n\n\n\n\n\nThese results can be saved to file, as previously described. Key objects are also saved for convenience.\n\n\n\nAnnotation is added using the detailRanges function, as previously described.\n\n\n\nThe top-ranked DB event will be visualized here. This corresponds to a simple DB event, as all windows are changing in the same direction, i.e., up in the WT. The binding region is also quite small relative to some of the H3K9ac examples, consistent with sharp TF binding to a specific recognition site.\n\n\n\n\n\nPlotting is performed using two tracks for each library – one for the forward-strand coverage, another for the reverse-strand coverage. This allows visualization of the strand bimodality that is characteristic of genuine TF binding sites. In Figure 12, two adjacent sites are present at the Gbe1 promoter, both of which exhibit increased binding in the WT genotype. Coverage is also substantially different between the WT replicates, consistent with the presence of a batch effect.\n\nBlue and red tracks represent forward- and reverse-strand coverage, respectively, on a per-million scale (capped at 5 in SRR1145788, for visibility).\n\n\n\nNote that that the gax and greg objects are the same as those used in the visualization of the H3k9ac data.\n\n\nSummary\n\nThis workflow describes the steps of a window-based DB analysis, from read alignment through to visualization of DB regions. All steps are performed within the R environment and mostly use functions from Bioconductor packages. In particular, the core of the workflow – the detection of DB regions – is based on a combination of csaw and edgeR. Analyses are shown for histone mark and TF data sets, with differences in parametrization that are appropriate to each data type. Readers are encouraged to apply the concepts and code presented in this article to their own data.\n\n\nSoftware availability\n\nThis workflow depends on various packages from version 3.1 of the Bioconductor project, running on R version 3.2.0 or higher. It requires a number of software packages, including csaw, edgeR, Rsubread, Rsamtools, Gviz, rtracklayer and ChIPpeakAnno. It also depends on the annotation packages org.Mm.eg.db and TxDb.Mmusculus.UCSC.mm10.knownGene. Version numbers for all packages used are shown below.\n\n\n\n\n\nFor the command-line tools, the fastq-dump utility (version 2.4.2) from the SRA Toolkit must be installed on the system, along with the MarkDuplicates command from the Picard software suite (version 1.117). Readers should note that the read alignment steps for each data set can only be performed on Unix or Mac OS. This is because the various system calls assume that a Unix-style command-line interface is present. In addition, Rsubread is not supported for Windows. However, downstream analyses of the BAM files can be performed using any platform on which R can be installed.",
"appendix": "Author contributions\n\n\n\nA.T.T.L. developed and tested the workflow on the H3K9ac and CBP data sets. G.K.S. provided direction on the design of the workflow. Both A.T.T.L. and G.K.S. wrote the article.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNational Health and Medical Research Council (Program Grant 1054618 to G.K.S., Fellowship to G.K.S.); Victorian State Government Operational Infrastructure Support; Australian Government NHMRC IRIIS.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank Prof. Stephen Nutt for his valuable insights on B-cell biology.\n\n\nReferences\n\nBallman KV, Grill DE, Oberg AL, et al.: Faster cyclic loess: normalizing RNA arrays via linear models. Bioinformatics. 2004; 20(16): 2778–2786. PubMed Abstract | Publisher Full Text\n\nBenjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Royal Stat Soc B. 1995; 57(1): 289–300. Reference Source\n\nEdgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002; 30(1): 207–210. PubMed Abstract | Publisher Full Text | Free Full Text\n\nENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414): 57–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffmann R, Seidl T, Neeb M, et al.: Changes in gene expression profiles in developing B cells of murine bone marrow. Genome Res. 2002; 12(1): 98–111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHumburg P, Helliwell CA, Bulger D, et al.: ChIPseqR: analysis of ChIP-seq experiments. BMC Bioinformatics. 2011; 12: 39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarmodiya K, Krebs AR, Oulad-Abdelghani M, et al.: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells. BMC Genomics. 2012; 13: 424. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKasper LH, Qu C, Obenauer JC, et al.: Genome-wide and single-cell analyses reveal a context dependent relationship between CBP recruitment and gene expression. Nucleic Acids Res. 2014; 42(18): 11363–11382. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKharchenko PV, Tolstorukov MY, Park PJ: Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol. 2008; 26(12): 1351–1359. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLandt SG, Marinov GK, Kundaje A, et al.: ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 2012; 22(9): 1813–1831. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLawrence M, Gentleman R, Carey V: rtracklayer: an R package for interfacing with genome browsers. Bioinformatics. 2009; 25(14): 1841–1842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiang K, Keles S: Detecting differential binding of transcription factors with ChIP-seq. Bioinformatics. 2012; 28(1): 121–122. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiao Y, Smyth GK, Shi W: The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013; 41(10): e108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLun AT, Smyth GK: De novo detection of differentially bound regions for ChIP-seq data using peaks and windows: controlling error rates correctly. Nucleic Acids Res. 2014; 42(11): e95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLund SP, Nettleton D, McCarthy DJ, et al.: Detecting differential expression in RNA-sequence data using quasi-likelihood with shrunken dispersion estimates. Stat Appl Genet Mol Biol. 2012; 11(5): 1544–6115, Article 8. PubMed Abstract | Publisher Full Text\n\nMcCarthy DJ, Chen Y, Smyth GK: Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 2012; 40(10): 4288–4297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPal B, Bouras T, Shi W, et al.: Global changes in the mammary epigenome are induced by hormonal cues and coordinated by Ezh2. Cell Rep. 2013; 3(2): 411–426. PubMed Abstract | Publisher Full Text\n\nPodojil JR, Sanders VM: Selective regulation of mature IgG1 transcription by CD86 and beta 2-adrenergic receptor stimulation. J Immunol. 2003; 170(10): 5143–5151. PubMed Abstract | Publisher Full Text\n\nRevilla-I-Domingo R, Bilic I, Vilagos B, et al.: The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis. EMBO J. 2012; 31(14): 3130–3146. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson MD, Oshlack A: A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11(3): R25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRosenbloom KR, Armstrong J, Barber GP, et al.: The UCSC Genome Browser database: 2015 update. Nucleic Acids Res. 2015; 43(Database issue): D670–681. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss-Innes CS, Stark R, Teschendorff AE, et al.: Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature. 2012; 481(7381): 389–393. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimes RJ: An improved Bonferroni procedure for multiple tests of significance. Biometrika. 1986; 73(3): 751–754. Publisher Full Text\n\nZhang Y, Liu T, Meyer CA, et al.: Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008; 9(9): R137. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu LJ, Gazin C, Lawson ND, et al.: ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics. 2010; 11: 237. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10876",
"date": "30 Oct 2015",
"name": "Lihua Julie Zhu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “From reads to regions: a Bioconductor workflow to detect differential binding in ChIP-seq data” clearly describes a comprehensive computational workflow of Differential Binding (DB) analysis of ChIP-seq data set, based primarily on R software packages from the open-source Bioconductor project. It provides readers with practical usage examples of DB analyses of two typical types of ChIP-seq data sets, that covers all steps of the analysis pipeline, from alignment of read sequences, normalization, DB identification, annotation to interpretation and visualization of putative DB regions. We believe that this well-written paper will greatly help users to apply the workflow to their own DB analysis of ChIP-seq data sets.In the following section, please explicitly explain that the two data sets were chosen to represent two common ChIP-seq use cases, one data set for dealing with wider peaks while the other data set is for working with sharp peaks\"The application of the methods in this article will be demonstrated on two publicly available ChIP-seq data sets. The first data set studies changes in H3K9ac marking between pro-B and mature B cells (Revilla-I-Domingo et al., 2012). The second data set studies changes in CREB-binding protein (CBP) binding between wild-type and CBP knock-out cells (Kasper et al., 2014). \"According to the Rsubread documentation about parameter type athttp://bioconductor.org/packages/release/bioc/manuals/Rsubread/man/Rsubread.pdf, type is an integer giving the type of sequencing data. Possible values include 0 (RNA- seq data) and 1 (genomic DNA-seq data such as WGS, WES, ChIP-seq data etc.). By default, it is set to 0. Therefore, please set type to 1 in the following section.\"library(Rsubread)bam.files <- paste0(names(by.group), \".bam\")align(index=“index/mm10\", readfile1=group.fastq, TH1=2,\n\ninput_format=“FASTQ\", output_file=bam.files)\"\"align(index=\"index/mm10\", readfile1=all.fastq, phredOffset=64,\n\ninput_format=\"FASTQ\", output_file=bam.files)\"Suggest adding file.exists check to see if temp.dir exists already before dir.create(temp.dir).For system call to fastq-dump and MarkDuplicates, suggest to add path information to these programs in case the path to these programs are not in the search path.\"For this analysis, reads are only used if they have a mapping quality score above 50.\" 50 is pretty high although we understand that this is for illustration purposes. To prevent readers from getting the idea that 50 is the recommended cutoff, could you please also provide a quality score threshold commonly used in the field?\"The filter threshold is defined based on the assumption that most regions in the genome are not marked by H3K9ac. Reads are counted into large bins and the median coverage across those bins is used as an estimate of the background abundance. Windows are only retained if they have abundances 3-fold higher than the background.\" In the example, the window size (width) for calculating background (bg) coverage is set to 2000 bp, and the width is set to 150 bp for win.data. When calculating fold enrichment over background, is the bg coverage scaled down using the ratio of the window size (2000 vs. 150)?\"The implicit assumption of non-linear methods is that most windows at each abundance are not DB. Any systematic difference between libraries is attributed to bias and is removed. This is not appropriate in situations where large-scale DB is expected, as removal of the difference would result in loss of genuine DB. However, there is no indication that such changes are present in this data set, so non-linear methods can be applied without too much concern.\"Could you please suggest a normalization method for situations where large-scale DB is expected (perhaps TMM as mentioned in the later section)?\"Note that only the trended dispersion will be used here – the common and tagwise values are only shown for diagnostic purposes\"Could you please comment on how common and tag wise values help with diagnosis of the data set?\"Determining the direction of DB is more complicated, as clusters could potentially contain windows that are changing in opposite directions.\"How about controlling this by allowing nearby windows to merge only if the changing directions are the same?\"The behaviour of ChIPpeakAnno complements that of detailRanges. The latter reports all overlapping and flanking genes, while the former reports only the closest gene (but in greater detail). Which is preferable depends on the proclivities of the user and the purpose of the annotation.\"Actually, ChIPpeakAnno can also report all overlapping and flanking genes by setting output=\"both\" (or output =\"overlapping\") and maxgap. For example, it outputs all overlapping and flanking genes within 5kb if set maxgap = 5000L and output =\"overlapping\". Here is an example using Txdb annotation data.library(TxDb.Mmusculus.UCSC.mm10.knownGene)ucsc.mm10.knownGene <- genes(TxDb.Mmusculus.UCSC.mm10.knownGene)anno.peaks <- annotatePeakInBatch(minimal, annotationData=ucsc.mm10.knownGene,\n\noutput=\"overlapping\", maxgap=5000L)\"Reads are then counted into sliding windows. For TF data analyses, smaller windows are necessary to capture sharp binding sites. A large window size will be suboptimal as the count for a particular site will be “contaminated” by non-specific background in the neighbouring regions. In this case, a window size of 10 bp is used.win.data <- windowCounts(bam.files, param=param, width=10, ext=frag.len)\"It seems that space is set to 50bp for both sharp peaks and broad peaks. Could you please comment on this? We notice that the width for local background is set to 2kb for broad peaks and 10kb for sharp peaks. Could you please justify? Would adding a fitted line in Figure 3 and Figure 4 help with the visualization? In addition, it would be great if you could provide recommendations on how to evaluate the quality of the BCV in Figure 5. Is there a range or specific shape we are targeting?",
"responses": [
{
"c_id": "1678",
"date": "11 Jan 2016",
"name": "Aaron Lun",
"role": "Author Response",
"response": "We believe that this well-written paper will greatly help users to apply the workflow to their own DB analysis of ChIP-seq data sets.Thanks Julie. In the following section, please explicitly explain that the two data sets were chosen to represent two common ChIP-seq use cases, one data set for dealing with wider peaks while the other data set is for working with sharp peaks Done. Therefore, please set type to 1 in the following section. Done. We note that, at the time of publication, the workflow was constructed using packages in BioC 3.1, at which time the \"type\" parameter was not available in Rsubread. This has now been changed, along with minor code updates to various other parts of the workflow for BioC 3.2. Suggest adding file.exists check to see if temp.dir exists already before dir.create(temp.dir). There's no need for the extra check. By default, \"dir.create\" gives a warning if the directory already exists. We want users to be able to run the code multiple times if they wish, overwriting earlier files if they exist. For system call to fastq-dump and MarkDuplicates, suggest to add path information to these programs in case the path to these programs are not in the search path. We believe that implementing this suggestion may be counter-productive for most readers; the absolute path to these programs on our machines will not be useful to the general audience, given that the installation directory will change on different machines. It is the user's responsibility to make sure that these system commands are available prior to analysis - how this is done is largely irrelevant to successful execution of the workflow. The Picard software suite and the SRA Toolkit are quite widely used, so we do not think that installation and access would pose a major problem for most users. To prevent readers from getting the idea that 50 is the recommended cutoff, could you please also provide a quality score threshold commonly used in the field? We use 50 for the first data set as the reads are very short (< 40 bp) such that strict filtering is required to avoid mapping errors, non-uniquely-mapped reads, etc. We use the same value for the second data set for consistency, though we concede that a lower value could be used in practice. We have added a comment regarding this to the workflow.We also note that the MAPQ values reported by (R)subread tend to be quite binary for long reads, i.e., either very low or very high. For example, in SRR1145790, there are 24212282 reads with a MAPQ score >= 50, and 24907156 with a MAPQ score >= 10. This equates to a (relatively minor) 3% increase in available reads from a large change in the MAPQ threshold. All in all, we believe that analyses based on Rsubread alignments are generally robust to the choice of threshold. When calculating fold enrichment over background, is the bg coverage scaled down using the ratio of the window size (2000 vs. 150)? Yes. This is done automatically within the \"filterWindows\" function. We have added a note to the workflow to point this out. Could you please suggest a normalization method for situations where large-scale DB is expected (perhaps TMM as mentioned in the later section)? As you have noted, this issue is addressed more comprehensively in the analysis for the CBP data set. We have added a comment here regarding the existence of alternative normalization strategies when large-scale DB is expected. Could you please comment on how common and tag wise values help with diagnosis of the data set? The common BCV provides a measure of the overall variability of the data set, averaged across all windows. The tagwise BCVs should be dispersed around the fitted trend to indicate that the fit was successful. These comments have been added to the workflow. How about controlling this by allowing nearby windows to merge only if the changing directions are the same? This possibility has not escaped us. However, the sign of the log-FC is not independent of the DB status of each window in this particular application. In fact, clustering windows based on the signs of the log-FCs will result in loss of detection power for DB events. To illustrate, consider a non-DB site with a number of overlapping windows. Due to stochasticity, the log-FCs of those windows fluctuate around zero. Now, assume that we only allow merging of adjacent windows where the signs of the log-FCs are identical. For our non-DB site, we end up with lots of small clusters, comprised of windows with positive or negative log-FCs due to chance. In contrast, for a genuinely DB site, all windows will have positive (or negative) log-FCs, as stochasticity will not change the sign of the log-FC. When this behaviour is considered on a genome-wide level, we can see that non-DB sites will generate several non-DB clusters, while DB sites will only generate one DB cluster. This results in an increase in the number of tests with large p-values, increasing the severity of the BH correction without any increase in the number of potential discoveries. Ultimately, this results in the loss of detection power for DB events. One might be tempted to overcome this by filtering out small non-DB clusters, but this will compromise FDR control and result in liberalness. We consider loss of power to be more problematic than the existence of complex DB events. The former means that you don't detect anything, whereas the latter just requires some more consideration during interpretation. In fact, proper identification of complex events is not irrelevant - the context of a change in binding will affect its interpretation (e.g., if it occurs adjacent to an opposing change in the same general region) and this would be lost if changes were reported separately. Of course, these theoretical issues are beyond the scope of this workflow article. However, considerations of proper FDR control across regions have been discussed in our methodological articles (Lun and Smyth, NAR 2014; Lun and Smyth, NAR 2015). Actually, ChIPpeakAnno can also report all overlapping and flanking genes by setting output=\"both\" (or output =\"overlapping\") and maxgap. For example, it outputs all overlapping and flanking genes within 5kb if set maxgap = 5000L and output =\"overlapping\". Thanks for letting us know. We have amended our comment regarding the differences between the two annotation strategies. We note that there are still some differences; in particular, for any given input region, \"annotatePeakInBatch\" with output=\"overlapping\" reports each overlapping feature as a separate entry of the output object, while \"detailRanges\" reports all overlapping features in one string. The former provides more detail and is easier to use for further analysis, while the latter is more convenient for annotating an existing DB list that needs to be saved to file. It seems that space is set to 50bp for both sharp peaks and broad peaks. Could you please comment on this? In general, the spacing governs the compromise between spatial resolution and computational convenience. Smaller spacings increase resolution, at the cost of increasing memory usage and runtime. However, for most ChIP-seq analyses, resolution is fundamentally limited by the width of the binding event and by the size of the post-sonication fragments. As long as the spacing is smaller than either of these two parameters (i.e., the window size and the extension length), there will be enough windows to profile each interval of the genome for a satisfactory analysis. Smaller spacings and additional windows will provide no benefit, and will only increase computational work. This is true for both TF and histone mark analyses, such that the choice of spacing interval does not need to be changed between them. We have added a brief remark about this to the workflow. We notice that the width for local background is set to 2kb for broad peaks and 10kb for sharp peaks. Could you please justify? A local background of 2 kbp can also be used for sharp peaks. We have used 10 kbp for convenience; the counts for large bins were already loaded for normalization in the previous step, so it makes sense to re-use them for filtering. In general, smaller bins provide a more accurate estimate of the background abundance, as unbound regions can be distinguished from binding sites more effectively. However, loss of spatial resolution is negligible for large background regions in the CBP data set. We have added a comment about this to the workflow. Would adding a fitted line in Figure 3 and Figure 4 help with the visualization? We believe that the trend in the log-fold changes is obvious enough without the need for a fitted line. In addition, it would be great if you could provide recommendations on how to evaluate the quality of the BCV in Figure 5. Is there a range or specific shape we are targeting? As a general rule, we expect to see a curve that decreases to a plateau with increasing average abundance. This reflects the increased reliability of the data at large counts, where the effects of stochasticity and technical artifacts (e.g., mapping errors, PCR duplicates) are averaged out. In Figure 5, the range of abundances is such that the plateau has already been reached; a more dramatic decrease can be observed by including more lower-abundance windows."
}
]
},
{
"id": "10877",
"date": "25 Nov 2015",
"name": "Cenny Taslim",
"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 useful workflow on detecting differential binding in ChIP-seq data with examples and codes from R packages and other softwares. This paper will help researchers on analyzing their ChIP-seq using R. However, it would be better if there was more description on the methods used in the workflow.“Reads are first aligned to the genome using the Rsubread package (Liao et al., 2013).” Please describe what algorithm is used to align the reads.\"Technical replicates are merged together prior to further processing.\" What does merged together mean? Concatenate? Or average? Or pick one randomly?\"Ideally, the proportion of mapped reads should be high, while the proportion of marked reads should be low.\" Proportion mapped is those reads that can be uniquely mapped to the genome?\"Thus, the marking status of each read will be ignored in the rest of the analysis, i.e., no duplicates will be removed in downstream steps.\" I believe generally people exclude duplicates in downstream steps?\"By default, windows with very low counts are removed to reduce memory use.\" What is the definition of very low counts? <=1? If it is described by the section filtering windows by abundance, please mention it.bins <- windowCounts(bam.files, bin=TRUE, width=2000, param=param)filter.stat <- filterWindows(win.data, bins, type=\"global\")min.fc <- 3keep <- filter.stat$filter > log2(min.fc)summary(keep)##\n\nMode\n\nFALSE\n\nTRUE\n\nNA's## logical\n\n906406 663218\n\n0 hist(filter.stat$back.abundances, xlab=\"Background abundance\", main=\"\", breaks=50)threshold <- filter.stat$abundances[1] - filter.stat$filter[1] + log2(min.fc)abline(v=threshold, col=\"red\") In code above, where is the background? I assumed it is filter.stat$filter[1]? However, it looks like the filter is background + log2(3)? If it’s at least 3-fold background, shouldn’t it be filter.stat$filter[1]*3? Figure 2 doesn’t make sense. Why is there windows with < 0 abundance? Normalizing for library-specific trended biases. Figure 3 only shows log-fold change between mature B and pro-B but there is more than one sample in mature B and pro-B. How do you apply the normalization? Do you take average of all mature B samples and average of all pro-B samples and then do the loess normalization? ##\n\nGene nWindows logFC.up logFC.down\n\nPValue\n\nFDR## 6 Ldlrap1\n\n19\n\n11\n\n0 0.224741404877 0.2705479855## 7 Mdn1\n\n29\n\n12\n\n11 0.000004447727 0.0001347924## 8 Pydc3\n\n8\n\n0\n\n6 0.051183399851 0.0781822366## 9 Wfdc17\n\n6\n\n0\n\n6 0.000069604922 0.0008738790## 10 Mfap1b\n\n19\n\n1\n\n10 0.107116609335 0.1440819313## 13 Gm15772\n\n30\n\n12\n\n7 0.085543435687 0.1192823092How do you interpret this results? For Mdn1, there are 29 DB windows? What are the logFC.up and logFC.down.",
"responses": [
{
"c_id": "1718",
"date": "11 Jan 2016",
"name": "Aaron Lun",
"role": "Author Response",
"response": "Thank you for your report. You asked for more details of the methodology in a number of places, and we have added further details as appropriate. We are puzzled about your approval \"with reservations\", as we did not find any criticisms in your report, apart from the Figure 2 x-label not being explicit about the log-scale used. “Reads are first aligned to the genome using the Rsubread package (Liao et al., 2013).” Please describe what algorithm is used to align the reads. The alignment algorithm in Rsubread package uses a seed-and-vote paradigm. We have added a very brief mention of this, though for a complete description of the algorithm, it would be advisable to consult the Liao et al. reference. We note that the differences between different aligners are not relevant to this workflow article. What is relevant is that the subread aligner is an appropriate aligner for ChIP-seq data, and that it is available as a native implementation in a Bioconductor package. \"Technical replicates are merged together prior to further processing.\" What does merged together mean? Concatenate? Or average? Or pick one randomly?Merging simply refers to pooling the reads from all replicates into a single library. We have altered the wording to make this more obvious.\"Ideally, the proportion of mapped reads should be high, while the proportion of marked reads should be low.\" Proportion mapped is those reads that can be uniquely mapped to the genome?Yes. By default, Rsubread only reports mapped reads as those with unique mapping locations. We have added a mention of this to the text.\"Thus, the marking status of each read will be ignored in the rest of the analysis, i.e., no duplicates will be removed in downstream steps.\" I believe generally people exclude duplicates in downstream steps? Duplicate removal is not recommended for routine DB analyses with edgeR. It will reduce detection power by capping read coverage at strongly bound sites, such that a region that is DB may not be detected if the coverage of that region gets capped to the same level in two libraries. It may also increase false positives for analyses involving different library sizes, when the same cap is applied across libraries; subsequent normalization for library size will result in a spurious difference in the capped heights. In theory, duplicate removal should only be necessary and appropriate when a large portion of duplicate reads arise from PCR duplicates of the same DNA fragment. This should seldom occur unless the amount of DNA is overly small, in which case there are likely to be more general problems with data quality. \"By default, windows with very low counts are removed to reduce memory use.\" What is the definition of very low counts? <=1? If it is described by the section filtering windows by abundance, please mention it.The internal filter in windowCounts removes windows with count sums less than 10 across all libraries, simply to reduce memory usage. Windows with such low counts will not provide sufficent evidence for detecting DB, so their removal does not seem like a major loss. We have added a mention of this threshold to the text.In code above, where is the background? I assumed it is filter.stat$filter[1]? However, it looks like the filter is background + log2(3)? If it’s at least 3-fold background, shouldn’t it be filter.stat$filter[1]*3?By default, abundances in edgeR are reported as log-CPMs. So, a 3-fold increase over the background coverage corresponds to a log2(3) increase in the abundance. This has been reworded for more clarity.Figure 2 doesn’t make sense. Why is there windows with < 0 abundance?Again, abundances are reported as log-CPMs, for which it is entirely possible to obtain negative values. We have clarified this on the x-axis label.Normalizing for library-specific trended biases. Figure 3 only shows log-fold change between mature B and pro-B but there is more than one sample in mature B and pro-B. How do you apply the normalization? Do you take average of all mature B samples and average of all pro-B samples and then do the loess normalization?The algorithm constructs an average library containing average counts across all samples for each window. It then performs loess normalization between each sample and this average sample. A full description is available in our recently published paper (doi: 10.1093/nar/gkv1191, to which we have added a reference), but is beyond the scope of this workflow article. How do you interpret this results? For Mdn1, there are 29 DB windows? What are the logFC.up and logFC.down. We have already described the meaning of these fields in the text following the first call to combineTests(), in the section \"Controlling the FDR across regions\". The same interpretation can be applied to the output of all combineTests() calls."
}
]
},
{
"id": "10879",
"date": "18 Dec 2015",
"name": "Rory Stark",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 comprehensive and useful presentation of a window-based differential binding analysis. Currently, most quantitative differential binding studies rely on a peak calling step; this demonstration of the benefits of avoiding such a step is of great interest. The workflow is a valuable complement to the author's related published discussions (Lun and Smyth 2014, 2015). It is comprehensive in that is takes the user from archived sequencing reads, through two core analyses, and includes annotation and visualization code as well. Notably the authors include interesting exploration of many important details, particularly in the area of normalization, that are frequently overlooked and can have a crucial impact on analytical results.I include below a number of questions for the authors, the answer to which may make the article even more useful.Computational resources. It would be useful to include some discussion of the computational resources required to perform this analysis (memory and compute time). How do memory/compute requirements change as a function of lowering the window size? Can this handle a large number of samples? Experimental design. Are there any guidelines of how many replicates should be included for a successful analysis? Duplication rates. Duplicates are included in the analysis, are there any guidelines for acceptable duplication rates? The authors say that \"ideally, the proportion of mapped reads should be high, while the proportion of marked reads should be low\" -- how high and how low? Is there some point where there are too many duplicates to expect a successful analysis? Blacklists. The workflow uses a published blacklist to mask areas of the genome where reads will be not be counted. Some of these are attributable repeat regions, but some are anomalous, and there may be tissue-specific issues. Given that the workflow includes only very limited use of control reads (such as Input) for filtering non-enriched windows, perhaps it would be good to use blacklists generated from the controls, such as those made by the GreyListChIP Bioconductor package.This is especially important for experiments that use multiple tissue types or cell lines. Read counting. The authors state that “the number of extended reads overlapping a window is counted”. As almost all reads will overlap more than one window (fragment lengths generally being longer than the window size), it would be helpful to be explicit regarding if a read is counted in more than one window. If so, do single reads resulting in multiple counts have an implication for the assumption of binomial distribution of reads? Filtering windows by abundance.By using read abundance to remove windows prior to testing, is there an issue with the same information being used to choosing which windows to compare as is used for the comparison itself? Can window filtering be compared to peak calling in that it uses read counts to reduce the proportion of the genome being considered for differential analysis?By using a fold measure as a threshold, in many cases, very small changes in the number of background reads can have a big impact on calculated fold change. How sensitive is this filter process? How important is it to final results? NormalizationIs there way to determine computationally if a trended-bias normalization is appropriate, or is this best done by visual examination of the mean-difference plot?If the scale of differential binding is not known a priori, what normalization method should be used? Is it safe to use a non-linear trended correction? Or TMM on large \"background\" windows? Merging windowsIt appears possible to create merged regions with higher FDR than the minimum FDR of constituent windows. Windows that would have an FDR lower than some “significance” threshold may be “lost” in a merged region. Is there a way to constrain the merging function to not create a “non-significant” region that contains “significant” windows (according to a specified FDR threshold)?Another reviewer commented that it would be nice to not merge regions that have windows with both positive and negative fold changes, this seems useful as well.Regarding regions with positive and negative fold changes, it seems worth referencing MMDiff (Schweikert et al BMC Genomics 2013), which is designed to detect differences in the gain/loss patterns of binding profiles. For the CBP example:One or more plots visualizing the batch effect (MDS/PCA) would be helpful!Is a batch effect the only possible explanation for large dispersion estimates and infinite prior d.f.? Can we always assume a batch effect if we see this?If the batch effect applies to a more than one sample, can this be modeled in a multi-factor design? If it applies only to one sample, perhaps this is a ChIP efficiency issue?",
"responses": [
{
"c_id": "1740",
"date": "11 Jan 2016",
"name": "Aaron Lun",
"role": "Author Response",
"response": "This article represents a comprehensive and useful presentation of a window-based differential binding analysis.Thanks Rory.It would be useful to include some discussion of the computational resources required to perform this analysis (memory and compute time). How do memory/compute requirements change as a function of lowering the window size? Can this handle a large number of samples?Including the alignment steps, the entire analysis of the two data sets takes approximately 7-8 hours. It uses around 10 GB of memory in total, most of which is spent in alignment or in processing the large numbers of windows in edgeR. Memory requirements will not change as a function of window size but will change linearly as a function of window spacing, as this determines the number of windows that are extracted from the genome. We recommend that, for large windows, the spacing can be increased to avoid unnecessary computational work (given that any additional loss of spatial resolution is irrelevant for large windows). As for the number of samples, we typically use csaw to analyze data from small contained experiments (5-10 samples). Large numbers (>50) of samples will probably require high-performance computing resources and some additional care, e.g., chromosome-by-chromosome processing. As suggested, we have added some expected time/memory requirements to the \"Software availability\" section.Are there any guidelines of how many replicates should be included for a successful analysis?edgeR requires at least two replicates in one group to estimate the NB/QL dispersion. We usually analyze data with two replicates in each of our biological conditions of interest. This ensures that the results for each condition are replicable and are not the result of technical artifacts such as PCR duplicates. We have added some comments regarding this to the text. Of course, the more replicates, the better, but we appreciate that ChIP-seq is one of the more technically challenging techniques and that obtaining high levels of replication is not always feasible.Duplicates are included in the analysis, are there any guidelines for acceptable duplication rates? The authors say that \"ideally, the proportion of mapped reads should be high, while the proportion of marked reads should be low\" -- how high and how low? Is there some point where there are too many duplicates to expect a successful analysis?We have added some ballpark figures to the workflow. In our experience, mapping proportions above 70 to 80% are quite satisfactory (with the missing reads probably lying in unmappable repeat regions), along with duplicate proportions below 20%. While some of the marked reads will inevitably correspond to non-duplicate fragments that happen to overlap in enriched regions, this is unlikely to explain very high proportions of marked reads (> 40%). Such cases are more likely to be caused by high levels of PCR duplication.Given that the workflow includes only very limited use of control reads (such as Input) for filtering non-enriched windows, perhaps it would be good to use blacklists generated from the controls, such as those made by the GreyListChIP Bioconductor package.This is especially important for experiments that use multiple tissue types or cell lines.Done.As almost all reads will overlap more than one window (fragment lengths generally being longer than the window size), it would be helpful to be explicit regarding if a read is counted in more than one window. If so, do single reads resulting in multiple counts have an implication for the assumption of binomial distribution of reads?Yes, reads are often counted in more than one window. We have added a statement regarding this to the workflow. Multiple counting of reads introduces technical correlations between windows, but this will not affect the downstream analysis. Estimation of the EB shrinkage statistics in edgeR are robust to correlations between features. Similarly, the BH correction used to control the FDR is robust to correlations between tests.By using read abundance to remove windows prior to testing, is there an issue with the same information being used to choosing which windows to compare as is used for the comparison itself? Can window filtering be compared to peak calling in that it uses read counts to reduce the proportion of the genome being considered for differential analysis?The use of an independent filtering criterion is the important concept here. In a NB model, the average abundance (i.e., the log-NB mean) is a filter statistic that is independent of the DB status of each test, i.e., knowing the average abundance doesn't tell you whether or not the window is DB. This ensures that the selection of high-abundance windows will not bias the analysis towards or against detecting DB in those windows, and maintains the validity of the downstream multiplicity correction procedures.Conceptually, filtering and peak calling have some similarities, as you have pointed out. However, from a statistical perspective, filtering on the average abundance is only equivalent to peak calling if all libraries were pooled together and the pooled library was used for peak calling (and even then, only if those libraries are of the same size). Indeed, most peak callers were not designed for processing multi-sample data sets, especially not in a manner that preserves the validity of downstream DB analyses.By using a fold measure as a threshold, in many cases, very small changes in the number of background reads can have a big impact on calculated fold change. How sensitive is this filter process? How important is it to final results?We protect against this effect by using large bins to compute the background abundance. This increases the number of reads in each bin and stabilizes the estimated abundance against stochastic changes in coverage. Further stability is provided by taking the median of the abundance across all bins. We find that random differences in coverage make little difference to the estimated background and to the filter threshold. Instead, filtering is more sensitive to the choice of the fold-increase over the background. Obviously, requiring a larger fold-increase will result in the loss of weak binding sites. This is an arbitrary decision that depends on what the user considers to be relevant and cannot be avoided, even with peak callers (in which case, the choice of threshold is that of the significance of each peak).Is there way to determine computationally if a trended-bias normalization is appropriate, or is this best done by visual examination of the mean-difference plot?This is best done with visual examination. Abundance-dependent normalization involves fitting a trend, and then adjusting the observations in order to \"flatten out\" the trend. This means that if you were to repeat the trend fitting process on the adjusted data, you would end up with a flat trend by definition. This would not be helpful in diagnosing problems in the normalization procedure.If the scale of differential binding is not known a priori, what normalization method should be used? Is it safe to use a non-linear trended correction? Or TMM on large \"background\" windows?We would suggest repeating the analysis with both normalization strategies. If they give similar results, then it doesn't matter which method is used. However, if they yield different results, this indicates that there are systematic differences in read coverage between some of the libraries. The origin of these differences cannot be conclusively determined from the data alone - they may be due to differences in IP efficiency, or due to systematic and genuine DB in one set of conditions. Thus, care will be required in the interpretation of the DB results. Regions detected with both approaches are most likely to be reliable.We note that systematic differences between replicates within a condition can be directly interpreted as efficiency biases. However, this does not guarantee that there are no systematic differences in binding between conditions. Applying normalization to remove efficiency biases will also remove any systematic and genuine DB between conditions. In short, an assumption about the underlying biology is still required in order to apply one method or the other. In practice, normalization of the efficiency biases is usually the lesser of two evils, as large differences between replicates will inflate the dispersions and interfere with DB detection.A third possibility is to use spike-ins (e.g., Drosophila chromatin) for normalization. This should be able to distinguish between genuine DB and efficiency bias, as only the latter should affect spike-in coverage. See the csaw user's guide for some guidelines on accommodating spike-in data in csaw.Is there a way to constrain the merging function to not create a “non-significant” region that contains “significant” windows (according to a specified FDR threshold)?As a general rule, the merging algorithm must be independent of the significance of the window in order to maintain statistical validity. This means that it is not allowed to know which windows are significant or not during its operation. If this is not true, FDR control across the regions cannot be guaranteed.That said, it may be useful in some applications to get tighter intervals containing only the significantly DB regions. This can be achieved by defining putative DB windows based on a window-level FDR threshold, and then clustering them to obtain DB regions. An informal estimate of the region-level FDR for these DB regions can be computed using the clusterFDR() function, and the window-level threshold can then be adjusted until the region-level FDR is below some desired threshold. This two-step procedure is necessary as the window-level and region-level FDRs can be quite different for many overlapping windows. However, it is less statistically rigorous than the default approach which is blind to the significance of each window.Another reviewer commented that it would be nice to not merge regions that have windows with both positive and negative fold changes, this seems useful as well.Such a merging procedure would result in loss of detection power - see our comments below.One or more plots visualizing the batch effect (MDS/PCA) would be helpful!Added. Note that we use a larger 'top' set of windows to make the MDS plot. This is simply to improve the visualization of the systematic differences between libraries that are not captured with the default top value (500).Is a batch effect the only possible explanation for large dispersion estimates and infinite prior d.f.? Can we always assume a batch effect if we see this?Very large dispersions in conjunction with infinite prior d.f. means there are large differences between the replicates that are too consistent to be random. In other words, there are systematic differences between the replicates, and that is almost the definition of a batch effect.If the batch effect applies to a more than one sample, can this be modeled in a multi-factor design? If it applies only to one sample, perhaps this is a ChIP efficiency issue?Yes, the batch effect can easily be modelled in the GLM, provided that it does not confound detection of DB between the conditions of interest.Batch effects and differences in ChIP efficiency are not mutually exclusive - the fact that samples are processed in separate batches is often the cause for a difference in efficiency. In this case, systematic differences in peak heights are observed between the two CBP WT replicates. This can be seen most clearly in the coverage tracks of Figure 12 (13 in version 2). This would suggest that the batch effect corresponds to an increase in ChIP efficiency for one of the WT libraries."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1080
|
https://f1000research.com/articles/4-1208/v1
|
03 Nov 15
|
{
"type": "Research Article",
"title": "Fascial Manipulation® for chronic aspecific low back pain: a single blinded randomized controlled trial",
"authors": [
"Mirco Branchini",
"Francesca Lopopolo",
"Ernesto Andreoli",
"Ivano Loreti",
"Aurélie M Marchand",
"Antonio Stecco",
"Francesca Lopopolo",
"Ernesto Andreoli",
"Ivano Loreti",
"Aurélie M Marchand",
"Antonio Stecco"
],
"abstract": "Background: The therapeutic approach to chronic aspecific low back pain (CALBP) has to consider the multifactorial aetiology of the disorder. International guidelines do not agree on unequivocal treatment indications. Recommendations for fascial therapy are few and of low level evidence but several studies indicate strong correlations between fascial thickness and low back pain. This study aims at comparing the effectiveness of Fascial Manipulation® associated with a physiotherapy program following guidelines for CALBP compared to a physiotherapy program alone.Methods: 24 subjects were randomized into two groups, both received eight treatments over 4 weeks. Outcomes were measured at baseline, at the end of therapy and at a 1 month and a 3 months follow-up. Pain was measured with the visual analogue scale (VAS) and the brief pain inventory (BPI), function with the Rolland-Morris disability questionnaire (RMDQ), state of well-being with the short-form 36 health-survey (SF-36). The mean clinical important difference (MCID) was also measured.Results: Patients receiving Fascial Manipulation® showed statistically and clinically significant improvements at the end of care for all outcomes, in the short (RMDQ, VAS, BPI) and medium term for VAS and BPI compared to manual therapy. The MCID show significant improvements in the means and percentage of subjects in groups in all outcomes post-treatment, in the short and medium term.Conclusion: Fascial tissues were implicated in the aetiology of CALBP and treatment led to decreased symptomatic, improved functional and perceived well-being outcomes that were of greater amplitude compared to manual therapy alone.",
"keywords": [
"low back pain",
"thoracolumbar fascia",
"myofascial pain",
"nonspecific pain"
],
"content": "Introduction\n\nChronic aspecific chronic low back pain (CALBP) is defined as pain and/or discomfort localised below the costal margin and above the gluteal folds with possible posterior thigh irradiations not extending below the knee; symptoms have to be present for over 3 months or longer than the expected normal healing time1. CALBP is one of the most common and costly syndromes of modern times, with a lifetime prevalence estimated at 70% and reaching 80% in Europe2. Negative impacts of CALBP include for the patient: pain, reduction in activities of daily living, reduced work productivity and/or work absence; for society: increased contacts with health care providers, high demands of medical investigations and related treatments3. Chronicity occurs in 5% of patients suffering from low back pain (LBP) but generates up to 80% of the total costs related to this disorder4.\n\nThe multifactorial aetiology of LBP creating pain and functional limitations has been investigated with particular emphasis on physiopathological mechanisms, neuropsychosocial factors and motor control alterations. Several regional, national and international guidelines on LBP treatment exist5–8 but the levels of evidence supporting these are not always optimal, and a strong recommendation for a specific physiotherapeutic approach is currently lacking. Moreover recent studies have demonstrated an association between thickness and disorganization of the connective tissue (fascia) and CALBP9–11 but no guidelines could be found regarding the recommendations of a therapeutic approach focused on fascial tissues. For these reasons, it was decided to conduct an experimental study comparing the effectiveness of Fascial Manipulation® (FM) versus the physiotherapeutic recommendations provided by guidelines (standard manual therapy) (MT).\n\nThe null hypothesis was that there would be no difference in short and medium term outcomes between patients undergoing standard physiotherapy only and those where FM was added to standard care.\n\n\nMethods\n\nThis study is a single blinded randomized controlled trial aiming at controlling the effectiveness of FM treatment added to a standard protocol of care in patients suffering from CALBP for the primary outcome of pain and secondary outcomes of function and perceived well-being. The study was submitted and approved by the Ethics Committee of “Azienda Ospedaliero-Universitario di Bologna – Policlinico S. Orsola-Malpighi” (n.46/2009/O/Sper, 21/04/2009) and it was conducted according to the principles expressed in the Declaration of Helsinki. Trial was registered in ClinicalTrials.gov with the identifier number NCT01269983. Participants were selected from patients presenting to the outday clinic of Physical Medicine and Rehabilitation Unit of “Azienda Ospedaliero-Universitario di Bologna”, Bologna, Italy. Inclusion criteria were diagnosis of chronic LBP or chronic lumbosciatic pain (above 3 months of duration), age between 20–60 years old, signed informed consent to the study. Exclusion criteria were neurological signs of spinal stenosis or reflex loss or dysesthesia, continued pharmacological treatment (non-steroidal anti-inflammatory, corticosteroids, painkillers), intake of drugs including antidepressants or anxiolytics or neuroleptics, structural lesions on imaging (spondylolysis or spondylolisthesis, vertebral canal stenosis, secondary vertebral lesions (neoplastic origin), vascular aetiology (i.e. abdominal aneurisms), systemic rheumatological pathologies (i.e. ankylosing spondylitis), comorbidities of the central or peripheral nervous system or cardiovascular or respiratory systems (inability to participate in the therapeutic protocol), insurance claim in act. Patients were screened for inclusion and exclusion criteria by two medical doctors (a physiatrist and an orthopedist). All the participants agreed to avoid any additional therapy or treatment during the study. It was recommended to patients to maintain the posology of their pharmacological therapy. Participants were free to leave the study in case of insurgence of new severe illness.\n\nPatients conforming to both inclusion and exclusion criteria were randomized into a study group (SG) or control group (CG) using a computer generated randomization list. Both groups shared the following treatment characteristics: duration of treatment (4 weeks), frequency (twice a week), duration of treatment session (45 minutes), experience of treating physiotherapists (students of physiotherapy in the third and final year of university training), and each physiotherapist exclusively treated patients belonging to either the SG or the CG. The CG patients followed a program of physiotherapy tailor-made in accordance to the national and international guidelines of CALBP. Treatment consisted of exercises on relaxation, control of diaphragmatic breathing, improved proprioception of the lumbar region, segmental and global stretching of the posterior back and lower limbs musculature, postural re-education, core stability, functional rehabilitation, home exercises. The repetitions and intensity of the manual therapy program was proportional to the patient improving and varied at each session. The SG alternated one treatment of FM and one treatment of MT per week.\n\nFM is a manual therapy that focuses on the deep muscular fascia. This method considers the fascia as a three-dimensional continuum. The mainstay of this manual technique lies in the identification of specific localised areas of the fascia, defined Centre of Coordination (CC) by Luigi Stecco12, where the gliding of the subcutis should be preserved to avoid biomechanical in-coordination of the surrounding muscles. The method is performed by applying a deep friction over the CCs that result more altered at the clinical palpation. The deep friction on these points aims at restoring the physiological gliding properties of the fascia and lead to immediate pain reduction, increased range of motion, improved function that may be objectively evaluated by the therapist13,14.\n\nThe primary outcome measure of pain was measured using the Visual Analogue Scale (VAS)15 and the sensorial, qualitative and emotional outcome was evaluated with the Brief Pain Inventory (BPI)16,17, functional outcome was evaluated with the Rolland and Morris Disability Questionnaire (RMDQ)18, quality of life outcome was evaluated with the Short-Form-36-Health Survey (SF-36)19. All of the above were measured at T0 (prior start of therapy). VAS was measured at the start and end of each treatment session whereas BPI, RMDQ and SF-36 were measured at the end of the (T15). The VAS was administered by the physiotherapists pre- and post-treatment in order to evaluate whether a trend was present between FM and MT treatments and between the SG and CG. The RMDQ is composed of 24 functional activities that may be affected by lumbar pain. The SF-36 is a generic psychometric questionnaire evaluating the levels of activity and feelings of well-being, it is composed of 8 scales with multiple questions (36 in total) measuring 8 sections: vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning and mental health. Scoring ranges from 0 to 100 with higher scores corresponding to higher quality of life.\n\nAll outcomes were measured at follow-up at one month (T16) and 3 months (T17) after end of care. The measurements at T0, T15, T16 and T17 were performed by the medical doctor who enrolled the participant in the study who remained blinded to treatment allocation. For the VAS, improvement of 1.5 was considered significant (improvement of or above 4 highly significant), for the BPI the cut-off was 1.5, for the RMDQ the cut-off was improvement of at least 30%20.\n\nData were encoded into a general database. The software of STATA v10 was used21. Descriptive statistics were used to evaluate differences in outcome measures between groups. Inferential statistics were used to investigate if homogeneity of groups was present at baseline. When interval data was normally distributed Student T-tests were performed, ordinal data were analysed using Mann-Whitney U test. The differences in all outcomes at T0, T16, T17 were analysed. RMDQ were analysed as percentage of improvement. Clinical significance was evaluated using the Minimal Clinical Important Difference (MCID)22,23 for the VAS, RMDQ and BPI.\n\n\nResults\n\nThirty-two patients were recruited from April to December 2009, 24 completed the trial, 6 participants were not enrolled for organizational reasons, 2 withdrew for reasons not directly related to treatment (one had a sport injury, one had a pulmonary infection). From the 24 participants who completed the trial 2 were lost at follow-up (T16, T17). After randomization the SG was composed of 11 patients (4 males, 7 females), the CG was composed of 13 patients (4 males, 9 females). At baseline the VAS was significantly higher in the SG, all other outcomes did not reach statistical significance (see Table 1).\n\nS.D. = standard deviation.\n\nFigure 1a–d shows the results of the measured outcomes at T0, T15, T16 and T17. All outcomes showed improvement for both groups. It can be observed that the improvement is maintained in the medium term (at one and three month follow up) for both groups, however results in the SG were significantly better compared to the CG as seen by the inclination of the trend lines.\n\na) VAS measures reported per time of evaluation b) BPI measures reported per time of evaluation c) RMDQ measures reported per time of evaluation d) SF-36 measures reported per time of evaluation.\n\nTable 2 shows the means, standard deviations, and statistical significance of each outcome for each group. VAS and BPI reached statistical significance for the SG. The outcome of the RMDQ did not reach statistical significance; however the percentage improvement reached significance when measured between T0-T15, T0-T16 a positive trend, not reaching significance, was observed between T0-17 for the group following FM. Table 3 shows the MCID along with the percentage of patients respecting its value for the VAS, RMDQ, BPI for T0-T15, T0-T16, T0-T17. It can be observed that both groups show clinical improvement. Strong clinical significance is reached in the SG for the VAS. In the same group both BPI and RMDQ reached higher clinical significance compared to patients following the program of MT.\n\nS.D. = standard deviation.\n\nThe values and trend lines of the VAS prior to each treatment are seen in Figure 2. Patients of the SG had a steeper trend, showing higher reduction of pain after each FM treatment. The drop in VAS was statistically significant after the first FM treatment (z=0.0239) for the SG, and reaching significance at the 8th manual therapy treatment (z=0.0405) for the CG. The mean difference in VAS value pre- and post-treatment was 2.9 during the first and 1.6 during the third session (Figure 3). The CG did not reach clinically significant improvement in VAS during treatment sessions. Table 4 shows the mean variation in VAS for the SG was 1.7 for FM treatment and 0.69 for MT treatment. The mean variation in VAS for the CG was 0.96. The trend is more linear for the CG (0.46–1.38 variation) and is steeper for the SG (0.11–2.92 variation).\n\n\nDiscussion\n\nFM treatment sessions, associated to four treatments of MT over 4 weeks in chronic aspecific low back pain, resulted in significant statistical and clinical improvement compared to eight treatments of MT alone at end of therapeutic intervention, at 1 month and at 3 months follow up. Therefore the null hypothesis was rejected.\n\nThis is the first randomized trial evaluating the effectiveness of FM for CALBP. The results observed in the MT group are similar to those reported in the literature24,25.\n\nWith regards to methodology it was decided to apply the exclusion criteria inherent to diagnostic criteria proposed in the specific guidelines for low back pain. These recommend a diagnostic triage of patients to exclude specific spinal pathologies or pain caused by compression on nerve roots. The outcome measure of pain was evaluated with the VAS and the BPI. The BPI was chosen as outcome measure because it is a complete and multidimensional test that evaluates the sensorial, qualitative and emotional aspects of pain. It initially started as a scale for oncological patients but its validity has also been proven in patients with chronic pain of non-neoplastic origin17. The SF-36, even though it is not specific for low back pain, was chosen as an outcome for perceived well-being.\n\nBoth groups were homogeneous at baseline except for the VAS value. Both followed a course of eight treatment sessions performed twice a week over 4 weeks. In both groups the therapists had the same level of education. Improvement in all outcomes was observed for both groups with the SG showing greater statistically and clinically significant improvements. The initial VAS value was higher for the SG which may have skewed the results towards a better improvement for the SG. However it has to be noted that both the VAS and the BPI reached statistical significance at all measurement times for the SG. This indicates that the FM treatment also created a significant improvement in another outcome that was similar at baseline. Therefore it may be considered that the VAS improvement reported in the SG is not solely related to the higher VAS at baseline.\n\nThe VAS values pre- and post- each treatment allow us to evaluate which intervention (MT or FM) showed the best improvement in the SG. It can be observed that the values post-FM treatment showed greater diminution compared to the CG and compared to the MT treatment performed in the SG. Indeed the first and third treatments in the SG showed a greater difference pre- and post-treatment. It can be observed that the first FM treatment lead to a statistically significant decrease in VAS compared to the CG. Such results can support the rationality of the FM method that, restoring the sliding of the fascia proximally and distally from the area of the pain, is able to recover the range of motion, decrease the stiffness and painful sensation perceived by the patient. With regards to the VAS improvement in the SG, it may be argued that, at the 8th treatment, statistically significant improvement would be more difficult to reach with a VAS value pre-treatment of 0.4, compared to 1.9 in the CG.\n\nThe perceived state of well-being showed significant improvement at the end of therapeutic intervention, in the FM and MT group, compared to the MT group only. At follow-up the values did not reach statistical significance, but showed a better trend for the SG. Functionality (of the RMDQ scale) reached statistical significance at the 1 month follow-up.\n\nClinical significance showed higher mean of improvement for all outcomes for the SG. Also the percentage of patients having higher values of MCID was higher than the CG and reaching 100% for BPI and RMDQ at T16 and T17. This indicates that FM treatment, when added to MT treatment, leads to clinically significant improvement in the short and medium term with regards to severity of pain and disability.\n\nThe rationale for including FM treatment for patients suffering from CALBP is based on the fact that fascia is a type of dense connective tissue on which about 30% of muscle fibres have either insertions or origins26. Fascia is therefore tensioned during any kind muscular activity in any direction of movement; it transmits tensions towards the perimysium and towards synergic muscular groups27,28. The deep fascia is composed of layers of dense connective tissue that are dedicated to transmitting the load and loose connective tissue that allows the gliding of the collagen layers. Different authors have demonstrated the rich innervation of the fascial tissue and in particular of the thoracolumbar fascia29–32. Langevin9,33 has demonstrated an increase of the thickness of the thoraco-lumbar fascia and a decrease of the gliding of the different layers. It is hypothesised that the layers of loose connective tissue are the ones causing the increase in thickness because fibrosis is not recognized in subject with CALBP. Recent studies have demonstrated that the major component of the loose connective tissue, the hyaluronan, by increasing concentration and/or size, begins to entangle into complex arrays, leading to a decrease in the gliding properties of the fascia34. Due to the viscoelastic properties of the loose connective tissue, allowing it to modulate the dynamic response of the mechanoreceptor, we hypothesize that a stiff thoracolumbar fascia can alter the receptor afferents35,36 providing another mechanism to the multifactorial aetiology of chronic back pain37.\n\nThe FM treatment utilises the non-Newtonian properties of the hyaluronan, by applying continuous shear to the fascial tissue, to increase the temperature of the fascial tissue. This leads to the destruction of the van der Waals and hydrophobic forces that hold the hyaluronan chains together. A temperature of 40°C is able to decrease the viscosity of the hyaluronan38 and increases its ability to glide39. A less viscous loose connective tissue allows fibroblasts to perceive the lines of tensions of the fascial layers and may therefore lead to the remodelling of their dense connective tissue with a deposition of collagen fibres in the correct lines of force40.\n\nThis study demonstrates that FM + MT reached clinical and statistical significance in outcomes compared to MT alone. However the sample size was small because, to avoid a long recruitment period that could generate bias in the data evaluation, it was difficult to increase the number of subjects treated in each group. Validated self-administered scales do not investigate variations in anatomical findings such as thickness of connective tissue as highlighted with diagnostic imaging including ultrasounds5. It was therefore not possible to evaluate whether pain changes reported by patients were related to anatomical changes in this study. Further, the discrepancy in baseline VAS score may have affected the statistical evaluation, but it has to be noted that, in the SG, both the VAS and the BPI reached statistical significance at all measurement times, indicating that FM treatment created significant improvement in another outcome that was similar at baseline. For this reason, in SG, the VAS improvement cannot be solely related to the higher VAS at baseline. Statistical correction for the different VAS value at baseline could be considered.\n\n\nConclusions\n\nThis study shows that the implementation of FM, within a course of MT following established guidelines, reaches statistically and clinically significant improvement in the outcomes of pain, function and quality of life in patients suffering CALBP both at the end of care as at one and three months follow-up compared to MT alone. Considering the costs of CALBP to health care systems it may be considered to implement FM treatment as part of routine care in existing physiotherapy programs. Considering the significant decrease in pain especially following the first two FM treatment sessions, it may be hypothesized that FM may reduce the number of treatments required for patients with CALBP and therefore reduced the overall costs to health care systems and patients.\n\n\nData availability\n\nF1000Research: Dataset 1. Experimental dataset from Branchini et al., ‘Fascial Manipulation® for chronic aspecific low back pain: a single blinded randomized controlled trial’, 10.5256/f1000research.6890.d100559\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained from the patients.",
"appendix": "Author contributions\n\n\n\nMB and FL conceived the study. EA and IL evaluated all the patients. MB carried out statistical analysis of the data. AS and AM 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\nThe authors Francesca Lopopolo, Ernesto Andreoli, Ivano Loreti declare that there is no conflict of interest regarding the publication of this manuscript. Antonio Stecco, Aurelie Marie Marchand and Mirco Branchini are members of Fascial Manipulation Association. The Association is non-profit, with the objective of promoting and supporting research in the field of pain relief in general, and in particular, the field of anatomy and physiopathology of the fasciae.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work.\n\n\nAcknowledgement\n\nThis study was made possible by the participation of students enrolled in the Physiotherapy Program of the University of Bologna who were in charge of treatments in the CG. We would like to acknowledge: Martina Berti, Morgan Bignami, Valentina Odaldi, Lucia Pratelli, Massimo Riccardi.\n\n\nReferences\n\nO'Sullivan P: Diagnosis and classification of chronic low back pain disorders: maladaptive movement and motor control impairments as underlying mechanism. Man Ther. 2005; 10(4): 242–55. PubMed Abstract | Publisher Full Text\n\nRaspe H: How epidemiology contributes to the management of spinal disorders. Best Pract Res Clin Rheumatol. 2002; 16(1): 9–21. PubMed Abstract | Publisher Full Text\n\nAndersson GBJ: The epidemiology of spinal disorders. In: Frymoyer JW, ed. The Adult Spine: Principles and Practice. 2nd ed. New York, NY: Raven Press; 1997; 93–141.\n\nAiraksinen O, Brox JI, Cedraschi C, et al.: COST B13 Working Group on Guidelines for Chronic Low Back Pain. Chapter 4. European guidelines for the management of chronic nonspecific low back pain. Eur Spine J. 2006; 15(Suppl2): S192–300. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPiano Nazionale Linee Guida. Come produrre, diffondere e aggiornare raccomandazioni per la pratica clinica. Manuale metodologico. Roma: PNLG; 2002. Reference Source\n\nChou R, Qaseem A, Snow V, et al.: Clinical Efficacy Assessment Subcommittee of the American College of Physicians; American College of Physicians; American Pain Society Low Back Pain Guidelines Panel. Diagnosis and treatment of low back pain: a joint clinical practice guideline from the American College of Physicians and the American Pain Society. Ann Intern Med. 2007; 147(7): 478–91. PubMed Abstract | Publisher Full Text\n\nSavigny P, Kuntze S, Watson P, et al.: London: National Collaborating Centre for Primary Care and Royal College of General Practitioners. Low Back Pain: Early Management of Persistent Non-specific Low Back Pain [Internet]. 2009. PubMed Abstract\n\nInstitute for Clinical Systems Improvement (ICSI). Adult low back pain. Bloomington (MN): Institute for Clinical Systems Improvement (ICSI); 2008; 66. Reference Source\n\nLangevin HM, Stevens-Tuttle D, Fox JR, et al.: Ultrasound evidence of altered lumbar connective tissue structure in human subjects with chronic low back pain. BMC Musculoskelet Disord. 2009; 10: 151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBednar DA, Orr FW, Simon GT: Observations on the pathomorphology of the thoracolumbar fascia in chronic mechanical back pain. A microscopic study. Spine (Phila Pa 1976). 1995; 20(10): 1161–1164. PubMed Abstract | Publisher Full Text\n\nYahia L, Rhalmi S, Newman N, et al.: Sensory innervation of human thoracolumbar fascia. An immunohistochemical study. Acta Orthop Scand. 1992; 63(2): 195–197. PubMed Abstract | Publisher Full Text\n\nStecco L: Fascial Manipulation for Musculoskeletal Pain. PICCIN, Italy, ISBN 88-299-1697-8. 2004; 11. Reference Source\n\nStecco A, Stecco C, Macchi V, et al.: RMI study and clinical correlations of ankle retinacula damage and outcomes of ankle sprain. Surg Radiol Anat. 2011; 33(10): 881–90. PubMed Abstract | Publisher Full Text\n\nStecco A, Meneghini A, Stern R, et al.: Ultrasonography in myofascial neck pain: randomized clinical trial for diagnosis and follow-up. Surg Radiol Anat. 2014; 36(3): 243–53. PubMed Abstract | Publisher Full Text\n\nPrice DD, McGrath PA, Rafii A, et al.: The validation of visual analogue scales as ratio scale measures for chronic and experimental pain. Pain. 1983; 17(1): 45–56. PubMed Abstract | Publisher Full Text\n\nBonezzi C, Nava A, Barbieri M, et al.: [Validazione della versione italiana del Brief Pain Inventory nei pazienti con dolore cronico]. Minerva Anestesiol. 2002; 68(7–8): 607–11. Italian. PubMed Abstract\n\nTan G, Jensen MP, Thornby JI, et al.: Validation of the Brief Pain Inventory for chronic nonmalignant pain. J Pain. 2004; 5(2): 133–7. PubMed Abstract | Publisher Full Text\n\nPadua R, Padua L, Ceccarelli E, et al.: Italian version of the Roland Disability Questionnaire, specific for low back pain: cross-cultural adaptation and validation. Eur Spine J. 2002; 11(2): 126–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nApolone G, Mosconi P: The Italian SF-36 Health Survey: translation, validation and norming. J Clin Epidemiol. 1998; 51(11): 1025–36. PubMed Abstract | Publisher Full Text\n\nRiddle DL, Stratford PW, Binkley JM: Sensitivity to change of the Roland-Morris Back Pain Questionnaire: Part 2. Phys Ther. 1998; 78(11): 1197–207. PubMed Abstract\n\nhttp://www.stata.com/stata10/.\n\nBeaton DE, Boers M, Wells GA: Many faces of the minimal clinically important difference (MCID): a literature review and directions for future research. Curr Opin Rheumatol. 2002; 14(2): 109–14. PubMed Abstract | Publisher Full Text\n\nCopay AG, Subach BR, Glassman SD, et al.: Understanding the minimum clinically important difference: a review of concepts and methods. Spine J. 2007; 7(5): 541–6. PubMed Abstract | Publisher Full Text\n\nBalthazard P, de Goumoens P, Rivier G, et al.: Manual therapy followed by specific active exercises versus a placebo followed by specific active exercises on the improvement of functional disability in patients with chronic non specific low back pain: a randomized controlled trial. BMC Musculoskelet Disord. 2012; 13: 162. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAjimsha MS, Daniel B, Chithra S: Effectiveness of myofascial release in the management of chronic low back pain in nursing professionals. J Bodyw Mov Ther. 2014; 18(2): 273–81. PubMed Abstract | Publisher Full Text\n\nHijikata T, Wakisaka H, Niida S: Functional combination of tapering profiles and overlapping arrangements in nonspanning skeletal muscle fibers terminating intrafascicularly. Anat Rec. 1993; 236(4): 602–10. PubMed Abstract | Publisher Full Text\n\nTurrina A, Martínez-González MA, Stecco C: The muscular force transmission system: role of the intramuscular connective tissue. J Bodyw Mov Ther. 2013; 17(1): 95–102. PubMed Abstract | Publisher Full Text\n\nHuijing PA: Epimuscular myofascial force transmission between antagonistic and synergistic muscles can explain movement limitation in spastic paresis. J Electromyogr Kinesiol. 2007; 17(6): 708–24. PubMed Abstract | Publisher Full Text\n\nStecco C, Gagey O, Belloni A, et al.: Anatomy of the deep fascia of the upper limb. Second part: study of innervation. Morphologie. 2007; 91(292): 38–43. PubMed Abstract | Publisher Full Text\n\nTesarz J, Hoheisel U, Wiedenhöfer B, et al.: Sensory innervation of the thoracolumbar fascia in rats and humans. Neuroscience. 2011; 194: 302–8. PubMed Abstract | Publisher Full Text\n\nYahia L, Rhalmi S, Newman N, et al.: Sensory innervation of human thoracolumbar fascia. An immunohistochemical study. Acta Orthop Scand. 1992; 63(2): 195–7. PubMed Abstract | Publisher Full Text\n\nHoheisel U, Rosner J, Mense S: Innervation changes induced by inflammation of the rat thoracolumbar fascia. Neuroscience. 2015; 300: 351–9. PubMed Abstract | Publisher Full Text\n\nLangevin HM, Fox JR, Koptiuch C, et al.: Reduced thoracolumbar fascia shear strain in human chronic low back pain. BMC Musculoskelet Disord. 2011; 12: 203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStecco C, Stern R, Porzionato A, et al.: Hyaluronan within fascia in the etiology of myofascial pain. Surg Radiol Anat. 2011; 33(10): 891–6. PubMed Abstract | Publisher Full Text\n\nSong Z, Banks RW, Bewick GS: Modelling the mechanoreceptor's dynamic behaviour. J Anat. 2015; 227(2): 243–54. PubMed Abstract | Publisher Full Text\n\nSuslak TJ, Armstrong JD, Jarman AP: A general mathematical model of transduction events in mechano-sensory stretch receptors. Network. 2011; 22(1–4): 133–42. PubMed Abstract | Publisher Full Text\n\nPanjabi MM: A hypothesis of chronic back pain: ligament subfailure injuries lead to muscle control dysfunction. Eur Spine J. 2006; 15(5): 668–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTømmeraas K, Melander C: Kinetics of hyaluronan hydrolysis in acidic solution at various pH values. Biomacromolecules. 2008; 9(6): 1535–40. PubMed Abstract | Publisher Full Text\n\nRoman M, Chaudhry H, Bukiet B, et al.: Mathematical analysis of the flow of hyaluronic acid around fascia during manual therapy motions. J Am Osteopath Assoc. 2013; 113(8): 600–610. PubMed Abstract | Publisher Full Text\n\nLangevin HM, Bouffard NA, Fox JR, et al.: Fibroblast cytoskeletal remodeling contributes to connective tissue tension. J Cell Physiol. 2011; 226(5): 1166–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStecco A, Branchini M, Lopopolo F, et al.: Dataset 1 in: Fascial Manipulation® for chronic aspecific low back pain: a single blinded randomized controlled trial. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11477",
"date": "09 Dec 2015",
"name": "Olavi Airaksinen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNice article. In general format it looks very good. The presentation style is clear – good and informative figures. The critical points that the article and the researchers have realized is the limitations of a sample size 24 to divided two groups – so 12 per group. The other critical issue will be the follow up time up to 3 months. So on that background I prefer to talk with these results as preliminary findings – which were very promising and will stimulate to work more with this and plan next trial with bigger population and longer follow up - up to one or two years (taking count that the patients were chronic LBP patients). Or one possibility will be add the phrase 'a pilot study' to the title?We have to be careful when making conclusions based on preliminary and pilot studies – the results seem to be promising – and I like to say that this way. As a conclusion I would prefer the indexation of this article with my suggested minor revisions.",
"responses": []
},
{
"id": "11667",
"date": "21 Dec 2015",
"name": "Heidi Prather",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWell written manuscript regarding a controversial yet very important topic: the management of axial low back pain.Comments:I am assuming people with a history of surgery were excluded. Is this an appropriate assumption? How long had these people experienced low back pain? Did they have pain every day or exacerbations? Myofascial manipulation requires the treating provider to be experienced with the evaluation of the patient that then directs treatment. How did you attempt to standardize this?",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1208
|
https://f1000research.com/articles/5-39/v1
|
08 Jan 16
|
{
"type": "Software Tool Article",
"title": "BioTapestry now provides a web application and improved drawing and layout tools",
"authors": [
"Suzanne M. Paquette",
"Kalle Leinonen",
"William J.R. Longabaugh",
"Suzanne M. Paquette",
"Kalle Leinonen"
],
"abstract": "Gene regulatory networks (GRNs) control embryonic development, and to understand this process in depth, researchers need to have a detailed understanding of both the network architecture and its dynamic evolution over time and space. Interactive visualization tools better enable researchers to conceptualize, understand, and share GRN models. BioTapestry is an established application designed to fill this role, and recent enhancements released in Versions 6 and 7 have targeted two major facets of the program. First, we introduced significant improvements for network drawing and automatic layout that have now made it much easier for the user to create larger, more organized network drawings. Second, we revised the program architecture so it could continue to support the current Java desktop Editor program, while introducing a new BioTapestry GRN Viewer that runs as a JavaScript web application in a browser. We have deployed a number of GRN models using this new web application. These improvements will ensure that BioTapestry remains viable as a research tool in the face of the continuing evolution of web technologies, and as our understanding of GRN models grows.",
"keywords": [
"Visualization",
"networks",
"open-source",
"gene regulatory networks",
"web applications",
"automatic network layout"
],
"content": "Introduction\n\nGene regulatory networks (GRNs) are responsible for driving the process of embryonic development1. This is an extremely complex process, and dedicated software tools are necessary to document both the network architecture and its dynamic evolution over time and space. Since a single static network figure does not adequately convey these complex behaviors, these tools also need to be highly interactive. The user should be able to explore the behavior of network subcircuits at particular points in time and space, and have access to relevant documentation of the underlying experimental evidence for each feature of the network. To be an effective and widely used tool, it must also be easy to share these interactive networks models over the web, rather than requiring users to download and install specialized software.\n\nBioTapestry2,3 is an open-source software application that was developed to fill the need for a GRN modeling tool that can share interactive models over the web. Figure 1 shows the desktop BioTapestry Editor displaying a Zebrafish developmental GRN4. BioTapestry has many notable features:\n\nThe early endoderm specification GRN for Zebrafish4 as it appears in the BioTapestry Java desktop Editor.\n\n• It represents a complex network using a model hierarchy in which each child model is constrained to contain a subset of the network elements present in its parent model. This central feature allows researchers to organize and maintain models that track development of complex embryos over time and space, and is also useful for organizing any large network model.\n\n• It represents the network with a level of abstraction that is appropriate for GRN models. General-purpose network visualization tools are not domain-specific enough to represent GRN clearly and effectively.\n\n• It uses colored, orthogonal directed hyperedges, i.e. “circuit traces” or “link trees”; see Figure 2. These link trees provide a compact and unambiguous representation of the GRN edges.\n\n• It allows users to associate experimental data or URLs with each network node or link.\n\nBy sharing link segments, hyperedges (right) create a more parsimonious representation of a set of directed links than standard edges (left).\n\nRecent BioTapestry development work, which has been released as Versions 6 and 7, focused on two distinct areas: new features to assist users to draw and layout networks, and a BioTapestry Viewer web application which runs entirely in a web browser.\n\nBigger networks need better drawing support. As noted above, BioTapestry represents the edges of the GRN using “link trees”. Experience working with users has demonstrated that it is helpful to provide tools that aid them in creating clean, well formed, unambiguous, and orthogonal link trees. It is also beneficial to provide automatic network layout tools, since BioTapestry has grown beyond its roots as a tool to draw small-to-medium sized networks, and can be used to visualize large networks as well.\n\nA new platform is needed. BioTapestry has always been a web-centric application. Both the BioTapestry Editor (which is used to build the GRN model) and original Viewer (a read-only client for sharing the model on the web) were written in Java using the Java2D graphics library and deployed using Java Web Start (JWS). These two separate packages are the same program behind the scenes, with the Viewer exposing a limited subset of the functionality in the Editor. However, in recent years developers have been moving away from JWS as a platform for web-enabled applications. Increased emphasis on Java security has made it more difficult for users to quickly and easily launch a JWS application. In response, since September 2012 we have distributed the BioTapestry Editor as a downloadable executable that does not require JWS to run. This continues to support the BioTapestry Editor’s primary use as a GRN model-building tool, yet for simple and easy sharing of interactive GRN models on the web, we needed to consider an entirely new approach.\n\nThe first wave of migration away from Java-based browser tools for network visualization applications relied heavily on Adobe Flash. The Flash-based Cytoscape Web platform5, STRING6, and the myGRV component of myGRN7 were examples of this trend. More recently, it has become possible to create rich 2D visualization web applications (software programs which run entirely within a web browser) using Scalable Vector Graphics (SVG) or HTML5 Canvas in combination with JavaScript, Hypertext Markup Language (HTML), and Cascading Style Sheets (CSS).\n\nExisting JavaScript/HTML5/SVG visualization efforts include Google Charts, Protovis8, and D3.js9. The BioJS repository10 is an example of a new resource that has been created to provide a framework for open-source browser components for biological data visualization. Two notable JavaScript libraries specifically targeted at network visualization are Cytoscape.js and Sigma.js. (Although D3.js can be used to visualize networks, it is not focused on that domain.) In particular, the current Version 3 of the Cytoscape Java desktop application11 can export a set of files that can be hosted on a web server to present a network in the web browser using Cytoscape.js.\n\nGiven the growth of the JavaScript/HTML5/SVG visualization platform, graphics-rich biomedical web applications using these technologies have been appearing, such as Regulome Explorer and the Personal Genome Browser12. These same technologies have also made it possible for us to now replace the JWS-based BioTapestry Viewer with a new web application.\n\n\nMethods\n\nEnhanced drawing support. Our experience has shown that creating small tools to help users draw link trees can make network creation more efficient. Two recently introduced drawing tools are illustrated in Figure 3: one tool takes a tree that was drawn with diagonal links and tweaks it to make the segments orthogonal, and the second tool reorganizes the tree to eliminate ambiguous overlapping segments. The orthogonality tool allows the network creator to quickly “rough-in” the path geometry to approximate the desired final overall organization and let the system clean it up. Note that this tool does not do de novo layout of the links, but shifts and splits the existing link segments as needed to make links orthogonal. Since the tool rejects paths that overlap other links and nodes, and does not consider 180 degree turns in the possible solutions, the user may need to add additional link corners for guidance. The overlap elimination tool is designed to clean up incorrectly formed link trees, e.g. trees that have self-crossing or overlapping segments. The latter situation is particularly problematic, since the link tree may visually appear correct, but users clicking on the overlapping segments can get ambiguous results. One advantage of this tool is that users can create well-formed link trees by quickly dragging tree elements around to new, albeit incorrect, configurations and then have the tool clean up the mess, as shown in Figure 3.\n\nThe new orthogonalization tool (here we use Layout->Fix All Non-Orthogonal Segments->Minimize Shifts) takes existing “roughed-in” hyperedge geometry (A) and tweaks it so it is orthogonal (B). The resulting geometry is not generally optimal, since corner points (e.g. those labeled red 1 and 2 in (A) are often retained. Gross features such as the 180-degree turn at red 3 must be provided to guide placement. In (C), the user can quickly drag segments (red arrow 4) and corner points (red arrows 5 and 6). Although these changes create a severely malformed link tree, the overlap tool can repair it, making this approach a quick and easy shortcut for editing tree geometry. Selecting Layout->Clean Up All Overlapping Link Tree Geometry produces the clean result in (D).\n\nVersion 6 also introduced a new automatic layout technique, the overlay-driven layout. This technique leverages BioTapestry’s Network Overlays, which allow the user to create layers that annotate a GRN with Network Modules. Network Modules are collections of boxes that contain sets of related nodes. Users can also draw links between Network Modules to represent how the modules interact. In this fashion, the network creator can illustrate the abstract organization of the network at a high level.\n\nMerging the network overlay feature with BioTapestry’s existing automatic layout feature is a powerful combination, since the user can provide an overall automatic layout organization which is informed by their biological domain knowledge. GRNs can be broken down into biologically relevant sets of nodes, and the network layout will use these groupings to create a meaningful visualization. The layout also uses the module links from the overlay to route the edges between the nodes. An example is shown in Figure 4.\n\nThis example is the end result of the overlay-driven layout use case described below. Using a simulated GRN data set (see Supplemental File 1 - https://f1000researchdata.s3.amazonaws.com/supplementary/7620/671ed832-fd5f-4130-a21e-c94b645b6720.sif), it groups genes into separate network modules. Two modules (upper and center left) contain “control genes”, while all other target genes are separated into modules based on their combination of inputs. Here the overlay intensity level is set near the minimum to de-emphasize the overlay features.\n\nIn order to create this feature, we added a new stacked layout strategy to the existing set of strategies that BioTapestry uses for automatic layout. Each BioTapestry layout strategy is designed to locate nodes and route the link trees in a fashion that avoids ambiguity and separates nodes and links in a rational fashion. For the stacked strategy, the goal was to create a simple rectangular organization containing rows of nodes. The difference between the stacked strategy and other traditional hierarchical layouts (e.g. the Sugiyama-styled layout13) is that links are routed horizontally in reserved tracks above each node row, and traverse vertically between the rows in a shared, dedicated track down the left side of the block. While this canonical approach to link routing is far from the most parsimonious use of “link ink”, it is highly organized and utterly predictable. The stacked strategy also conforms to BioTapestry’s approach of treating genes as first-class citizens, with non-gene nodes preferentially grouped near the genes they are associated with. The network module groups in Figure 4 are all laid out using the stacked strategy.\n\nIn addition to its use as a building block in the overlay-driven layout, the stacked strategy works well for automatic layout of both selected subsets of nodes and single BioTapestry regions, so both of those new features were introduced in Version 6 as well. Regions are used in BioTapestry to represent different developmental domains, or more fundamentally, different regulatory states, in submodels of the model hierarchy. More details are available in 2,3 and the BioTapestry Quickstart Tutorial.\n\nNew architecture: A hybrid desktop/web design. GRN models are most often publicly shared as a set of static images in a journal article, but the BioTapestry Viewer is intended to provide a richer and more interactive way of disseminating the GRN model if the creator chooses to do so. With an online model, users can explore the full model hierarchy, search for the sources and targets of genes, examine alternative paths between genes, and zoom in and out to more closely examine various aspects of the model’s architecture. An online model can also interactively provide experimental data and relevant citations. However, we still characterize the predominant use case for BioTapestry as a researcher using the full-featured BioTapestry Editor as a desktop Java application to create GRN models that are saved as local files on their computer. Given this situation, the redesigned BioTapestry needed to support the new read-only Viewer web application, while also continuing to support the existing Java desktop Editor. One approach is to have the Java application export the network as JavaScript Object Notation (JSON), and then create a standalone browser framework for rendering it; this is similar to the approach currently used by Cytoscape 3 and Cytoscape.js.\n\nHowever, our development roadmap for the browser-based BioTapestry goes well beyond just viewing a completed, published network. Our ultimate goal is to support a full-featured browser-based Editor web application, enabling a distributed research community to collaborate on a shared GRN model using the browser-based tool. Thus, although our first step has been to create a browser-based Viewer, all the architectural decisions made were driven by this long-term goal of creating a browser-based BioTapestry Editor.\n\nThese requirements argued for a heavyweight server-side component driving the web application. We redesigned the BioTapestry architecture so the desktop Editor’s existing Java code base could also be hosted by a Java Servlet which supports a client interface running in an HTML5 web browser. This provides us with a migration pathway that continues to support existing users while allowing us to transition to a fully web-based user interface, all while maintaining as much of a common code base as is practical. The architecture we describe here has been used to produce the new Version 7 BioTapestry Viewer web application, and forms the basis for our work towards building a future BioTapestry Editor web application. This architecture can also serve as a roadmap for other development teams who are contemplating moving from Java Web Start to web applications.\n\nIssues addressed by the new architecture\n\nPer-user application state: Moving the code base from one that supported a single user on the desktop to multiple simultaneous users in a server required us to focus on separating out program state so that it could be stored in a per-session object. In the original single-user code, it was convenient to use Singleton objects14, implemented as static class members, to provide globally accessible resources. However, we removed all these static variables, and now a separate per-session state object is maintained for each user; this session state argument was then added to many method signatures in the application. With the desktop Editor, a single instance of this state object is sufficient, but the web application creates, retrieves, and maintains a unique instance for each separate user session.\n\nFlow of control: The original code base followed standard practice for writing an application built using Java Swing, where commands executed by selecting a menu item were written to extend Swing’s AbstractAction class, with the actionPerformed(ActionEvent e) method being overridden to implement the command. Whenever user input was needed to guide subsequent processing decisions, this method (or a subroutine) would make a call to display a modal dialog. The user’s inputs would become available for further processing once that dialog was dismissed. To move to an architecture where the server does not have to manage a separate thread on a per-session basis, we split these methods into a series of separate functions that maintain their state in the per-session state object. Each function in this series is called to update the current state based on user inputs, and return a request for any inputs needed by the next function in the chain. In the desktop case, these requests are still fulfilled by calling modal dialogs, but in the server case the thread launched for the request completes by returning the request for user information to the web browser.\n\nAs a result of this reorganization, the commands previously implemented as Swing AbstractActions are now implemented as ControlFlows, and we created two separate ControlFlowHarness implementations, which are frameworks to execute these commands from either a Swing desktop or web server context.\n\nDual rendering pipeline: The rendering process had to be abstracted so that the same rendering code could either drive a Java2D rendering layer for the desktop application, or send a description over the wire to a remote client renderer. Originally, the rendering code drew directly to the screen using Java2D commands. The new Version 7 multi-renderer architecture now uses a layer of indirection, and all objects are rendered by first generating a stream of low-level geometric primitive shapes. These low-level shape streams can then be rendered on a variety of different platforms by simply implementing a thin platform-specific rendering layer for each platform. The architecture of our new split pipeline is shown in Figure 5.\n\nThis is the architecture of the rendering pipeline. (A): Each drawing class (e.g. a Gene) knows how to render the final glyph from primitive geometric shapes; additional shapes are also used to render the “selected” version. The classes also encapsulate information needed to perform intersection testing. (B): In the desktop application, the logic for determining point intersections is provided by drawing class functions. In the server application, the rendering pipeline gets a list of intersection boundaries from each drawing class instance, and stores them in the CacheGroup. (C): The concrete CacheGroup implementations have differing interfaces, depending on what needs to be exported to the web client while running as a server application. Shapes are added to the CacheGroups, which are then added to the ModelObjectCache; the concrete type of the ModelObjectCache depends on the target platform, as illustrated. (D): In the server application, the addGroup() method of the VisitableObjectCache stores concrete CacheGroups to an array matching the appropriate DrawLayer. (DrawLayers are used to organize the overall order of what is drawn first to last.) The CacheGroups are stored intact for serialization when the web client requests a model rendering update. The accept() method enables iterating through stored CacheGroups per DrawLayer. Alternately, in the desktop application, the addGroup() method of the ConcreteGraphicsCache extracts the primitive shapes from the CacheGroup and stores the shapes to an array matching the appropriate DrawLayer. The render() method then draws the stored shapes to a Graphics2D object given a DrawLayer. (E): CacheGroup instances are not directly serialized for transmission. Instead, a helper class is used depending on the CacheGroup’s type; that is then serialized to JSON format.\n\nIn the new architecture, the primitives generated for a particular element such as a node are bundled into a CacheGroup object. For the desktop application, these CacheGroups are then used directly to drive the execution of Java2D rendering commands. Alternatively, for the web application, the CacheGroups are used by the model export logic to serialize the data into JSON, which is exported to the web browser for rendering. In addition to the rendering geometry, the exported CacheGroups also contain geometric primitives that, in combination with the network element ID stored in the CacheGroup, are used by the web client to perform mouse-click intersection testing that returns the element ID.\n\nTo reduce the latency of certain operations on the browser, such as clicking on a node so that it displays its orange “selected” highlighting, the server pre-generates certain rendering elements, such as all of these orange selected highlighting shapes, and ships these out in the CacheGroups along with the basic model geometry. The network module shapes that can be used in BioTapestry to annotate the network, which the user can toggle on and off, are also pre-generated for the web application. Both these pre-generating operations are only done for the web application; in the desktop, those shapes are only generated on the fly when they are needed.\n\nFor the web client, we needed to decide which technology to use for our rendering layer. As discussed above, HTML5 Canvas or SVG are the two main options for creating the rendering layer in the web browser, and we chose to use HTML5 Canvas. We based our decision on performance concerns (the Cytoscape.js project reported they decided to move from using SVG to HTML5 Canvas for performance reasons), and on the high degree of similarity between the HTML5 Canvas Application Programming Interface (API) and the Java2D API we already are using in BioTapestry.\n\nWe did encounter some issues while using Canvas. Perhaps the most onerous were font size inconsistencies. Certain nodes (e.g. box nodes) have their size set using the size of the enclosed text, and this calculation occurs on the server. Yet the same point size and font family produces a string of different dimensions on the server and different browser types; even the server operating system was a factor in this mix. We needed to implement logic in the web client renderer to use the HTML5 Canvas text metrics facility to calculate the required affine transform to match the text token dimensions provided by the server. Other problems we encountered were with dashed link rendering, since Internet Explorer 9 does not provide the API for setting dashed line rendering; we needed to implement a workaround using bitmaps. We also encountered some differences in compositing operators, since HTML5 Canvas implementations do not currently support a “clear” rule.\n\nCross-platform specification classes: The original desktop application created user interface (UI) elements, e.g. menus and toolbars, directly using available Swing components. To support a web application deployment, we created abstract, cross-platform descriptors to specify the contents of UI elements. These descriptors can then be used to create a Swing component for the desktop, or be serialized to JSON and sent to the browser, where a corresponding set of JavaScript UI components is created.\n\nCross-platform dialog factories: In the new architecture, dialogs implement a common interface that the web and desktop clients interact with. They are generated by Factory classes which return instances appropriate for the deployment context. On the desktop, the Factory returns a Swing dialog, while the web application context provides the information needed by the web client to construct the dialog, including a description of any information, including user inputs, the web application needs to collect and return to the server. This design approach allows future dialog modifications to take place in a single Java source code file and helps to prevent the divergence of the web and desktop clients.\n\nWeb client implementation\n\nChoosing Dojo: To build the BioTapestry web client, we needed a JavaScript framework that had API documentation that was consistently available and kept up to date, an active community addressing bugs and producing new features, and a quick development time. To inform our decision of which framework to use, we built small BioTapestry prototypes in Ember.js, AngularJS, and the Dojo Toolkit. Based on our criteria and the experience of building these prototypes, including time to learn the framework’s API, the Dojo Toolkit covered all of our needs. It comes with a large library of user interface widgets that incorporate accessibility standards and manage browser/OS differences via a single API, all of which can be extended to perform as required. Dojo modules can also be loaded asynchronously on an as-needed basis [Asynchronous Module Definition (AMD)]. This ensures the web application will only use those network and client resources that are required as defined by how the user makes use of the application. The result is shown in Figure 6; compare this representation to the Java desktop Editor version in Figure 1.\n\nThe early endoderm specification GRN for Zebrafish4 as it appears in Google Chrome v47, Windows 7, served by Apache Tomcat version 7.0.55. Compare to desktop Editor version in Figure 1.\n\nVirtual scrolling system: Users can pan, scroll, and zoom GRN models in the network view panel. With the Java2D desktop implementation there is no severe performance penalty for using a JPanel matching the size of the whole model at the specified zoom level, which can often be quite large. However, this becomes a problem in the web client, because Canvas elements in the browser use a significant amount of memory at larger sizes, enough that a very large Canvas element can cause the browser to malfunction or crash. To address this problem, in the web client we implemented a ‘virtual’ scrolling and zooming system that restricts the Canvas element’s size to the drawing viewport’s display area. Panning, zooming, and scrolling events are handled as drawing functions, rather than using the native browser functions. This keeps the Canvas using the minimum amount of memory required while providing the same zooming, scrolling, and panning functionality users would expect.\n\n\nOperation\n\nThe Java Runtime Environment (JRE) Version 1.5 or newer is required to use the Java desktop Editor. JRE 1.5 and Apache Tomcat Version 6 or 7 are required to host a model on a web server. Chrome 4.0+, Firefox 3.5+, Safari 4.0+, and IE 9.0+ with JavaScript enabled are required to use the web application client. Any operating system that supports these technologies can be used to host a model, view it in the web client, or run the desktop Editor.\n\nBecause we redesigned BioTapestry in Version 7 in a manner that allows the traditional desktop Editor to continue to operate as it always has, there is no change to the way a user would create a GRN model using BioTapestry. The online “Quickstart” tutorial is still the best way to learn how to use the tool, and existing additional online documentation shows how to use other more advanced features.\n\nSharing a GRN model over the web using the new Viewer web application is significantly different than previous methods. The BioTapestry Viewer web client is distributed as a Java Web Application Archive (WAR) file that contains the BioTapestry Version 7 Viewer clients in both Java and JavaScript, and all required libraries (flexjson 2.1, the Dojo Toolkit 1.10, underscore.js, put-selector, dgrid, and xstyle). The WAR file can be placed in an active Apache Tomcat Web Application deployment directory (the server default is $CATALINA_HOME/webapps/), and by default Tomcat will automatically unpack the WAR file and deploy the application into a folder that matches the WAR file’s name. This will also dictate the URL at which the BioTapestry web application is available. For example, on a locally running instance of Apache Tomcat, which is accessible on port 8080 by default, BioTapestry.war will deploy to http://localhost:8080/BioTapestry/.\n\nThe base BioTapestry web application distribution comes with a plain GRN model for testing the application’s deployment. There are two methods for loading a new GRN model file into your deployment: editing the already deployed application directly, or, editing the contents of the WAR file and (re)deploying it. For an active BioTapestry web application, place the new GRN model file into the WEB-INF/data/ folder of the deployed web application and edit the modelfile entry of the configuration.txt file to reflect the name of the new file, then reload the web application from the Apache Tomcat Web Application Manager. To edit the BioTapestry web application WAR file itself, use Oracle’s jar utility, or a compression utility capable of working with ZIP format archives. Make the same changes as you would to a live deployed BioTapestry web application, and then redeploy the WAR file.\n\nDetailed instructions for working with the web application, including installing and customizing it, are available from the project web site.\n\n\nUse cases\n\nAs an example of how to use the overlay-driven layout, we want to create a layout where the “control” genes with outputs are broken out separately, and “target” genes with only inputs are grouped into blocks based upon common sets of inputs. This allows us to clearly see the control circuitry, while providing a useful way to break the large number of targets into similar groups. To do this:\n\n1) Import a tab-delimited Cytoscape .sif file using BioTapestry’s File->Import->Import Root Network From SIF... command; this particular example uses a simulated GRN data set (see Supplemental File 1 - https://f1000researchdata.s3.amazonaws.com/supplementary/7620/671ed832-fd5f-4130-a21e-c94b645b6720.sif). Using a stacked strategy, keep the defaults except for setting the maximum row size to 20 and the target grouping strategy to Order Targets by Source. The resulting network is shown in Figure 7, and illustrates how the new stacked strategy organizes a full network.\n\n2) Using the command Edit->Manage Network Overlays and Modules->Add Network Overlay…, create an overlay with a presentation style of Transparent and named e.g. “Target Groups”.\n\n3) Drag the genes that will go into different modules around so each set can easily be contained in a bounding box. Then, using Edit->Manage Network Overlays and Modules->Draw a New Network Module…, draw a One Box type module around each group of nodes (the other module types do not support the overlay-driven layout). Modules boxes are drawn by clicking to start the box, and then clicking to end it.\n\n4) Drag each module to the desired location by right-clicking on the module name or boundary and selecting Move Module. With the One Box type module, dragging the module name or the genes to reposition them will cause the module boundaries to expand if needed to still enclose all module contents. For best auto-layout results if the module boundaries have been automatically expanded in this way, right-click on the module name or boundary and select Resize Core Module Definition to Current Visible Bounds if that option is enabled.\n\n5) Using Edit->Manage Network Overlays and Modules->Draw a New Network Module Link…, draw orthogonal Promote module links trees between all the modules that have interactions. See Figure 8. (Note: the link trees here have been reduced from the original arrangement in Figure 7 for clarity, but this is not necessary.)\n\n6) Select Layout->Apply Auto Layouts->Per Current Overlay. If there are problems with the overlay (e.g. a required link between interacting modules is missing, or a module link is crooked), an error dialog will appear; these problems must be fixed before trying again. When the layout is complete, the default settings create the final result that was shown above in Figure 4. Figure 4 has the overlay Intensity level dialed down to near the minimum to better show the inter-module links.\n\nNote that the module boxes are enlarged as needed to contain the stacked layouts, since the system tries to maintain the relative horizontal and vertical orderings of the module features (i.e. edges and link positions). Thus, some module boxes may become larger than necessary, and manual tweaking may improve the aesthetics. It also helps to draw the original overlay with this ordering in mind. Finally, the modules are not resized to best position the module label, which often needs to be relocated for best results.\n\nImporting a network from a tab-delimited .sif file, and using the stacked automatic layout strategy, produces the result shown here. This shows how this new layout strategy can be applied to the whole network.\n\nAfter the user creates a network overlay, and drags genes into piles, they draw a network module around each. This action automatically adds the genes to the modules. Then, after moving each module to the desired location, the user draws directed network module links between interacting modules. The next step will be to apply the overlay-driven layout algorithm.\n\nMTB Network Portal. The first deployment of the BioTapestry Viewer web application was an interactive network model for the Mycobacterium tuberculosis (MTB) Network Portal15. This portal provides resources for computational modeling of host/pathogen interactions in Mycobacterium tuberculosis, and the BioTapestry web client handles the Environment-specific Gene Regulatory Network. This deployment of the web client needed to support experimental data hosted on external servers and sourced from web links embedded in the BioTapestry file. Thanks to BioTapestry’s implementation of an experimental data display plugin API, we were able to produce a custom Java plugin to retrieve this data and display it when users open experimental data pages.\n\nEctoderm/Endomesoderm Networks. The BioTapestry Java Web Start Viewer was originally developed to host an interactive model of the GRN controlling the first 30 hours of development of the endomesoderm in Strongylocentrotus purpuratus16. This model has now been completely ported to the new Viewer platform. Additionally, the recently added GRN model for ectoderm development, based upon 17–19, is hosted using our new BioTapestry Viewer as well.\n\nNew BioTapestry model repository. In order to provide access to other GRN models produced with BioTapestry using the new Viewer platform, and to provide a hosting site for models that were previously available only via Java Web Start, we have created a model repository. This site hosts some previously published models4,19–25, including some models that were “orphaned” when their original websites were shut down. It is our hope that this repository will continue to grow and serve as a reliable method for accessing GRN models via a web browser. The site provides a “Quickstart” user guide, complete with screenshots, to orient new users to the BioTapestry web client's functionality.\n\n\nConclusions\n\nAs GRN models continue to grow larger and more complex, we will continue to add features to BioTapestry that will aid researchers in building them. The layout tools we have described here are the most recent examples of such improvements. Our new BioTapestry Viewer web application ensures that users will continue to have access to dynamic, interactive GRN models online. Since this new architecture provides a richer server/browser interaction that only downloads data to the client when it is requested, we expect the new system to be better at handling very large and complex model hierarchies as GRN models grow in the future.\n\nWe are continuing to refactor the Java desktop Editor to use the new architecture, and plan to implement a web application of the Editor that can be used by research communities to collaborate on GRN models online. Given the cross-platform nature of our design, we will be able to do this while continuing to fully support the BioTapestry Java desktop Editor. Our GRN website will foster community involvement with GRN models as we continue to build it out. The site’s current compliment of models has amply demonstrated how the new web application Viewer can support interactive exploration of GRN models.\n\n\nSoftware availability\n\nhttps://github.com/BioTapestry\n\nhttp://dx.doi.org/10.5281/zenodo.3544726\n\nhttp://dx.doi.org/10.5281/zenodo.3566427\n\nBioTapestry source code (Java, JavaScript, HTML, CSS): GNU Lesser General Public License (LGPL) V 2.1. Some of the toolbar image files are freely distributed under a separate license from Sun Microsystems, now Oracle. Other libraries are also used in the server and client. The Dojo Toolkit, dgrid, xstyle, and put-selector are distributed under a Modified Berkeley Software Distribution (BSD) License. Flexjson is distributed under Apache License Version 2.0, and underscore.js is distributed under an MIT License.",
"appendix": "Author contributions\n\n\n\nSMP developed the web client, and contributed to the manuscript. KL developed the Canvas/Java2D rendering implementation, and contributed to the manuscript. WJRL conceived of and led the project, developed the server-side architecture, wrote much of the server/desktop Java code, and contributed to the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe work described here was supported by the National Institute of General Medical Sciences under Award Number R01GM061005, (Eric Davidson, PI) and by the Eunice Kennedy Shriver National Institute Of Child Health & Human Development of the National Institutes of Health under Award Number R01HD073113, (W.J.R. Longabaugh, PI). This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors are grateful to Dr. Hamid Bolouri and the late Prof. Eric H. Davidson for their vision in initiating the BioTapestry project.\n\n\nSupplementary materials\n\nSupplemental File 1.\n\nThis is the mockup GRN definition file referenced in the overlay-driven use case. The file is in tab-delimited Cytoscape Simple Interaction Format (SIF), i.e. of the form source node<tab>interaction label<tab>target node. Networks in this format can be imported into a number of network visualization programs, such as Cytoscape, BioTapestry, and BioFabric.\n\nClick here to access the data.\n\n\nReferences\n\nPeter IS, Davidson EH: Genomic Control Process: Development and Evolution. Amsterdam: Academic Press; 2015. Publisher Full Text\n\nLongabaugh WJ, Davidson EH, Bolouri H: Computational representation of developmental genetic regulatory networks. Dev Biol. 2005; 283(1): 1–16. PubMed Abstract | Publisher Full Text\n\nLongabaugh WJ, Davidson EH, Bolouri H: Visualization, documentation, analysis, and communication of large-scale gene regulatory networks. Biochim Biophys Acta. 2009; 1789(4): 363–374. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChan TM, Longabaugh W, Bolouri H, et al.: Developmental gene regulatory networks in the zebrafish embryo. Biochim Biophys Acta. 2009; 1789(4): 279–298. PubMed Abstract | Publisher Full Text\n\nLopes CT, Franz M, Kazi F, et al.: Cytoscape Web: an interactive web-based network browser. Bioinformatics. 2010; 26(18): 2347–2348. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFranceschini A, Szklarczyk D, Frankild S, et al.: STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013; 41(Database issue): D808–D815. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBacha J, Brodie JS, Loose MW: myGRN: a database and visualisation system for the storage and analysis of developmental genetic regulatory networks. BMC Dev Biol. 2009; 9: 33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBostock M, Heer J: Protovis: a graphical toolkit for visualization. IEEE Trans Vis Comput Graph. 2009; 15(6): 1121–1128. PubMed Abstract | Publisher Full Text\n\nBostock M, Ogievetsky V, Heer J: D3: Data-Driven Documents. IEEE Trans Vis Comput Graph. 2011; 17(12): 2301–2309. PubMed Abstract | Publisher Full Text\n\nGómez J, García LJ, Salazar GA, et al.: BioJS: an open source JavaScript framework for biological data visualization. Bioinformatics. 2013; 29(8): 1103–1104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJuan L, Teng M, Zang T, et al.: The personal genome browser: visualizing functions of genetic variants. Nucleic Acids Res. 2014; 42(Web Server issue): W192–197. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSugiyama K, Tagawa S, Toda M: Methods for visual understanding of hierarchical system structures. IEEE T Syst Man Cyb. 1981; SMC-11(2): 109–125. Publisher Full Text\n\nGamma E, Helm R, Johnson R, et al.: Design Patterns: Elements of Reusable Object Oriented Software. Reading MA: Addison-Wesley; 1995. Reference Source\n\nPeterson EJ, Reiss DJ, Turkarslan S, et al.: A high-resolution network model for global gene regulation in Mycobacterium tuberculosis. Nucleic Acids Res. 2014; 42(18): 11291–11303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavidson EH, Rast JP, Oliveri P, et al.: A genomic regulatory network for development. Science. 2002; 295(5560): 1669–1678. PubMed Abstract | Publisher Full Text\n\nLi E, Cui M, Peter IS, et al.: Encoding regulatory state boundaries in the pregastrular oral ectoderm of the sea urchin embryo. Proc Natl Acad Sci U S A. 2014; 111(10): E906–E913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBen-Tabou de-Leon S, Su YH, Lin KT, et al.: Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs, and cell fate lockdown. Dev Biol. 2013; 374(1): 245–254. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCui M, Siriwon N, Li E, et al.: Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo. Proc Natl Acad Sci U S A. 2014; 111(47): E5029–E5038. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRabinowitz AH, Vokes SA: Integration of the transcriptional networks regulating limb morphogenesis. Dev Biol. 2012; 368(2): 165–180. PubMed Abstract | Publisher Full Text\n\nVokes SA, Ji H, McCuine S, et al.: Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning. Development. 2007; 134(10): 1977–1989. PubMed Abstract | Publisher Full Text\n\nPaul L, Wang SH, Manivannan SN, et al.: Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila. Proc Natl Acad Sci U S A. 2013; 110(13): 5058–5063. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBonneau R, Facciotti MT, Reiss DJ, et al.: A predictive model for transcriptional control of physiology in a free living cell. Cell. 2007; 131(7): 1354–1365. PubMed Abstract | Publisher Full Text\n\nGeorgescu C, Longabaugh WJ, Scripture-Adams DD, et al.: A gene regulatory network armature for T lymphocyte specification. Proc Natl Acad Sci U S A. 2008; 105(51): 20100–20105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKueh HY, Rothenberg EV: Regulatory gene network circuits underlying T cell development from multipotent progenitors. Wiley Interdiscip Rev Syst Biol Med. 2012; 4(1): 79–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLongabaugh WJ, Paquette SM: WebApplication: BioTapestry now provides a web application and improved drawing and layout tools. Zenodo. 2015. Data Source\n\nPaquette SM, Leinonen K, Longabaugh WJ: BioTapestry/Production. Zenodo. 2015. Data Source"
}
|
[
{
"id": "11875",
"date": "18 Jan 2016",
"name": "Matthew Loose",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBioTapestry is the pre-eminent viewer for building and analysing developmental gene regulatory networks. Although many other GRN viewers are available, very few provide the ability to link developmental processes and the concepts of tissues and cell types emerging over time with the underlying GRN. BioTapestry was the first to really formalise this process (I believe building on the work of Schilstra and colleagues with NetBuilder) and provide a method of displaying GRNs incorporating the logic first proposed by Eric Davidson. The ongoing development and maintenance of tools like BioTapestry is very important to the understanding of GRNs in development. This manuscript is clearly written, accurately described and focuses on key improvements made in BioTapestry versions 6 and 7. I approve of the migration to a more web centric view for BioTapestry future development and look forward to the ability to edit GRNs in a web based environment. The focus on layout tools (the 'hyper edges' are an example) is extremely useful for cleaning up a visualisation. The network overlay-driven layout features seem to work well for clustering groups of genes in perhaps biologically meaningful ways.The option to right click on a network link to visualise all the data behind that particular interaction is very helpful indeed.The source code for BioTapestry is appropriately available from github and the documentation accompanying its release is clear and easy to follow.One possible problem with BioTapestry is with respect to its deployment. The BioTapestry target audience is \"...a researcher using the full-featured BioTapestry Editor as a desktop Java application to create GRN models that are saved as local files on their computer.\" (Page 6, Paragraph 1). This user will also tend to be a wet lab experimentalist collating data from direct observation as well as pooling other sources of knowledge, be that from the literature or - perhaps - other bioinformatics pipelines. The process by which such an individual shares their network becomes complex. The user is expected to set up an Apache Tomcat installation and configure the package. At the same time, the authors present a model repository (Page 12, Para 3). These concepts should perhaps be linked. It would be great if users could submit models quickly and easily to the model repository which - itself - could be searched by others or a direct link to it could be shared with others. Of course, it may not be a single experimentalist working on the model either - perhaps there is a larger team at work who all wish to work on the model at once.Perhaps a hinderance to this process at the moment is that it appears each model visualised via the web viewer requires its own full install of the BioTapestry WAR file. A possible future improvement to the deployment method might be to pass the appropriate configuration files to the BioTapestry package at launch. This could then simplify the process of hosting a GRN model server visualised in BioTapestry.A minor comment that might improve usability is with respect to the search features for nodes(genes) of interest. The default behaviour currently is \"Match Full Name\". This usually failed for me when browsing the endomesoderm network (as an example) whereas switching to \"Match part of name\" found the required gene or pathway (nice that it highlights pathway members in the case of signalling molecules!) straight away. An auto suggest function or simply switching the viewer to defaulting to \"Match part of name\" would increase usability.A final broader comment with respect to the viewer. As far as I can see there is no way to export a model file (either .sif or any other format) from a network model. Whilst BioTapestry is the clearest and easiest way to draw developmental GRNs, many other tools exist for the analysis of networks. To have an easy way to export a collection of interactions from a BioTapestry web view would be helpful. This would enable users to analyse a network they were viewing in other tools if they so wished without having to redraw or otherwise reconstruct the network.Overall I thank the authors for their continued work on BioTapestry - a tool which is incredibly useful for all those interested in the study of developmental GRNs.",
"responses": [
{
"c_id": "1776",
"date": "22 Jan 2016",
"name": "William Longabaugh",
"role": "Author Response",
"response": "Dear Prof. Loose:Thank you very much for taking the time to review our paper. I believe that your review contains four excellent points about current BioTapestry shortcomings, to wit:1) Current model deployment is too complex:We agree completely that the requirements for publishing an online model need to be greatly simplified. Our new model repository is a small, first step in this direction. Our current BioTapestry development proposal (for post Version 8, which is scheduled for 2016 release) is to significantly enhance grns.BioTapestry.org to allow models to be easily uploaded, searched, and shared. Combined with the online editor we are currently developing, it will make collaboration by distributed groups of researchers possible.2) Current requirement for full WAR install:The current requirement of running a separate servlet for each model is indeed far too restrictive. Your suggestion to make multiple models available from a single WAR deployment is spot on. This is also on our development roadmap, and is dependent on us completing a detailed thread-safety analysis followed by refactoring of the main control flow to improve parallelism of the servlet. Running multiple servlets is currently our workaround to improve overall concurrency.3) \"Match full name\" as current search default:Absolutely true; thanks for pointing this out. We plan to include this suggestion in our upcoming 7.0.1 bug-fix release.4) No way to export network as, e.g., a SIF file:The lack of a download/export option in Version 7 can trace its roots back to the original no-permissions, security-sandboxed Java WebStart implementation. You are absolutely correct that since we are now operating in a browser, those restrictions are removed, and we need to open up our thinking! It should be possible to provide this feature in the upcoming Version 8. But this feature is likely to be specified on a per-model basis, since we have found some labs have preferred to provide the underlying model files through more traditional one-on-one email correspondence.Finally, though this paper did not dwell on the origins of BioTapestry [Longabaugh, Davidson & Bolouri, Dev Biol. 283(1), 2005], you are absolutely correct that it was a follow-on to the original NetBuilder project [Schilstra, M.J., Bolouri, H.: Logical Modeling of Developmental Genetic Regulatory Networks with NetBuilder [abstract]. In: Proceedings of the Second International Conference on Systems Biology (ICSB2001); 2001 Nov 4-7, Pasadena CA, abstract 112. Available at http://icsb2001.net/Posters/112_schilstra.pdf; current project home page is http://homepages.stca.herts.ac.uk/~erdqmjs/NetBuilder%20home/NetBuilder/]. Thanks for pointing that out.Bill Longabaugh"
}
]
},
{
"id": "12950",
"date": "29 Mar 2016",
"name": "Tanja Muetze",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 version 6 and 7 of BioTapestry in which they introduced improved network layout and drawing capabilities and added an in-browser JavaScript web editor functionality to BioTapestry as a hybrid between desktop and web app. BioTapestry aims to visualize and analyze gene regulatory networks and make it easy for users to share them. The article is clearly written and has a good flow, despite being occasionally a bit informal. The article follows a clear structure with a descriptive title as well as a well-described abstract and conclusion that summarize the work. It links to documentation/tutorials as well as the code. As another referee noted, the authors followed a recent trend for more web security by using a HTML Canvas, CSS and JavaScript approach instead of deploying the app via Java Web Start which runs Java code as previously done, a design decision that I support. The following suggestions could further improve the article.The proposed software was created about two years ago. I would encourage a timelier publication of this article to promote awareness of the software as well as to be a resource and reference guide for users. On similar terms, the Github code repository seems to be a bit outdated. The last commit was 2 years ago. Consider how version updates of dependent software might impact BioTapestry despite version backward compatibility. Currently, Java 8 is distributed while this update to BioTapestry was initially written for Java 5. Integration with other (network) tools, such as Gephi, would be desirable. The website mentions that export to SBML and SIF (for Cytoscape) are currently supported. It might be of interest to open a voting on the website (e.g. based on IP address and/or browser session to discourage multiple votes) to find out which import/export capabilities are most desired by most users. This would provide a fluent integration of BioTapestry with other bioinformatics, network or general software and would be an incentive for users of other programs to start using BioTapestry. More on a side note, while being very descriptive and conversational, the article could have been more concise and separated future development from current design choices. Overall, the authors did a very good job at developing and clearly presenting an update to BioTapestry, an important tool for gene regulatory network analysis and distribution.",
"responses": [
{
"c_id": "1933",
"date": "25 Apr 2016",
"name": "William Longabaugh",
"role": "Reader Comment",
"response": "Thank you very much for taking the time to review our paper and provide feedback.We agree that more frequent publications would help to promote awareness of BioTapestry! This is our first time publishing an F1000Research Software Tool article, and it looks like a promising venue for perhaps more timely future submissions. With GitHub, since our user base is virtually 100% non-developers, and our small developer team is all in-house, we have been able to follow a more \"traditional\" open-source model of publicly releasing the source code with each new version every 12-18 months. But we would be happy to provide a development branch if we gain external development collaborators.It's true that BioTapestry only requires Java 5, as it has always been a project goal to make sure the user does not have to worrying about upgrading their Java installation to get BioTapestry running. Since the only dependency for the desktop BioTapestry Editor has been Java itself, we have fortunately only had rare instances (e.g. Apple Java release 1.6.0_15) where Java upgrades have briefly broken things. But you are correct that with our new web application, and a more complex software stack, we may run into more dependency issues that would benefit from more frequent source code releases. Of course, always plan to respond to any critical bugs arising from e.g. faulty backward compatibility with a point release and refreshed public code repositories.Triggered by your observation, we checked the latest platform usage statistics and note that it appears that Java 5 has been completely flushed out of the worldwide installed base, with Java 6 now also close to disappearing. So we are considering bumping the minimum requirement to Java 6. Yet we always work to make sure we do not depend upon any recent Java language or platform features to make BioTapestry run well.The question of integration with other network tools (e.g. Gephi) remains a challenging one given BioTapestry's unique enforced model hierarchy framework. This means that while the user can import network definitions from other tools into BioTapestry's root-level \"Full Genome\" model, or export a single BioTapestry submodel, populating a complete BioTapestry model hierarchy from another network tool can be a complex procedure. There is also the issue of needing to map generic imported nodes onto BioTapestry's specific node types (e.g. a gene, signal, or protein bubble node). Ultimately, the nuances involved in importing networks to BioTapestry has typically involved building custom scripts on a case-by-case basis that can create a BioTapestry comma separated value (CSV) import file (see http://www.biotapestry.org/tutorials/csv/CSVTutorial.html). Our current development road map includes a plan to make this CSV import feature much more capable.On the plus side, for the simple \"flat\" network import case, we did slightly upgrade the existing Cytoscape .sif file input feature in Version 7, so that the link descriptor field can now be used to specify link signs using the \"pos\", \"neg\", and \"neu\" keywords. But it remains the case that with .sif files, all node imports are still mapped to gene nodes. For continuing to improve our compatibility with other tools, your suggestion of providing an online poll for getting input on what input files the user community would like to see is a good one, and we will explore that option.Finally, it's true that our treatment may have made it difficult to parse apart our discussion about current design choices versus future development issues. Much of our Version 7 redesign work was directed towards getting ready for those next steps, so the two have tended to blend together in our thinking.Again, thanks for your helpful comments!"
}
]
},
{
"id": "12952",
"date": "01 Apr 2016",
"name": "Morris Maduro",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGene Regulatory Networks describe the causal relationships, in space and time, among transcription factors and signaling events that drive processes under genetic control. The most frequent application of GRNs is to describe developmental processes, such as the specification of germ layer fates in the sea urchin embryo.This article describes the general functionality of BioTapestry, in use since 2005, and its usefulness in describing the dynamic architecture of gene regulatory networks. In particular, improvements the tools used for drawing the connectivity in a network are described. The drawing tools are useful and intuitive and the authors justify the use of a \"stacked layout strategy\" to break down a large network into sets of functionally important nodes. Changes to the software are detailed as they apply to versions 6 and 7 of BioTapestry. Improvements include updating the software to run in a browser. For specific details about usage and installation, users are referred to online tutorials. By way of example, instructions are included for using the 'network overlay-driven' layout.Migrating to a newer, interactive web-based platform presents itself with a number of logistical issues which are described in the article in detail, with a view toward eventually migrating BioTapestry to a web-based, collaborative platform. Logistical issues include the separation of functions to the server or the user's desktop, and cross-platform consistency in font and line rendering.The text itself is well-written, and contains active links to many applications in context.Comments:The simulated GRN set is useful for demonstrating the modeling diagrams, but it could be made much simpler if it had fewer downstream targets, i.e. to simplify Figs. 4, 7, 8. Some parts have a bit too much detail regarding how the authors dealt with specific problems, but these are not too distracting. The Microsoft browser is now Edge. Edge seems to support the web-based viewer for the networks available via http://grns.biotapestry.org. This could be indicated in the first paragraph of the 'Operation' section. As a final comment, while the authors consider evolving web technologies, they may also consider creating an \"App\" that can be installed on tablets and portable devices. This might be easiest to develop for the Android platform as this uses Java.The authors have presented a very nice article that informs readers of updates to BioTapestry, which continues to be a terrific tool for visualization of gene network interactions.",
"responses": [
{
"c_id": "1934",
"date": "25 Apr 2016",
"name": "William Longabaugh",
"role": "Reader Comment",
"response": "Thanks for taking the time to review our paper and provide feedback.Your point that Figures 4, 7, and 8 could have been made simpler with fewer downstream targets is right on the mark. Though our paper did not dwell on it, one of the big improvements we made to Version 7 was to make the automatic layout engine much more efficient, so it can reasonably handle much larger networks (e.g. thousands of nodes). The example was built with an eye towards also illustrating that BioTapestry can be used to advantage for larger networks, but you are right that this ancillary goal burdened the example with too much complexity.Also, you are right that we occasionally go into the weeds in some parts. It was also our original intent to make the manuscript a comprehensive guide for bioinformatics software developers who also need to make the jump from a Java application to a web-based browser version. But when it became clear that the result was far too long and technical, we chose to trim it down to the version you see here, yet some of that in-depth detail still snuck through. Thanks for making sure that our web application works with Microsoft's new Edge browser. Making sure that a web application runs correctly on all the popular web browsers is an ongoing challenge, and the introduction of Edge will add to that complexity as long as Internet Explorer remains in the installed base as well. We will make sure it gets tested as well in future releases.Finally, thanks for suggesting we create an App for tablets! Though Android uses Java, the user interface (UI) library we use for the desktop version (Java Swing) is not part of Android. But this is a situation where our Version 7 redesign, where we abstracted away the UI from the rest of the code, can help us immensely. After we complete moving the Editor to our new design in order to create the web version, it should be much easier to then support an alternative tablet UI for the Editor as well.Thanks again. We appreciate your comments!"
}
]
}
] | 1
|
https://f1000research.com/articles/5-39
|
https://f1000research.com/articles/5-38/v1
|
08 Jan 16
|
{
"type": "Research Note",
"title": "Rituximab efficiently depletes B cells in lung tumors and normal lung tissue",
"authors": [
"Albane Joly-Battaglini",
"Clara Hammarström",
"Branislava Stankovic",
"Henrik Aamodt",
"Johan Stjärne",
"Odd Terje Brustugun",
"Åslaug Helland",
"Inger Øynebråten",
"Alexandre Corthay",
"Albane Joly-Battaglini",
"Clara Hammarström",
"Branislava Stankovic",
"Henrik Aamodt",
"Johan Stjärne",
"Odd Terje Brustugun",
"Åslaug Helland",
"Inger Øynebråten"
],
"abstract": "Rituximab is a monoclonal antibody that targets the CD20 B-cell-specific antigen and is widely used as therapy for B-cell lymphoma. Since rituximab depletes both malignant and normal B cells, it is increasingly being used to treat various conditions in which normal B cells have a pathogenic role, such as rheumatoid arthritis and multiple sclerosis. It is well-established that rituximab efficiently eliminates B cells in blood, lymph nodes, and spleen. In contrast, the effect of rituximab in non-lymphoid tissues remains poorly documented and is debated. Here, we report a rheumatoid arthritis patient who was treated with rituximab before receiving thoracic surgery for non-small cell lung cancer. Using flow cytometry and immunohistochemistry, we show that rituximab efficiently depleted CD20-positive B cells in a primary lung tumor, in lung-associated lymph nodes, and in normal lung tissue. We conclude that rituximab may be very efficient at depleting normal B cells in the lungs. This property of rituximab may potentially be exploited for the treatment of conditions in which pathogenic B cells reside in the lungs. On the other hand, the clearance of lung B cells may provide an explanation for the rare cases of severe non-infectious pulmonary toxicity of rituximab.",
"keywords": [
"rituximab",
"B cells",
"depletion",
"monoclonal antibody",
"lungs",
"tumor",
"lymph node",
"non-small cell lung cancer"
],
"content": "Introduction\n\nRituximab was the first monoclonal antibody to be approved for the treatment of cancer and it is estimated that >4 million people have been treated with rituximab worldwide1. Rituximab is a depleting chimeric anti-CD20 monoclonal antibody routinely used for the treatment of B-cell lymphoma2–4. The B cell-specific antigen CD20 is expressed on all normal B cells, except for early B cell precursors and antibody-secreting plasma cells, and by nearly all B-cell lymphomas. Since rituximab depletes both malignant and normal B cells, its use has been extended to non-cancerous conditions in which normal B cells are believed to play a central role in pathogenesis. Significant clinical benefits have been reported for the treatment of autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, vasculitis, Sjögren’s syndrome, and scleroderma5–9. The mechanism whereby rituximab depletes B cells is not fully understood but there is evidence for complement-dependent cell lysis and for antibody-dependent cellular cytotoxicity2,10,11. It has been shown that rituximab efficiently eliminates normal and malignant B cells in blood and in lymphoid organs such as lymph nodes, spleen, and bone marrow12–14. In contrast, the effect in non-lymphoid tissues remains poorly documented. Here, we report the effect of rituximab in the lungs of a patient who was treated with rituximab because of rheumatoid arthritis before receiving thoracic surgery for non-small cell lung cancer.\n\n\nMethods\n\nThe Regional Committee for Medical and Health Research Ethics (Oslo, Norway) has approved the study (permit number: REK S-05307). Written informed consent for publication of the clinical details was obtained from all patients included in the study.\n\nPatient blood was sampled from a central venous catheter before the start of surgery and collected into ethylenediaminetetraacetic acid (EDTA)-containing tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using a gradient (Lymphoprep, Axis-Shield, Oslo, Norway; cat. no. 1114544). Fresh biopsies from the tumor, a lung-associated lymph node, and normal lung tissue, were sampled under sterile conditions in the operating room, after the removal of the lung lobe by the surgeon. Samples were treated enzymatically with 2 mg/ml collagenase A and 50 units/ml DNase (both from Roche, Basel, Switzerland; collagenase A, cat. no. 10103586001; DNase, cat. no. 11284932001) and incubated for 1 h on a magnet stirrer at 37°C. Single-cell suspension was obtained by squeezing the dissolved tissue through a 100 μm mesh and centrifuging at 300g for 7 min. Nonspecific binding was blocked by incubation with 12.5 μg/ml IgG purified from pooled mouse sera (Sigma-Aldrich, St. Louis, Missouri, USA; cat. no. I8765). Cells were stained in a 96-well plate for 20 min on ice with fluorochrome-labeled monoclonal antibodies diluted 1:10 in phosphate-buffered saline (Sigma-Aldrich, cat. no. D8537) supplemented with 10% foetal bovine serum (Sigma-Aldrich cat. no. F7524). The following monoclonal antibodies were used (all from BioLegend, San Diego, California, USA): anti-CD3 (clone UCHT1, cat. no. 300415); anti-CD4 (clone OKT4, cat. no. 317409); anti-CD8 (clone SK1, cat. no. 344713); anti-CD14 (clone HCD14, cat. no. 325617); anti-CD19 (clone HIB19, cat. no. 302227); anti-CD45 (clone HI30, cat. no. 304029); anti-HLA-DR (clone L243, cat. no. 307610). Stained cells were analyzed with a BD LSRFortessaTM Cell Analyzer instrument (BD Biosciences, Franklin Lakes, New Jersey, USA, model no. 647794E6) and FlowJo software version 10 (FlowJo, Ashland, Oregon, USA).\n\nFor light microscopy, 4 μm thick sections from formalin-fixed paraffin-embedded tissue were automatically stained with hematoxylin and eosin in a Sakura Tissue-Tek Prisma instrument (Sakura Finetek, Torrance, California, USA). The immunostainings were done on a Dako Autostainer instrument (Dako, Agilent Technologies, Santa Clara, California, USA, model Link 48), and the incubation time for the primary antibodies was 20 min. CD3 was immunostained by clone SP7 (a monoclonal rabbit antibody, diluted 1:150; Thermo Scientific, Waltham, Massachusetts, USA; cat. no. RM-9107), and CD20 was immunostained by clone L26 (a mouse IgG2a antibody, diluted 1:600; Dako, cat. no. M0755). The secondary detection was performed with Dako EnVisionTM Flex (Dako, cat. no. K8000) for 20 min, followed by diaminobenzidine (DAB) staining for 10 min. The slides were thereafter treated with CuSO4 for 5 min before contrastaining with hematoxylin. Samples were examined with a Nikon Eclipse model Ni-U microscope (Nikon, Tokyo, Japan) equipped with Nikon Plan-Fluor objective lenses (2×, 20×, and 40×) and images were taken with an Infinity 2 digital camera (Lumenera Corporation, Nepean, Ontario, Canada).\n\n\nResults\n\nA 62-year-old woman with seronegative rheumatoid arthritis was diagnosed in 2015 with lung adenocarcinoma, stage IIB (pT3N0Mx, TNM 7th edition). The patient, a former heavy-smoker with a smoking history of 30 pack-years, underwent right lower lobectomy. The patient had been treated with Prednisolone (usually 5 mg daily since 2005), as well as several different drugs (Methotrexate 10 mg/week for 3 weeks in 2005, Metoject 1×10 mg in 2005 and 2×10mg in 2007, Plaquenil 400 mg/day in January-February 2006, Arava 10 mg/day for 8 days in 2006, and Enbrel 50 mg/week from January 2008 to April 2009), all discontinued due to side-effects or inefficiency. Over the past six years before lung cancer diagnosis (2009–2014), the patient received seven cycles of rituximab (MabThera, 6 cycles of 2×1000mg and 1 cycle of 2×500mg) with only moderate clinical effect. Serum immunoglobulin (Ig) levels were normal before initiation of the rituximab treatment (IgG=6.1g/L; IgA=1.3g/L; IgM=2.1g/L), excluding any B-cell immunodeficiency. Serum IgA and IgM levels remained normal (IgA≥0.9g/L; IgM≥1.2g/L), whereas low IgG levels (4.2–5.6g/L) were observed several times over the past three years. The last rituximab cycle (2×1000mg) was given 6 months pre-operatively.\n\nUpon informed consent, the patient was included in a research project. A pre-operative blood sample and biopsy samples from the tumor, a lung-associated lymph node, and normal lung were collected for flow cytometric analysis. For comparison, samples from a control patient with lung adenocarcinoma (not treated with rituximab) were analyzed. The control patient had CD19+ B cells in blood and tumor (Figure 1A,B). In the rituximab-treated patient, CD19+ B cells were virtually absent from the blood (Figure 1C) and strongly reduced in the tumor (0.2% of all CD45-positive leukocytes, Figure 1D). The remaining CD19+ B cells in the tumor were mostly HLA-DR-negative (Figure 1H) indicating that they were plasma cells which typically lack the CD20 antigen. Rituximab did not deplete other types of immune cells, such as monocytes/macrophages, CD4+ T cells, or CD8+ T cells (Figure 1I–P). Flow cytometric analysis of a lung-associated lymph node and normal lung tissue revealed virtual absence of CD19+ B cells in the rituximab-treated patient (Figure 2).\n\nUpon chest surgery for removal of a lung tumor from a patient previously treated with rituximab, tumor biopsy and serum samples were analyzed by flow cytometry. Results from a control lung cancer patient (not treated with rituximab) are shown for comparison. Both patients were diagnosed with lung adenocarcinoma. CD45-positive leukocytes were gated and analyzed further for expression of CD19/CD3 (A–D), HLA-DR/CD14 (E–L), and CD4/CD8 (M–P). The dot plots E–H show expression of HLA-DR and CD14 by CD19-positive B cells only (red gates in A–D). Numbers in quadrants indicate the percentage of cells detected. PBMCs, peripheral blood mononuclear cells.\n\nUpon chest surgery for removal of tumor-containing lung lobe from a patient previously treated with rituximab, a lung-associated lymph node and normal lung tissue samples were analyzed by flow cytometry. Results from a control lung cancer patient (not treated with rituximab) are shown for comparison. Both patients were diagnosed with lung adenocarcinoma. Live leukocytes (CD45-positive, propidium iodide-negative) were gated and analyzed further for expression of CD19 (B cells) and CD3 (T cells). Numbers indicate the percentage of cells detected in each gate.\n\nImmunohistochemistry of formalin-fixed paraffin-embedded routine specimen was performed by staining for CD3 and CD20. In the control patient, the inflammatory infiltrate in and around the tumor contained both CD20+ B cells (Figure 3A,C) and CD3+ T cells (Figure 3E,G). In contrast, the inflammatory infiltrate in the rituximab-treated patient contained T cells (Figure 3F,H) but virtually no B cells (Figure 3B,D), in accordance with the flow cytometry data.\n\nLung tissue sections from a control patient (left) and from a rituximab-treated patient (right) were stained with anti-CD20 (A–D) or anti-CD3 (E–H) antibodies, and contrastained with hematoxylin. Both patients were diagnosed with lung adenocarcinoma. Arrowheads delineate the border of the tumor. Small boxes in A,B,E, and F indicate magnified areas in C,D,G, and H, respectively. Tu, tumor tissue. A,B,E, and F: 20× magnification; scalebar = 1 mm. C,D,G, and H: 200× magnification; scalebar = 100 μm.\n\nThe same pattern was observed in normal lung and in lung-associated lymph nodes. In the control patient, normal lung tissue contained peribronchial lymphoid foci with both CD20+ B cells (Figure 4A) and CD3+ T cells (Figure 4C). In contrast, the peribronchial lymphoid foci from the rituximab-treated patient contained T cells (Figure 4D) but virtually no B cells (Figure 4B). In control lung-associated lymph nodes, a normal lymphocyte distribution was observed with typical germinal centers with a high density of CD20+ B cells (Figure 5A), whereas CD3+ T cells were mostly present outside the germinal centers (Figure 5C). In sharp contrast, lung-associated lymph nodes from the rituximab-treated patient contained virtually no B cells (Figure 5B) and were homogenously and densely populated by T cells (Figure 5D). The black dots in Figure 5B represent anthracotic pigment in macrophages. Thus, rituximab therapy resulted in efficient depletion of CD20-positive B cells throughout the lungs, including in a lung tumor, in normal lung tissue, and in lung-associated lymph nodes.\n\nNormal lung tissue sections from a control patient (left) and from a rituximab-treated patient (right) were stained with anti-CD20 (A,B) or anti-CD3 (C,D) antibodies, and contrastained with hematoxylin. Both patients were diagnosed with lung adenocarcinoma. Small boxes indicate areas that are magnified in the upper right inserts. LF, peribronchial lymphoid focus. Main images: 20× magnification; scalebar = 1 mm. Upper right inserts: 200× magnification.\n\nA lung-associated lymph node from a control patient (left) and from a rituximab-treated patient (right) were stained with anti-CD20 (A–B) or anti-CD3 (C–D) antibodies, and contrastained with hematoxylin. Both patients were diagnosed with lung adenocarcinoma. The small box in B indicates the area that is magnified in the upper right insert. The black dots in B represent anthracotic pigment in macrophages. Main images: 20× magnification; scalebar = 1 mm. Upper right insert in B: 400× magnification.\n\n\nDiscussion\n\nRituximab was initially developed with the goal of eradicating B-lymphoma cells which typically reside in blood and lymphoid organs1–4. It is now well established that rituximab efficiently eliminates normal and malignant B cells in those anatomical locations12–14. In contrast, current knowledge on the effect of rituximab therapy in non-lymphoid tissues remains fragmentary. This is problematic because rituximab is being considered as a therapeutic option for a number of non-malignant conditions such as autoimmune diseases5–9 and myalgic encephalopathy/chronic fatigue syndrome15. In autoimmune diseases, depletion of pathogenic B cells in inflamed tissues is likely to be required to obtain clinical benefits. In the cerebrospinal fluid of patients with multiple sclerosis, rituximab therapy was shown to result in 90–95% depletion of B cells16,17. In the salivary glands of patients with Sjögren’s syndrome, the efficiency of rituximab remains controversial because both complete and partial depletion of B cells have been reported8,18. Similarly, the effect of rituximab on synovial B cells is debated since various levels of depletion have been reported in patients with rheumatoid arthritis12,19,20.\n\nOur case report illustrates that rituximab may efficiently deplete B cells in the lungs, including lung tumor, normal lung tissue, and lung-associated lymph nodes. This property of rituximab is of particular interest for the treatment of conditions in which pathogenic B cells reside in the lungs, such as antisynthetase syndrome, granulomatosis with polyangiitis, and scleroderma-associated interstitial lung disease9,21,22. On the other hand, the strong B cell-depleting effect in the lungs may provide an explanation for the rare cases of severe non-infectious pulmonary toxicity of rituximab23,24. Rituximab-associated lung disease is a rare but potentially fatal complication of rituximab therapy, whose pathogenic mechanism remains to be elucidated23.\n\nRituximab therapy was associated with virtual absence of tumor-infiltrating B cells in a patient with lung adenocarcinoma. Non-small cell lung cancer (NSCLC) tumors typically contain tertiary lymphoid structures with a high frequency of CD20+ follicular B cells25. Tumor-infiltrating immune cells, including B cells, may represent an ongoing protective immune response against the malignant cells26,27. In fact, it has recently been reported that a high density of follicular B cells correlated with longer patient survival in NSCLC25. Therefore, rituximab-mediated depletion of tumor-infiltrating normal B cells may potentially have a detrimental impact on the antitumor immune response, particularly in NSCLC.",
"appendix": "Author contributions\n\n\n\nHA, OTB, IØ, and AC conceived the study. AC supervised the study. JS and ÅH managed the patient. AJB, CH, BS, JS, and ÅH acquired the data. AJB, CH, BS, JS, and AC analyzed the data and prepared the figures. AC 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\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by grants from the Research Council of Norway, Norway Grants 2009-2014 under project contract NFI/R/2014/051 (to A. Corthay), and the Norwegian Cancer Society.\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 Ingjerd Solvoll, Inger Johanne Ryen, and Kahsai Beraki for technical help.\n\n\nReferences\n\nLim SH, Levy R: Translational medicine in action: anti-CD20 therapy in lymphoma. J Immunol. 2014; 193(4): 1519–1524. PubMed Abstract | Publisher Full Text\n\nReff ME, Carner K, Chambers KS, et al.: Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994; 83(2): 435–445. PubMed Abstract\n\nMaloney DG, Liles TM, Czerwinski DK, et al.: Phase I clinical trial using escalating single-dose infusion of chimeric anti-CD20 monoclonal antibody (IDEC-C2B8) in patients with recurrent B-cell lymphoma. Blood. 1994; 84(8): 2457–2466. PubMed Abstract\n\nMaloney DG, Grillo-López AJ, White CA, et al.: IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood. 1997; 90(6): 2188–2195. PubMed Abstract\n\nEdwards JC, Szczepanski L, Szechinski J, et al.: Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med. 2004; 350(25): 2572–2581. PubMed Abstract | Publisher Full Text\n\nHauser SL, Waubant E, Arnold DL, et al.: B-cell depletion with rituximab in relapsing-remitting multiple sclerosis. N Engl J Med. 2008; 358(7): 676–688. PubMed Abstract | Publisher Full Text\n\nStone JH, Merkel PA, Spiera R, et al.: Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med. 2010; 363(3): 221–232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPijpe J, Meijer JM, Bootsma H, et al.: Clinical and histologic evidence of salivary gland restoration supports the efficacy of rituximab treatment in Sjögren's syndrome. Arthritis Rheum. 2009; 60(11): 3251–3256. PubMed Abstract | Publisher Full Text\n\nDaoussis D, Liossis SN, Tsamandas AC, et al.: Experience with rituximab in scleroderma: results from a 1-year, proof-of-principle study. Rheumatology (Oxford). 2010; 49(2): 271–280. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeng WK, Levy R: Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol. 2003; 21(21): 3940–3947. PubMed Abstract | Publisher Full Text\n\nAnolik JH, Campbell D, Felgar RE, et al.: The relationship of FcgammaRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus. Arthritis Rheum. 2003; 48(2): 455–459. PubMed Abstract | Publisher Full Text\n\nTeng YK, Levarht EW, Hashemi M, et al.: Immunohistochemical analysis as a means to predict responsiveness to rituximab treatment. Arthritis Rheum. 2007; 56(12): 3909–3918. PubMed Abstract | Publisher Full Text\n\nCioc AM, Vanderwerf SM, Peterson BA, et al.: Rituximab-induced changes in hematolymphoid tissues found at autopsy. Am J Clin Pathol. 2008; 130(4): 604–612. PubMed Abstract | Publisher Full Text\n\nAudia S, Samson M, Guy J, et al.: Immunologic effects of rituximab on the human spleen in immune thrombocytopenia. Blood. 2011; 118(16): 4394–4400. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFluge Ø, Risa K, Lunde S, et al.: B-Lymphocyte Depletion in Myalgic Encephalopathy/ Chronic Fatigue Syndrome. An Open-Label Phase II Study with Rituximab Maintenance Treatment. PLoS One. 2015; 10(7): e0129898. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCross AH, Stark JL, Lauber J, et al.: Rituximab reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol. 2006; 180(1–2): 63–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPiccio L, Naismith RT, Trinkaus K, et al.: Changes in B- and T-lymphocyte and chemokine levels with rituximab treatment in multiple sclerosis. Arch Neurol. 2010; 67(6): 707–714. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPers JO, Devauchelle V, Daridon C, et al.: BAFF-modulated repopulation of B lymphocytes in the blood and salivary glands of rituximab-treated patients with Sjögren's syndrome. Arthritis Rheum. 2007; 56(5): 1464–1477. PubMed Abstract | Publisher Full Text\n\nVos K, Thurlings RM, Wijbrandts CA, et al.: Early effects of rituximab on the synovial cell infiltrate in patients with rheumatoid arthritis. Arthritis Rheum. 2007; 56(3): 772–778. PubMed Abstract | Publisher Full Text\n\nKavanaugh A, Rosengren S, Lee SJ, et al.: Assessment of rituximab's immunomodulatory synovial effects (ARISE trial). 1: clinical and synovial biomarker results. Ann Rheum Dis. 2008; 67(3): 402–408. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarie I, Dominique S, Janvresse A, et al.: Rituximab therapy for refractory interstitial lung disease related to antisynthetase syndrome. Respir Med. 2012; 106(4): 581–587. PubMed Abstract | Publisher Full Text\n\nHenderson SR, Copley SJ, Pusey CD, et al.: Prolonged B cell depletion with rituximab is effective in treating refractory pulmonary granulomatous inflammation in granulomatosis with polyangiitis (GPA). Medicine (Baltimore). 2014; 93(27): e229. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHadjinicolaou AV, Nisar MK, Parfrey H, et al.: Non-infectious pulmonary toxicity of rituximab: a systematic review. Rheumatol (Oxford). 2012; 51(4): 653–662. PubMed Abstract | Publisher Full Text\n\nBitzan M, Anselmo M, Carpineta L: Rituximab (B-cell depleting antibody) associated lung injury (RALI): a pediatric case and systematic review of the literature. Pediatr Pulmonol. 2009; 44(9): 922–934. PubMed Abstract | Publisher Full Text\n\nGermain C, Gnjatic S, Tamzalit F, et al.: Presence of B cells in tertiary lymphoid structures is associated with a protective immunity in patients with lung cancer. Am J Respir Crit Care Med. 2014; 189(7): 832–844. PubMed Abstract | Publisher Full Text\n\nHaabeth OA, Bogen B, Corthay A: A model for cancer-suppressive inflammation. Oncoimmunology. 2012; 1(7): 1146–1155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCorthay A: Does the immune system naturally protect against cancer? Front Immunol. 2014; 5: 197. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "12242",
"date": "03 Feb 2016",
"name": "Bertrand Huard",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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/abstract, content, conclusions, data are all appropriate.As a minor revision, I would ask authors to speculate on what could be the function of B cells in non infectious lung.",
"responses": []
},
{
"id": "12922",
"date": "29 Mar 2016",
"name": "Hilde Schjerven",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis well-written paper by Joly-Battaglini et al. describes a case study on the effect of Rituximab on B cells in the lung tissue and tumor microenvironment of a rheumatoid arthritis patient with lung adenocarcinoma. The authors employ the complementing techniques of flow cytometry and immunohistochemistry to show that Rituximab efficiently eliminates CD19+ B cells in the lungs and a majority of CD19+ B cells in the tumor microenvironment, without depleting other lymphoid cells which are not CD20+. Only a low percentage (0.2 %) of CD19+ cells were found in the tumor, and these were suggested to be plasma cells due to lack of HLA-DR by flow cytometry. This paper confirms the efficiency of Rituximab, and adds new knowledge on its ability to eliminate B cells in lung tissue and tumor microenvironment. The questions asked by the study were approached using appropriate methods, and the paper is clearly written. Suggestions for minor revisions; It would help the clarity of the paper if the title would indicate that it was a case study. Also, in the title, lung tumor should be corrected to singular, not plural (current title: “lung tumors”) It would help the reader if the authors would comment on the life span of B cells with respect to the duration of Rituximab treatment. It would be interesting to see a discussion on whether the low percentage of presumed plasma cells observed in the tumor were from post- or pre- Rituximab treatment. Does Rituximab reach and actively deplete intra-tissue B cells, or is the depletion a result of decreased levels of circulating B cells able to infiltrate tissues, and reflects a decrease due to the life-span (or resident time) of tissue-resident B cells? What could the significance of the 0.2% of plasma cells be with respect to B cell implicated diseases? This could be discussed in light of serum IgG levels and statement of the patient displaying “only moderate clinical effect”. It is interesting in regards to the discussion of use of Rituximab for treatment of B-cell mediated diseases (non-cancer) and tissue-resident B cells as opposed to circulating of lymphoid tissue B cells (see point above). What is the statement “The black dots in Figure 5B represent anthracotic pigment in macrophages” based on? Although plausible, a suitable control could for instance be isotype control stain to display this as background, or CD19 (vs e.g. CD11b or similar) stain using a different secondary detection method (that would not detect anthracotic pigment). Although such added experiments might be feasible (if more material is left), it is not required, as it will not affect the main message of the paper in regards to the observed major depletion of B cells.Overall, the article is well-written, scientifically sound, and presents a case study observation that warrants indexing.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-38
|
https://f1000research.com/articles/5-34/v1
|
07 Jan 16
|
{
"type": "Case Report",
"title": "Case Report: Efficacy of dobesilate in insertional Achilles tendinopathy",
"authors": [
"Pedro Cuevas",
"Tomás Fernández Jaén",
"Pedro Guillén",
"Javier Angulo",
"Guillermo Giménez-Gallego",
"Tomás Fernández Jaén",
"Pedro Guillén",
"Javier Angulo",
"Guillermo Giménez-Gallego"
],
"abstract": "Achilles tendinopathy is an overuse syndrome, common among runners, with sometimes considerable negative impact on their performance, overall health, and well-being. Our report shows that local injection of an aqueous solution of the diethylammonium salt of dobesilate, an inhibitor of fibroblast growth factor with significant anti-angiogenic and anti-inflammatory effects, is effective in reducing vascular density and pain in insertional Achilles tendinopathy.",
"keywords": [
"Achilles tendinopathy",
"neovascularization",
"inflammation",
"fibroblast growth factor",
"dobesilate"
],
"content": "Case description\n\nA 37-year-old healthy Caucasian male runner presented with a 3 month history of chronic pain and swelling on top of his left heel. There was no history of direct trauma to his left heel. He experienced a constant dull pain when walking. He had to discontinue sport because of the severity of his pain. At the beginning of symptomatology, the patient initiated sporadic treatments with over-the-counter analgesic and anti-inflammatory drugs. One week before presentation, following recommendations of his physician, the patient initiated, unsuccessfully, a treatment with paracetamol (1g twice a day) and ibuprofen (400mg three times per day). At presentation heel pain was rated as 6 out of 10 on the visual analogue scale (VAS). Colour doppler ultrasound examination at the insertional site of the left Achilles tendon revealed significant neovascularity, mainly at intratendinous mass (Figure 1). After discussing the various treatment options, the patient opted to try a dobesilate injection to the Achilles tendon and gave informed consent. Lidocaine was infiltrated into the skin overlying the Achilles tendon insertional junction. Dobesilate (2 ml of diethylammonium salt formulation; etamsylate, Dicynone®, Sanofi, France) was peritendinously injected under ultrasound guidance into the Achilles tendon. The procedure was uneventful. The patient was advised to perform some gentile range of motion exercises the following day. He was back to his regular life the day after injection. At the 1 month follow-up visit, the patient reported a marked reduction of his pain, and the VAS was rated as 1. Colour doppler ultrasound scans revealed a significant reduction of tendon hypervascularity at the time (Figure 1). The patient was able to return to running and his previous level of sport without any restrictions. No adverse events were observed during treatment and the 2 month follow-up period.\n\nLong-axis colour doppler ultrasound scans taken at three different planes before and after one month of treatment. Note the reduction of hypervascularity after treatment. The large vessels were persistently observed in real time ultrasonography examination. Achilles tendon mass was delimited by discontinuous line.\n\n\nDiscussion\n\nTendinopathy is a common health problem affecting nearly 8% of middle and long distance runners under the age of 45. The consequences of this medical condition include pain, disability, early retirement from sport and work, mental distress and health care cost1. The treatment of tendinopathies is a significant challenge for sport medicine physicians wishing to avoid surgery, since there is no obvious non-surgical options as efficacious therapeutic treatments2.\n\nInflammation and angiogenesis are two cardinal biological processes which cause tendinopathies3. Consequently, control of inflammation and neovascularization seem two obvious targets to develop new treatments for management of tendinopathies.\n\nThe anti-inflammatory treatments, by themselves, do not seem to achieve a significant success in the case of Achilles tendinopathies, in addition they seem less effective in patients with Achilles insertional tendinopathies than in those with mid-portion tendinopathies4.\n\nHypervascularity has been reported in human and animal Achilles tendinopathies, as well as in patellar disease, long head biceps tendons and in the rotator cuff5,6. Furthermore, the tendon area of hypervascularization coincides with the most common localisation for tears5,6. Healthy tendons are not painful, and have no detectable blood vessels, as assessed by ultrasonography9. However, pain is a common symptom which accompanies neovascularization in chronic Achilles tendinopathies. Furthermore, tendon neovascularization is accompanied with nerve in-growth facilitating pain transmission in Achilles tendinopathy10. Consequently, strategies to destroy neovessels (i.e local application of sclerosing agents as polidocanol) cause pain amelioration11,12, despite the associated side effects13,14. Inhibiting angiogenesis in addition to inflammation seems, thus, a reasonable strategy for development of new therapies against Achilles tendinopathies4,7,8,15.\n\nFibroblast growth factor (FGF) is nowadays considered a pro-inflammatory and pro-angiogenic protein16–18. FGF can be inhibited with dobesilate19. This old drug, with a high safety profile20 but with considerably vague pharmacological and therapeutic targets until its anti-FGF activities were described, has been shown, since this precise point in time, to relieve inflammation and prevents undesirable neovessel formation in many different pathological scenarios21–25. The data presented in this case report show that local administration of dobesilate seems also effective in reducing neovessel formation and inflammation in the case of insertional Achilles tendinopathies. Recently, it has been reported that FGF is a nociceptive modulator26. Since target inhibition of FGF in tissues undergoing pathological angiogenesis is safe without significant off-target effects on non-diseased tissues27, dobesilate seems an attractive drug for treating tendinopathies.\n\nLarge-scale therapeutic trials are obviously needed for more solidly establishing the efficacy of dobesilate in the treatment of Achilles tendinopathy. The results presented in this report seem a reasonable support for undertaking these trials.\n\n\nConsent\n\nWritten informed consent for publication of the clinical details and images was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nPC and GGG wrote the paper. TFJ and PG were the physicians responsible for the patient in this case report. All authors have participated in the concept and design/analysis and interpretation of data, drafting and revising the manuscript, and they have given final approval for the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKujala UM, Sarna S, Kaprio J: Cumulative incidence of Achilles tendon rupture and tendinopathy in male former elite athletes. Clin J Sport Med. 2005; 15(3): 133–5. PubMed Abstract | Publisher Full Text\n\nSussmilch-Leitch SP, Collins NJ, Bialocerkowski AE, et al.: Physical therapies for Achilles tendinopathy: systematic review and meta-analysis. J Foot Ankle Res. 2012; 5(1): 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlfredson H, Cook J: A treatment algorithm for managing Achilles tendinopathy: new treatment options. Br J Sports Med. 2007; 41(4): 211–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlfredson H: Chronic tendon pain--implications for treatment: an update. Curr Drug Targets. 2004; 5(5): 407–10. PubMed Abstract | Publisher Full Text\n\nChansky HA, Iannotti JP: The vascularity of the rotator cuff. Clin Sports Med. 1991; 10(4): 807–22. PubMed Abstract\n\nBlevins FT, Hayes WM, Warren RF: Rotator cuff injury in contact athletes. Am J Sports Med. 1996; 24(3): 263–7. PubMed Abstract | Publisher Full Text\n\nPetersen W, Pufe T, Zantop T, et al.: Expression of VEGFR-1 and VEGFR-2 in degenerative Achilles tendons. Clin Orthop Relat Res. 2004; (420): 286–91. PubMed Abstract\n\nFowble VA, Vigorita VJ, Bryk E, et al.: Neovascularity in chronic posterior tibial tendon insufficiency. Clin Orthop Relat Res. 2006; 450: 225–30. PubMed Abstract\n\nOhberg L, Lorentzon R, Alfredson H: Neovascularisation in Achilles tendons with painful tendinosis but not in normal tendons: an ultrasonographic investigation. Knee Surg Sports Traumatol Arthrosc. 2001; 9(4): 233–8. PubMed Abstract | Publisher Full Text\n\nKnobloch K: The role of tendon microcirculation in Achilles and patellar tendinopathy. J Orthop Surg Res. 2008; 3: 18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhberg L, Alfredson H: Ultrasound guided sclerosis of neovessels in painful chronic Achilles tendinosis: pilot study of a new treatment. Br J Sports Med. 2002; 36(3): 173–5; discussion 176–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMousavizadeh R, Khosravi S, Behzad H, et al.: Cyclic strain alters the expression and release of angiogenic factors by human tendon cells. PLoS One. 2014; 9(5): e97356. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuffy DM: Sclerosants: a comparative review. Dermatol Surg. 2010; 36(Suppl 2): 1010–25. PubMed Abstract | Publisher Full Text\n\nMarrocco-Trischitta MM, Guerrini P, Abeni D, et al.: Reversible cardiac arrest after polidocanol sclerotherapy of peripheral venous malformation. Dermatol Surg. 2002; 28(2): 153–5. PubMed Abstract | Publisher Full Text\n\nSavitskaya YA, Izaguirre A, Sierra L, et al.: Effect of angiogenesis-related cytokines on rotator cuff disease: the search for sensitive biomarkers of early tendon degeneration. Clin Med Insights Arthritis Musculoskelet Disord. 2011; 4: 43–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPresta M, Andrés G, Leali D, et al.: Inflammatory cells and chemokines sustain FGF2-induced angiogenesis. Eur Cytokine Netw. 2009; 20(2): 39–50. PubMed Abstract | Publisher Full Text\n\nAndrés G, Leali D, Mitola S, et al.: A pro-inflammatory signature mediates FGF2-induced angiogenesis. J Cell Mol Med. 2009; 13(8B): 2083–108. PubMed Abstract | Publisher Full Text\n\nThomas KA, Rios-Candelore M, Giménez-Gallego G, et al.: Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1. Proc Natl Acad Sci U S A. 1985; 82(19): 6409–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFernández IS, Cuevas P, Angulo J, et al.: Gentisic acid, a compound associated with plant defense and a metabolite of aspirin, heads a new class of in vivo fibroblast growth factor inhibitors. J Biol Chem. 2010; 285(15): 11714–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRabe E, Ballarini S, Lehr L, et al.: A randomized, double-blind, placebo-controlled, clinical study on the efficacy and safety of calcium dobesilate in the treatment of chronic venous insufficiency. Phlebology. 2015 (publication ahead of print); pii: 0268355515586097. PubMed Abstract | Publisher Full Text\n\nCuevas P, Angulo J, Giménez-Gallego G: Topical treatment of contact dermatitis by pine processionary caterpillar. BMJ Case Rep. 2011; 2011: pii: bcr0620114351. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAngulo J, Cuevas P, Cuevas B, et al.: Diacetyloxyl derivatization of the fibroblast growth factor inhibitor dobesilate enhances its anti-inflammatory, anti-angiogenic and anti-tumoral activities. J Transl Med. 2015; 13: 48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCuevas P, Hinojosa M, Angulo J, et al.: Efficacy of dobesilate in allergic rhinitis: case report. J Develop Drugs. 2015; 4: 139. Reference Source\n\nCuevas P, Outeiriño LA, Angulo J, et al.: Treatment of Stargardt disease with dobesilate. BMJ Case Rep. 2012; 2012: pii: bcr2012007128. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCuevas P, Outeiriño LA, Angulo J, et al.: Chronic cystoid macular oedema treated with intravitreal dobesilate. BMJ Case Rep. 2012; 2012: pii: bcr2012006376. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu H, Wu QF, Li JY, et al.: Fibroblast growth factor 7 is a nociceptive modulator secreted via large dense-core vesicles. J Mol Cell Biol. 2015; 7(5): 466–75. PubMed Abstract | Publisher Full Text\n\nOladipupo SS, Smith C, Santeford A, et al.: Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis. Proc Natl Acad Sci U S A. 2014; 111(37): 13379–84. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "17347",
"date": "02 Nov 2016",
"name": "Nicola Maffulli",
"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 having allowed me to review this manuscript. I am very concerned at the fact that the authors try and generalise from one single case report.\nThere is little modern evidence that neovascularity is the primum movens of tendinopathy, and that it should be a therapeutic target.\n\nThe enrollment of the patient after such a short period of symptoms is questionable.\n\nThe follow up is much too short to be able to make recommendations.",
"responses": [
{
"c_id": "2333",
"date": "29 Nov 2016",
"name": "Pedro Cuevas",
"role": "Author Response",
"response": "In summary, our report has been considered not to be of an acceptable scientific standard by the reviewer Nicola Maffulli. This blunt disqualifier statement is surprising. The report submited to F1000 has been elaborated according the same scientific standards employed in the rest of our scientific papers, quoted thousands of times (according the statistics of the WOK), sometimes published in journals of the widest international diffusion. Keystone for the scientific disqualification seems an article published by the reviewer in 2012, in the journal \"Knee Surg Sports Traumatol Arthrosc\" (20:1891–1894), which has been quoted eleven times along these last four years, that defends that detecting neovessels has no additional value for the diagnosis, no firmly confirmed prognostic value, and no proven relation with symptoms in Achilles tendinopathy, a statement that seems contradictory with some studies quoted in the same article: In (a series of) Swedish studies, the investigators were able to detect neovessels in 100 % of the symptomatic tendons [1, 26, 37, 38]..... other researchers, ... reported a percentage varying from 47 to 88 % [13, 41, 42, 54]. Our report does not aim to engage in any sort of controversy about the relevance of neovascularization of the Achilles tendinopathy and whether it is a primum movens or not in such diseases, a discussion that still seem widely open, according to the results of bibliographical searches of recent publications. Accordingly, the report should not be judged from such a point of view. We, exclusively, intend to report the case of a tendinopathy the vascularization of which healed very efficiently, accompanied of a considerable relief of pain, after local treatment with an inhibitor of fibroblast growth factor (FGF), a main angiogenic and inflammatory protein. The treatment was applied after three months of unsuccessfully treated chronic pain and swelling on top of his left heel that made the patient had to discontinue sport practices. This does not seem a questionable short period of symptoms, before the enrolment in the alternative treatment (point 2 of the reviewer considerations), taking into account the quite undesiderable consequences of a prolonged inflammation and abnormal vascularization [Carmeliet, Nature 438 (2005) 932]. We did not try to report a long term healing process, but the fast disappearance of neovascularization and pain relief by inhibiting FGF, a treatment supported by a solid rationale. To this purpose a two-month follow up seems reasonable. A different question is if we had intended to report on a long-term cure. But this is the objective of a different-aimed observational study that we are carrying out at this moment (point 3 of the reviewer comments). The treatment was carried out, as detailedly described in the report, according to the requirements of a proper science, which, surprisingly, was straight on globally disqualified without pointing out at any major mistake in the followed procedure."
}
]
},
{
"id": "14970",
"date": "09 Jan 2017",
"name": "Andreas Bikfalvi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article where the authors showed the efficacy of dobesilate for the treatment of Achilles tendinopathy.\nDobesiltate is an inhibitor of Fibroblast Growth factors. Inhibition of FGF in this pathology reduced angiogenesis in the tendon and pain-related symptoms.\nThe report shows that it could be used safely in patients.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-34
|
https://f1000research.com/articles/5-33/v1
|
07 Jan 16
|
{
"type": "Case Report",
"title": "Case Report: Acute obstructive hydrocephalus associated with infratentorial extra-axial fluid collection following foramen magnum decompression and durotomy for Chiari malformation type I",
"authors": [
"Sunil Munakomi",
"Binod Bhattarai",
"Pramod Chaudhary",
"Binod Bhattarai",
"Pramod Chaudhary"
],
"abstract": "Acute obstructive hydrocephalus due to infratentorial extra-axial fluid collection (EAFC) is an extremely rare complication of foramen magnum decompression (FMD) and durotomy for Chiari malformation type I. Presence of infratentorial EAFC invariably causes obstruction at the level of the fourth ventricle or aqueduct of Silvius, thereby indicating its definitive role in hydrocephalus. Pathogenesis of EAFC is said to be a local arachnoid tear as a result of durotomy, as this complication is not described in FMD without durotomy. Controversy exists in management. Usually EAFC is said to resolve with conservative management; so hydrocephalus doesn’t require treatment. However, in this case EAFC was progressive and ventriculo-peritoneal shunting (VPS) was needed for managing progressive and symptomatic hydrocephalus.",
"keywords": [
"Foramen Magnum Decompression",
"Chiari Malformation",
"Extra Axial Fluid Collection",
"Ventriculo-Peritoneal Shunt"
],
"content": "Introduction\n\nChiari malformation type I is not associated with other congenital conditions. Typically it presents in adulthood with varying features of increased intracranial pressure, headache, progressive cerebellar ataxia, progressive spastic quadriparesis, segmental amyotrophy and sensory loss, with or without pain. The latest management protocol is foramen magnum decompression (FMD) with or without durotomy. Several complications such as cerebrospinal fluid (CSF) fistula, cerebellar hematoma, and worsening neurological status have been described, but acute obstructive hydrocephalus due to infratentorial subdural hygroma is very rare1,3–6. Herein we discuss one such case and review the literature regarding the current knowledge of such a rare complication in the posterior fossa.\n\n\nCase report\n\nA 30-year-old Tharu lady from Birgunj, Nepal was referred to the outpatient clinic at the College of Medical Science (Nepal) with symptoms of chronic dull neck pain without radiation for the previous two years. There was no history of trauma. There was no significant past medical or surgical illnesses. The patient was taking over the counter pain medications (Diclofenac 75 mg PO SOS) for the pain. Due to further aggravation of her symptoms, she opted for medical review. On general examination, she had short neck (distance between external occipital protuberance and C7 spinous process was only 60 mm) with low hair line. There was cape-like dissociative sensory loss starting from C2-T4 level. Muscle tone was of Ashworth grade I. She had exaggerated deep tendon reflexes in upper and lower limbs without appreciable clonus. Magnetic resonance imaging (MRI) of the cervical and thoracic spine was suggestive of Chiari malformation type I with syringohydromyelia extending from C2 to T8 level (Figure 1 and Figure 2). There was no evidence of hydrocephalus. The patient was thoroughly counselled of her medical condition and surgical intervention was advised.\n\nFMD and durotomy were performed after receiving patient consent. Improvement in spasticity was seen immediately from the 1st postoperative day. The patient’s hospital stay was uneventful and she was discharged after suture removal on the 7th postoperative day.\n\nThree weeks following surgery, the patient returned with complaints of headache and dizziness. She had no added neurological deficits and fundus examination was normal. A CT head scan showed minimal infratentorial EAFC and rounded third ventricle and prominent temporal horn. Initially she was managed conservatively with tablet acetazolamide 250 mg PO every 8 hours and strict monitoring for features of raised intracranial pressure such as persistent vomiting, hypertension and bradycardia. On the 6th day of her admission, she deteriorated with severe headache and persistent vomiting Early papilloedema was evident on fundoscopy and a repeat CT scan showed an increase in EAFC and triventricular hydrocephalus (Figure 3). Ventriculo-peritoneal shunting (VPS) was done for the same. Ventricular tapping revealed high pressure clear CSF. Her symptoms subsequently disappeared and she was discharged on the 7th day. She continues to follow up in the outpatient department every 6 months with no new symptoms and better resolution of her previous ailments. She is able to differentiate the sense of temperature in the areas where she had dissociative sensory loss prior to the management. Her exaggerated deep tendon reflexes have gradually resolved over 2 months of her surgery. Her VPS is functioning well to-date (2 years after surgery).\n\n\nDiscussion\n\nFMD with or without durotomy is the most widely used surgical technique for Chiari malformation type I1. Some surgeons prefer to perform a wide midline suboccipital craniectomy and not to open the dura with a view that duramater can slowly stretch so as to accommodate posterior fossa structures. Although some authors advocate performing a simple lax duraplasty alone, others provide a rationale for performing arachnoidolysis subsequent to duraplasty2.\n\nWith our improved insights on the pathogenesis of Chiari malformation, our surgical nuances and techniques on the management of the condition have improved significantly. Complication rates are reported to be as low as 2.3%1. Among these complications subdural hygroma or EAFC causing acute obstructive hydrocephalus is very rare1,3–6. The exact cause of subdural hygroma is still not clear. It is believed that during durotomy or arachnoidolysis, a small hole may be created on the arachnoid which will cause CSF egress in the subdural space. This tear acts like a one way valve causing more CSF to collect in the subdural space. Initially, CSF accumulates in and around the foramen magnum. With pressure created, CSF will slowly flow towards the cisterna magna, bilateral cerebellar convexity, below tentorium cerebelli and finally through the tentorial hiatus to the supratentorial subdural space3. Some authors have advocated the probable role of increased permeability of the intracranial vessels, which seems to be logical in cases of traumatic brain injury only7. Subdural hygroma under tension causes compression at the level of the fourth ventricle or aqueduct of Silvius, thus causing acute hydrocephalus5.\n\nAs seen from the literature review, this complication is usually seen in patients above 10 years of age and has special preponderance to females. No clear hypothesis or mechanisms has been proposed for this, however a tear or breach in the dura seems to be the most likely. Age varied from 10 years to 55 years. Until now, including our study, nine cases have been reported, of which seven describe female patients.\n\nThe site of maximal collection of extra-axial fluid collections differ between case reports. Such extra-axial collections may only be confined to infratentorial area, may extend to supratentorial area or only be prominent in supratentorial areas. Infratentorial extra-axial fluid collections usually cause compression at the level of fourth ventricle or aqueduct of Silvius and cause triventricular hydrocephalus. It may also result in direct compression of the brain stem and cranial nerves, causing acute symptoms. Supratentorial subdural effusion (EAFC) acts like decompensated chronic subdural hematoma thus causing relevant symptoms such as hemiparesis or signs of raised intracranial pressure.\n\nEAFC may resolve spontaneously or aggravate causing acute hydrocephalus. Acute hydrocephalus may be transient as EAFC might slowly resolve. Patients usually are symptomatic requiring hospital admission from 3–21 days after FMD.\n\nThis is a rare complication so the ideal management protocol is not known. Effort should be made to prevent it by avoiding durotomy and arachnoidolysis whenever possible and opening the arachnoid widely whenever deemed necessary. Some authors suggest re-surgery for closing the tiny hole in the arachnoid or opening the arachnoid widely and suturing it to the dura4,9,10. Widely opening the arachnoid routinely during surgery might prevent this complication as pinhole dural tear may even occur during closure3.\n\nAfter complication has already occurred, and is not life threatening, minimal measures such as burr hole and external ventricular drain (EVD) should be undertaken. In most cases reported till date, permanent VPS is not usually required as EAFC usually resolves6,8,10. However, if there is evidence of significant mass effect and raised intracranial pressure, such patients should be managed surgically either with placement of appropriate burr holes or by performing VP shunting for CSF diversion. VPS should be considered for progressive neurological deterioration and increasing ventricular size. A subduroperitoneal shunt should be employed for recurrent supratentorial EAFC. There is inadvertent risk of slit ventricular syndrome due to over-drainage of CSF following low pressure VP shunting. Repeated EVD (more than twice) increases the risk of infection as well as the odds of upward central herniation in case of CSF over-drainage. So, VPS should be considered in such cases.\n\n\nConclusions\n\nEAFC complicating acute obstructive hydrocephalus is a very rare complication following FMD and durotomy for Chiari malformation type I; but should be suspected in post operative cases presenting with headache and vomiting. We can avoid this by only undertaking osseous decompression or in cases where there is need for durotomy, widely opening the arachnoid and suturing it to the duramater. Whenever applicable, attempts should be made to manage these cases without VPS.\n\n\nConsent\n\nBoth written and verbal informed consent for publication of images and clinical data related to this case was sought and obtained from the patient.",
"appendix": "Author contributions\n\n\n\nSM and PC reviewed the literature and formatted the paper. BB revised and edited the final format.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nTubbs RS, McGirt MJ, Oakes WJ: Surgical experience in 130 pediatric patients with Chiari I malformations. J Neurosurg. 2003; 99(2): 291–6. PubMed Abstract | Publisher Full Text\n\nSiasios J, Kapsalaki EZ, Fountas KN: Surgical management of patients with Chiari I malformation. Int J Pediatr. 2012; 2012: 640127. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBahuleyan B, Menon G, Hariharan E, et al.: Symptomatic posterior fossa and supratentorial subdural hygromas as a rare complication following foramen magnum decompression for Chiari malformation Type I. J Neurosurg. 2011; 114(2): 510–3. PubMed Abstract | Publisher Full Text\n\nElton S, Tubbs RS, Wellons JC 3rd, et al.: Acute hydrocephalus following a Chiari I decompression. Pediatr Neurosurg. 2002; 36(2): 101–4. PubMed Abstract | Publisher Full Text\n\nKabir SM, Jennings SJ, Makris D: Posterior fossa subdural hygroma with supratentorial chronic subdural haematoma. Br J Neurosurg. 2004; 18(3): 297–300. PubMed Abstract | Publisher Full Text\n\nMarshman LA, Benjamin JC, Chawda SJ, et al.: Acute obstructive hydrocephalus associated with infratentorial subdural hygromas complicating Chiari malformation Type I decompression. Report of two cases and literature review. J Neurosurg. 2005; 103(4): 752–5. PubMed Abstract | Publisher Full Text\n\nNishizaki T, Tamaki N, Fujiwara H, et al.: Posterior fossa subdural effusion due to head trauma. Neurosurgery. 1988; 23(1): 81–4. PubMed Abstract | Publisher Full Text\n\nPerrini P, Rawlinson A, Cowie RA, et al.: Acute external hydrocephalus complicating craniocervical decompression for syringomyelia-Chiari I complex: case report and review of the literature. Neurosurg Rev. 2008; 31(3): 331–5. PubMed Abstract | Publisher Full Text\n\nZakaria R, Kandasamy J, Khan Y, et al.: Raised intracranial pressure and hydrocephalus following hindbrain decompression for Chiari I malformation: a case series and review of the literature. Br J Neurosurg. 2012; 26(4): 476–81. PubMed Abstract | Publisher Full Text\n\nRanjan A, Cast IP: Symptomatic subdural hygroma as a complication of foramen magnum decompression for hindbrain herniation (Arnold-Chiari deformity). Br J Neurosurg. 1996; 10(3): 301–3. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "12295",
"date": "29 Feb 2016",
"name": "Lekhjung Thapa",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 attempted to demonstrate a rare manifestation of operative intervention on adult type Chiari malformation. This kind of case report would undoubtedly help surgeons to understand the current status of managing this, which is still controversial. They have highlighted the importance of post operative headache and vomiting to identify a rare yet a treatable cause like EAFC. Comments:Please mention how you defined the low hair-line. It would be better for authors to mention why they chose ventriculoperitoneal shunting and not external ventricular drain to deal with the complication, as we know they may resolve spontaneously. (Marshman et al. 20051) As the literature review seems less, I would rather say \"Herein we report a case of Chiari malformation type I with postoperative infratentorial subdural hygroma\"",
"responses": []
},
{
"id": "13971",
"date": "25 May 2016",
"name": "Murat Şakir Ekşi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report is informative about obstructive hydrocephalus development secondary to extradural fluid collection in the posterior fossa following decompression surgery for Chiari malformation type I. The authors mentioned about presence of scarce amount of cases in the literature. It would be better to delineate the prior cases in a table to give more information about the nature of the disease.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-33
|
https://f1000research.com/articles/5-26/v1
|
06 Jan 16
|
{
"type": "Research Note",
"title": "Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway",
"authors": [
"Xiaojia Guo",
"Rong Ju",
"Charles Cha",
"Michael Simons",
"Xiaojia Guo",
"Rong Ju",
"Charles Cha"
],
"abstract": "Adrenomedullin 2 plays diverse physiological roles such as regulating cardiovascular functions and blood pressure. It was reported that adrenomedullin 2 can activate protein kinase C in murine ventricular myocytes to augment cardiomyocyte contractile function. Using a protein kinase Cα knockout mouse model, we show here that adrenomedullin 2 activates extracellular-signal-regulated kinase in a protein kinase Cα-independent mechanism in endothelial cells.",
"keywords": [
"adrenomedullin 2 (ADM2)",
"intermedin (IMD)",
"protein kinase C (PKC)",
"endothelial cells",
"signal transduction"
],
"content": "Introduction\n\nAdrenomedullin 2 (ADM2), also known as intermedin, is a secreted peptide that belongs to the calcitonin gene-related-peptide family1,2. It has been reported that ADM2 regulates intracellular calcium levels and contractile function in protein kinase C (PKC) - and protein kinase A (PKA) - dependent mechanisms in cardiomyocytes3. ADM2 activates the cAMP/PKA signaling pathway, which mediates inactivation of contractility and strengthening of cell-cell adhesion in endothelial cells4. ADM2 activates extracellular-signal-regulated kinase (ERK), a key signaling molecule for cell proliferation in endothelial cells5. To investigate whether ADM2 activates ERK through PKCα, which is a major upstream activator of ERK in endothelial cells we examined the effect of phosphorylation of ERK on ADM2 stimulation in endothelial cells isolated from PKCα null mice or wild type (wt) counterpart mice.\n\n\nMethods\n\nAnimal care and experimental procedures were performed under protocol # CC0004 approved by the Institutional Animal Care and Use Committees of Yale University. Endothelial cells were isolated from wild type (C57BL/6J, The Jackson Laboratory, Cat # 000664) and PKCα-/- mice (Prkcatm1Jmk, The Jakson Laboratory, Cat # B6;129-Prkcatm1Jmk/J) and maintained as previously described6. Briefly, the arteries of both wild-type and knockout mice were harvested, finely minced with scissors, and digested with 25 ml collagenase (2 mg/ml) at 37°C for 45 min under gentle agitation. The crude preparation was triturated, passing it 12 times through a cannula needle, and was then filtered on a 70-μM sterile cell strainer. The filtered preparation was spun at 400 × g, and the pellet was resuspended in 2 ml of 0.1% BSA. Magnetic beads (Invitrogen) coated with anti-mouse CD31 (BD Biosciences) were added to the cell suspension and incubated with rotation at room temperature for 15 min. The bead-bound cells were recovered with a magnetic separator and washed with DMEM containing 20% FBS. Cells were suspended in 10 ml of complete DMEM and seeded on cell culture plates (Catalog # 353003, Corning Inc., Corning, NY). Subconfluent cells were serum-starved for 16h followed by incubation with 10 ng/ml ADM2 peptide (Pheonix Pharmaceuticals, Burlingame, CA) for the indicated time length: 0, 5, and 30min . Cells were lysed in RIPA buffer (Catalog # R0278, Sigma-Aldrich, St Louis, MI), supplemented with protease inhibitor cocktail (Catalog # 11 873 580 001, Roche Diagnostics, Mannheim, Germany) and phosphatase inhibitor cocktails (Catalog # P0044 and P5726, Sigma-Aldrich, St Louis, MI) as instructed by manufactures. Total cell lysates were subjected to immunoblotting analysis as described previously2. The membranes were hybridized with antibodies recognizing phospho-ERK (at 1:2,000 dilution of Catalog # 4370, Cell Signaling Technologies, Danvers, MA), total ERK (at 1:1,000 dilution of Catalog # 4695, Cell Signaling Technologies, Danvers, MA), PKCα (at 1:500 dilution of Catalog # 610108, BD BioSciences, San Jose, CA), and β-actin (at 1:10,000 dilution of Catalog # sc-47778, Santa Cruz Biotechnology Inc., Dallas, Texas). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG (Zymed Laboratories Inc., San Francisco, CA). Western signals were visualized with enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA).\n\n\nResults and conclusion\n\nAs shown in Figure 1, ADM2 increased phosphorylation of ERK in endothelial cells. However, there was no difference in ERK phosphorylation levels in wt versus PKCα null endothelial cells (Figure 1). Our results indicate that ADM2 activates ERK in endothelial cells via a PKCα – independent pathway.\n\nRepresentative immunoblot showing that ADM2 increased phosphorylation of ERK, via a PKCα-independent pathway, in endothelial cells. Mouse endothelial cells (mEC) were isolated from wild type (wt) and Protein kinase Cα knockout mice (PKCα-/-). Cells were serum-starved overnight followed by stimulation with ADM2 synthetic peptide (10ng/ml) for indicated time and cell lysates were analyzed by immunoblotting for ERK activation.\n\n\nData availability\n\nF1000Research: Dataset 1. Gel images for ‘Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway’ by Guo X., et al., 10.5256/f1000research.2420.d1106897",
"appendix": "Author contributions\n\n\n\nXG cultured and treated cells and performed Western analyses, and prepared the manuscript; RJ isolated endothelial cells from mice, CC and MS directed the study.\n\n\nCompeting interests\n\n\n\nThere is no disclosure of competing interests.\n\n\nGrant information\n\nThis work is supported by OHSE fund (Department of Surgery, Yale School of Medicine) funding to XG.\n\n\nReferences\n\nKobayashi Y, Liu YJ, Gonda T, et al.: Coronary vasodilatory response to a novel peptide, adrenomedullin 2. Clin Exp Pharmacol Physiol. 2004; 31(Suppl 2): S49–50. PubMed Abstract | Publisher Full Text\n\nGuo X, Schmitz JC, Kenney BC, et al.: Intermedin is overexpressed in hepatocellular carcinoma and regulates cell proliferation and survival. Cancer Sci. 2012; 103(8): 1474–1480. PubMed Abstract | Publisher Full Text\n\nDong F, Taylor MM, Samson WK, et al.: Intermedin (adrenomedullin-2) enhances cardiac contractile function via a protein kinase C- and protein kinase A-dependent pathway in murine ventricular myocytes. J Appl Physiol (1985). 2006; 101(3): 778–784. PubMed Abstract | Publisher Full Text\n\nAslam M, Pfeil U, Gündüz D, et al.: Intermedin (adrenomedullin2) stabilizes the endothelial barrier and antagonizes thrombin-induced barrier failure in endothelial cell monolayers. Br J Pharmacol. 2012; 165(1): 208–222. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith RS, Gao L, Bledsoe G, et al.: Intermedin is a new angiogenic growth factor. Am J Physiol Heart Circ Physiol. 2009; 297(3): H1040–1047. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCorti F, Finetti F, Ziche M, et al.: The syndecan-4/protein kinase Cα pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo. J Biol Chem. 2013; 288(18): 12712–12721. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuo X, Ju R, Cha C, et al.: Dataset 1 in: Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway. F1000Research. 2015. Data Source"
}
|
[
{
"id": "12575",
"date": "19 Feb 2016",
"name": "James G. Taylor VI",
"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 “Adrenomedullin 2 activates extracellular-signal-regulated kinase in endothelial cells via a protein kinase C α-independent pathway” presents an elegant mutational analysis of cellular activation by the enigmatic adrenomedullin 2. Endothelial cells harvested from PKC-α knock out mice still demonstrate ERK phosphorylation, suggesting that the current paradigm of ADM2 intracellular activation (ADM2 signalling occurring through a PKC receptor pathway) is not completely understood. While this work is limited in scope, it does set a foundation for needed work in this field. There are some technical and context specific elements which would significantly strengthen this research note.Major points:Which specific ADM2 peptide was used? It would be helpful to mention this in the methods; ADM2-47 is believed to be the more potent agonist compared to the -40 peptide. Perhaps it would also be appropriate to test both ADM2 peptides (both -47 and -40) for comparison. The abstract and introduction sets the context for this work by describing ADM2 signalling in cardiomyocytes, but this research note presents data for arterial endothelial cells. The addition of the same phospho-Westerns for the same PKC-α knock out mouse cardiomyocytes would address this criticism. The results section is clearly presented, but a brief discussion and conclusion to put these results in a physiologic context would again enhance the impact of this research note. Specifically, this reviewer could cite a review by Hong et al. (2012) where it is stated that the receptors that mediate ADM2 action are not completely understood (i.e. the authors results suggest that perhaps the calcitonin like receptor pathway for ADM2 might activate endothelial cells independent of protein kinase). Presenting an updated version of Figure 4 from this review would also further work in this field and put the results in context.",
"responses": []
},
{
"id": "13696",
"date": "04 May 2016",
"name": "Hajime Takizawa",
"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 purpose of the current study was to investigate whether ADM2 activates ERK through PKCα, which is a major upstream activator of ERK in endothelial cells. The authors examined the effect of phosphorylation of ERK on ADM2 stimulation in endothelial cells isolated from PKCα null mice or wild type (wt) counterpart mice. The findings obtained in their experiments were clear. However, they studied by only a single method. They need to study, for example, by siRNA method to certify the independency on PKCα. Another concern is that the authors are expected to, at least, discuss the crucial signal pathway to activate ERK in this cell system. In this context, they need to study Ras-Raf-MAPK/ERK kinase (MEK).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-26
|
https://f1000research.com/articles/4-33/v1
|
02 Feb 15
|
{
"type": "Research Article",
"title": "The Dutch Techcentre for Life Sciences: Enabling data-intensive life science research in the Netherlands",
"authors": [
"Lars Eijssen",
"Chris T. Evelo",
"Ruben Kok",
"Barend Mons",
"Rob W.W. Hooft",
"and the other founding members of DTL Data (see Acknowledgements)",
"Chris T. Evelo",
"Ruben Kok",
"Barend Mons",
"Rob W.W. Hooft"
],
"abstract": "We describe a new national organisation in scientific research that facilitates life scientists with technologies and technological expertise in an era where new projects often are data-intensive, multi-disciplinary, and multi-site. The Dutch Techcentre for Life Sciences (DTL, www.dtls.nl) is run as a lean not-for-profit organisation of which research organisations (both academic and industrial) are paying members. The small staff of the organisation undertakes a variety of tasks that are necessary to perform or support modern academic research, but that are not easily undertaken in a purely academic setting. DTL also represents the Netherlands in the ELIXIR ESFRI, and the office supports this task. The organisation is still being fine-tuned and this will probably continue over time, as it is crucial for this kind of organisation to adapt to a constantly changing environment. However, already being underway for several years on the path to professionalisation, our experiences can benefit researchers in other fields or other countries setting up similar initiatives.",
"keywords": [
"Data",
"technologies",
"initiative"
],
"content": "Introduction\n\nIn this introduction we will explain the origin of DTL, the change in Dutch funding of life science technology that led to the start of the DTL foundation, and how the efforts of the DTL Data programme fit in the parallel development of professional data stewardship and knowledge structuring initiatives in science overall.\n\nDuring the preparatory phase of the ELIXIR ESFRI (elixir-europe.org), in 2012, several high profile bioinformatics and systems biology representatives started an initiative called DISC, The Data Integration and Stewardship Centre. They met several times to discuss the implementation of ELIXIR in the Netherlands. In parallel to this, the initiative to establish the Dutch Techcentre for Life Sciences was launched on the 31st of October 2012. The DTL organisation was started as a platform of leading universities, research institutes, university medical centres, science funders, government funding sectors (‘topsectoren’ in the Netherlands) and private companies from the health, nutrition, agrigenomics and industrial microbiology and information engineering sectors. We soon discovered that there was a significant overlap in the goals of the two initiatives, and it was decided to merge DISC into DTL as its Data programme. Starting from the 1st of January 2014, organisations have been signing up for formal membership of DTL.\n\nThe initiative for DTL was based on the growing data challenge as well as the changing funding landscape in the Netherlands. From 2003 to 2013 significant funding in the Netherlands went to institutes developing technical services and techniques for life sciences. For example, the Netherlands Bioinformatics Centre (NBIC) operated between 2004 and 2014 as a nation-wide initiative of bioinformatics experts in academia and industry. These institutes were expected to foster technology research, drive the exchange of methodology among labs and translate these into technical services that other scientists could use. Around 2012 it became clear that in the future the development and use of these technologies would no longer receive similar direct funding, and that research projects that apply a technology would need to budget for that. Rather than letting the previous investment in the technological institutes go to waste and let life scientists each replicate the experience at their own institutes, the technology institutes decided to develop a work form in which they would continue to exchange expertise across technology disciplines, build up a collective and well-accessible research infrastructure (RI) and deliver the services required. This has led to the formation of DTL.\n\nNext to the historical perspective, there are also forward-looking reasons for the start of the DTL organisation:\n\nTechnology programmes working together in a single organisation enable the application of what we call integrated life science research requiring the use of multiple technologies in a single research project, and the integration of generated and already available data.\n\nMembers of DTL can collaboratively draw attention to the fact that the fundamental developments in the technology fields require more attention of both the collaborating research organisations as well as the national funding agencies. Together we can look for solutions to tackle these challenges.\n\nEstablishing a collective technology platform of the major research organisations in the Netherlands provides further chances to establish international partnerships for individual member organisations or as a collective.\n\nThe organisational structure of DTL comprises three main programmes, as described below.\n\nDTL is governed by a board that is advised by a scientific advisory committee, and operations are monitored by a board of representatives from the partner institutes. DTL has organised its actions in three areas, Data, Technologies and Learning, which run as individual but cross-connected programmes within the organisation.\n\nDTL Learning, to start with the third area, manages an inventory of all training needs and offerings in life science technologies. It forms the bridge to the national Research School on Bioinformatics and Systems Biology (BioSB, biosb.nl) and other related research schools, and maintains contacts with all academic institutes that offer bioinformatics bachelor and master programs or postdoctoral training. DTL Learning also bundles expertise available in the DTL network, and organises both ad hoc and repeated training and courses on diverse subjects related to developments in the Data and Technologies programmes.\n\nDTL Technologies bundles more than 100 research labs that offer support to life scientists with different technologies (so called technology hotels). These technology hotels include a wide coverage of a variety of experimental (e.g. next generation sequencing, proteomics, metabolomics, bioimaging) technologies as well as bioinformatics and systems biology expertise. DTL Technologies facilitates the contact between the technology hotels and external researchers as potential customers e.g. through the organisation of funding calls that encourage new collaborative projects. In the DTL Technologies programme we will also work on harmonising and optimising access to hotels to make it easier for life scientists to use the latest technological opportunities and access multiple facilities in parallel.\n\nDTL Data brings together experts on every aspect of data stewardship, tools and databases, and e-Infrastructure. DTL Data builds relations for the people involved in the other DTL programs and partner organisations and connects to international initiatives such as ELIXIR, the pan-European life science research data infrastructure. The setup of DTL Data has gone hand in hand with more generic developments related to data and knowledge handling in the life sciences that we will address first.\n\nThe rise and wide application of modern data-intensive technological approaches in the life sciences has led to pressure on funders to provide support to keep acquired data around for longer than a project lasts. As such, initially in the US, and later in Europe, funding agencies have started demanding data stewardship to be an integral part of all scientific research projects. This is important because present-day research projects collect much data that intrinsically has more value than the first project will extract. Acquiring such data a second time is unnecessarily expensive, and this makes data stewardship a good investment. Furthermore, good data stewardship is required to make the work reproducible. In addition, proper structuring of knowledge sources that represent the aggregated and possibly curated findings of the body of previous research is of equal importance to fully enable integrative research.\n\nDTL facilitates data stewardship and knowledge structuring in all associated projects through participation in the development and deployment of the FAIR initiative. The FAIR acronym stands for Findable, Accessible, Interoperable, and Reusable (datafairport.org). To allow data and knowledge sources to be findable and accessible by both humans and computer systems requires a standardised description of metadata and study capturing as well as long-term storage and proper licensing. Interoperability and reusability require the representation of data and knowledge in such a way that they can be easily combined and used for further analytical processing1.\n\nTo support practical implementation of good data stewardship, DTL and its Data programme are on a mission to bring together all experts that can help life scientists with different aspects of their data management, and to show life scientists that it is not efficient to do everything in house using local solutions.\n\nThe remainder of this paper describes the organisational structure and approaches of the DTL Data programme in more detail.\n\n\nContent of the DTL Data programme\n\nDTL-associated scientists and engineers are responsible for data integration and stewardship in various life science initiatives in different life science sectors. They bring expertise, reusable tools and databases that have been developed in the Netherlands or elsewhere, and have access to a shared e-infrastructure.\n\nDTL brings together experts with a very diverse professional expertise in life science data management. This expertise is classified along four independent dimensions:\n\nThe life science sector: current activities are in health, agri/food, nutrition, and industrial biotechnology.\n\nLocation: even though the Netherlands is a relatively small country, a local expert is sometimes preferred for an advice or in a collaboration.\n\nPhase in the data lifecycle: we distinguish expertise in planning an experiment, collecting data, data processing, data analysis, data and knowledge integration, and modelling. There is also underlying expertise in biostatistics, systems biology, instrumentation, data security, computing infrastructure, and computer science approaches.\n\nTechnical discipline and type of data: e.g. genomics, proteomics, metabolomics, bioimaging, biobanking, knowledge representation.\n\nAll expertise can be classified along those four dimensions. To make all of this available to life scientists everywhere, we are working on setting up a network of local expert centres at different sites. Such expert centres can function as help desks: places where information can be obtained about the expertise available locally as well as elsewhere. Representatives of the expert centres are involved in frequent contact with each other to learn about new developments and learn of each other’s experience (both in techniques and in organisation). Over time, DTL will also extend its own help desk that can guide people to the right expert centres.\n\nA very important mission of DTL Data is to prevent projects from running into problems because of unconscious incompetence; we want to facilitate early interaction between life scientists with a specific plan and experts in all the technical fields that they need to engage, to avoid underestimating technological tasks or risks.\n\nMany of the experts collaborating in the DTL Data programme have (co)developed reusable tools and databases. For such tools there is ample experience to implement their use in different projects. Such tools can often be reused by a new project in an existing shared deployment with dedicated help for users. In other cases, specialised installations of the software can be made, tailored to the project. DTL has a strong preference for reuse of existing tools, which have proven their value in earlier national or international projects. Advantages of such tools are that they have overcome their teething problems, that their continued development benefits multiple projects, and that the reuse increases interoperability with other tools and existing data.\n\nIn the past, many life science labs have each been taking care of their own needs for computing. More and more, however, the need for data processing becomes too large to handle. Furthermore, server system maintenance is not a core competence of a life scientist, and keeping a local cluster running should not be the task of a PhD candidate. Computing and data storage are becoming an infrastructure: equipment that nobody can do without, and which is inefficient to duplicate for every project. Many groups are therefore no longer willing to maintain the needed infrastructures themselves, and set up institutional services together employing specialised people for maintaining the computing equipment. Additional benefits of such centralisation efforts are flattening-off peak demands and allowing individual projects to be run at relatively short notice. Also, it reduces the need for synchronising new equipment purchase with the start of new projects, which without central facilities results in waste for short projects and the use of outdated computing resources for longer projects. DTL brings experience from centralisation efforts together, and ensures alignment with the national centres for computing. Together, these people work on harmonising the computer centres so that migration of computing work and federation of resources become easier. When a new data intensive life science project is started with new demands for computing or storage, the best solution for the location of such computing is found in collaboration.\n\nThe e-Infrastructure that can be shared is not limited to the computer racks (Infrastructure as a Service, IaaS). We also investigate possibilities for sharing higher level platforms (Platforms as a Service, PaaS), for example the workflow supporting software Galaxy2, which has been supported by the Netherlands bioinformatics centre in the past, and potentially other shared infrastructures for systems biology. We are also working together on a shared data publishing infrastructure based on experience from the Open PHACTS project3.\n\n\nOrganisation of the DTL Data programme\n\nThe DTL Data programme is coordinated by a programme manager from the DTL Office. All projects are executed by DTL partners, outside of the office. The primary organisation of DTL Data is per sector of life science research (Figure 1). We organise several kinds of meetings for different target groups, which we have identified as fulfilling an urgent need: project leader meetings, programmer meetings and so-called focus meetings. We also identify people with similar interest and facilitate interest groups and working groups with their own meetings. Each of these types of events will be described in more detail.\n\nDTL has a small office that organises and coordinates programme management, community and communication for the different programmes. The actual DTL Data projects are run by the partners outside of the DTL Office. The coordination structure is primarily divided into four sectors.\n\nProject leader meetings\n\nWithin each of the life science Sectors, DTL Data brings project leaders together who are each functionally coordinating the progress in a particular project.\n\nFor the healthcare sector, this is a continuation of a weekly project leader meeting that has been running since 2009, and involves 10 project leaders meeting 60 minutes every week. These meetings are conducted as teleconferences where the participants collaboratively edit the meeting notes. This style of focused reporting of what has been accomplished and what is planned builds trust between the project leaders and leads to many accidental discoveries of potential synergy between their projects. This results in cost savings for the projects and does not stand in the way of healthy competition. These meetings also provide a direct connection to TraIT, the IT project for the Dutch translational medicine project CTMM.\n\nThe other sectors (agrigenomics, nutrigenomics and industrial biotechnology) are now in the process of setting up similar meetings. The principle project leaders who will be leading these meetings have been identified. These four principal project leaders will be meeting together on a monthly basis to discuss progress and to identify synergies between the sectors.\n\nProgrammers meetings\n\nMany of the programmers involved in the bioinformatics projects in the different sectors of DTL Data are so-called embedded programmers, often the only bioinformatician in a biology or medical setting. Others work together in groups. In DTL Data, we call programmers from both settings together every two months for lectures and workshops on topics ranging from programming techniques to biological applications. Sometimes we invite external speakers, but most topics are presented by members of the group. This way they keep each other informed. At these meetings we also encourage interactions between programmers in smaller groups.\n\nFocus meetings\n\nDuring our work we regularly recognise similar problems or solutions being raised in more than one context. For such topics we organise focus meetings. A focus meeting brings together a group of people that preferably have never met in that composition, to discuss a subject that is either crossing borders between technologies or between sectors. Focus meetings are not only organised by DTL Data, but also by the DTL Technologies and DTL Learning programmes. A focus meeting often contains a few short lectures, followed by a well-prepared discussion that engages the whole audience. After the meeting, a white paper is written by the organisers of the meeting that is published on the DTL website.\n\nInterest and working groups\n\nIf a group of people, e.g. after a focus meeting, feels the need to exchange experience more often, they can form a so-called interest group within DTL. DTL facilitates these interest groups with meeting rooms, and tries to find a young researcher as a champion of the group to keep it going. This is modelled after “Project and Area Liaisons” (PALs) from earlier EU and UK projects4. PALs are rewarded for introducing new ways of working: they are provided with extra support for their work and direct influence on the development of the new working methods.\n\nAn interest group that has identified an issue they want to work on together can form a working group. A working group needs to be supported by a part-time project leader to take the practical work out of the hands of the principal investigators. Each working group must deliver a practical result (deliverable) after a limited time. DTL is looking for ways to support the working groups by providing resources for the project leaders.\n\nBoth interest groups and working groups can be supported with a good software development environment, mailing lists, a website and a wiki to exchange information.\n\nThe data programme interacts with many organisations, both internal to DTL (other programmes and partners) as well as external (for instance IMI projects and RIs under the EC ESFRI scheme).\n\nHelp desk, training and education\n\nIn the day to day operations of the Data programme, we frequently come across needs for training: both training for data scientists to broaden their knowledge with newly developed technologies, as well as training for life scientists to make them aware of and teach them how to use solutions that are being developed in DTL Data projects. This is expected to become even more important once the development of local data desks in different institutions will be realised. The setup of these data desks will bring together experienced data scientists from different institutes, and they will find out that others have complementary expertise that they sometimes need to replicate. Also, life scientists with less experience will have a low barrier to approach their local data desk for advice, bringing in more demand for basic data awareness training. All of these training needs will be developed with the DTL training Programme, which is very well connected to people and organisations that can support this effort.\n\nData-related technology hotels\n\nMany of the people involved in DTL Data offer their services to life scientists as a Data hotel in the DTL Technologies Programme. DTL Data works with DTL technologies to define the needs of and requirements for these data-specific hotels. An overview of current DTL hotels is available at www.dtls.nl/expertise-services/hotels.\n\nELIXIR\n\nSynchronous with the development of the DTL organisation, bioinformatics institutes and laboratories all over Europe have set up the European research infrastructure for life science data and bioinformatics, ELIXIR. ELIXIR is organised as a hub hosted at the EBI in Hinxton, UK, and nodes in each of the member countries. In the Netherlands, DTL hosts the ELIXIR node (ELIXIR-NL). Association with ELIXIR gives us the possibility to reach out to experts and tools all over Europe.\n\nDTL and ELIXIR have developed the concept of so-called Bring Your Own Data (BYOD) parties as a platform to bring together data owners and data experts. Also biological domain experts are invited where relevant. The main goal of these meetings is to get data owners acquainted with the possibilities to connect and functionally interlink their data with other datasets and knowledge resources by applying FAIR principles. Researchers can suggest a BYOD party and DTL will assist with the logistics and invite data experts.\n\nOther ESFRI programmes and national projects\n\nEurope has many other Research Infrastructures in the life sciences, each with their own special focus. Also in the Netherlands several larger project organisations are active in life science research. All of these have their own research data and associated challenges. In the Netherlands we make sure that the people working with that data are co-developing and steering the DTL Data Programme. This ensures that the methods and tools they use are compatible with the ELIXIR choices and avoids unnecessary duplication of development efforts.\n\n\nConclusion\n\nLife science research becomes more and more data intensive and cross-disciplinary at unprecedented scales. Individual research groups do not have the resources and the interest to keep in contact with all expert providers and keep informed of the progress of other related projects at such scales. In the Netherlands we have developed a networked approach to accommodate for the challenges posed by modern data-intensive life science research. The establishment of DTL as a collective platform that brings together experts in various technological disciplines across life science domains, facilitated by a small core team, allows projects to run efficiently. Already in the preparatory period and in the first year of operations we have identified synergies between parallel running research projects and found common interests from surprisingly differently focused researchers. The growing community of experts involved in DTL Data makes sure required data-related expertise can be located for any researcher in the life sciences starting on any new project. At the publication date of this article DTL had over twenty confirmed member organisations. The current partner list can be found at www.dtls.nl/about/partnership/.\n\nTo find out how DTL Data can support your challenges or for more inquiries about the setup of the organisation, contact Rob Hooft (programme leader) at rob.hooft@dtls.nl\n\nFor further information on the other programmes of DTL contact Ruben Kok (director DTL) at ruben.kok@dtls.nl\n\nWebsite: www.dtls.nl",
"appendix": "Author contributions\n\n\n\nAll authors have been involved in the setup of DTL Data and have contributed to the text of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has been funded by the author’s home institutes.\n\n\nAcknowledgements\n\nNext to the authors, the following people have played instrumental roles in setting up DTL Data, and all share a DTL affiliation:\n\nJan-Willem Boiten, CTMM-TraIT, Eindhoven\n\nLuiz Olavo Bonino, Dutch Techcentre for Life Sciences (Foundation office), Utrecht\n\nJildau Bouwman, TNO, Zeist and Netherlands Metabolomics Centre, Leiden\n\nRichard Finkers, WUR, Wageningen and Plant Research International, Wageningen\n\nFemke Francissen, Dutch Techcentre for Life Sciences (foundation office) and BioSB research school\n\nCelia van Gelder, Dutch Techcentre for Life Sciences, Utrecht and Radboudumc, Nijmegen\n\nMartien Groenen, WUR, Wageningen\n\nJaap Heringa, VU University Amsterdam and BioSB research school (Deputy Head of Node ELIXIR-NL)\n\nIrene Nooren, SURFsara, Amsterdam\n\nMerlijn van Rijswijk, Dutch Techcentre for Life Sciences (foundation office), Utrecht and Netherlands Metabolomics Centre, Leiden\n\nMarco Roos, LUMC, Leiden\n\nMorris Swertz, UMCG, Groningen\n\n\nReferences\n\nMons B, van Haagen H, Chichester C, et al.: The value of data. Nat Genet. 2011; 43(4): 281-3. PubMed Abstract | Publisher Full Text\n\nGoecks J, Nekrutenko A, Taylor J, et al.: Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams AJ, Harland L, Groth P, et al.: Open PHACTS: semantic interoperability for drug discovery. Drug Discov Today. 2012; 17(21–22): 1188-98. PubMed Abstract | Publisher Full Text\n\nGoble C, Wolstencroft K, Owen S, et al.: SysMO-DB: A pragmatic approach to sharing information amongst Systems Biology projects in Europe. In: Proceedings of the UK e-Science All Hands Meeting. 2009. Reference Source"
}
|
[
{
"id": "7548",
"date": "25 Feb 2015",
"name": "Steffi Suhr",
"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 drivers and motivation behind setting up the Dutch Techcentre for Life Sciences and describe the organisational setup so that the experiences gained can benefit researchers in other fields or countries setting up similar initiatives. As the number of initiatives seems to be steadily growing, this is helpful and could indeed provide some useful background against which other efforts could model themselves.However, trying to come at this from the perspective of someone who might want to implement such an infrastructure, there are some bigger and smaller points that would need to be addressed to be able to give this paper an \"approved\".The focus is slightly unclear: the abstract and introduction describe the DTL in its entirety, including its history, while more than three quarters are focused on the services related to the last area of activity listed, \"DTL Data\". The manuscript would benefit hugely from a reorganisation that makes the main focus clearer right at the beginning. It might also be worth considering to put the description of the background and origin (timeline, why was DTL started) at the end of the manuscript. Alternatively, the authors might want to consider leaving the history out of the manuscript completely: at present, the first point that seems to be mentioned is that funding became more scarce, rather than focusing on the benefits. Abstract: as mentioned under (1), the focus on data-related issues should be reflected. It would also be helpful to (very briefly) give examples of \"tasks ... that are not easily undertaken in a purely academic setting\", as this seems to be one of the core drivers behind DTL. Examples for why and how the environment is changing, and how DTL can address this, would be good in the introduction (rather than leaving this hanging in the abstract). The part about \"governance and organisational programmes\" is confusing: governance structures are not really described in any detail (who is on the board, and why?). In general, it is unclear that this information adds value to the manuscript - as organisational structures will often heavily depend on local framework conditions, the paragraph on the board and SAB could be left out and the section simply renamed \"DTL programme areas\" or similar. Section \"parallel developments..\": the opening paragraph on open data is quite weak and possibly incorrect (e.g. the push towards open data does not originate from the application of data-intensive technologies - arguably, data intensive technologies provide a challenge for open data). For the purpose of this manuscript, less would be more. Going into the Data programme, section on Tools & databases: which tools are supported by DTL? How are they selected and/or prioritised? What is the DTL contribution to these tools? e-infrastructure: It might be interesting to explain if there any interactions with European-wide e-infrastructures. Meetings described: There is an odd level of detail. What is missing is who is involved and why, in particular in the case of the project leader meetings. If these are a continuation of TraiT meetings it is clear that there would be a spirit of collaboration, but what about the \"other sectors\" mentioned - how were participants identified, how will the aims be stated, how will members be motivated? Focus meetings and working groups: who prioritises the topics, and based on what? Relations with external programmes: ELIXIR - \"..gives us the possibility to reach out to experts and tools all over Europe\" - how about the other way around, can needs also be identified across DTL that would better be met at the European level?Minor points (mostly language):Page 2: \"...and let life scientists each replicate the EXPERTISE at their own institutes, the technology institutes decided to develop a FRAMEWORK in which they...\" Page 3:\"..., and Reusable (datafairport.org). ALLOWING data and knowledge sources..\"\"All expertise can be classified along those four dimensions\" - delete sentence (redundant). Next sentence, change to: \"To make expertise fulfilling all four dimensions available,..\"\"Representatives of the expert centres are in frequent contact..\" (delete \"involved\").Change to: \"More and more, however, the data to be processed becomes too large to handle.\" Conclusion: \"...and found common interests across researchers with a focus in surprisingly different disciplines. ... makes sure NECESSARY data-related expertise..\" ELIXIR should be listed by its name (without \"ESFRI\"), with the full name at first mention",
"responses": [
{
"c_id": "1743",
"date": "06 Jan 2016",
"name": "Lars Eijssen",
"role": "Author Response",
"response": "Thank you for the extensive and detailed feedback. Herewith a point-by-point response.We rephrased the abstract to directly introduce the Data programme of the DTL, instead of DTL as a whole. We still kept some background on DTL in the introduction, since this gives the context in which DTL Data was set up and lives, but also there we adapted the first lines to mention the programmes, including Data, right away. Furthermore, we put the timeline in a separate box to improve the structure of the text. Also, we rephrased the text to indicate the benefits that clearly underlie both changes in funding policies as well as the Data programme. Data-related issues get more focus in the adapted abstract, as described in the response to the first point. We also added a phrase to exemplify the tasks referred to (“DTL Data takes care of such tasks related to data stewardship, facilitating exchange of knowledge and expertise, and brokering access to e-infrastructure.”). We now also already briefly mention the generic environmental changes right at the opening of the introduction: “The initiative for DTL was based on the growing complexity of life sciences projects requiring multidisciplinary collaboration, coinciding with an increase in variety and volume of data.” For DTL Data specifically, this is further detailed in the ‘Parallel developments: data stewardship and knowledge structuring’ subsection right after the introduction of the three programmes. We have adapted the paragraph in accordance with these comments. We renamed the section and removed the opening line on the board. At the end, we included a short paragraph and a figure on the governance structure, but more focused at its relationship to the programmes including Data. We agree this was not clearly formulated. It is an indirect ‘push’: because of the more generic value such data has, making the data open gets even more important. We rephrased the opening sentence to: “Driven by the rise and wide application of modern data-intensive technological approaches, present-day research projects in the life sciences collect much data that intrinsically has more value than the project itself will extract”. Also, we reformulated the start of the paragraph to indicate that this is the reason for data stewardship (and for funders to ask for this) rather than funder’s requirements being its original cause. DTL, at this moment, is not performing any selection or endorsement of tools and databases. DTL Tools are supported by DTL partners that have developed the tools or by power users in the organisation. DTL, however, is actively involved in the inventory of Dutch tools for the ELIXIR tools registry (https://bio.tools/). Selection does take place only when the initiative is taken to start supporting the tools or databases in an international context. In such cases the tools are verified to be one of a kind, best of breed and have sufficiently scalable support infrastructure. DTL can then suggest to include the tools in the ELIXIR programme. To be accepted into the ELIXIR programme, tools have to be part of the node plan, which must be accepted by the ELIXIR SAB. No tools have been added to the ELIXIR NL portfolio yet. A shortened version of this text has been included in the ‘Tools & databases’ subsection. To name some important initiatives, we added the clause: “…as well as international e-infrastructure projects like the European Grid Initiative (EGI, www.egi.eu) and EUDAT (www.eudat.eu).” We removed a substantial part of the detailed description, including the background related to the project leader meetings. Also we added a short line on who initiates the topics for focus meetings (“These common interests can be signalled by the programme managers or brought up by DTL scientists”), but not in too much detail to keep this part short. Yes, obviously it would be a two-way interaction, as is the basic idea of ELIXIR. To make this more clear we added: “Each of the nodes contributes specific expertise to the ELIXIR network. The Dutch node contributes expertise related to interoperability, learning, and computer and network infrastructure. Through the network, DTL-associated scientists can benefit from all European contributions.”Minor points: all adapted according to suggestion."
}
]
},
{
"id": "7547",
"date": "16 Mar 2015",
"name": "Karin Verspoor",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper describes the structure and role of the Dutch Techcentre for Life Sciences (DTL), with a specific emphasis on the DTL Data programme. The DTL is an important initiative in the Netherlands to support effective and efficient data-intensive life science research throughout the Dutch research community by facilitating the connection of researchers with computational tools, expertise, and infrastructure.As the first reviewer has identified, there are some structural issues in the presentation of this paper that could improve its focus and clarity. There are also some more minor issues, for instance a lack of background on certain referenced programs when first mentioned (ELIXIR is introduced in the second paragraph of the Introduction, but not defined until several paragraphs later; not all readers may be familiar with the program).Elaborating on the first reviewer's point about e-infrastructures, it would be helpful for the authors to relate the model described in the paper, as developed for the DTL, to other possible models that exist, for instance the National Centers for Biomedical Computing in the US (see e.g., http://jamia.oxfordjournals.org/content/jaminfo/19/2/151) or bioinformatics core facilities in place at a more local level (see e.g., the discussion at http://bioinformatics.oxfordjournals.org/content/27/10/1345). Given the emphasis on data management and computing infrastructure of the DTL Data programme, cloud-based generic e-research infrastructure supported at a national level (e.g. for social sciences research or the Australian National eResearch Collaboration Tools and Resources (NECTAR), e.g., the Genomics Virtual Laboratory and other Australian research infrastructure programs) are also relevant. I believe that understanding how the DTL Data model is different or unique in the global context is important to support the authors' goals of providing insight to new efforts. Furthermore, it would be helpful to more clearly distinguish the role of the DTL from the national centres for computing to which the DTL is aligned (p. 4).There are a few minor issues with wording choices that the authors may wish to revisit, e.g. the phrase \"the path to professionalisation\" -- in what sense is the organisation being \"professionalised\"? (this recurs in the phrase \"professional data stewardship\") -- and the phrase \"unconscious incompetence\" which sounds perhaps more severe than the authors intend.",
"responses": [
{
"c_id": "1744",
"date": "06 Jan 2016",
"name": "Lars Eijssen",
"role": "Author Response",
"response": "Thank you for your feedback. Herewith our response.Response to \"As the first reviewer...familiar with the program).\":We improved the introduction of ELIXIR in the text. In the abstract we added context by: “ELIXIR, the European infrastructure for life science data”. Furthermore both in the abstract and the timeline we removed the mentioning of the term ‘ESFRI’, to only introduce this (but now written in full) in the ‘Relation with external programmes’ section by: “…and infrastructures under the European Strategy Forum on Research Infrastructures (ESFRI) scheme, including ELIXIR”.Response to \"Elaborating on ... DTL is aligned (p. 4).\":An important distinction between DTL and other institutes with similar functions in other countries is that DTL was not set up as an institute by a (national) funding organisation (like e.g., the National Centers for Biomedical Computing in the US, and NECTAR in Australia), but as a collaboration institute funded primarily by partner organisations. Where such bottom-up efforts to set up a supporting organisation is seen, they are often localised to a single research institute and rarely started as a public-private partnership. We have also included this text in the manuscript, including suggested references, in the ‘Why was DTL started?’ section.Related to the last point, DTL has more a role as an orchestrator between research projects, generic computing initiatives, and national computing centres. As suggested by the first reviewer, we added some international initiatives to this phrase, widening the scope. Furthermore, we modified the next phrase (“Together these people work…”) to “DTL links to and between the people that work…” to make DTL’s role as a connector more clear.Response to \"There are a few minor...the authors intend.\":We removed both occurrences of professionalisation/professionalised, as they were superfluous.The phrase ‘unconscious incompetence’ was a deliberate choice, as a term coined to indicate the issue not being aware of not doing something according to standards. We added a reference to a paper by Kruger et al. on this topic."
}
]
},
{
"id": "7550",
"date": "24 Mar 2015",
"name": "Bryn Williams-Jones",
"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 outlines the background, formation and to an extent the operation of the Dutch Techcentre for Life Sciences (DTL) with a focus on the data programme, DTL-Data. Given this type of organisation is of relevance to data researchers in other nations, researchers in other fields, and set against a background of increasing data complexity and institutional networking in the wider community, this could provide a model for other inititatives. In common with the other reviewers, there are some issues with the presentation which would help readers new to DTL to understand the background, structure and operation of this entity. To encourage similar organisations in other nations, the historical background is relevant, but could be split to a separate section - as a text box/diagram or similar to aid the flow of the description of DTL operations as they are now.Whilst DTL-Data is discussed in some detail, the other sections are briefly mentioned and its challenging to gain a picture of the holistic DTL and the competencies necessary to establish a similar organisation in other nations. Again, as noted by the other reviewers, an outlook comparing other national efforts, their relation to DTL, what may have particularly aided development of DTL in the Netherlands from both a structural/funding and cultural/operational perspective would be useful. Similarly examples of FAIR data principles, information on some of the tools and databases mentioned but not named could help researchers in other nations contextualise the assertions made. This may be of more value to those less familiar with the operations of DTL than detailed description of the meetings which would be better summarised in less detail.",
"responses": [
{
"c_id": "1745",
"date": "06 Jan 2016",
"name": "Lars Eijssen",
"role": "Author Response",
"response": "Thank you for your feedback. Herewith our responses to the points raised.Response to \"In common with...as they are now\":According to the suggestion, we have added a separate ‘timeline’ box to describe the historical context of DTL. Furthermore we have made more clear right from the opening of the paper that it is mainly about the Data programme, and described the remaining DTL context more in connection to this focus.Response to \"Whilst DTL-Data...in other nations\":Even when trying to keep the text on the other parts of DTL brief, and making more clear in the text that this paper specifically addresses the DTL Data programme, we have added an extra short paragraph and a diagrammatic figure (Figure 1 and the paragraph directly preceding it) on the organisation of DTL as a whole.Response to \"Again, as noted by...summarised in less detail\":We rephrased the ‘Why was DTL started’ subsection to more clearly introduce its benefits and reasons for setup and to extend the description of the projects that were running in the Netherlands before DTL, to better sketch the national operational and funding perspectives. We have substantially reduced the level of detail in our description of the meetings. For (stewardship and other) tools and databases, we deliberately had not included examples in order not to select some above others, since DTL partners are the entities that are offering and supporting tools, rather than DTL centrally, which does not enforce specific ones. We now included a paragraph to the manuscript (also in response to a similar point made by the first reviewer) clarifying this aspect and mentioning the relationship between DTL and ELIXIR with respect to tool selection, referring to the latter’s tool registry (‘Tools & databases’ subsection)."
}
]
}
] | 1
|
https://f1000research.com/articles/4-33
|
https://f1000research.com/articles/3-116/v2
|
21 Apr 15
|
{
"type": "Review",
"title": "Post-operative rehabilitation and nutrition in osteoarthritis",
"authors": [
"Giuseppe Musumeci",
"Ali Mobasheri",
"Francesca Maria Trovato",
"Marta Anna Szychlinska",
"Rosa Imbesi",
"Paola Castrogiovanni",
"Ali Mobasheri",
"Francesca Maria Trovato",
"Marta Anna Szychlinska",
"Rosa Imbesi",
"Paola Castrogiovanni"
],
"abstract": "Osteoarthritis (OA) is a degenerative process involving the progressive loss of articular cartilage, synovial inflammation and structural changes in subchondral bone that lead to loss of synovial joint structural features and functionality of articular cartilage. OA represents one of the most common causes of physical disability in the world. Different OA treatments are usually considered in relation to the stage of the disease. In the early stages, it is possible to recommend physical activity programs that can maintain joint health and keep the patient mobile, as recommended by OA Research Society International (OARSI) and European League Against Rheumatism (EULAR). In the most severe and advanced cases of OA, surgical intervention is necessary. After, in early postoperative stages, it is essential to include a rehabilitation exercise program in order to restore the full function of the involved joint. Physical therapy is crucial for the success of any surgical procedure and can promote recovery of muscle strength, range of motion, coordinated walking, proprioception and mitigate joint pain. Furthermore, after discharge from the hospital, patients should continue the rehabilitation exercise program at home associated to an appropriate diet. In this review, we analyze manuscripts from the most recent literature and provide a balanced and comprehensive overview of the latest developments on the effect of physical exercise on postoperative rehabilitation in OA. The literature search was conducted using PubMed, Scopus, Web of Science and Google Scholar, using the keywords ‘osteoarthritis’, ‘rehabilitation’, ‘exercise’ and ‘nutrition’. The available data suggest that physical exercise is an effective, economical and accessible to everyone practice, and it is one of the most important components of postoperative rehabilitation for OA.",
"keywords": [
"Osteoarthritis",
"Rehabilitation",
"Exercise"
],
"content": "Introduction\n\nOsteoarthritis (OA) is a degenerative disease of load-bearing synovial joints1–5. Knee OA is the most common type of OA6 and represents one of the most common causes of physical disability in the world7–9. Deterioration of the articular cartilage is the main problem associated with OA with consequent chronic pain and functional restriction10. OA can be caused by previous traumas (fractures, ligament tears and meniscal injury), wrong kinematics, obesity, genetics and age, which lead to alterations in the joint cartilage2,8,10. Traumatic injury to synovial joints is increasingly considered an important risk factor for the development of post-traumatic OA (PTOA). Traumatic injuries sustained during the lifetime of an individual, combined with normal age-related wear and tear, may conspire to facilitate the progression of degenerative joint diseases and may lead to chronic disability. OA is an insidious disease that typically develops gradually over the years with several symptoms including pain, stiffness, limited range of motion (ROM) in the joint and localized swelling. OA pain usually worsens after physical activity3,11–13, while stiffness arises after sitting for prolonged periods of time. As OA progresses, symptoms generally become more severe and then pain can become continuous. Generally, OA occurs when the dynamic steady-state between destructive forces and repair mechanisms alters the joint homeostasis7,14. For example, the tibiofemoral mechanics and loading patterns, during walking, influence the regional development of the articular cartilage8. Alterations in normal gait mechanics due to trauma, acute injury, ligamentous laxity, weight gain and improper footwear can shift the loading patterns to areas of the articular cartilage not well adapted to accept improper loads8. If patients do not improve with non-invasive therapies or have excessive pain and loss of mechanical function, OA treatment consists of surgical intervention15,16 and subsequent rehabilitation2,17,18,19. All patients with hip and knee OA should be informed about the objectives of the treatment and educated to the importance of all the measures that unload the damaged joint (lifestyle changes, regular exercise, weight reduction and other). The initial focus should be on self-help and patient-driven treatments rather than on passive therapies delivered by health professionals. Subsequently, emphasis should be placed on encouraging adherence to the regimen of non-pharmacological therapy20, as widely promoted during the last years (Table 1). Recently, the European League Against Rheumatism (EULAR) proposed 11 evidence-based recommendations for the non-pharmacological management of people with hip or knee OA21. Moreover, the OA Research Society International (OARSI) proposed other evidence-based recommendations, providing guidance to patients and practitioners on the treatments applicable to all individuals with knee OA, as well as therapies that can be considered according to specific patient needs and preferences22. In the present review, we analyze the effects of postoperative rehabilitation exercise program in OA patients treated with surgical procedures. The aim of this review was to underline the importance of exercise combined with an appropriate daily diet in postoperative rehabilitation for OA patients, and to present exercise as an effective and economical accessible to everyone.\n\n\nMaterials and methods\n\nIn this review, we analysed articles from the most recent literature, providing a balanced and comprehensive overview of the most important discoveries on pathogenesis and therapeutic approaches for osteoarthritis in to the context of post-operative rehabilitation and nutrition. Subsequently, the selected articles were divided in “Morphological aspects of osteoarthritis”, “First step postoperative rehabilitation in OA”, “Second step postoperative rehabilitation in OA” and “Nutrition in postoperative rehabilitation in OA”, to provide interested researchers with a detailed and schematic overview of all the recent studies on osteoarthritis. The literature search was conducted from in March 2014 to April 2015 on PubMed, Scopus, Web of Science and Google Scholar using appropriate keywords (osteoarthritis, rehabilitation, exercise and nutrition). Of approximately 160 papers (original articles, systematic and meta-analysis reviews) only 67 have been chosen and considered appropriate for the purpose of the review. The other papers, have not been considered as they resulted outside the scope of the research.\n\n\nMorphological aspects of osteoarthritis\n\nCartilage is the most commonly studied tissue in the joint in the context of OA research. It is a unique load-bearing connective tissue with viscoelastic and compressive properties that are largely due to the presence of extracellular matrix, mainly composed of collagen type II and the proteoglycan aggrecan9,17,18,23. OA is a degenerative process involving the progressive loss of structural features and functionality of the articular cartilage caused by an imbalance between anabolic and catabolic processes in the cartilage tissue, so that cartilage degradation exceeds reparative processes and OA progresses10,14,24. Generally, the surface of healthy hyaline cartilage appears white, shiny, elastic and firm. In contrast, OA cartilage shows a dull and irregular surface with discoloration, softening, and often with increased production of synovial fluid25. In advanced OA the cartilage shows signs of rupture; the cartilage surface is rough and broken by fissures and cracks which can reach down to the calcified zone26, and chondrocytes are arranged in clusters (especially around fissures) or disappear. The organization of cartilage is widely disordered and replaced by fibro-cartilaginous, scar-like tissue with fibroblast-like cells27. As described in detail by several authors, the development of a rheumatoid-like ‘pannus’ of various extents can overlay the damaged cartilage tissue,28,29. The extent of damage to the articular cartilage depends on the joint surface area, which is exposed to different loading patterns and conditions in distinct regions25.\n\n\nFirst step post-operative rehabilitation in OA\n\nPostoperative rehabilitation is crucial for the success of any surgical procedure30. It has the purpose of recovering muscle strength, range of motion, coordination in walking and mitigation of the pain. The postoperative rehabilitation program usually starts 48 hours after the surgery procedure as a result of the clinical evaluation of each specific case of OA. The rehabilitation is often long because of the time necessary for the cartilage cells to adapt and mature into repair tissue. Cartilage is a slow adapting tissue, indeed it undergoes 75% adaptation in approximately 2 years. When the rehabilitation period is too short, the cartilage repair might be put under too much stress, causing the repair to fail30. The type of postoperative exercise program depends on the injury. Experimental and clinical studies demonstrate that early, controlled mobilization is superior when compared to immobilization for primary treatment of acute musculoskeletal soft-tissue injuries and postoperative management. Early mobilization helps return the patients more quickly to physical activity, reduces persistent swelling, restores stability, restores ROM, and improves patient satisfaction with the rehabilitation outcome31.\n\nA postoperative rehabilitation exercise program should be personalized and based on the type of surgical procedure, location, size and depth of the lesion, in order to facilitate the healing process32, as well as on the age and medical condition. Arthroscopic procedures, such as chondroplasty or microfracture, may resolve faster than osteochondral autograph transplantation (OATS) or autologous chondrocyte implantation (ACI) that involve larger incisions, requiring a slower exercise rehabilitation program32,33. Since immobilization and unloading result in proteoglycan loss in articular cartilage and gradual weakening, controlled weight bearing and ROM are essential to facilitate the healing process and to prevent degeneration24,34,35. Furthermore, controlled compression and decompression forces during weight bearing nourish the articular cartilage and induce molecular signals necessary to produce an optimal extracellular matrix34. A force platform is a useful tool in the rehabilitation program to perform limited weight-bearing activities facilitate a normal gait pattern and enhance strength, proprioception, and balance32. The postoperative rehabilitation exercise program includes performance of motion exercises and muscle strengthening with any ambulatory aids (walker, sticks, forearm crutches), training in postural changes and in the execution of stairs. During rehabilitation, the passive range of motion (PROM) activities, in a limited ROM, are also indicated to nourish the healing articular cartilage and prevent the formation of adhesions36. Continuous passive motion (CPM) enhances cartilage healing and long-term outcomes following articular cartilage procedures36. As the lesion heals and symptoms decrease, the ROM is modified to allow greater muscle strengthening over a greater range of movement32. With surgical procedures, particularly with the OATS and ACI, because of the large incision and extensive soft tissue trauma, arthrofibrosis could take place and rehabilitation can avoid this event32. When the surgical procedure has implanted a prosthesis, depending on the type of prosthesis, the use of special machines for the passive flexion-extension of the joint is advisable.\n\nSymptoms, such as pain and effusion, could cause the inhibition of the muscles, so electrical muscle stimulation and biofeedback are complementary with the rehabilitation exercise program to promote the active contraction of musculature36.\n\nStretching exercises should be included as the patient progresses to advanced phases of rehabilitation32. As the patient returns to functional activities, it is important to increase gradually the amount of stress applied to the treated joint, to provide a stimulus for healing to cartilage tissues without causing damage32. The rehabilitation exercise program following surgical procedures for OA is fundamental to the long-term success and functional outcome of patients involved32 (Table 2).\n\nSchematic representation of primary rehabilitation activities that should be included in post-operative rehabilitation program soon after the surgery.\n\n\nSecond step post-operative rehabilitation in OA\n\nFollowing hospital discharge, the patient should continue the rehabilitation exercise program at home. The physiotherapist will indicate and teach the exercises to be carried out independently, aimed at maintaining a good muscular and articular quality. Patients surgically treated for OA often suffer from pain and have problems during everyday activities, and physical activity could attenuate these deficits37. Strengthening exercises, aerobic exercises or both together, show positive effects for both pain and physical function37,38. However, data from literature show that the long-term benefits of exercise have no significant effect on pain or physical function after 6 months, except when booster sessions are implemented39.\n\nResistance exercise decreases pain and increases physical function, reducing disability40. It includes loads, repetitions, movement speed and frequency of sessions, and often is supported by the use of machines or free weights41. Strength, ROM, pain throughout the range of motion and the possibility of patient to have access to the necessary equipment for exercise should be considered for a resistance exercise program41. When access to machines is too expensive for the patient, an exercise program should still be performed at home42–44. The resistance exercise program should be performed 3 days per week, with 2–3 sets per exercise at 8–15 repetitions per set41, and loads should vary from high to low44. The patient’s tolerance should take into account the initial resistance loads and the joint ROM41. The resistance loads or number of sessions per week should increase as the patient acquires strength and confidence41. Resistance exercise increases muscle strength42,43 and in a period of 2–9 months of progressive exercise, pain could decrease by 42–43%42,43. Isokinetic torque can increase further after greater resistance exercise intensity42. These data support the idea that improvements in symptoms and function are directly related to exercise intensity and that higher intensity resistance exercise sustains muscle strength and preserves functionality41.\n\nAerobic exercise includes several activities such as walking, cycling or the use of a seated stepper machine. It has beneficial effects on joint mobility and pain, and it improves the functional status of their general mobility and respiratory capacity30,45. Although modality and dosage are currently not well defined, aerobic exercise program should take into account age, mobility, co-morbidities and preferences30. The exercise bike is a helpful tool for exercising at home. Aquatic exercise seems not to have effects on walking ability or joint ROM46, so it should be considered as an optional activity for exercise program30,46. Land-based exercise and aerobic exercise show higher beneficial effects for pain and function compared with aquatic exercise and strengthening exercise47. A combination of both aerobic training and strengthening exercise could be an optimal choice to decrease impairments48. The beneficial effects of exercise programs are mostly related to the adherence and constancy of patients to the program and the number of sessions, while variations in the delivery, content and dosage do not influence the outcome37. Data from literature show that exercise programs have short-term benefits in reducing pain and improving physical function, but they do not persist in the long term without adherence to the program37,39,45. Strategies to increase long-term adherence to exercise are necessary to maximize the benefits of exercise program37. Self-efficacy is also associated with higher adherence and better outcomes45. Finally, the exercise program should be combined with education and behavioral strategies to promote a positive lifestyle change and increase physical activities30 (Table 3).\n\nSchematic representation of rehabilitation activities that the patients, once discharged from hospital, should keep on at home.\n\n\nNutrition in postoperative rehabilitation in OA\n\nAs mentioned above, cartilage is a connective tissue with viscoelastic and compressive properties, largely due to the extracellular matrix, mainly composed of collagen type II and the proteoglycan aggrecan9,17,18,23. In OA, a progressive loss of structural features occurs because of an imbalance between anabolic and catabolic processes in the cartilage tissue10,14,24. Therefore, one of the goals of OA postoperative rehabilitation, in addition to the restoration of joint function, is the metabolic homeostasis of cartilage tissue, also obtainable through an appropriate diet. The exercise program in OA postoperative rehabilitation would surely have a greater efficacy if combined with a nutritional education in order to promote a healthier lifestyle. There are numerous foods containing natural anti-inflammatory compounds, which are able to reduce some important symptoms of OA, such as pain. These foods are known as natural painkillers and some of them are illustrated in Figure 1.\n\nThe Mediterranean Diet (Med Diet) is the traditional dietary pattern of the Mediterranean areas in the early 1960s49. Olive oil (OO) is the principal source of fat of Med Diet. It is extracted from Olea europaea fruits and is rich in monounsaturated fatty acids (MUFAs). The beneficial effects of OO are ascribed to its phytochemicals such as phenolic compounds, tocopherol and carotenoids, that have antimicrobial, antioxidant and anti-inflammatory properties50. Some epidemiological studies reported an association between consumption of diets rich in polyphenols and protection against chronic diseases51, but few studies investigated the effects on cartilage tissue of such compounds that seem to have a potential protective role52. The phenolic compounds present in OO may interact with the inflammatory cascade preventing cellular damage thank to their antioxidant action. In rheumatoid arthritis patients the dietary supplementation with OO improves joint pain and morning stiffness53. Both leaves and fruit of the olive plant are rich in beneficial polyphenols54, among which the most bioactive are oleuropein and hydroxytyrosol52. Oleuropein is a secoiridoid and represents the most important microconstituent of virgin OO for its health implications. It has high antioxidant activity in vitro, and its hydrolysis product, oleuropein aglycone, ameliorates resistance to the development of arthritis55. Indeed oleuropein reduces the release of proinflammatory cytokines and leukocytes infiltration in the joints affected by collagen induced arthritis, thus reducing the progression of chronic joint inflammation55. Moreover, when administered after the clinical onset of arthritis, oleuropein reduces swelling and the other clinical manifestations, as well as the histological severity of the disease55. This compound reduced the bone loss and improved inflammation, showing a bone sparing effect, in an animal model of senile osteoporosis56.\n\nAnother important phenolic compound is oleocanthal (OLC) that shows anti-inflammatory and neuroprotective properties57. OLC inhibits the cyclooxygenase enzymes in the pathway of prostaglandin biosynthesis in a more potent manner than ibuprofen58. Rutin (quercetin-3-O-rutinoside) is a flavonoid ubiquitously found in plants. Quercetin, the circulating aglycone form of rutin, has the ability to scavenge free radicals59,60 and the association with oleuropein induces interesting metabolic and structural effects on OA cartilage and synovium, supporting their use in human trials52. The fruits of Elaeagnus angustifolia is similar to those of Olea europaea, and although belonging to another botanical family, possesses the same anti-inflammatory potential and was showed to be active in female arthritis patients61.\n\nGiven its known anti-inflammatory properties, we have recently studied the possible benefits of extra-virgin OO, in association with physical activity on joint disease, in order to evaluate the inflammation and the expression of lubricin in articular cartilage after injury and the consequent occurrence of OA16. In our study, we highlighted that Med Diet and extravirgin OO consumption may help attenuate and resolve inflammation in articular cartilage after injury, preventing OA16.\n\nMoreover, deficiencies of vitamins D62,63 and K64 increase the risk of development and progression of OA. A recent controlled trial on arthritis patients comparing the exercise and the nutritional interventions, according to the MyPyramid and MyPlate approaches65, showed an improvement also in the nutritional program group, probably due to the weight loss and the increase of motivation to leisure time physical activity66. Indeed, lowering the fat content typical of the Western diet increases daily physical activity and resting energy expenditure, affecting also the mood, in particular anger and hostility67. Thus, a high consumption of saturated fats might reduce the motivation for physical activity leading to the individual's propensity for weight gain, which is detrimental especially in patients affected by OA. Therefore, a healthy diet combined to a rehabilitation exercise program could improve the quality of life and the mood of post-surgery patients.\n\n\nConclusions\n\nThe articular joint is a highly complex ‘organ system’ that requires regular maintenance. The immobilization of the joints results in a number of negative physiologic consequences. Severity, mobility, pain, stiffness and radiographic progression may be partly mediated by the level of chronic inflammation in OA patients. In the most severe cases of OA, surgical intervention is necessary. It is essential to combine a postoperative rehabilitation exercise program with surgical interventions in order to restore full function of the involved joint. This is crucial for the success of any articular cartilage surgery procedure, and has the purpose to improve muscle strength, ROM, coordination in walking and mitigate of the pain. After hospital discharge, patients should continue the rehabilitation exercise program at home with strengthening exercises, aerobic exercises or both, combined together with a correct diet, so that positive benefits may be gained in terms of pain control and quality of life. Furthermore, regular physical activity combined with a healthy diet improves physical function, muscular strength and endurance, reduces some OA symptoms and leads to psychological and mood benefits. The goal of the postoperative rehabilitation program in OA is to restore joint function, prevent functional limitations and mitigate the progression of the disease, but it is evident that it would surely have a greater efficacy if combined with a nutritional education in order to promote a healthy lifestyle.",
"appendix": "Author contributions\n\n\n\nAll authors have made substantial intellectual contributions to the conception and design of the study. GM conceived the study design and supervised, wrote and structured the review. AM reviewed and edited the paper. FMT and PC researched the area and identified papers, wrote and structured the review. MAS and RI structured the paper and literature search. All authors have approved the final version submitted.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by grants provided by FIR 2014–2016, (cod. 314509), University of Catania, Italy. A. Mobasheri has received wishes to acknowledge funding from the European Union Seventh Framework Programme (FP7/2007–2013, FP7-PEOPLE-2013-IEF) under grant agreement numbers 305815 and 625746.\n\n\nAcknowledgements\n\nThe authors would like to thank Prof. Iain Halliday for commenting and making corrections to the paper.\n\n\nReferences\n\nSchroeppel JP, Crist JD, Anderson HC, et al.: Molecular regulation of articular chondrocyte function and its significance in osteoarthritis. Histol Histopathol. 2011; 26(3): 377–394. PubMed Abstract\n\nSinusas K: Osteoarthritis: diagnosis and treatment. Am Fam Physician. 2012; 85(1): 49–56. PubMed Abstract\n\nMusumeci G, Castrogiovanni P, Leonardi R, et al.: New perspectives for articular cartilage repair treatment through tissue engineering: A contemporary review. World J Orthop. 2014; 5(2): 80–88. 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PubMed Abstract | Publisher Full Text\n\nHollman PC, van Trijp JM, Mengelers MJ, et al.: Bioavailability of the dietary antioxidant flavonol quercetin in man. Cancer Lett. 1997; 114(1–2): 139–40. PubMed Abstract | Publisher Full Text\n\nSakanashi Y, Oyama K, Matsui H, et al.: Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment. Life Sci. 2008; 83(5–6): 164–9. PubMed Abstract | Publisher Full Text\n\nNikniaz Z, Ostadrahimi A, Mahdavi R, et al.: Effects of Elaeagnus angustifolia L. supplementation on serum levels of inflammatory cytokines and matrix metalloproteinases in females with knee osteoarthritis. Complement Ther Med. 2014; 22(5): 864–9. PubMed Abstract | Publisher Full Text\n\nZhang FF, Driban JB, Lo GH, et al.: Vitamin D deficiency is associated with progression of knee osteoarthritis. J Nutr. 2014; 144(12): 2002–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSanghi D, Mishra A, Sharma AC, et al.: Elucidation of dietary risk factors in osteoarthritis knee-a case-control study. J Am Coll Nutr. 2015; 34(1): 15–20. PubMed Abstract | Publisher Full Text\n\nMisra D, Booth SL, Tolstykh I, et al.: Vitamin K deficiency is associated with incident knee osteoarthritis. Am J Med. 2013; 126(3): 243–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilcox S, McClenaghan B, Sharpe PA, et al.: The steps to health randomized trial for arthritis: a self-directed exercise versus nutrition control program. Am J Prev Med. 2015; 48(1): 1–12. PubMed Abstract | Publisher Full Text\n\nCatalano D, Trovato GM, Pace P, et al.: Mediterranean diet and physical activity: an intervention study. Does olive oil exercise the body through the mind? Int J Cardiol. 2013; 168(4): 4408–9. PubMed Abstract | Publisher Full Text\n\nKien CL, Bunn JY, Tompkins CL, et al.: Substituting dietary monounsaturated fat for saturated fat is associated with increased daily physical activity and resting energy expenditure and with changes in mood. Am J Clin Nutr. 2013; 97(4): 689–97. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "8515",
"date": "07 May 2015",
"name": "Ivana Gadjanski",
"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 review article by Musumeci et al. under the title \"Post-operative rehabilitation and nutrition in osteoarthritis\" is well written, with appropriate title and abstract. The article largely accomplishes to underline importance of exercise, combined with an appropriate daily diet, in postoperative rehabilitation for osteoarthritis (OA) patients. However, I have several comments which I hope will help the authors to bring across this important message even more efficiently.Major points:It is not very clear how the selection of articles was performed. The authors state: \"The literature search was conducted from in March 2014 to April 2015 on PubMed, Scopus, Web of Science and Google Scholar using appropriate keywords (osteoarthritis, rehabilitation, exercise and nutrition). Of approximately 160 papers (original articles, systematic and meta-analysis reviews) only 67 have been chosen and considered appropriate for the purpose of the review. The other papers, have not been considered as they resulted outside the scope of the research.\" However, out of these 67, only 7 are from 2014 and 3 from 2015. The rest range from 1995-2013 (References). The authors should clarify the selection criteria and perhaps include more of the more recent papers. Suggestions of the papers to include:OARSI Clinical Trials Recommendations: Design and conduct of clinical trials of lifestyle diet and exercise interventions for osteoarthritisKnee osteoarthritis: Clinical connections to articular cartilage structure and functionExercise for osteoarthritis of the kneeRegular Exercises in Knee and Hip OsteoarthritisExercise, nutrition and managing hip fracture in older persons The authors state: \"OA is a degenerative process involving the progressive loss of structural features and functionality of the articular cartilage caused by an imbalance between anabolic and catabolic processes in the cartilage tissue, so that cartilage degradation exceeds reparative processes and OA progresses\" - this is not sufficiently precise, since the subchondral bone is also involved in pathogenesis of OA. The authors should also mention this and further on, comment on the effects of weight loss/gain and exercise training on bone mineral density in OA patients.Suggested paper: The independent and combined effects of intensive weight loss and exercise training on bone mineral density in overweight and obese older adults with osteoarthritis.Minor points:\"Cartilage is a slow adapting tissue, indeed it undergoes 75% adaptation in approximately 2 years.\" - Reference for this statement? The paragraph under title “Second step post-operative rehabilitation in OA” - the whole paragraph is very repetitive, with the same kind of information provided in the table as well. Can be shortened. It would be good to give short overview of the available surgical procedures to treat OA. The authors mention “Arthroscopic procedures, such as chondroplasty or microfracture, may resolve faster than osteochondral autograph transplantation (OATS) or autologous chondrocyte implantation (ACI) that involve larger incisions, requiring a slower exercise rehabilitation program”, but it would be better if they gave a short description of each procedure (not more than a sentence long).",
"responses": [
{
"c_id": "1418",
"date": "06 Jan 2016",
"name": "Giuseppe Musumeci",
"role": "Author Response",
"response": "Thank you for your review and for the comments. Comments from reviewers are all valuable and very helpful for revising and improving our paper. We have studied the comments carefully and have made corrections which we hope meet with your approval. Point by point repliesReviewer: It is not very clear how the selection of articles was performed. The authors state: \"The literature search was conducted from in March 2014 to April 2015 on PubMed, Scopus, Web of Science and Google Scholar using appropriate keywords (osteoarthritis, rehabilitation, exercise and nutrition). Of approximately 160 papers (original articles, systematic and meta-analysis reviews) only 67 have been chosen and considered appropriate for the purpose of the review. The other papers, have not been considered as they resulted outside the scope of the research.\" However, out of these 67, only 7 are from 2014 and 3 from 2015. The rest range from 1995-2013 (References). The authors should clarify the selection criteria and perhaps include more of the more recent papers.Response: The authors thank the reviewer for this comment. Perhaps we have not been clear, we meant to say that we have made the literature search in the period March 2014 - April 2015, but the period considered in the selection of the references is much larger (1995 – 2015). Thank you for letting us know about the possible misinterpretation. We have corrected the text to be clearer. Thank you for the papers suggested, we included most of them in the text. Reviewer: The authors state: \"OA is a degenerative process involving the progressive loss of structural features and functionality of the articular cartilage caused by an imbalance between anabolic and catabolic processes in the cartilage tissue, so that cartilage degradation exceeds reparative processes and OA progresses\" - this is not sufficiently precise, since the subchondral bone is also involved in pathogenesis of OA. The authors should also mention this and further on, comment on the effects of weight loss/gain and exercise training on bone mineral density in OA patients.Response: We have provided to modify the text with comment on the involvement of the subchondral bone in pathogenesis of OA, and on the effects of weight loss/gain and exercise training on bone mineral density in OA patients. Thank you for the paper suggestion. Reviewer: \"Cartilage is a slow adapting tissue, indeed it undergoes 75% adaptation in approximately 2 years.\" - Reference for this statement?Response: We inserted the following reference: Hambly K, Griva K: IKDC or KOOS? Which measures symptoms and disabilities most important to postoperative articular cartilage repair patients? Am J Sports Med. 2008;36(9):1695-704. Reviewer: The paragraph under title “Second step post-operative rehabilitation in OA” - the whole paragraph is very repetitive, with the same kind of information provided in the table as well. Can be shortened.Response: We have provided for modifying the text and making it shorter. Reviewer: It would be good to give short overview of the available surgical procedures to treat OA. The authors mention “Arthroscopic procedures, such as chondroplasty or microfracture, may resolve faster than osteochondral autograph transplantation (OATS) or autologous chondrocyte implantation (ACI) that involve larger incisions, requiring a slower exercise rehabilitation program”, but it would be better if they gave a short description of each procedure (not more than a sentence long).Response: We have provided for modifying the text with a short description of each procedure.The Authors hope that the reviewer will be satisfied with the changes and revisions made. Thank you for your time and for considering our paper."
},
{
"c_id": "1747",
"date": "06 Jan 2016",
"name": "Giuseppe Musumeci",
"role": "Author Response",
"response": "Thank you for your review and for the comments. Comments from reviewers are all valuable and very helpful for revising and improving our paper. We have studied the comments carefully and have made corrections which we hope meet with your approval. Point by point replies ReviewerIt is not very clear how the selection of articles was performed. The authors state: \"The literature search was conducted from in March 2014 to April 2015 on PubMed, Scopus, Web of Science and Google Scholar using appropriate keywords (osteoarthritis, rehabilitation, exercise and nutrition). Of approximately 160 papers (original articles, systematic and meta-analysis reviews) only 67 have been chosen and considered appropriate for the purpose of the review. The other papers, have not been considered as they resulted outside the scope of the research.\" However, out of these 67, only 7 are from 2014 and 3 from 2015. The rest range from 1995-2013 (References). The authors should clarify the selection criteria and perhaps include more of the more recent papers. ResponseThe authors thank the reviewer for this comment. Perhaps we have not been clear, we meant to say that we have made the literature search in the period March 2014 - December 2015, but the period considered in the selection of the references is much larger (1995 – 2015). Thank you for letting us know about the possible misinterpretation. We have corrected the text to be clearer. Thank you for the papers suggested, we included most of them in the text. ReviewerThe authors state: \"OA is a degenerative process involving the progressive loss of structural features and functionality of the articular cartilage caused by an imbalance between anabolic and catabolic processes in the cartilage tissue, so that cartilage degradation exceeds reparative processes and OA progresses\" - this is not sufficiently precise, since the subchondral bone is also involved in pathogenesis of OA. The authors should also mention this and further on, comment on the effects of weight loss/gain and exercise training on bone mineral density in OA patients. ResponseWe have provided to modify the text with comment on the involvement of the subchondral bone in pathogenesis of OA, and on the effects of weight loss/gain and exercise training on bone mineral density in OA patients. Thank you for the paper suggestion. Reviewer\"Cartilage is a slow adapting tissue, indeed it undergoes 75% adaptation in approximately 2 years.\" - Reference for this statement? ResponseWe inserted the following reference: Hambly K, Griva K: IKDC or KOOS? Which measures symptoms and disabilities most important to postoperative articular cartilage repair patients? Am J Sports Med. 2008;36(9):1695-704. ReviewerThe paragraph under title “Second step post-operative rehabilitation in OA” - the whole paragraph is very repetitive, with the same kind of information provided in the table as well. Can be shortened. ResponseWe have provided for modifying the text and making it shorter. ReviewerIt would be good to give short overview of the available surgical procedures to treat OA. The authors mention “Arthroscopic procedures, such as chondroplasty or microfracture, may resolve faster than osteochondral autograph transplantation (OATS) or autologous chondrocyte implantation (ACI) that involve larger incisions, requiring a slower exercise rehabilitation program”, but it would be better if they gave a short description of each procedure (not more than a sentence long). ResponseWe have provided for modifying the text with a short description of each procedure. The Authors hope that the reviewer will be satisfied with the changes and revisions made. Thank you for your time and for considering our paper."
}
]
},
{
"id": "11704",
"date": "29 Dec 2015",
"name": "Carolien H. G Bastiaenen",
"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\nIt is an well-written and informative paper on the topic. The paper underlines the importance of physical expertise on the treatment of OA, combined with an appropriate diet in post-operative situations. Nevertheless I have serious comments on the scientific basis of the review.The review is not performed in line with a scientific accepted methodology regarding systematic reviews. The search terms only are focused on the topic OA and not on methodological terms. There are no in-and exclusion criteria for the papers formulated. Also, there is no quality assessment of the papers regarding methodological quality or a systematically presented evidence synthesis. Therefore the presentation of the results, discussion and the conclusion is difficult to interpret.",
"responses": [
{
"c_id": "1746",
"date": "06 Jan 2016",
"name": "Giuseppe Musumeci",
"role": "Author Response",
"response": "Point by point replies ReviewerIt is an well-written and informative paper on he topic. The paper underlines the importance of physical expertise on the treatment of OA, combined with an appropriate diet in post-operative situations. Nevertheless I have serious comments on the scientific basis of the review. ResponseThe authors thank the reviewer for this comment. ReviewerThe review is not performed in line with a scientific accepted methodology regarding systematic reviews. The search terms only are focused on the topic OA and not on methodological terms. There are no in-and exclusion criteria for the papers formulated. Also, there is no quality assessment of the papers regarding methodological quality or a systematically presented evidence synthesis. Therefore the presentation of the results, discussion and the conclusion is difficult to interpret. ResponseWe thank the reviewer for this comment. Probably, the misunderstanding came from the scope of this paper. Authors intentions were to write a Narrative Review, as now specified in the methodological section, although this type of review has some limitations. There are two kinds of review articles commonly present in the scientific literature as shown in the following editorial: Rother, ET: Systematic literature review X narrative review. Acta paul. enferm. 2007; 20(2): 5-6. Publisher Full Text. In summary this editorial says: Narrative literature review articles are contributions which describe and debate the state of scientific knowledge of a specific field with a critical analysis of the literature published.These types of review articles do not list the types of databases and methodological approaches used to carry out the review nor the evaluation criteria for inclusion of retrieved articles during databases search, anyway we added in the methodology section more information and details as kindly suggested (inclusion and exclusion criteria and so on) to improve our review and to help readers better understand.Narrative literature review articles have a key role in continuous learning/training because they provide readers with update knowledge about a specific field of interest, although it does not describe the methodological approach that would permit reproduction of data nor answer to specific quantitative research questions. Results are of a qualitative rather then a quantitative meaning.Moreover, systemic literature review \"is a well planned review to answer specific research questions using a systematic and explicit methodology to identify, select, and critically evaluate results of the studies included in the literature review\". Therefore, systematic literature review utilize methodological rigor to prevent the prejudice of a narrative review. The Authors hope that the reviewer will be satisfied with the changes and revisions made. Thank you for your time and for considering our paper."
}
]
}
] | 2
|
https://f1000research.com/articles/3-116
|
https://f1000research.com/articles/4-142/v1
|
04 Jun 15
|
{
"type": "Systematic Review",
"title": "Is MTHFD1 polymorphisms rs 2236225 (c.1958G>A) associated with the susceptibility of NSCL/P? A systematic review and meta-analysis",
"authors": [
"Huaxiang Zhao",
"Jieni Zhang",
"Mengqi Zhang",
"Feng Deng",
"Leilei Zheng",
"Hui Zheng",
"Feng Chen",
"Jiuxiang Lin",
"Huaxiang Zhao",
"Jieni Zhang",
"Mengqi Zhang",
"Feng Deng",
"Leilei Zheng",
"Hui Zheng"
],
"abstract": "Aims: To investigate the association between the methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) polymorphism rs 2236225 (c.1958G>A) and susceptibility to non-syndromic cleft of the lip and/or palate (NSCL/P).Methods: An extensive literature review has been conducted using PubMed, Web of Science, Cochrane Library, Google Scholar, the China National Knowledge Infrastructure (CNKI), and Wanfang Database for eligible researches. The terms for searching were “cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC” AND “methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD”. Two independent researchers screened, evaluated and extracted the data of included studies. The pooled odds ratios (OR) with 95% confidence intervals (95% CI) were calculated by random effects model under five gene models. Subgroup, sensitivity analysis and publication bias were also assessed.Results: Ten case-control studies have been included in the systematic review and eight studies have been considered for the meta-analysis. Overall, the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P showed pooled OR (95% CI) of 1.02 (0.86-1.21) under allelic model. A higher degree of heterogeneity was observed in Asian countries (I2 = 75.6%) compared to non-Asian countries (I2 = 48.9%). Similar consequence appeared in the subgroup of children (I2 = 78.6%) compared with that of mothers (I2 = 0.0%). There was no significant difference in the publication bias by the Begg’s funnel plot (P = 0.711) and Egger’s regression test (P = 0.746).Conclusion: Our assessment suggested there was no significant association between the MTHFD1 polymorphism rs 2236225 (c.1958G>A) and the susceptibility to NSCL/P. Further investigations using a large sample size and a more advanced technique should be adopted to reach a more precise conclusion in the future.",
"keywords": [
"MTHFD1",
"Polymorphisms",
"NSCL/P susceptibility",
"Meta-analysis"
],
"content": "Introduction\n\nCleft of the lip and/or palate (CL/P) is one of the most common facial malformations1–3 and a societal burden, affecting the patient ability to eat and speak and influencing social integration4. Non-syndromic CL/P, accounting for about 70% of CL/P, is considered closely related to genetic and environmental factors5. Recent studies suggested that using folic acid could reduce the rates of oral clefts6,7 and single nucleotide polymorphisms of some genes such as MTHFR8,9, MTR40 and MTRR involved in the metabolism of folic acid have been associated to high risk of NSCL/P8,9. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), a key gene associated with three sequential enzymatic reactions in the metabolism of folic acid, might play a potential role in the risk of NSCL/P, especially the polymorphism rs2236225 (c.1958G>A)10. Indeed, different observations that linked the polymorphism rs2236225 to the risk of NSCL/P have been reported11,12. The suggestion of a link between rs2236225 polymorphism and susceptibility to NSCL/P might be result of the limitations in sample size, different ethnic populations and other environmental factors. Therefore, we conducted a systematic review and meta-analysis of eligible case-control studies to reveal a more precise connection between the MTHFD1 polymorphism rs2236225 and the risk of NSCL/P.\n\n\nMaterials and methods\n\nA systematic search based on the principle of evidence-based medicine13 was performed in PubMed, Web of Science, Cochrane Library, Google Scholar, China National Knowledge Infrastructure (CNKI) and WanFang Database. The final update was made on April 5th, 2015. In line with our knowledge background, the Medical Subject Headings (MESH) terms in PubMed and the known aliases of the genes of interests in the National Center of Biotechnology Information (NCBI), the following terms were used for searching: “cleft lip OR cleft palate OR CLP OR CL/P OR oral facial cleft OR OFC” AND “methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 OR methenyltetrahydrofolate cyclohydrolase formyltetrahydrofolate synthetase OR MTHFD1 OR MTHFD”, which were slightly adjusted to optimize search results (Table S1; PubMed). We didn’t limit the search depending on publication types, data and language. Of course, the review of the published literature was examined carefully and manual search was conducted to avoid missing potential data. Two of the authors (Huaxiang Zhao and Mengqi Zhang) were in charge of the search independently and a third author (Jieni Zhang) conducted a random inspection.\n\nResearches included in our systematic review and meta-analysis meet the following criteria: (1) evaluating the association between the NSCL/P and MTHFD1 polymorphism rs2236225, (2) focusing on humans, (3) case-control studies. Exclusion criteria were: (1) no association between NSCL/P and MTFHD1, (2) not focusing on humans but animal models or in vitro studies, (3) duplication of previous researches, (4) not original literature such as reviews, meta-analyses, comments and editorials.\n\nData from eligible studies were extracted by two independent researchers (Huaxiang Zhao and Mengqi Zhang) in accordance with the inclusion and exclusion criteria. In case of any discrepancies, the third chief author (Feng Chen) would make a further investigation or bring it into a group-discussion. A special table was used for collecting information from the selected articles and the following entries were recorded: authors (year), country, location of geography, subjects, methods for genotyping, sample size of cases/controls, descriptions of samples rolled in the study, P for HWE (Hardy-Weinberg equilibrium) of control group, whether included in the meta-analysis or not.\n\nA methodological quality assessment adapted from previous studies14–16 was carried on included studies (Table S2). Cases, source of controls, sample sizes and Hardy-Weinberg equilibrium (HWE) were considered as important aspects in this systematic review.\n\nThe PRISMA checklist (Supplementary material S3) was used as a protocol in our meta-analysis17. Odd ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the association between the susceptibility to NSCL/P and MTHFD1. Five genetic models were used in the process of pooling the OR and 95% CIs: allelic comparison (A versus G), heterozygote model (AG versus GG), homozygote model (AA versus GG), dominant model (AA + AG versus GG), recessive model (AA versus AG + GG). The significance of the pooled effects was determined by Z-test with P value less than 0.05. The Q-statistic and the I2 test were used to evaluated; P < 0.05 in Q statistic or I2 > 50%18,19, would indicate a significant heterogeneity. When P > 0.05 in Q statistic or I2 < 50%, the fixed pooling model (Mantel-Haenszel) was conducted; if not, the random pooling model (M-H heterogeneity) was used. We also carried subgroup analyses in which different subjects (mothers or children), location of geography (non-Asian countries or Asian countries) were considered potential source of heterogeneity. A sensitivity analysis was conducted by omitting each study in turn to evaluate the single study’s influence on the overall estimation. We used Begg’s funnel plot and Egger’s linear regression test to find out the publication bias of the included studies20–22. The studies with disequilibrium of HWE among control group were added into a supplementary meta-analysis as described previously23. Meanwhile, as for the studies included but not carried into the meta-analysis, to achieve a qualitative analysis we adopted a method described by others24. Results were considered significant when P < 0.05. Stata 12.0 (Stata Corp, College Station, TX, USA) was used for the analysis.\n\n\nResults\n\nA total of 251 articles resulted from the search described above (PubMed: 86, Web of Science: 8, Google Scholar: 135, Cochrane Library: 0, CNKI: 18, Wanfang: 4). After being imported into EndNote X6 software (Thomson Corporation, Stamford), a screening process was conducted among 102 articles– that is, duplicates were removed using the ‘Discard Duplicates’ function as well as by handwork. Following paper selection by two independent researchers, 15 studies were then thoroughly reviewed. Of these, five studies were excluded, among which two had no control groups25,26, one no relation to MTFHD127, and the other two presented data previously published28,29. Finally, 10 studies that met the criteria were included in the systematic review (Table 1)10–12,30–36 and mathematic data from eight studies were used for reference to carry out the meta-analysis10–12,31–33,35,36. The selection process is shown in Figure 1.\n\nHWE*: Hardy-Weinberg equilibrium.\n\nNMψ: Not mentioned in the study.\n\nPCR-RFLPγ: PCR-restriction fragment length polymorphism.\n\nMALDI-TOF MSξ: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.\n\nEventually, all 10 studies containing 6216 samples (2959 cases and 3257 controls) were analyzed in our review. The characteristics of every study can be seen in Table 1. To summarize briefly, there were four studies from European groups, four from Asian groups and two from South American groups, among which two studies focused on the genotype of patients’ mothers only, four on children’ s genotype only and four on both of them. PCR-restriction fragment length polymorphism (PCR-RFLP) was the major method of genotyping, while other techniques had been used as well.\n\nThe association between MTHFD1 polymorphism rs2236225 (c.1958G>A) and NSCL/P susceptibility was analyzed through a meta-analysis and qualitative analysis. In the meta-analysis, since significant heterogeneity had been identified by Q-test and I2 statistic in every genetic model, the random effect models were used. Overall, a significant association was not found in any genetic model (A versus G: OR = 1.02, 95% CI 0.86–1.21, PH = 0.010, Figure 2; AG versus GG: OR = 0.97, 95% CI 0.75–1.26, PH = 0.019, Figure 3A; AA versus GG: OR = 1.07, 95% CI 0.70–1.65, PH = 0.005, Figure 3B; AA + AG versus GG: OR = 1.00, 95% CI 0.76–1.31, PH = 0.006, Figure 3C; AA versus AG + GG: OR = 1.05, 95% CI 0.71–1.53, PH = 0.014, Figure 3D). On the other hand, no association was found in the genotypes of children, mothers or fathers in the qualitative analysis30,34.\n\n(A) Heterozygote model, AG versus GG. (B) Homozygote model, AA versus GG. (C) Dominant model, AA + AG versus GG. (D) Recessive model, AA versus AG + GG.\n\nNext we conducted the subgroup analysis using allelic A versus G model according to the location of geography and subjects (mothers or children). It turned out that there was no significant difference between Asian (OR = 1.03, 95% CI 0.75–1.40, PH = 0.003) or non-Asian population (OR = 1.06, 95% CI 0.86–1.30, PH = 0.118). However, a higher degree of heterogeneity was observed in the Asian countries compared to non-Asian countries (Figure 4A). A similar result was observed in the subgroup analysis between mothers and children. The heterogeneity was much higher in the children group (OR = 0.99, 95% CI 0.72–1.36, PH = 0.001) than in the mothers’ group (OR = 1.11, 95% CI 0.98–1.27, PH = 0.630), while no significant difference was observed in both groups (Figure 4B).\n\nSubgroup analysis by locations of geography (A) and subjects (B) under allelic comparison of MTHFD1 polymorphism rs2236225 (c.1958G>A).\n\nTo access the influence of each individual study on the pooled ORs, a sensitivity analysis was performed by omitting each study at a time. The results of sensitivity suggests that no individual study affects the pooled ORs of the associations between MTHFD1 polymorphism rs2236225 (c.1958G>A) and NSCL/P risk under allelic model (Figure 5).\n\nWe used the Begg’s funnel plot and Egger’s regression test (both used the allelic A versus G model) to estimate the publication bias. Our results indicate that there is no significant publication bias both in the symmetry of Begg’s funnel plot (P = 0.711, Figure 6) and Egger’s regression test (P = 0.746).\n\n\nDiscussion\n\nCL/P is one of the most common facial malformations, affecting approximately 1.7/1000 people around the world with ethnic and geographic variation1. Although CL/P is not considered one of the major causes of infant mortality, individuals affected by CL/P it may have difficulties in feeding, speaking, difficult social integration4. Approximately 70% of CL/P cases are considered to be non-syndromic37,38, and their susceptibility has been linked to the expression of various candidate genes through twin studies, familial clustering studies and genome-wide studies39.\n\nRecent studies suggest that using folic acid could reduce the rates of oral clefts6,7. Some genes involved in the metabolism of folic acid such as MTHFR8,9, MTR40, and MTRR41 have been identified. MTHFD1, a crucial gene associated with three sequential enzymatic reactions among 5,10-methylenetetrahydrofolate, 5,10-methenyltetrahydrofolate, 10-formyltetrahydrofolate, tetrahydrofolate, might play a potential role in NSCL/P10. However, controversial results about the MTHFD1 polymorphism rs2236225 (c.1958G>A) have been reported in different articles10,12.\n\nIn this systematic review, 10 independent case-control studies were included (eight studies for meta-analysis and two studies qualitatively analyzed) containing 6216 samples (2959 cases and 3257 controls). All the eligible studies of meta-analysis and qualitative analysis showed no significant association of MTHFD1 rs2236225 to the risk of NSCL/P, whether in the whole analysis of five model (A versus G, AG versus GG, AA versus GG, AA + AG versus GG, AA versus AG + GG) or in the subgroup of subjects (mothers or children) and the location of geography (non-Asian countries or Asian countries). Meanwhile, high heterogeneity was observed, which might be the reason for the genetic drift and natural selection among different ethnic groups42. Also, small sample size of different studies might be a possible reason for the disparate results. Our findings suggest that the MTHFD1 polymorphism rs2236225 (c.1958G>A) might not be an appropriate biomarker in predicting the susceptibility of an individual to NSCL/P.\n\nSome limitations of this systematic review and meta-analysis should be noted. Firstly, the choice of retrospective studies has its own limitations, as we may encounter selection bias and influence the results of our analysis43. However, a bigger size of cohort study cannot be conducted easily because of the relatively low morbidity44. Secondly, only 10 studies were included in our review, a small sample size that might not provide sufficient evidence to estimate the connections between the MTHFD1 polymorphisms and the risk of NSCL/P.\n\nNSCL/P is also associated with gene-gene and gene-environment interactions45. Although no correlation was observed between MTFHD1 polymorphism rs2236225 (c.1958G>A) and the risk of NSCL/P, in view of MTFHD1 gene’s key role in folic acid metabolism, we cannot draw a definite conclusion that there is no association between MTFHD1 and NSCL/P’s susceptibility. The use of larger sample size studies, different techniques and considering gene-gene or gene-environment interactions should be explored in future investigations.",
"appendix": "Author contributions\n\n\n\nH Zhao, F Chen, J Lin were responsible for study conception and design of the study. H Zhao, J Zhang, M Zhang acquired the data. H, Zhao, F Chen F, Deng, L Zheng, H Zheng analyzed the data. H Zhao and J Zhang wrote the main manuscript text. Prof. J Lin and Prof. F Chen had full access to all of the data in this review and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by grants 81200762 from National Natural Science Foundation of China; Program for the National Clinical Key Subject, Natural Science Foundation of China (81271183, 81470772); the Medical Scientific Research Project of Chongqing (20141013).\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nMossey PA, Little J, Munger RG, et al.: Cleft lip and palate. Lancet. 2009; 374(9703): 1773–85. PubMed Abstract | Publisher Full Text\n\nWehby GL, Goco N, Moretti-Ferreira D, et al.: Oral cleft prevention program (OCPP). BMC Pediatr. 2012; 12: 184. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMossey PA, Shaw WC, Munger RG, et al.: Global oral health inequalities: challenges in the prevention and management of orofacial clefts and potential solutions. Adv Dent Res. 2011; 23(2): 247–58. PubMed Abstract | Publisher Full Text\n\nWehby GL, Cassell CH: The impact of orofacial clefts on quality of life and healthcare use and costs. Oral Dis. 2010; 16(1): 3–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLidral AC, Murray JC: Genetic approaches to identify disease genes for birth defects with cleft lip/palate as a model. Birth Defects Res A Clin Mol Teratol. 2004; 70(12): 893–901. PubMed Abstract | Publisher Full Text\n\nYazdy MM, Honein MA, Xing J: Reduction in orofacial clefts following folic acid fortification of the U.S. grain supply. Birth Defects Res A Clin Mol Teratol. 2007; 79(1): 16–23. PubMed Abstract | Publisher Full Text\n\nJohnson CY, Little J: Folate intake, markers of folate status and oral clefts: is the evidence converging? Int J Epidemiol. 2008; 37(5): 1041–58. PubMed Abstract | Publisher Full Text\n\nHan Y, Pan Y, Du Y, et al.: Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and nonsyndromic orofacial clefts susceptibility in a southern Chinese population. DNA Cell Biol. 2011; 30(12): 1063–8. PubMed Abstract | Publisher Full Text\n\nMurthy J, Gurramkonda VB, Karthik N, et al.: MTHFR C677T and A1298C polymorphisms and risk of nonsyndromic orofacial clefts in a south Indian population. Int J Pediatr Otorhinolaryngol. 2014; 78(2): 339–42. PubMed Abstract | Publisher Full Text\n\nMurthy J, Gurramkonda VB, Lakkakula BV: Significant association of MTHFD1 1958G>A single nucleotide polymorphism with nonsyndromic cleft lip and palate in Indian population. Med Oral Patol Oral Cir Bucal. 2014; 19(6): e616–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMostowska A, Hozyasz KK, Jagodzinski PP: Maternal MTR genotype contributes to the risk of non-syndromic cleft lip and palate in the Polish population. Clin Genet. 2006; 69(6): 512–7. PubMed Abstract | Publisher Full Text\n\nYanbo Z, Xinhua L, Xinrong N: Association between rs2236225 polymorphism in MTHFD1 gene and non-syndromic cleft lip with or without cleft palate in Shanxi population. Shi Yong Kou Qiang Yi Xue Za Zhi. 2013; (4): 521–524. Reference Source\n\nCook DJ, Mulrow CD, Haynes RB: Systematic reviews: synthesis of best evidence for clinical decisions. Ann Intern Med. 1997; 126(5): 376–80. PubMed Abstract | Publisher Full Text\n\nMoher D, Jones A, Lepage L: Use of the CONSORT statement and quality of reports of randomized trials: a comparative before-and-after evaluation. JAMA. 2001; 285(15): 1992–5. PubMed Abstract | Publisher Full Text\n\nda Costa BR, Cevallos M, Altman DG, et al.: Uses and misuses of the STROBE statement: bibliographic study. BMJ Open. 2011; 1(1): e000048. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuo J, Jin M, Zhang M, et al.: A genetic variant in miR-196a2 increased digestive system cancer risks: a meta-analysis of 15 case-control studies. PLoS One. 2012; 7(1): e30585. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med. 2009; 151(4): 264–9, W64. PubMed Abstract | Publisher Full Text\n\nJackson D, White IR, Riley RD: Quantifying the impact of between-study heterogeneity in multivariate meta-analyses. Stat Med. 2012; 31(29): 3805–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeters JL, Sutton AJ, Jones DR, et al.: Comparison of two methods to detect publication bias in meta-analysis. JAMA. 2006; 295(6): 676–80. PubMed Abstract | Publisher Full Text\n\nZintzaras E, Ioannidis JP: HEGESMA: genome search meta-analysis and heterogeneity testing. Bioinformatics. 2005; 21(18): 3672–3. PubMed Abstract | Publisher Full Text\n\nBegg CB, Mazumdar M: Operating characteristics of a rank correlation test for publication bias. Biometrics. 1994; 50(4): 1088–101. PubMed Abstract | Publisher Full Text\n\nEgger M, Davey G, Smith M, et al.: Bias in meta-analysis detected by a simple, graphical test. BMJ. 1997; 315(7109): 629–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H, Tie K, Hu N, et al.: Association of two polymorphisms rs2910164 in miRNA-146a and rs3746444 in miRNA-499 with rheumatoid arthritis: a meta-analysis. Hum Immunol. 2014; 75(7): 602–8. PubMed Abstract | Publisher Full Text\n\nThomas J, Harden A: Methods for the thematic synthesis of qualitative research in systematic reviews. BMC Med Res Methodol. 2008; 8: 45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPalmieri A, Masiero E, Martinelli M, et al.: The MTHFD1 gene is not involved in cleft lip with or without palate onset among the Italian population. Ann Hum Genet. 2008; 72(Pt 3): 297–9. PubMed Abstract | Publisher Full Text\n\nBlanton SH, Henry RR, Yuan Q, et al.: Folate pathway and nonsyndromic cleft lip and palate. Birth Defects Res A Clin Mol Teratol. 2011; 91(1): 50–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu Y, Liu Q, Xu W, et al.: TGFA and IRF6 contribute to the risk of nonsyndromic cleft lip with or without cleft palate in northeast China. PLoS One. 2013; 8(8): e70754. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi R: Association between genetic polymorphism of MTHFD1 and MTR adn non-syndromic oral cleft with or without cleft palate in Shanxi, China. Shanxi Medical University. 2013.\n\nZhao Y: Research of association between rs22362225 polymorphism in MTHFD1 and non-syndromic cleft lip with or without cleft palate in Shanxi population. Shanxi Medical University. 2012.\n\nBoyles AL, Wilcox AJ, Taylor JA, et al.: Folate and one-carbon metabolism gene polymorphisms and their associations with oral facial clefts. Am J Med Genet A. 2008; 146A(4): 440–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMills Jl, Molloy AM, Parle-McDermott A, et al.: Folate-related gene polymorphisms as risk factors for cleft lip and cleft palate. Birth Defects Res A Clin Mol Teratol. 2008; 82(9): 636–643. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBufalino A, Paranaiba LMR, de Aquino SN, et al.: Maternal polymorphisms in folic acid metabolic genes are associated with nonsyndromic cleft lip and/or palate in the Brazilian population. Birth Defects Res A Clin Mol Teratol. 2010; 88(11): 980–986. PubMed Abstract | Publisher Full Text\n\nMostowska A, Hozyasz KK, Wojcicki P, et al.: Associations of folate and choline metabolism gene polymorphisms with orofacial clefts. J Med Genet. 2010; 47(12): 809–15. PubMed Abstract | Publisher Full Text\n\ndeAquino SN, Hoshi R, Bagordakis E, et al.: MTHFR rs2274976 Polymorphism Is a Risk Marker for Nonsyndromic Cleft Lip with or without Cleft Palate in the Brazilian Population. Birth Defects Res A Clin Mol Teratol. 2014; 100(1): 30–35. PubMed Abstract | Publisher Full Text\n\nLi R, Ma X, Nan X: Association between genetic polymorphism of methylenetetrahydrofolate dehydrogenase 1, mehionine synthase and non-syndromic cleft lip with or without cleft palate in Shanxi, China. Guo Ji Kou Qiang Yi Xue Za Zhi. 2013; (2): 144–147. Publisher Full Text\n\nYuan G: Nuclear family study of the relationship between MTHFD1, MTR polymorphism and non-syndromic cleft lip with or without cleft palate in Shanxi Province. Shanxi Medical University. 2013. Reference Source\n\nJones MC: Etiology of facial clefts: prospective evaluation of 428 patients. Cleft Palate J. 1988; 25(1): 16–20. PubMed Abstract\n\nFitzPatrick DR, Raine PA, Boorman JG: Facial clefts in the west of Scotland in the period 1980–1984: epidemiology and genetic diagnoses. J Med Genet. 1994; 31(2): 126–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDixon MJ, Marazita ML, Beaty TH, et al.: Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet. 2011; 12(3): 167–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBezerra JF, Oliveira GH, Soares CD, et al.: Genetic and non-genetic factors that increase the risk of non-syndromic cleft lip and/or palate development. Oral Dis. 2014; 21(3): 393–9 . PubMed Abstract | Publisher Full Text\n\nChorna LB, Akopian HR, Makukh HV, et al.: [Allelic polymorphism of MTHFR, MTR and MTRR genes in patients with cleft lip and/or palate and their mothers]. Tsitol Genet. 2011; 45(3): 51–6. PubMed Abstract\n\nSerre D, Montpetit A, Pare G, et al.: Correction of population stratification in large multi-ethnic association studies. PLoS One. 2008; 3(1): e1382. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStroup DF, Thacker SB, Olson CM, et al.: Characteristics of meta-analyses related to acceptance for publication in a medical journal. J Clin Epidemiol. 2001; 54(7): 655–60. PubMed Abstract | Publisher Full Text\n\nKling RR, Taub PJ, Ye X, et al.: Oral clefting in China over the last decade: 205,679 patients. Plast Reconstr Surg Glob Open. 2014; 2(10): e236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMurray JC: Gene/environment causes of cleft lip and/or palate. Clin Genet. 2002; 61(4): 248–56. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "8906",
"date": "17 Jun 2015",
"name": "Jingtan Su",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNon-syndromic cleft of the lip and/or palate (NSCL/P) is considered closely related to genetic and environmental factors. As a key gene in the metabolism of folic acid which is associated to high risk of NSCL/P, methylenetetrahydrofolate dehydrogenase (MTHFD1) polymorphisms rs 2237225 (c. 1958G>A) may be associated with the susceptibility of NSCL/P. However, controversial conclusions on this association have been reported by different groups. This paper conducted a systematic review and meta-analysis of eligible case-control studies which is necessary before we go forward to larger sample size studies. This paper is scientifically sound, clear and well-organized. However, these following revisions may make it more interesting.Page 6, Figure 1, Eligibility section, the number of full-text articles excluded with reasons should be 5. The lack of folic acid will result in many health problems such as neural tube defects, macrocytic anemia, mental depress and so on, not only NSCL/P. MTHFD1 is important in the metabolism of folic acid. Why is it suggested that MTHFD1 polymorphisms may be associated with the susceptibility of NSCL/P, but not neural tube defects (Meng et al., 2015) or other diseases (Weiner et al., 2014; Silva et al., 2011)? It is mentioned in the Discussion section that controversial results about the MTHFD1 polymorphism rs2236225 (c. 1958G>A) have been reported in different articles. A brief description about the controversial results and their conclusions will make the discussion more interesting. MTHFR, MTR, AND MTRR which are involved in the metabolism of folic acid are reported to be associated with high risk of NSCL/P. Why MTHFD1 which is also involved in the metabolism of folic acid shows no significant association with susceptibility to NSCL/P? It’s due to the sample size or the limit of technique, or it is the truth? A brief discussion on this will be interesting.",
"responses": [
{
"c_id": "1709",
"date": "06 Jan 2016",
"name": "Feng Chen",
"role": "Author Response",
"response": "Thank you. We have studied your valuable comments and revised the manuscript according to your suggestions. The point to point responds are listed as following:We have corrected the error, thank you; Neural tube defects and NSCL/P is similar in the origin of development, while the different relationship with MTHFD1 may suggest the different pathogenesis. Maybe each part of organ has a specific different folic acid metabolic pathway in their development. We are so glad to follow your advice. In all of the included 8 studies, 7 did not show significant difference between the case and control groups. 1 study reported the case group showed closer relationship with MTHFD1 polymorphisms rs 2236225 (c.1958G>A). A brief description of this controversy is also included in this article. All 10 studies containing 6216 samples (2959 cases and 3257 controls) were analyzed in our article. Limited to the size of simple, our result cannot give a certain conclusion about the relationship of MTHFD1 and NSCL/P and won’t deny the possibility of an actual relationship. The heterogeneity exists in the research method, sample source area and maternal or children genotypes. The incident rate and clinical manifestation differ in various areas. Different research method would lead to diverse accuracy even controversial conclusion. Thus we are still not sure about the association between MTHFD1 and NSCL/P and further exploration is needed. We also discussed this matter in our article."
}
]
},
{
"id": "9328",
"date": "06 Jul 2015",
"name": "Jose Suazo",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general terms, this article contributes to unraveling the complex genetic architecture of NSCL/P. This type of systematic reviews are always relevant due to the fact that they pooled several singles trials, and therefore, the sample sizes have an important increase. However, this article needs some modifications that avoid to be published in its current version:All of the gene acronyms may be written italics. The title says \"Is MTHFD1 polymorphisms...\" but it refers to only one SNP. The Materials and Method section includes the quality analysis of each paper included in this meta-analysis, but Results section did not mentioned anything about this analysis. The publication bias analysis when < 10 articles is not statistically resolved (see Egers's paper), so for this meta-analysis it is necessary at least a comment about this issue. The first two paragraph of the Discussion section are almost the same as the Introduction. I think that the Discussion could include a comment about maternal genotype effects in order to explain the negative results of this analysis.",
"responses": [
{
"c_id": "1708",
"date": "06 Jan 2016",
"name": "Feng Chen",
"role": "Author Response",
"response": "Thank you so much for your precious advice. We have studied your comments carefully and revised the manuscript. The point to point responds are listed as following:We have corrected all the inappropriate format of gene acronyms; Our title says “Is MTHFD1 polymorphisms rs 2236225 (c.1958G>A)…”, which refers to a specific SNP in accordance with the content; The traditional meta-analysis focuses on the clinical RCTs, which aims to reveal the relevance between specific interruption and clinical effects. A complete assessment standard for the included RCT reports has been widely accepted and used. While our study material is totally different from RCTs thus there is no clear assessment standard to refer. So we do not discuss the quality analysis. But in order to provide the readers an assessment standard we provide the tables 2 in supplementary materials for reference. We agree that the usual method to evaluate publication bias is not suitable for our study because of the limited amount, which reduces the reliability of our conclusion. We have declared that in the discussion of our updated version. Actually the Discussion is an overview of the article, which includes and further explains the content above. While in avoidance of repetition we modified and simplified the first and second paragraph of the Discussion. Two of our included studies only analyzed the maternal genotype, one study reported the mother and children genotype and three took samples from children and their parents. The gene samples from mother were too scarce to be representative and to explain our results. We do recommend more samples from parents in the future studies, which is significant for the early stage diagnose, as the current technology can only diagnose CLP in the midterm even later in the pregnancy."
}
]
}
] | 1
|
https://f1000research.com/articles/4-142
|
https://f1000research.com/articles/5-20/v1
|
05 Jan 16
|
{
"type": "Review",
"title": "Harnessing the protective potential of HIV-1 neutralizing antibodies",
"authors": [
"S Abigail Smith",
"Cynthia A Derdeyn",
"S Abigail Smith"
],
"abstract": "Recent biological, structural, and technical advances are converging within the HIV-1 vaccine field to harness the power of antibodies for prevention and therapy. Numerous monoclonal antibodies with broad neutralizing activity against diverse HIV-1 isolates have now been identified, revealing at least five sites of vulnerability on the envelope (Env) glycoproteins. While there are practical and technological barriers blocking a clear path from broadly neutralizing antibodies (bNAb) to a protective vaccine, this is not a dead end. Scientists are revisiting old approaches with new technology, cutting new trails through unexplored territory, and paving new roads in the hopes of preventing HIV-1 infection. Other promising avenues to capitalize on the power of bNAbs are also being pursued, such as passive antibody immunotherapy and gene therapy approaches. Moreover, non-neutralizing antibodies have inhibitory activities that could have protective potential, alone or in combination with bNAbs. With a new generation of bNAbs, and a clinical trial that associated antibodies with reduced acquisition, the field is closer than ever to developing strategies to use antibodies against HIV-1.",
"keywords": [
"human immunodeficiency virus type 1",
"HIV-1 neutralizing antibodies",
"broadly neutralizing antibody",
"antibody-mediated protection",
"antibody-mediated therapy"
],
"content": "Introduction\n\nNeutralizing antibodies against HIV-1 were first described early in the HIV-1 epidemic1, raising hopes that a protective vaccine was in the immediate future. Yet 30 years later, only one vaccination regimen has shown any efficacy in humans to date2, essentially in the absence of functional neutralizing antibody activity3,4. Indeed, most vaccine approaches tested so far mainly elicit antibodies that can only neutralize T-cell line-adapted strains and the minor fraction of naturally occurring strains that exhibit a highly sensitive tier 1 neutralization phenotype4–8. This is because most naturally occurring HIV-1 isolates tend to mask vulnerable surfaces, making them much more difficult to neutralize, classified as possessing a tier 2 or 3 phenotype9–11. Stopping these neutralization-resistant, genetically diverse HIV-1 strains, which constitute the majority of the current global pandemic, will require an approach that successfully targets one or more regions of the trimeric envelope glycoprotein (Env) spike that are vulnerable to neutralization on most tier 2 viruses. However, all broadly neutralizing antibodies (bNAbs) identified thus far possess one or more unusual features12, suggesting that they will be difficult to generate via vaccination. To overcome this and other potential obstacles, the HIV-1 research community is now moving forward along two major paths: (1) developing novel strategies to elicit antibodies capable of broadly and potently neutralizing clinically relevant, genetically diverse HIV-1 strains and (2) combining knowledge of bNAbs with modern technologies to create protective alternatives and therapeutic options (Figure 1).\n\nEach area of pursuit is shown, and potential obstacles are listed below. For vaccination, there are two approaches for generating immunogens that will face common barriers. Env, envelope glycoprotein; NHP, non-human primate.\n\n\nInnovative application of bNAbs for HIV prevention and therapy\n\nDuring natural HIV-1 infection, most individuals develop antibodies with some level of heterologous neutralization breadth13,14, although only a small fraction of these individuals go on to develop high titers of the most extraordinary bNAbs15. A focused effort to recover monoclonal antibodies from these rare individuals with very potent and broad plasma-neutralizing activity has led to a collection of a few dozen bNAbs, defined as having the ability to neutralize more than 60% of the variants in multi-clade HIV-1 Env panels at a concentration of less than 50 μg/ml12,16. In turn, these discoveries have led to the identification of five regions on the viral Env trimer that are accessible to bNAbs: the CD4-binding site (CD4bs), the gp120 V1V2 hyper-variable domain, a glycan patch centered on the base of the gp120 V3 domain, the gp120 and gp41 interface, and the membrane proximal external region (MPER) of gp41. These discoveries have collectively bolstered optimism that bNAbs could also be elicited by vaccination.\n\nHowever, genetic and structural characterizations of the most promising bNAbs have revealed substantial obstacles to eliciting them through vaccination. Although HIV-1 bNAbs can arise from several immunoglobulin germline precursors, they have unusual features such as high levels of somatic hypermutation, uncharacteristically long CDR H3 (complementarity determining region H3) domains, and in some cases polyreactivity12. This may be because bNAbs must recognize complex epitopes that exhibit variation across HIV-1 strains and are often composed of both glycan and peptide17–21. Moreover, HIV-1 Env protein immunogens often do not readily bind and activate the germline precursors that have been associated with bNAb development, complicating immunogen design22–26. To gain more insight into the ontogeny of bNAbs, recent studies identified individuals who developed neutralizing antibody breadth early in infection and tracked co-evolution of the autologous virus and antibody lineages27–30. Key features that drive bNAb development appear to be the presence of a specific antibody germline precursor, a viral Env that can engage it, and temporal exposure of the antibody lineage to an array of highly related Env variants presenting different versions of the same epitope. The proficiency with which an antibody can neutralize autologous escape variants seems to be directly correlated with its ability to neutralize genetically diverse isolates29. Yet even within a successful bNAb lineage, there are antibodies that share the features associated with bNAb activity but do not acquire breadth29,31,32. The observation that some antibodies follow an evolutionary path toward acquiring neutralization breadth, while others do not, has yet to be explained. Incorporating information from these longitudinal studies of bNAb development into the design of Env immunogens that mimic this naturally occurring, but rare, process will be challenging, since it is likely that other host and viral factors also influence bNAb development12,33.\n\nNow that the development of two bNAb lineages has been traced in natural infection and the initiating and mutated Env variants determined, panels of Env immunogens based on these pathways can be developed and tested. Then again, the most important considerations for evaluating these novel Env immunogen panels are ambiguous. Should the indication to move forward with a series of immunogens be marked by successful stimulation of a particular bNAb germline, or a bNAb-related specificity with any germline? What level of heterologous tier 2 neutralization should be considered a success? Should protection from viral challenge also be demonstrated? Although transgenic mice have been used to model bNAb induction34,35, the rhesus macaque model remains the most relevant for HIV vaccine studies. But this model is not ideal. There is limited information about the development of neutralization breadth in HIV-1 Env-immunized or chimeric simian-human immunodeficiency virus (SHIV)-infected rhesus macaques36–40, and the antibody germline repertoire of this species is similar but not identical to that of humans39,41. Interestingly, Env-specific antibodies from immunized rhesus macaques that exhibited the highest somatic hypermutation levels arose from a single VH (immunoglobulin variable domain heavy-chain segment) gene37. Furthermore, a high level of somatic hypermutation in VH was not associated with serum neutralization breadth. Studies of immunized rhesus macaques have also demonstrated that CD4bs antibodies can be elicited, but these antibodies do not exhibit breadth, possibly because their angle of approach differs from that of the VRC01-like CD4bs bNAbs42,43. Thus, it will be important to carefully consider similarities and differences among non-human primates and humans when evaluating strategies to elicit bNAbs.\n\nAnother uncertainty regarding testing naturally occurring bNAb-associated Env immunogens is what form of Env should be administered. Do immunogens that retain the native trimeric Env structure present more relevant epitopes than monomeric gp120 or gp140 proteins for eliciting antibodies with breadth? The best-characterized Env trimer to date, BG505 SOSIP.664, is capable of activating inferred germlines from bNAbs in vitro26. However, thus far, immunization of rhesus macaques with the BG505 SOSIP.664 trimer has not induced cross-reactive tier 2 neutralizing antibody responses8. The BG505 trimer immunogen did elicit low titers of antibodies that neutralized the tier 2 autologous pseudovirus and higher titers of antibodies that neutralized tier 1 Env variants. In addition, rhesus macaques immunized with a different trimer produced an antibody response that was polyclonal even within those directed against a single target42. Additional Env trimers from different HIV-1 genetic subtypes (clades) of the virus are being generated and characterized44 and this will lead to a greater selection of immunogens to build upon.\n\nAnother matter to consider is that a precursor B cell that is capable of initiating a bNAb lineage may be relatively rare within the immune repertoire. To highlight the low chance of activating a particular heavy chain (VH) germline, there are approximately 76 to 84 functional VH possibilities in the human genome. The VH3-30 family, which is found in the V1V2-targeted bNAb lineage CAP256-VRC26 described by Doria-Rose et al.27 and Bhiman et al.29, contains 19 different alleles45. Furthermore, there are inherent biases for and against heavy chain families and even individual alleles within families. Stimulating a specific B cell out of so many possibilities, in a genetically diverse population, is a daunting task. Compounding the rarity of the initial B cell, even the unmutated common ancestor, may need to have acquired unique features, such as a long CDR H3, or unusual insertions/deletions. The CAP256-VRC26 bNAb lineage was initiated by an unmutated precursor that had an extraordinarily long CDR H3 region27,29. At 35 to 37 amino acids, the CAP256-VRC26 CDR H3 regions are more than twice the average length for human antibodies, approximately 15 amino acids46. These inherent obstacles will need to be taken into consideration when designing a vaccine strategy. Finally, a specific Env variant that is capable of activating this unique germline must be present. It seems unlikely that a randomly selected Env immunogen will elicit a bNAb lineage. In the case of CAP256-VRC26, a variant that evolved from a superinfecting virus, but not the primary infecting virus quasispecies, initiated the bNAb lineage29. The site of recognition on the antigen(s) by that initial antibody precursor also seems to be important, as different pathways to neutralization breadth were described for V1V2- and CD4bs-targeted bNAbs, the latter of which required a second helped antibody lineage27,28.\n\nEven with more knowledge about bNAbs and how they develop, the barriers standing in the way of an HIV vaccine are formidable. Avenues that are being explored independently of a vaccine are passive administration of bNAbs for immunotherapy and prevention. With a newer generation of bNAbs, an older idea such as passive immunotherapy might be more effective. The process of selecting bNAbs to advance into the clinical pipeline will require a careful assessment of multiple antibody properties, including their stabilities, pharmacokinetic profiles, and safety. One phase I trial of bNAb VRC01, which targets the CD4bs, has been conducted evaluating both intravenous and subcutaneous administration. This study indicated that both delivery routes were safe; however, subcutaneous delivery may be limited by the dosing required for adults and may be more suitable for infants47. Caskey et al. infused variable doses of 3BNC117, which also targets the CD4bs, into healthy controls and HIV+ patients48. This monoclonal antibody therapy was well tolerated, and all of the HIV+ patients who received the highest dose experienced significant decreases in viral load. Predictably, in some patients, a familiar specter emerged: mutations present within the patients’ viral quasispecies facilitated escape from 3BNC117 neutralization. Thus, like the scenario that successful antiretroviral drug therapy requires the use of several different inhibitors, passive immunotherapy will likely require the use of a cocktail of bNAbs that target distinct regions of Env. An additional consideration is that bNAbs do not always exhibit complete neutralization in vitro. In some cases, a subfraction of virions exists that is refractory to inhibition. This “incomplete neutralization” phenomenon has been attributed to heterogeneity in virion-associated Env glycosylation, or conformation within the genetically clonal virus population, or both49,50. bNAbs that are more tolerant of this type of variation are more likely to achieve complete neutralization, but this is also dependent upon the properties of the Env variant itself49. Recently, for bNAbs that are capable of complete viral neutralization, it was shown that the slope of the neutralization curve could be an important determinant of the therapeutic potential of a particular bNAb and should be considered along with breadth and potency50. This study also revealed that CD4bs and V3 base/glycan-targeted bNAbs, because of their high inhibitory potential, could be especially beneficial for immunotherapy.\n\nBesides viral escape, other roadblocks to the successful and widespread implementation of passive immunotherapy are the cost and logistics associated with the mass production of a sufficient amount of bNAbs. Researchers are now using tobacco plants to produce an arsenal of HIV-1 bNAbs51,52. The tobacco plants have been genetically modified to modulate antibody glycosylation and sulfation and allow antibody production to be ramped up to an industrial scale. A cocktail of monoclonal antibodies produced in this manner was used as an emergency treatment for Ebola during the 2014 epidemic53,54. With the recovery of so many different HIV-1 bNAbs, some with extraordinary potency and breadth, immunotherapy could also have an important role to play in treating HIV-1 infection.\n\nGiven the uncertainty of whether bNAbs can be induced via a vaccine, researchers are also venturing into the uncharted territories of gene therapy, which combined with the new generation of bNAbs has opened up new options. Gene therapy protocols are being developed to potentially deliver bNAb over long periods of time; the idea is that a sufficient amount of bNAbs present at the time of exposure might be able to prevent HIV-1 infection altogether. This concept is supported by numerous observations that passive administration of bNAbs to rhesus macaques at high concentrations can prevent mucosal SHIV infection55. One question that remains is whether bNAbs generated by passive administration, gene therapy, and immunization will all have the same protective potential56–59. As proof of concept, adeno-associated virus (AAV) has recently been used as a vector to deliver the genetic material that encodes VRC07, a CD4bs bNAb, into humanized mice and rhesus macaques. Both animal models showed that this mode of antibody delivery could protect from a mucosal challenge60,61. Gardner et al. took this concept a step further, creating a novel chimeric antibody that is more potent and broad than even the best bNAbs62. This construct, eCD4-Ig, combines the broad recognition ability of CD4 (a receptor essential for all HIV-1, HIV-2, and simian immunodeficiency virus [SIV] variants) with a sulfated peptide that mimics the co-receptor CCR5. When AAV was used to deliver eCD4-Ig to rhesus macaques, the animals showed no sign of infection for the duration of the study (40 weeks post-therapy), despite escalating intravenous challenges. This represents a stellar example of how a strong basic research program can lead to innovative and promising approaches63–67.\n\nGiven the difficulties in delivering bNAbs discussed above or in eliciting them by vaccination, some researchers are staying the course to a vaccine by developing approaches to protect against HIV-1 without bNAb. One alternative is to generate antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Although the RV144 vaccine trial generated antibody responses with minimal neutralization capabilities, these same antibodies were capable of mediating ADCC68. Thus, it is worth revisiting the protective potential of non-neutralizing antibodies, which could be more amenable to elicitation by a vaccine57,69–71. Another option is to delve into the antibody ontogeny of more HIV-1-infected patients, looking for more attainable antibody goals as well as identifying viral and immunological roadblocks that prevent the development of neutralization breadth. Rather than trying to coax the immune system into making rare antibodies, one could focus on strategies to elicit antibodies with moderate breadth, limited somatic hypermutation, typical CDR H3 lengths, and commonly used VH germline precursors72. Perhaps eliciting combinations of these more common types of antibodies would provide some level of protection in the end.\n\n\nSummary\n\nThe past several years have seen major advances in terms of antibody-mediated protection and therapy against HIV-1. bNAbs, though rare, are generated in multiple HIV-1-infected individuals. Insight into bNAb ontogeny during natural infection has revealed novel strategies for vaccination, but at the same time these studies have highlighted the difficulties that will need to be overcome. Other avenues for passive therapy and protection have become possible because of the new generation of bNAbs, some of which can neutralize up to 90% of genetically diverse isolates tested and could be even more effective when combined optimally73. Innovative design of Env immunogens, chimeric antibodies, and enhanced bNAbs may also yield protective or therapeutic benefits. Finally, bNAb-independent approaches that involve more moderate neutralization breadth or non-neutralizing effector functions may also hold promise.\n\n\nAbbreviations\n\nAAV, adeno-associated virus; ADCC, antibody-dependent cellular cytotoxicity; bNAb, broadly neutralizing antibody; CD4bs, CD4-binding site; CDR H3, complementarity determining region H3; Env, envelope glycoprotein; SHIV, chimeric simian-human immunodeficiency virus; V1V2, gp120 hyper-variable domains 1 and 2; V3, gp120 variable domain 3; VH, immunoglobulin variable domain heavy-chain segment.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nCAD and SAS are supported by NIH-R01-AI58706.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWeiss RA, Clapham PR, Cheingsong-Popov R, et al.: Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients. Nature. 1985; 316(6023): 69–72. PubMed Abstract | Publisher Full Text\n\nRerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al.: Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med. 2009; 361(23): 2209–20. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHaynes BF, Gilbert PB, McElrath MJ, et al.: Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 2012; 366(14): 1275–86. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nShingai M, Donau OK, Schmidt SD, et al.: Most rhesus macaques infected with the CCR5-tropic SHIVAD8 generate cross-reactive antibodies that neutralize multiple HIV-1 strains. Proc Natl Acad Sci U S A. 2012; 109(48): 19769–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSundling C, Li Y, Huynh N, et al.: High-resolution definition of vaccine-elicited B cell responses against the HIV primary receptor binding site. Sci Transl Med. 2012; 4(142): 142ra96. PubMed Abstract | Publisher Full Text\n\nSundling C, Forsell MN, O'Dell S, et al.: Soluble HIV-1 Env trimers in adjuvant elicit potent and diverse functional B cell responses in primates. J Exp Med. 2010; 207(9): 2003–17. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSundling C, Zhang Z, Phad GE, et al.: Single-cell and deep sequencing of IgG-switched macaque B cells reveal a diverse Ig repertoire following immunization. J Immunol. 2014; 192(8): 3637–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPhad GE, Vázquez Bernat N, Feng Y, et al.: Diverse antibody genetic and recognition properties revealed following HIV-1 envelope glycoprotein immunization. J Immunol. 2015; 194(12): 5903–14. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTran K, Poulsen C, Guenaga J, et al.: Vaccine-elicited primate antibodies use a distinct approach to the HIV-1 primary receptor binding site informing vaccine redesign. Proc Natl Acad Sci U S A. 2014; 111(7): E738–47. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJulien J, Lee JH, Ozorowski G, et al.: Design and structure of two HIV-1 clade C SOSIP.664 trimers that increase the arsenal of native-like Env immunogens. Proc Natl Acad Sci U S A. 2015; 112(38): 11947–52. 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J Transl Med. 2014; 12: 346. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11485",
"date": "05 Jan 2016",
"name": "David C. Montefiori",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11484",
"date": "05 Jan 2016",
"name": "Michael Seaman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-20
|
https://f1000research.com/articles/5-18/v1
|
05 Jan 16
|
{
"type": "Opinion Article",
"title": "The life cycle of a genome project: perspectives and guidelines inspired by insect genome projects",
"authors": [
"Alexie Papanicolaou"
],
"abstract": "Many research programs on non-model species biology have been empowered by genomics. In turn, genomics is underpinned by a reference sequence and ancillary information created by so-called “genome projects”. The most reliable genome projects are the ones created as part of an active research program and designed to address specific questions but their life extends past publication. In this opinion paper I outline four key insights that have facilitated maintaining genomic communities: the key role of computational capability, the iterative process of building genomic resources, the value of community participation and the importance of manual curation. Taken together, these ideas can and do ensure the longevity of genome projects and the growing non-model species community can use them to focus a discussion with regards to its future genomic infrastructure.",
"keywords": [
"Genome sequencing",
"Bioinformatics education",
"opinion in bioinformatics",
"insect genomics",
"biocuration"
],
"content": "Introduction\n\nIn this perspectives and opinion article, created from the viewpoint and experience of an insect genome informatician, I seek to explain how the generation, maintenance and publication of genome projects has now reached a stage that requires the community to pause for thought. Each genomics community is at a crossroads while it decides on what is the best strategy for generating and using high quality genome projects. There are undoubtedly leaders who have been pursuing specific strategies but the community is not necessarily on the same path. For over a decade, dozens of genome projects have been completed and this number is increasing exponentially. Due to my experience around insect genomics and participation in the i5k activities (an international consortium providing leadership and resources for insect genome projects1), I shall present my views centred around the insect community but hopefully these views are broadly applicable. Previously, a co-author and I explained how the new, cheaper technologies changed the landscape by which genome projects are conceived and organised in the hope we can guide a burgeoning insect genomics community2. We also argued that individual researchers can produce a genome reference sequence for their favourite species and the large consortia are no longer needed. Since then, we have seen first-hand how an incredible worldwide effort has managed to shed light on why and how we can initiate hundreds (if not thousands) of insect genome projects1. The i5k’s aim is to educate and support individual scientists as they seek to acquire genomics skills. It has also produced the first tranche of key insect genomes, mainly picked due to their place in the phylogenetic tree, an achievement which may be currently underutilised but whose importance cannot be understated. After involvement with multiple genome projects, a diverse array of transcriptomic projects and at least one widely used genomics software, I have come to conclude that in our rush to initiate the genomes projects of a larger part the tree of life we have neglected some important issues. First, the only reason our throughput is so high is because all published genome papers present merely just a draft. It is a useful draft but still only an initial effort. Second, this draft does indeed contain many of the instructions of how to generate an organism, but a genome sequence alone does not decipher it. It merely transcribes so we can conduct experiments with it. Deciphering will require both good experimental design and the capability to integrate such experiments. Third, the research community is not just the end-user but also part of the project team; we have, on the whole, neglected to bring them up to speed. These issues may seem intuitive but much of the community’s leadership is not conscious of it. Such issues cannot be readily resolved without substantial education. In this opinion article, I present some of the insights I gained from my own journey that I believe can help inspire the community to follow us as we hack our way through the multi-species genome sequencing trail.\n\n\nInsight 1: A brave new, informatics-led, capability\n\nDuring the last few years, genomics has moved from being a capability led by – and limited by – wet lab techniques to one led by computational science. One of the first eukaryotic genomes ever assembled was Drosophila melanogaster3, chosen partly because its genome architecture is much simpler than that of the human one. At the time, there was much discussion on whether sheer computational power would have been able to solve such a puzzle. The history of how this affected the human genome project is well known. There were two camps with many inspiring individuals in each. One side was the publicly-funded effort to sequence the human genome who believed that ‘clone-at-a-time’, overlap-based sequencing was the answer. One the major forces of innovation from that group was Eric Lander. Originally a mathematician, his team at the MIT was responsible for creating a completely automated sequencing pipeline4. As the pressure to publish was looming, the team realised that they did not have the computational software to generate the final genome assembly (until the skill and leadership of Jim Kent saved the day). The privately-funded side (TIGR and Celera), was led by a computational approach from the onset. Eugene Myers - having already worked on efficient computational approaches for biology (e.g. BLAST, suffix arrays and assembly) - proved that new computer science algorithms had the power to solve this problem much more efficiently5. Their computational capability drove the development for a new wet-lab technique called Whole Genome Shotgun (WGS) sequencing. The first camp, in other words, built computational approaches to match the wet lab technique. The second one built a wet-lab capability to match a novel computational approach. The majority of the molecular biologists at the time did not support this WGS approach as it was rightly considered to be of inferior quality. At the end of the day, however, it was the cross-talk of the two capabilities that not only produced a comprehensive human genome but also allowed many other species to have their genome sequencing completed. Indeed, the human genome project completely changed our perception of how biological research can scale in a world that transcends borders and how it should maintain its science-led focus (see 6). Even though we still follow most of these tenets, as shown from the vast majority of genome project publications, genome sequencing is an enterprise inherently focused on building resources. That does not mean that it is no longer science-led, it is just the science is not always the fields of biochemistry, ecology or genetics.\n\nFor the human genome project, computer science approaches were used to not only convert the wet-lab data to something the research community could make use of but to also guide the wet-lab techniques. By now, an entire coterie of associated industries such as information technologies (IT), informatics (the use of IT to handle large datasets) and data science is underpinning much of genomics. These sciences, often lumped into the rather over-used but useful umbrella definition of ‘bioinformatics’, have not only allowed us generate genomes but have now become an integral part of any downstream experiment using a reference sequence. Generally, the wider community is not aware of the true potential of informatics as it could be. The benefits go beyond being able to analyse a dataset: The epistemological understanding that comes with studying statistics and informatics can provide the skills for integrating the multi-disciplinary and ever-increasing amounts of data and the framework to make sense of a more synthesized knowledge. If we – as educators - allowed for aspects of programming and data science to become an integral part of the undergraduate curriculum - rather than the lip service that is currently common in most institutions – then we not only equip the next generation with a set of skills but we inspire a uniquely effective way of dissecting complex problems. Further, the high-throughput analysis of data is now a core requirement for any genomic experiment, yet often the analysis is delegated to computer programs (bioinformatic software), which are, effectively, “black boxes”. Such software are great for enhancing productivity but they ought not to be used before we understand all assumptions made on our behalf and explore their parameter space for each particular dataset7. Perhaps a way to resolve this is for the software engineering community to invest beyond core algorithms and produce high quality protocol papers that seek to explain what and how a software works while simultaneously providing a user-friendly interface that focuses on productivity (see Haas et al. 2013 for an example from a popular RNA-Seq assembly software8). A final point is that genomics – being data-rich - is ideal for exploratory research (i.e. generating new hypotheses). The varying quality of genomes and the inherent noise present in biology can actually be accommodated by approaches residing within the information science field (colloquially known as “big data science”). The information science field has been widely used in other disciplines and there are tangible and immediate benefits in experiments such as those using expression data (c.f. see a perspectives article by Hudson et al. 20129). The only caveat is that, like all experiments, the quality of any such outcomes will depend on the quality of our resources. In order to avoid surprises, before we proceed with such experiments, we ought to first understand the process of generating such resources.\n\n\nInsight 2: A “life cycle” and a grand experiment\n\nFrom a pedagogical perspective, one can compare genome projects to the life cycle of an insect (Figure 1). Like a developing insect, genome projects go through several stages of development: project design (often of an underestimated importance); DNA and RNA library preparation and sequencing (with rapidly evolving protocols); genome and transcriptome assembly (initially more than one before a consensus one is decided), structural annotation (e.g. “where are the genes and other features?”); functional annotation (e.g. “what does this gene do?”); manual curation of these two annotation types (often the most time-consuming step); and data dissemination (i.e. the steps that are visible to the public and perhaps the most important stage). Viewing this process as a life cycle provides not only the basis of an improved educational narrative but also some immediate insights. For example, genome project can go through multiple iterations of this “life cycle”. Further, like insects, the fitness of each stage depends on the quality of all of the previous stages. Also notable is the fact that one cannot proceed unless a stage is completed and “frozen” (in sequencing centre jargon, i.e. no longer manipulated). For example, the annotation process cannot begin unless the assembly is completed. Again, this insight may seem intuitive but having it at the forefront of our thoughts while undertaking a genome project will afford us with some important advantages.\n\nBefore elaborating on that, it is important to first point out that creating a draft genome sequence is a scientific experiment. There is at least one question (often a biological one, the nature of which depends on the discipline of the research leaders), but at the very least involves investigating an organism’s genetic blueprint. From a computer science point of view the question is straightforward: what is the correct genome sequence for this species and what are the parts that are important for its function. Further, there are a variety of possible methods and approaches that can be used, there is a risk of failure and at the end there will hopefully be more questions than answers. Therefore, like all scientific experiments, a good project design is essential. This ought to be led by someone - or a team - possessing in-depth knowledge of every step of the process. A review by Richards and Murali 201510 outlines many of the common issues a team has to consider when working with insect genome projects. For example, DNA availability and quality, genome size and polymorphism are some of the most important aspects that have led to the poor quality of a number of genome projects. As we complete more genome projects, further capturing and sharing that knowledge is something that the community sorely needs.\n\nBy perceiving genome projects as an experiment with a life cycle, one can begin identifying a number of useful insights. For the sake of brevity I will expand on only a couple of the most important ones that can help steer genome projects to be more likely to succeed.\n\nFirst, most genome projects want to address a particular question which varies between disciplines. It may be to perform a quantitative genetic study, fully ascertain a gene family which hosts a number of recently duplicated and near-identical members, or to build a more accurate phylogenetic framework and identify genes that are key innovations. Each one of these aims requires a genome of a different quality and therefore the project design ought to focus on those outcomes. For example, quantitative genetic studies depend on long scaffolds so markers can be associated with causal genotypes. Gene family ascertainment needs not only a high base-level accuracy but also characterisation of any gene family member turnover (i.e. Copy Number Variants) that may exist within a species11. Phylogenomic studies, on the other hand, require neither of these two characteristics: rather multiple species have to be analysed and putative key genes need to be painstakingly curated and characterized. Striving for perfection by achieving all these characteristics could be attempted in the first iteration but rarely do genome project teams have the diversity of skill, time and money to achieve it in a timely fashion.\n\nSecond, striving for perfection often manifests as a lack of discipline in keeping with the original experimental design when faced with access to new technological advances. This is even more critical when one considers that the speed of innovation in this field is extraordinary and that new technologies are less well-tested both in terms of wet-lab techniques and the dry-lab algorithms meant to analyse them. For example, even though new approaches such as long read sequencing12,13, linkage maps14,15 or chromatin interaction data16 are an under-used approach and can be of great value to improving genome sequence contiguity (i.e. scaffolding), they are high-risk, time consuming and expensive. Unless the original design included them, in practice they will end up delaying genome projects by months if not years. If one accepts the iterative nature of genomes, one can strive for timely incremental improvements.\n\nThird, we ought to remember that the life cycle moves forward only when we are satisfied with the quality and therefore when we are not satisfied we have to backtrack. Most genome sequencing groups learn early on to appreciate the need to complete each stage to a satisfactory level before proceeding to the next stage. Quality assessment using pre-defined metrics is standard practice in data science. More experienced workers also learn that once a stage is satisfactorily completed (“frozen”) and the next one started, one must under no circumstances go back. For example, once the structural annotation is completed, any perturbation of the assembly will invalidate the genome sequence and co-ordinates that gene models depend on. Certainly, if the stage is not satisfactory then it is expected that we go back one step (or even back to stage 1, project design) and start over. For example during the Helicoverpa genome project, we found that when there are high levels of polymorphism, the error levels of the 454 technology were prohibiting in completing an assembly at an N50 higher than 30 kb; short Illumina reads (ca. 100 bp at the time) were far more suitable and could be coupled with 454 mate pair libraries to produce a genome assembly of an N50 exceeding 1,000 kb. Had we decided to continue with the life cycle then we would have invested enormous effort in annotating a fragmented genome that was not suitable for our aims.\n\nFourth, the life cycle enforces the notion that quality of any one step is dependent on the outcomes of the previous step. For example, using the best possible starting material and extensive quality control of the sequencing data can add far more value to an assembly than any wet-lab or even dry-lab investment. This may also appear self-evident but, as mentioned by Richard and Murali, many non-model insect projects have limitations due to biology or availability of samples which no assembly algorithm can account for. For example within the Heliconius community, a multi-species genome project undertaken by the Discovar team, showed that even this new approach provides results inferior to the well tested Allpaths-LG approach (created by the same team), even in the hands of the authors. This is postulated to be due to these butterflies exhibiting high levels of complex polymorphism and repeat structures (Owen McMillan pers. comm. September 2015). Instead, a current protocol using a more traditional Allpaths-LG17 strategy coupled with a dense linkage map has allowed for a far superior assembly. At the end of the day, a combined understanding of the computational approaches used in genomics and the genomic architecture of a species is required to generate an excellent assembly; this will likely require more iterations of the life cycle.\n\n\nInsight 3: Sharing is caring\n\nSignificant resources and team effort are required for updating a genome version. In my experience, the greater of the challenges is how to co-ordinate the release of a new genome so that the community has access to the latest science and the genome team is rewarded for their contribution in way appreciated by their funding bodies and employers. There are not many insect genomes that have completed the life cycle multiple times but a flagship example is that from the silkworm, Bombyx mori, which had three publications. The first paper provided an early draft of the genome that turned out to be of limited broad utility but was published in Science18. The second was published shortly afterwards by a second, competing, group19. Even though it was of higher overall accuracy, that paper appeared in the journal of DNA Research (Oxford University Press). The third publication was the result of intense political activity, leadership and labour from both teams. It delivered a genome resource that was of higher quality than any other non-model species published at the time (and for the time being, it still is). However, this last iteration was published in the domain-specific journal of Insect Biochemistry and Molecular Biology20. Further, it is unlikely that any funding body would support any such activity today: once a genome is “published” it is deemed complete. Even though some of the raw data was made available through GenBank, it is important to note that each silkworm paper came with its own data repository and database. Indeed, there is still no “one stop-shop” for silkworm genomics and there is no support for the community to provide feedback for particular scaffolds or genes (i.e. “curation”). By all accounts, even though silkworm genomics is very much alive, it seems that the relevant informatic community is no longer active and I fear that most insect genome projects are – by default rather than choice - following this protocol.\n\nSo we are faced with two issues: how to provide informatic support beyond that initial publication and how to create a sustainable publishing model that allows for a genome project life cycle. In this particular case, I believe these two issues have solutions that can address both. One option, currently undertaken by the community, is for the papers exhibiting a new genome version to be submitted as a technical advance to a low impact factor but useful journal. This may, however, prevent engaging the best bioinformaticians and also undervalues the contribution of bioinformatics. Another option is for genome project teams to address a different, novel and important research question. That way the value of an improved genome resource can be properly showcased. This is time-consuming, however, and will therefore result in significant delays for making the data accessible to the community. A third option is to decouple publications from resources while at the same time respecting the value of genomics. We can achieve that if we shifted our focus from the “impact factor route” (21; c.f. the San Francisco Declaration on Research Assessment from the American Society for Cell Biology) and focus on “real world impact”. This is what an increasing number of government and research institutions (such as universities) are being asked to focus on. In genomics, we can make use of an updated version of the “Fort Lauderdale Agreement”. First, the data is made available before publication (in a controlled fashion) and the community is offered the opportunity to edit and improve it. Importantly, the community should be able to use it for downstream experiments and - if a journal editor agrees and their work does not fall under genomics but say biochemistry or molecular ecology – publish their findings (e.g. see 22 for an example from the Helicoverpa Genome Project). The assembly and annotation are benchmarked by this process and the community acquires both awareness and training. Eventually the first “genome paper” is submitted to a journal. This showcases not a new technical capability or a competition for being “the first but not the best” but rather a broad body of work from a relative large section of the stakeholders. The subsequent genome versions can be linked to either new experimental work on this one species or multi-species comparative genomic insights. Under the leadership of the Baylor College of Medicine, this is exactly the model that the i5k community has chosen. Even though a considerable number of genome projects have been “completed but unpublished”, they are nonetheless available either freely (e.g. the Mediterranean fruit fly is on NCBI) or upon request (for example see https://www.hgsc.bcm.edu/i5k-pilot-project-summary) and there are significant real world impacts as scientists across the world are collaborating using these new resources.\n\nOverall, our community is excellent at producing and disseminating the outcomes of top quality research, however, what the broader community values most is the dissemination and maintenance of data. Except terminal data (e.g. assemblies), primary (e.g. sequencing reads) and annotation data are also extremely valuable for conducting further experiments. A reliable and user-friendly IT platform for dissemination is the most effective way to reduce the bioinformatic bottleneck that is manifesting in many labs. Traditionally, data dissemination occurred in tandem with publication (e.g. via GenBank). Sadly, this is often limited to what occurred to get a particular paper accepted in that one publication and we cannot rely on journal editors to ensure that up-to-date data are available. Informatics has certainly empowered the community by providing it with a number of tools such as those based on “GMOD toolkit” and Content Management Systems23,24, Ensembl25, InterMine26 or even entire infrastructures that can support the knowledge discovery process from beginning to the end27. Provision of resources is also not limited by a lack of effort (c.f. the Nucleic Acid Research and Database journals) but issues such as lack of funding, maintenance, exchange of data from other resources or communication with the relevant community. As a consequence, their utility or life-span can be limited and the invested informatic effort wasted. We need a system that has the interoperability of the UCSC Browser (the version developed for cancer research; 28), the web-services of InterMine (created originally for Drosophila; 26), the data richness of the ENSEMBL project25 and the ecosystem of iPlant27. At the same time it hosts a dedicated team knowledgeable on insect biology and tasked with not only managing the data for the insect community but also building awareness for best practices, providing training and enforcing quality control. Without this resource, every new insect genome that is funded will be of limited value. The major issue for ensuring long-term sustainability is that sequencing centres and science leaders cannot guarantee the long-term provision of the required computational infrastructure. Even though centres such as the NCBI can host raw data and finalised gene models, they cannot provide a community portal with domain specific tools and resources. This is another area where the i5k consortium has shown leadership: in collaboration with the National Agricultural Library (NAL) of the USDA the insect community has now access to a dedicated team which is deploying an increasing number of tools (including the GMOD toolkit) and provides basic computational resources and training29.\n\n\nInsight 4: The human touch\n\nThe NAL team goes beyond merely hosting data and developing tools: they provide a platform for the community to assess the quality of genomes and edit the results of the automated bioinformatic processes of annotation. This manual checking and editing, i.e. “curating”, is an important check on the automated approaches on the underlying data that any experiment will end up relying on and it has featured in all major genome projects. In the early days of genome projects, the automated annotation ‘freeze’ was the stage where significant community outreach and involvement was sought. This often took the form of Annotation Jamborees and these were driven – and funded - by the leaders of the consortium. There, community members would meet and edit the computational predictions using the Apollo annotation system30, discuss research questions and co-ordinate project activities. These events are now mostly associated with the Sanger era where the costs to create a genome sequence were orders of magnitude larger than the costs associated with hosting a meeting. However, these meetings played a critical role in not only improving on the computational predictions but also forming a genomics community and educating researchers on how to use the genome31. As genome project costs have been driven down we had to invent new ways of co-ordinating work. One solution has been the International Arthropod Genomics Workshop but that lacks the immediacy and cannot deal with the enormous volume of data and diversity of species in a timely manner. Perhaps not surprising, informatics came to the rescue with a number of ‘community curation’ tools developed. In the insect world, the clear winner has been the Web Apollo software (also known as WebApollo) a plugin of the JBrowse genome browser32,33. Except for offering a real-time, internet-enabled implementation of genome viewing and editing, this informatic capability is underpinning NAL’s effort to help in forming, educating and maintaining genomic communities. Our greatest challenge in this space, however, is that we are one step behind: even though we are excellent in collecting and curating genomic data, we will still have to learn how to efficiently collect and curate a vast amount of new types of information such as those derived from epigenetics, population genetics and even ecology.\n\n\nFuture directions\n\nIf there was one final take home message it would be that while the genomics community is currently reaping the benefits of a number of technological advances, it is also about to be faced with a paradigm shift due to not only the number of genome sequences being made available but also the types of data that are becoming cheaper and increasingly common. Certainly, we need more scientists to learn how these data are derived and how to work with them more effectively but we – the informatics community - also need to educate more of them of how to produce high-quality, long-living projects that meet best practice. In my opinion, the challenge to deliver such an outreach activity is the development and the delivery of a high quality, unified course that will perhaps be tailored for each taxonomic domain or discipline. It is true that genome analysis and bioinformatics is a research discipline that takes years to master but – like statistics – it is also a useful set of tools that empowers everyone who chooses to invest the time to acquire some basic knowledge. Even further, it is such an exciting time to be a biological data scientist that those who decide to view computational biology as a skill to be mastered, while excelling in their chosen biological discipline, may also drive many of the next generation of synthesis in biology.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author is supported by the Hawkesbury Institute for the Environment (Western Sydney University); no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI would like to thank Scott Cain, Alex Feltus, Monica Munoz-Torres, Nassib Nassar, Konrad Paszkiewicz, Gil Smith, Rob Waterhouse, Jennifer Wortman, Yannick Wurm and many others for inspiring me to write this review. I am also grateful to Alex Watson-Lazowski and Stephen Richards for comments on an earlier version of this paper.\n\n\nReferences\n\ni5K Consortium: The i5k Initiative: advancing arthropod genomics for knowledge, human health, agriculture, and the environment. J Hered. 2013; 104(5): 595–600. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEdwards OR, Papanicolaou A: A Roadmap for Whitefly Genomics Research: Lessons from Previous Insect Genome Projects. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoff SA, Vaughn M, McKay S, et al.: The iPlant Collaborative: Cyberinfrastructure for Plant Biology. Front Plant Sci. 2011; 2: 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoldman M, Craft B, Swatloski T, et al.: The UCSC Cancer Genomics Browser: update 2015. Nucleic Acids Res. 2015; 43(Database issue): D812–D817. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoelchau M, Childers C, Moore G, et al.: The i5k Workspace@NAL--enabling genomic data access, visualization and curation of arthropod genomes. Nucleic Acids Res. 2015; 43(Database issue): D714–D719. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLewis SE, Searle SM, Harris N, et al.: Apollo: a sequence annotation editor. Genome Biol. 2002; 3(12): RESEARCH0082. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElsik CG, Worley KC, Zhang L, et al.: Community annotation: procedures, protocols, and supporting tools. 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}
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[
{
"id": "11905",
"date": "12 Jan 2016",
"name": "Stephen Richards",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent review of the genome project process and life cycle, that most valuably shows the reader how a genome project fits into the larger goal of biological research around a species. Dr. Papanicolaou's insights remind us how genome scale data \"completely changed our perception of how biological research can scale in a world that transcends borders\" and provides passionate enthusiasm and advice for those researchers and communities without genomes who wish to join this new world.Insight 1 tells us about the power and necessity of bioinformatics for not just assembly and annotation, but the genome wide analyses required to gain the most biological insight, but additionally warns us against relying on the \"black box\" that software can become without understanding how it works. I for one have fallen for this by following the conventional wisdom about what a particular software package does, but finding out only by inspection of the code that something else entirely is going on.Insight 2 places the genome project in it's rightful place as part of the experimental life cycle. The emphasis on experimental design in genomics is important - all to often in the past this critical step is ignored either due to cost reasons in collecting a sufficient data set compared with the urge to do something and call it preliminary data or simply out of bravado and not planning, with the result of very poor genome assembly quality and unreliable or un-interpretable downstream analyses. Dr. Papanicolaou outlines the importance of things like community curation to simply enable the community to look closely at the data and gene models - something that is vital to get an idea of how much to trust any conclusions coming out. but at the same time to match genome quality to the desired experimental requirements, to freeze genomes and annotations, and to get on with it and publish. This is excellent advice, and many a genome projects publication has been stalled for multiple years in the pursuit of better quality without the contaminant investment of resources, or in slow transition between the various steps of analysis dues to poor planning and training for the entire process of the life cycle. This part of the review is required reading for anyone contemplating a genome project, directing the thoughts of the reader to consider the longer term plan for the genome for his or her's lab, experiment or even for a larger community, and tailoring the experimental plan to fit.The insights on Data Sharing, and the requirement for pre-publication data sharing are critical, and point the reader to resources that will enable placing new genome datasets in public repositories with long funding horizons, stable futures, and academic reach around the board. More interesting to this reviewer, was the discussion on the difficulties in funding and publishing the improvement of draft genomes in the future. Although this is getting technically easier with he advent of longer read sequencing technologies, the manuscript is correct in noting the difficulties in publishing a fourth improved draft genome compared with the third - it is hard to say it is a significant improvement to our state of knowledge when closing say 75% of the gaps. I believe in the future we will still be interested in \"effectively finished\" archival genomes, and that these will be worth data notes in lower impact journals, but the option of \"decouple publications from resources while at the same time respecting the value of genomics \" to me seems like the correct way forward as we one day hope to have sequenced all species on the planet - i.e. to read the primary biological data for life on earth. Whilst we realize the genome sequence of the 10,000th bird species may not make the highest profile journal, not to have this sequence in the natural history museums of the future seems unthinkable.The human touch insight is dedicated to the need for researchers to look at data to correct gene models, to understand the limits of the dataset. New tools allow this to be done in a co-ordinated manner with groups of researchers from around the world, with the result that research can be accelerated around the world with the sharing of a single genome. This is particularly true today with the use of RNAi and Crispr gene manipulation techniques. In the milkweed bug community RNAi was the mainstay of comparative developmental research, but relied on degenerate PCR to identify genes and design probes. A draft genome quickly gave this research community the information to design all the probes they needed, but human curation was still needed to checkoff the number of genes in a family had changed from the Drosophila model, or that the automated gene model had got the sequence right before committing to a wet lab experiment, and that phylogenetic trees had confirmed that the researcher was manipulating the gene he or she thought she was, and not a paralog or a gene from a different but related family.Overall Dr. Papanicolaou has written an excellent guide to the genome project, the reading of which will profit anyone contemplating a genome project. It is well written, and whilst I have a few differences of opinion on minor points, they are in no means enough to prevent indexation. Overall I believe this manuscript merits immediate indexation with no modification necessary.Bonus points for remembering and reminding us of the role of Jim Kent.",
"responses": []
},
{
"id": "11819",
"date": "15 Jan 2016",
"name": "John W. Davey",
"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 draws attention to several important issues related to the production and use of reference genomes and associated data sets, particularly gene annotations. It outlines the typical process for a genome project in the era when large consortia could acquire substantial funding for such a project and produce high-impact genome papers. It correctly notes that the process of generating genomes is rapidly changing, as sequencing costs are falling and tools are improving, allowing small groups to produce genomes for a fraction of previous costs, but also with reduced impact. This change has many scientific and political implications for the production of genomes, which the article attempts to summarise. However, I do not think it does a good job of summarising or addressing these implications. I hope the following criticisms will help to bring these important issues into the clearest possible light.The article highlights the conflict between the nature of a reference genome as a resource that requires long-term, communal effort and infrastructure, and the nature of science funding, which requires the repeated completion of substantial short-term goals leading to high-impact first- and last-author papers. It claims that genome projects are best undertaken with particular biological questions in mind, in order to deliver the most relevant possible resource at the time and to avoid perfectionism and the distraction of new sequencing technologies and assembly tools. It also rightly insists that genome projects require complex computational analyses that need to be understood to some extent by those producing and using the genome so that potential errors in analyses using the genome are well understood and can be fixed where possible. It calls for the active building and maintenance of communities of researchers working on particular genomes, partly through the training of scientists in genome assembly and annotation techniques. It also calls for the decoupling of publications from resources by rewarding efforts for their real world impacts, and for increasing the likelihood of real world impacts by making genomes available early and encouraging communities of researchers to use them. Finally, it highlights the importance of core teams such as that at the National Agricultural Library for disseminating training, hosting data, and providing tools and platforms in order to support individual groups working on genome projects, and the need to secure long-term funding for these teams.All of these points are important and worth making strongly (with a few caveats), both for those already involved in genome assembly and annotation and those new to the field. But they do not come across clearly in the article, as they are hobbled by inconsistent use of various concepts and by several bad examples that hurt the case being made.Firstly, the concept of a community is very unclear. There are 44 references to communities in the article, including the non-model species community, the genomics community, the insect community, the insect genomics community, the research community, the wider community, the software engineering community, the i5k community and the informatics community, but mostly just to 'the community'. There are then 39 references to what 'we' are doing or should do. Who are we? Which community or communities does 'we' refer to in each case, and who is the article addressed to? It is not clear who needs to do what differently in order to improve the situation, or where the problems really lie. To give one example, \"Third, the research community is not just the end-user but also part of the project team; we have, on the whole, neglected to bring them up to speed. These issues may seem intuitive but much of the community’s leadership is not conscious of it.\" Who have neglected to bring the research community up to speed? i5k? Informaticians? Which community's leadership is not conscious of the problems, the i5k community, the informatics community, the research community? The article at points seems to be attempting to address a general audience, but other times is directed to the i5k community, and at points seems to be saying i5k should be doing things differently, but at others recommending the i5k model as best practice. While these things are not necessarily incompatible, the article would be much easier to read if it was much clearer about the groups it is addressing, and the social structures that would improve the process of generating genomes.Secondly, the contrast between using genomes to answer questions and providing genomes as resources is passed over, with both being claimed as important while not addressing the conflict between them. 'Genomics' is sometimes used to refer strictly to the production of genome sequences and perhaps annotations, but sometimes to research done using these sequences and annotations. The concept of a genome project being an experiment is confused in the same way; sometimes it seems to refer to the genome assembly as an experiment itself, and sometimes to the genome as used to conduct an experiment that answers a biological question (which can drive the genome project design). And the same confusion arises over the encouragement for scientists to learn 'data science' and related fields; sometimes this is directly related to genome assembly, sometimes to research using the genome.Clarity on these issues is important, because at present the confusion obscures some hard problems. For example, while it is highly desirable to direct genome projects towards particular biological questions (to maximise the chance of funding and high-profile papers, and to circumscribe the limits of the genome project itself), and to engage the widest possible relevant community in the production of genome resources (to make sure the genomes are used correctly, to increase impact, and hopefully to increase quality of assembly and annotation), the article doesn't bring out explicitly the fact that these goals are antithetical. As more groups become involved in a genome project, the number of relevant biological questions increases, and the quality of the genome must increase to accommodate them, making it harder to design and manage the project (especially if the entire community is to be involved in not only the annotation but also the assembly, and do research on the genome along the way, as recommended in Insight 3).Also, while the article makes several welcome calls for better genomics education, the confusions over what the relevant communities are and the distinction between use and provision of genomes make it very unclear what the nature and extent of this education should be. Is the article arguing that bioinformaticians should do the assemblies but educate biologists in the limitations of genomes they deliver; or that bioinformaticians should train biologists to assemble and annotate genomes themselves; or that the role of bioinformatician should disappear and biologists should do it all themselves, given that all biology is computational these days; or that biologists should use more data science techniques on their research but leave the genome assembly up to dedicated bioinformaticians? The article seems to be arguing for variations on these possibilities at different points.I don't know what the answers are to these problems, but at least they should be brought out clearly in the article, rather than left obscure. With this in mind, I will turn to individual comments on the insight sections.Insight 1: there is an important point here, which is that results may vary greatly depending on how software is used, and a more basic one, which is that computation is (and always has been) required to produce genomes and so those who wish to produce a genome need to engage with computational analyses. But these points are obscured by more confusions and irrelevant or inaccurate points.The opening point, that genomics has moved from being led by and limited by wet lab techniques to being led by computational science, is highly debatable, and I personally don't agree, unless perhaps if 'genomics' here means biology in general. Genomics in the sense of genome assembly continues to be led by the available sequencing technologies, not by computation - the two current assembly methods referred to in the paper, Allpaths and Discovar, were both designed to fit an available sequencing technology, they did not prompt the development of the technology. In long read sequencing too, the technology is driving the algorithms, not the other way around. While the Celera assembler is a great achievement and is being heavily used to assemble long read sequences, it is far from the case that Pacific Biosciences and Oxford Nanopore are designing their machines to fit the design of the Celera assembler. And the human genome example doesn't support the case at all, given that, as noted, 'the WGS approach [was] rightly considered to be of inferior quality' and the private genome ended up incorporating a lot of the public mapping data; the article ends up concluding that it was the 'cross-talk of the two capabilities' that was important, contradicting the initial point of the paragraph.The second paragraph attempts to make the case for biologists to develop computational skills, but the range of terms used just further obfuscates the issue. What is the difference between information technologies, informatics, data science, information science and \"big data science\"? How exactly are they related to genomics and bioinformatics? What distinct 'epistemological understanding' does statistics and informatics provide that biology does not, and what is a 'framework to make sense of a more synthesized knowledge' (and why should a researcher want it)? If the point is to say biologists would benefit in general if they improved their computational skills, that may be true, but is isn't really relevant to an article about genome assembly (and it is mildly insulting to say biologists need to improve their statistical skills, given that they invented statistics). If the point is to say biologists need to engage with genome assembly and annotation, that's quite a different issue, and doesn't need to be backed up by the general case for computational training. Reducing the generalities about computation and increasing the specificities about how biologists need to engage with genome assembly and annotation would help here.Insight 2: again, several very different points are mixed up into one here. Genome projects to date do tend to follow a life cycle as described, and can be iterated. But the points that follow, especially those about experiments, are confused. It is true that a genome sequence can be used to test hypotheses, and that the relevant hypotheses can often direct the design of a genome project. But in what sense is 'creating a draft genome sequence' an experiment? What is the hypothesis being tested by the assembly process itself? In what sense is 'investigating an organism's genetic blueprint' a hypothesis-driven experiment? It's possible to make the analogy (perhaps every time an assembler compares two reads, it conducts an experiment to test whether the reads overlap or not?) but it is not very enlightening, and it is not necessary for making the case that good project design is essential, that a variety of methods can be used and that there is a risk of failure - many things other than experiments share these properties. The sentence about the computer science point of view is even more confusing; the question \"what is the correct genome sequence for this species\" does not require an experiment in the traditional sense, and \"what are the parts that are important for its function\" isn't really a computation-only question at all. Further, the advice here is quite convoluted - \"when we are not satisfied we have to backtrack\", but \"More experienced workers also learn that once a stage is satisfactorily completed... one must under no circumstances go back\", however, \"if the stage is not satisfactory that... we go back one step\". Clearly satisfaction is key here, but our satisfaction can change - and if our satisfaction about an earlier stage is changed by what we discover at a later stage, does that mean \"one must under no circumstances go back\"?While genome projects to date have followed a life cycle as described, and perhaps initial versions of a genome may need to follow this process, I'm not convinced that a strict adherence to this model for future iterations is helpful. Insisting that every stage of the life cycle is completed by the whole community step by step in order to lead to a paper of lower and lower impact is surely the model we want to get away from. There is decades of research in software engineering refining or rejecting completely this kind of waterfall model in favour of more incremental approaches; while there is still controversy over this, it seems likely that genomics could benefit from moving in this direction as well.In theory, there is no reason why genomes can't be patched and updated piecemeal as small assembly errors are fixed, or scaffolds are ordered, or single gene families are annotated, with infrequent major releases rolling together these patches. This is standard practice in the software industry and for the human genome. I don't claim this is the only way to do things, or that there aren't problems with this approach, and it is true the infrastructure is not in place to do this efficiently for non-model species. But that doesn't mean we should restrict ourselves to the existing life cycle model; adhering to this model is one of the causes of the problems the article is trying to address (big version releases lead to problems in acquiring funding, managing large communities, rewarding individual contributors, deciding on publication strategy...). Why not just change the model?Finally, the point about genome assembly often being limited by the biology of the organism is valid, but the example is a poor fit and should be removed. The Heliconius Discovar assemblies were never intended to provide reference-quality assemblies, as the Allpaths-LG assembly was, and the biology of the organism was not ignored, as the paragraph implies; in fact, the Discovar assemblies were specifically intended to test the Discovar assembler on a set of highly heterozygous genomes, and improve the assembler to deal with this data. The assemblies were preliminary and were never optimised because the Discovar team left the Broad and did not complete the project, so it isn't fair to compare the assemblies. A better example to support this point would be the Plutella xylostella genome, where considerable heterozygosity remained after ten generations of inbreeding and thorough fosmid sequencing was required to produce a genome of reasonable quality.Insight 3: the issues described here (how to provide informatic support beyond the initial publication and how to create a sustainable publishing model that allows for a genome project life cycle) are real, but the solutions provided are not very realistic, are already fairly standard practice, or do not address the issues. Three options are presented: submit new genome versions as low-impact technical papers; use the new version to address a new biological question, or (the preferred option) to decouple publications from resources and respect the value of the genomic resources. This last option might well be a good idea, but the proposals for achieving it fall short.Most of the points made (releasing data early, engaging the community and allowing them to publish before the genome is published in its own right, showcasing a wide variety of analyses in the eventual genome paper) are to do with the initial release of the genome, not how to maintain the genome beyond its initial publication. There isn't much new in these points, given that this is the template set by the human genome project, but that doesn't necessarily mean it's not worth highlighting them again. However, it should be noted that this very fluid use of data, where the community edits and improves the assembly and annotation, makes maintaining a strict life cycle with frozen stages even harder.The only point this paragraph does make about later versions of the genome is that they should be linked to new experimental work or multi-species comparative genomic insights - which is just the second option that was passed over earlier. Also, the first option is passed over because it is unappealing to the best bioinformaticians, but why should a bioinformatician working under the standard publishing model where first-author papers are required be more interested in the proposed model where a large range of community analyses, some perhaps previously published (and so lowering their impact or making them inadmissable for further publication), are put into one paper?The problem is correctly identified as the conflict between the publishing model for individual scientists and the need to build communal resources, but the text doesn't propose anything meaningful to address this, beyond insisting that it would be good to separate publications from resources. But how is this to be done? Which communities need to change what they are doing, and how they value work, to achieve this? What metrics should we be using and recommending to faculty in hiring computational biologists, other than publications? While touching on this issue, the article does not really address it, and does not extended 'real world impact' beyond the use of the data by other researchers. If this is the limit, what is wrong with the current system where impact is measured by the proxy of citations?Finally, the whole manuscript would benefit from more attention to detail. For example, \"Second, this draft does indeed contain many of the instructions of how to generate an organism, but a genome sequence alone does not decipher it. It merely transcribes so we can conduct experiments with it. Deciphering will require both good experimental design and the capability to integrate such experiments.\" - what do the two consecutive 'it's refer to? The organism, then the genome sequence? How does a genome sequence transcribe? What is being deciphered? What are the experiments being integrated with? \"this number is increasing exponentially\" - is it exponential? \"an achievement which may be currently underutilised but whose importance cannot be understated\" - surely overstated, but the hyperbole doesn't help here anyway. It's not convincing to just say the work is important; why is it so important?I am sorry to be so critical, especially in public. I hope this level of detail will be taken as a mark of respect for Dr Papanicolaou's expertise and passion for this subject, which I agree is a very important topic that needs to be engaged with by all involved. I thank him for stepping forward to raise these issues and hope that this review will be taken constructively and lead to improvements in the piece.The following typos or omitted words should be fixed:the genomes projects of a larger part the tree of lifeOne the major forces of innovationdataset: Theexplain what and how a software works For example, genome project can go throughallowed us generate genomesRichard and Muralifor their contribution in way appreciated byon the automated approaches on the underlying data that lacks the immediacy and Except for offering a real-time -> Because?may also drive many of the next generation of synthesis in biology. -> syntheses?",
"responses": []
},
{
"id": "11817",
"date": "15 Mar 2016",
"name": "Ian Holmes",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 nice opinion piece surveying the current community practices around genome projects and the shortcomings thereof. Using the metaphor of the insect lifecycle, the paper discusses the “genome lifecycle” (sequencing, assembly, annotation… re-sequencing, re-annotation, etc) and various lessons drawn from real-life case studies (e.g. informatics is key, perfection is the enemy of the good, we need to decouple data dissemination from publication to some extent, we need plans for sustained computational infrastructure, we need new collaborative tools).I agree with the positions espoused here and I find this piece a very insightful distillation of the challenges facing the community as funding pay-lines become tighter and we transition from an era of quick genome-project headlines to one in which the community can (with luck) collectively curate and maintain data, rather than letting data-silos decay.I had one minor suggested edit which is that the line “until the skill and leadership of Jim Kent saved the day” could use a citation (presumably to Kent & Haussler, 20011).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-18
|
https://f1000research.com/articles/5-3/v1
|
04 Jan 16
|
{
"type": "Case Report",
"title": "Case Report: Ischaemic appendicitis post mesenteric biopsy",
"authors": [
"Marianna Zukiwskyj",
"June Tun",
"Shashank Desai",
"June Tun",
"Shashank Desai"
],
"abstract": "A common indication for laparoscopic mesenteric lymph node biopsy is to provide a tissue diagnosis in the absence of palpable peripheral nodes via a minimally invasive approach. There are no reports to date of ischaemia to the appendix as a complication of this procedure.\n\nWe report the case of a 34-year-old lady who underwent a mesenteric biopsy for a lesion found incidentally on CT to investigate longstanding abdominal pain, and 2 days later required an appendicectomy for ischaemic appendicitis.",
"keywords": [
"Ischaemic appendicitis",
"biopsy",
"Carcinoma",
"mesenteric lymph node"
],
"content": "Introduction\n\nAppendicitis is the most common intra-abdominal surgical emergency. The classical pathophysiology of appendicitis is that of obstruction of the lumen and subsequent bacterial overgrowth3,8. Appendicitis arising as a complication of a surgical procedure has not been reported in the literature.\n\nA common indication for mesenteric lymph node biopsy, in the setting of enlarged mesenteric nodes or a mesenteric mass on imaging, is to provide a tissue diagnosis in the absence of palpable peripheral nodes via a minimally invasive approach1,5. Where this cannot by achieved by percutaneous techniques, laparoscopic mesenteric biopsy has been shown to be a safe and effective alternative, when compared with open techniques1,2,5. As laparoscopy is a minimally invasive technique, the reduction in wound healing time decreases the delay to therapeutic interventions such as chemotherapy2. Reasons for conversion to laparotomy include adhesions, poor intra-operative exposure and bleeding1. There are no reports to date of ischaemia to the appendix as a complication of this procedure.\n\nWe describe a case of appendicitis in the post operative period in a 34-year-old lady who underwent a mesenteric biopsy of a lesion found incidentally on CT.\n\n\nCase report\n\nA 34-year-old Caucasian lady, with no significant medical history, presented to her local GP with persistent epigastric pain and nausea. Investigation of the pain included an abdominal CT, which revealed an incidental mass in the right iliac fossa measuring 17mm suspicious for a carcinoid tumour, and no other features of concern or metastatic disease. She underwent a diagnostic laparoscopy and excisional biopsy of this mesenteric mass near the junction of the ileum and caecum, with a Harmonic scalpel. She had an uneventful recovery, and was discharged day 1 postoperatively. She later presented to the emergency department on the same day of discharge, with worsening abdominal pain and fevers. She was taken back to theatre the following day and was found to have a necrotic appendiceal tip, and underwent an appendicectomy.\n\nThe histology from the mesenteric biopsy showed a 10×15mm nodule of carcinoid tumour, as well as 2 of 4 lymph nodes containing metastases. The histology from the appendicectomy showed extensive mucosal necrosis, but no evidence of malignancy.\n\nThe patient has since undergone an open, uncomplicated right hemicolectomy which showed an 8mm grade 1 neuroendocrine tumour at the region of the ileocaecal valve with clear margins and 11 lymph nodes negative for metastatic disease. She is currently under the care of an oncologist and receiving lanreotide 60–120mg monthly, the duration of which is currently ongoing.\n\n\nDiscussion\n\nAppendicitis due to primary ischaemia, or as a complication of mesenteric biopsy, is not described in the literature. Acute torsion of the appendix and subsequent appendicitis has been described infrequently in the paediatric population4. The appendiceal artery is the terminal branch of the ileocolic artery, and runs in the free edge of the mesoappendix. Varying descriptions of anatomical locations and incidence of accessory appendiceal arteries are described in the literature6.\n\nWe hypothesise that this presentation of acute appendicitis on the second post-operative day following mesenteric biopsy was secondary to inadvertent vascular injury, and subsequent ischaemia, given the proximity of the initial operative field to the ileocaecal junction. One possible mechanism is that appendiceal artery, or a possible aberrant course of the appendiceal artery or accessory appendiceal artery, was not identified during initial laparoscopic survey and was divided during dissection. Another possible mechanism is that the appendiceal artery was indirectly affected by secondary thermal injury during harmonic dissection of the mesenteric lesion. Whilst uncommon, unexpected thermal injuries to surrounding structures have been reported during use of Harmonic dissection7.\n\n\nConclusion\n\nLaparoscopic mesenteric biopsies are becoming increasingly more common as histological diagnosis is required to confirm disease processes. This case illustrates an unusual complication of a procedure often viewed as straightforward and highlights the diligence required during mesenteric dissection to prevent inadvertent injury to nearby structures, as well as the possibility of unusual circumstances resulting in unexpected injuries when using the Harmonic scalpel as an energy source for dissection.\n\n\nConsent\n\nConsent for publication of information from this clinical case was sought and obtained from the patient.",
"appendix": "Author contributions\n\n\n\nJT and MZ wrote this initial drafts, and the final draft was reviewed and edited by SD. All authors have seen and agreed to the final draft of this manuscript.\n\n\nCompeting interests\n\n\n\nNone\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBhandarkar DS, Shah RS, Katara AN, et al.: Laparoscopic biopsy in patients with abdominal lymphadenopathy. J Minim Access Surg. 2007; 3(1): 14–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCasaccia M, Torelli P, Cavaliere D, et al.: Laparoscopic lymph node biopsy in intra-abdominal lymphoma: high diagnostic accuracy achieved with a minimally invasive procedure. Surg Laparosc Endosc Percutan Tech. 2007; 17(3): 175–8. PubMed Abstract | Publisher Full Text\n\nNunes FC, Silva AL: [Acute ischaemic appendicitis in rabbits: new model with histopathological study]. Acta Cir Bras. 2005; 20(5): 399–404. PubMed Abstract | Publisher Full Text\n\nDarbyshire D, Sutton PA, Kosai NR, et al.: Torsion of the vermiform appendix – a review of the literature. West London Med J. 2011; 3(1): 14–18. Reference Source\n\nDurai R, Mir N, Ng PC: Laparoscopic retroperitoneal/mesenteric lymph node sampling: a safe and effective technique. Singapore Med J. 2011; 52(10): 758–62. PubMed Abstract\n\nKulkarni U, Kulkarni D: Variations in arterial supply of vermiform appendix. IJAV. 2011; 4: 52–54. Reference Source\n\nVetere PF, Apostolis C: Ureteral injury due to a Harmonic scalpel during laparoscopic salpingo-oophorectomy. JSLS. 2010; 14(1): 115–119. PubMed Abstract | Free Full Text\n\nWangensteen OH, Dennis C: Experimental proof of the obstructive origin of appendicitis in man. Ann Surg. 1939; 110(4): 629–647. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "12127",
"date": "01 Feb 2016",
"name": "Heidi K Chua",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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. Zukiwsky, Tun and Desai has described ischemia as a potential complication of mesenteric biopsy. This certainly highlights the need to be cautious in performing what might be considered a minor procedure. I do have 2 requests:Can they be more descriptive in how the actual excisional biopsy was done - was there an attempt to identify the vascular supply to the nearby structures? Can they suggest ways of preventing ischemic injuries while performing similar procedure?",
"responses": []
},
{
"id": "11815",
"date": "11 Feb 2016",
"name": "Brian Keith Bednarski",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a case report of a complication from a laparoscopic mesenteric excisional biopsy. Following an excision of an suspected carcinoid node in the mesentery, the patient suffered an ischemic event to the appendix.My comments/suggestions for the article are as follows:The authors describe that the indications for the laparoscopic excision are for those areas not amenable to percutaneous biopsy. This should also include lesions that have failed image guided biopsy. To that end, please comment on the patient's course in that regard. Did they have an attempted biopsy? Did they have a biopsy that failed to yield the diagnosis? Did the patient have a colonoscopy? With a suspicious carcinoid, an endoscopic evaluation should be considered to assess for primary tumor. Also, please comment on the intraoperative findings in more detail. Carcinoid tumors, and specifically its associated lymphadenopathy, have a very distinctive appearance that can clue in the operative surgeon to the diagnosis. Did you consider intraoperative pathologic assessment? With the possibility of completing the hemicolectomy in the first operation? Lastly, I agree with the other review that additional information regarding recommendations/suggestions to avoid mesenteric vascular injury during these procedures would be helpful.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-3
|
https://f1000research.com/articles/5-2/v1
|
04 Jan 16
|
{
"type": "Opinion Article",
"title": "A pocket guide to electronic laboratory notebooks in the academic life sciences",
"authors": [
"Ulrich Dirnagl",
"Ingo Przesdzing",
"Ingo Przesdzing"
],
"abstract": "Every professional doing active research in the life sciences is required to keep a laboratory notebook. However, while science has changed dramatically over the last centuries, laboratory notebooks have remained essentially unchanged since pre-modern science. We argue that the implementation of electronic laboratory notebooks (eLN) in academic research is overdue, and we provide researchers and their institutions with the background and practical knowledge to select and initiate the implementation of an eLN in their laboratories. In addition, we present data from surveying biomedical researchers and technicians regarding which hypothetical features and functionalities they hope to see implemented in an eLN, and which ones they regard as less important. We also present data on acceptance and satisfaction of those who have recently switched from paper laboratory notebook to an eLN. We thus provide answers to the following questions: What does an electronic laboratory notebook afford a biomedical researcher, what does it require, and how should one go about implementing it?",
"keywords": [
"Code of Federal Regulations Title 21",
"Documentation",
"Data storage",
"Good Scientific Practice",
"Good Laboratory Practice",
"Laboratory information management systems",
"Software"
],
"content": "Introduction\n\nIn this article we argue that the implementation of electronic laboratory notebooks (eLN) in academic research is overdue, and we provide researchers and their institutions with the background and practical knowledge to select and initiate the establishment of an eLN in their laboratories. Based on our own extensive experience in moving from laboratory notebooks (LN) to eLN, we try to answer the following questions: What does it afford you, what does it require, and how should you go about implementing it?\n\nEvery professional doing active research in the life sciences is required to keep a LN. This is imperative for group leaders, post-docs, students, as well as technicians. LNs are the core element of record keeping, data management, and initial analysis and interpretation of results in research. Details of its specifications, storage, etc. are laid down in institutional, national, as well as international codes of conduct for research integrity and good laboratory practice1. These codes usually stipulate sequentially numbered and bound pages, use of permanent ink, storage for a minimum of 10 years; they often require that entries be signed and dated by a witness. The use of LN has a long history, which parallels the development of modern science since the Renaissance. However, while science has changed dramatically over the last centuries, LNs have remained essentially unchanged since pre-modern science2 (Figure 1). This is highly remarkable for a number of reasons. For one, most of the data gathered is no longer analog, but digital. Gone are the days when researchers read numbers from instrument for transfer to the LN. Today there is a complex mixture of (often repetitive) protocols, digital images, links to large data files, etc. In addition, the recent realization that there is a ‘reproducibility’ crisis in the life sciences, and an increasing number of high profile cases of research misconduct and subsequent retraction of publications has put record keeping in the spotlight. It is therefore not surprising that the pharmaceutical industry, with its superior resources and regulatory pressures (e.g. Code of Federal Regulations Title 213) has moved to eLNs. Many researchers and institutions in academia now realize that the implementation of eLNs is overdue. However, only a tiny fraction of university laboratories are using them. Major hurdles for implementation appear to include ignorance about practical issues, perceived scarcity of available options, and a lack of resources. As part of the implementation of an ISO 9001-certified quality management system, our department (Department of Experimental Neurology) has recently moved from LNs to an eLN. At this department with approximately 100 students, researchers, technicians we carry out multi-professional academic research in preclinical biomedicine with such standard approaches and techniques as in vivo and and in vitro modeling of disease, cell biology, molecular biology, biochemistry, as well as imaging (from multi-photon microscopy to magnetic resonance imaging). We therefore believe that our experience is applicable to a wide range of research operations in the life sciences.\n\nA: Page from the laboratory notebook of the father of experimental electrophysiology, Emil Dubois-Raymond (7 November 1818 – 26 December 1896). [Staatsbibliothek Berlin, 1865–1868, XIII, 22. VII. 65–9. VI. 68, reproduced with permission]. B: Pages from a contemporary laboratory notebook from the laboratory of the authors.\n\n\nWhy you will switch to an eLN\n\nWe believe that the question is not whether eLNs will become standard or even required in the academic life sciences, but when. The advantages of an eLN are as obvious as the disadvantages of the conventional LN4. Most of the original data obtained in laboratories worldwide is already digital and can easily be integrated or linked to the eLN. eLNs foster collaboration, as protocols, data and concepts can be shared within or between groups. Entries can be time stamped, changes are recorded, versions controlled. Protocols used frequently can simply be integrated as templates. Project progress and eLN use can be easily monitored by group or project leaders. eLNs are searchable, archiving is simple, and copies are easily made for the institution and the individual researcher, many of whom will leave the institution at some point. These features include just a few of the functionalities which are already available in eLNs and are completely absent in a LN. Future eLNs will provide further benefits, including direct data links to standard laboratory hardware through an application programming interface (API) and automatic alerts when instruments are malfunctioning or not calibrated, or direct links to open data repositories (such as Figshare or Dryad). LNs, on the other hand, tend to get lost, must stay within the institution, which in turn has to keep track of them and is charged with keeping reliable records of LNs, storing them and enabling access for at least 10 years.\n\n\nSelecting an eLN\n\nIf you are contemplating a switch to eLNs, you first need to decide what you expect from it, and match this with your resources (see also below). Table 1 summarizes the principal features of three different categories of eLNs. The simplest form (‘do-it-yourself’ – DIY - type) is a word processor or note-taking system5. It is cheap, easy to use, and has many of the features a conventional LN; its major drawback is its lack of any kind of audit trail or certification. Such DIY-eLNs thus do not even conform to the standards of classical LNs, and therefore are not a serious option for their replacement. Dedicated eLNs have many additional features. Importantly most commercially available eLNs are compliant with the Code of Federal Regulations Title 21 (CFR Title 21) of the US Food and Drug Administration (FDA). CFR Title 21 part 11 sets rigorous specifications for electronic record keeping, including electronic signatures and version control. CFR Title 21 is a must if protection of intellectual property or use of the records for regulatory processes (such as FDA) is a factor6. Dedicated eLNs also allow complex rights management within institutes and workgroups, and can integrate original data. High-end systems include all the features of an eLN, but also function as laboratory information management systems (LIMS), facilitating inventory management or direct link to laboratory equipment (such as microscopes, sequencers, etc.). Not surprisingly, while DIY-eLNs are very easy to use, the increasing functionality of dedicated eLNs and eLNs integrated into LIMS comes at the price of growing complexity in its use. This might be a particular concern when non-academic personnel need to work with the eLN. Another issue is language – the user menus and help functions of practically all commercially available eLNs are in English; only a few allow the user to switch to other languages. Again, this may, in combination with a complex functionality, pose problems, and hamper the acceptance of the eLN in non-academic and less tech-savy work environments. Several articles have reviewed and compared various eLNs7.\n\nNote that ease of use and the availability as well as power of features of eLNs are inversely related.\n\nAbbreviations: API, Application programming interface; 21 CFR 11, code of federal regulations title 21 part 11; LIMS, Laboratory Information Management System. *Indicates that this feature is available in some systems of this category only.\n\n\nWhat you need to get started\n\nFor the individual researcher planning to move to an eLN very few requirements exist. Several open source eLNs are freely available (e.g. 8). Some companies offer basic eLN versions for a limited number of users and only as cloud based solutions free of charge (e.g. Labfolder), but for full feature commercial solutions license fees will apply. If a whole workgroup, department, or institution wants to set up an eLN, it gets more complicated. First and foremost, one needs to make sure that the eLN will be accepted by the users. This is not trivial, as many researchers and technicians have been socialized using a conventional LN. They may not be familiar with the many additional useful features provided by an eLN, and are confronted with the challenge and potential distraction of learning how to use a new tool.\n\nTo investigate the willingness of staff in a large academic research institution to switch from paper LN to ELN, and to find out what they expect from an ELN, we have surveyed students, technicians, and scientists. We also queried the staff of a research department in the process of switching from LN to ELN. Across professions and career stages the preference was for an intuitive and easy to use interface, a better integration of digital content, use of templates, and the ability to structure notes better. Features considered much less relevant were annotation and freehand drawing, the ability to use mobile devices, or saving time. On an individual level, user expectations and ratings did not substantially change when they progressed from eLN-naive to eLN. More than 70% of those not using an eLN were eager to start working with one, while almost 82% of those already using an eLN now prefer it over the paper version. For details and full results of the surveys, see Supplementary materials 1–4. Although our survey revealed that users of paper laboratory notebooks had a strong motivation for switching to an eLN and a high satisfaction rate for eLNs among those using it, we recommend not to enforce the switch to an eLN. Rather, it should be offered as an opportunity to those who are interested, and scale up its implementation as more group members join in. Sceptics will be able to observe its use, and will very likely want to become users within a short time period.\n\nAnother important issue relates to information technology (IT). For workgroups and institutes, the program and data storage will need to run on a server, with local clients, or through a browser interface. Obviously, every lab member using the eLN needs access to a computer. Most eLNs can run on mobile devices and can therefore be taken to the bench or site of experiment even if no computer is present at the site. This requires a wireless connection (WLAN) covering the laboratory or institution. Data can be linked to the eLN by assigning the file and drive name where it is stored. More conveniently, clickable links can directly connect to the data, but this requires that the eLN is physically integrated into the data management structure of the institution. All of this means that in most cases the selection and installation of an eLN from a group level on needs the support of the institutional IT department. They will also be responsible for upgrades, backups, etc. For large-scale installations within whole departments and institutions, training and support contracts need to be considered. Table 2 gives an overview of the requirements.\n\nNote that prerequisites vary with type of eLN and number of users (see Table 1).\n\n\nObstacles and pitfalls\n\nAt present no standard exists for eLNs, and the market is still evolving, so that none of the software makers can guarantee support and further development of their eLN beyond a couple of years. As of now there are no standards for data annotation and integration, therefore migration between different platforms may be difficult or even impossible. In a worst-case scenario (eLN provider goes out of business, no further development or support), the existing eLNs must be saved to pdf-format (including time stamps, addresses linking to stored data etc.), or to html/xml formats, as this will help retain some of the functionality. Such a feature should be mandatory, and is provided by most eLNs. This would essentially mean reverting to a conventional lab book, but the pdf would still provide extra features such as searchability and ease of copying and storage. For eLNs evolving on an open source platform, termination of support of proprietary software is not an issue. However, development or bug fixing of open source software may also be terminated. In addition, such systems may have less support than commercial systems, and support be restricted to tech-savy users or environments with programming capabilities. Another issue relates to the complexity and wealth of functions provided in particular by the high-end eLNs (often part of a LIMS). If using the eLN becomes too complex or restrictive, users may start recording their work outside the eLN. Finally, committing to long-term license fees may be a problem, in particular for individual researchers who may have only fluctuating financial or institutional support.\n\n\nConclusion and recommendations\n\nHow biomedical scientists take notes and document their work has not changed much over the last 200 years: They write with a pen in a bound, paginated laboratory notebook. The only major modification is that today, printouts or images of results are often attached (Figure 1). Data, however, is meanwhile almost exclusively digital, and digital technology provides a plethora of tools for recording, annotating, sharing, processing, and storing all the information that cumulatively drives progress in the life sciences. Scientists use computers for everything and everywhere, privately and professionally, except for documenting their research, experiments, and laboratory procedures. Several reasons may account for the astounding survival of the paper LN. It is a robust and easy to handle ‘technology’, which has been handed down over generations of scientists. At the same time, the emerging eLN market has been dominated by expensive solutions for research and development in large life science companies. Standards for data annotation, exchange or export between different eLN platforms have not yet evolved. There is a hesitation to commit to a specific product that may no longer be supported when the company goes out of business. The inertia of scientists to abandon their cheap and time-honored record keeping system, despite its numerous disadvantages and despite the obvious advantages of electronic solutions, has hampered the development of mature and affordable products for the academic sector. This has led to a vicious circle: Lack of interest on the part of scientists has frustrated the development of dedicated software. Over the last few years, the situation has slowly but substantially changed, and mature and affordable (or even free) eLNs are available. Scientists who overcome their reservation and exchange their LN for an eLN regularly become avid supporters after a short learning period, praising functionalities like group collaboration, use of templates, embedding of data, scheduling, access to the eLN from any computer world-wide, etc. In addition, group leaders and organizations value enhanced documentation and version control, improved supervision of record keeping, as well as backup and archiving of records.\n\nEnsure willingness of staff to use the novel record keeping technology (ease of use, language of menus).\n\nClearly define the functionalities that you expect from the eLN. Do not get lost in the almost limitless portfolio of potential functionalities. Remember: You probably just want to replace your paper LN, and not install a new word processor, graphics editor, or groupware system.\n\nAlthough you may not be aware of it now, you probably want a product which complies to legal requirements like 21CFR11, as well as good scientific practice (full audit trial, restriction on deletion of data, timestamps, ability to freeze and sign entries, among others).\n\nUnless you will be the only user, flexible and hierarchical rights management is very important.\n\nThe system needs to be able to tag, filter and search entries. Organization of data in eLN in projects, subprojects, milestones, etc. is a must.\n\nMake sure that all entries, imported data, and links can be exported to a generic format (pdf, zip, xlm, etc.) for backup and reporting as well as allowing a bailout in case the maker of the software stops development, or your funds to pay for licence fees run dry.\n\nEnlist the support of your IT department at an early stage (selection of particular eLN).\n\nBeware of hidden costs (hardware like server, backup devices; on-site support and user training, if applicable etc.)\n\nBesides serving all the obvious functions of a paper LN, eLNs facilitate scientists’ workflow (quick creation and editing of experiments) and collaboration (sharing and reusing information, independent of location, harmonization of work practices). They allow the integration of data, images, files, etc. and can already read data directly from instruments. They eliminate the need to transcribe or cut-paste data from one system to another, thereby avoiding transcription errors. Templates and boilerplate text modules prevent tedious rewriting. eLNs facilitate the retrieval of data or information over long periods of time, improve data quality (legibility), and allow the detailed reconstruction of individual experiments. They facilitate the mobility of researchers. Last, but not least, eLNs promote compliance with Guidelines on Good Laboratory Practice (GLP) and Good Scientific practice, and help intellectual property protection by their compliance to 21CFR11. We have no doubt that eLNs will become standard in most life science laboratories in the near future.",
"appendix": "Author contributions\n\n\n\nUD prepared the first draft of the manuscript. UD and IP designed the survey, which was conducted and analyzed by IP. UD and IP 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\nSupported by the German Research Foundation (Exc 257), the Federal Ministry of Education and Research (01 EO 08 01), the Herman and Lilly Schilling Foundation, and intramural funding by the Berlin Institute of Health (all to UD).\n\n\nAcknowledgments\n\nWe thank Dr. Niko Offenhauser and Sebastian Major for their help in setting up the eLN.\n\n\nSupplementary material\n\nSupplementary material 1.\n\nResults of an anonymous survey at the Department of Experimental Neurology.\n\nClick here to access the data.\n\nSupplementary material 2.\n\nResults of an anonymous survey at Charité Universitätsmedizin Berlin.\n\nClick here to access the data.\n\nSupplementary material 3.\n\nPrintout of the questions of the survey at the Department of Experimental Neurology.\n\nClick here to access the data.\n\nSupplementary material 4.\n\nPrintout of the questions of the survey at Charité Universitätsmedizin Berlin.\n\nClick here to access the data.\n\n\nReferences\n\nThe European Code of Conduct for Research Integrity. Retrieved Oct 2015. Reference Source\n\nKanare HM: Writing the Laboratory Notebook. Amercian Chemical Society, Washington DC, 1985. Retrieved Oct 2015. Reference Source\n\nCFR Title 21. US FDA. Retrieved Oct 2015. Reference Source\n\nNussbeck SY, Weil P, Menzel J, et al.: The laboratory notebook in the 21st century: The electronic laboratory notebook would enhance good scientific practice and increase research productivity. EMBO Rep. 2014; 15(6): 631–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWalsh E, Cho I: Using Evernote as an electronic lab notebook in a translational science laboratory. J Lab Autom. 2013; 18(3): 229–34. PubMed Abstract | Publisher Full Text\n\nNickla JT, Boehm MB: Proper laboratory notebook practices: protecting your intellectual property. J Neuroimmune Pharmacol. 2011; 6(1): 4–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRubacha M, Rattan AK, Hosselet SC: A review of electronic laboratory notebooks available in the market today. J Lab Autom. 2011; 16(1): 90–8. PubMed Abstract | Publisher Full Text\n\nVoegele C, Bouchereau B, Robinot N, et al.: A universal open-source Electronic Laboratory Notebook. Bioinformatics. 2013; 29(13): 1710–2. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11787",
"date": "05 Jan 2016",
"name": "Thomas A. Kent",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 valuable description of the author’s experience with electronic laboratory notebook implementation within their department. There can be much value in educating the broader scientific community with respect to both their experience, the experience of those they surveyed and the available options for the researcher.The manuscript can be improved through better definition of terms and procedures related to the eLN with which the average laboratory scientist may not be familiar. Some examples follow:The title is appropriate and the abstract is a good summary.Introduction:The authors department pursued ISO 9001-certified laboratory quality management system. Could they expound on what this means, why they chose to pursue it and what it entails? What kind of resources did they devote to this and what did they hope to gain in return?“Why you will switch to an eLN”While suitably provocative, perhaps reword the section title as “Why you will switch to an eLN” seems to put the answer before the research is presented. Perhaps a more neutral statement might not appear that the conclusion has been pre-judged.In this section, the authors list several advantages to an eLN, including for example, that original data can be “easily integrated or linked to the eLN”. I assumed this meant a direct link to the eLN from the laboratory equipment, but later in this paragraph, the authors describe such a direct link as in the future. Could they then explain how data is easily integrated in the eLN? This question may be a major one for those used to using written notebooks where printouts are often physically attached. And if the data is not directly loaded into the eLN, how is it searchable? Are they scanning data through OCL or some other method? Some details would help the reader visualize how this system might help them.“What you need to get started”This is a well written section with a nice summary Table of the different options. If possible, it might be beneficial to discuss costs, perhaps within an order of magnitude for the different systems, although not if this is proprietary information. With respect to the surveys, while the heat maps are useful, it is not easy to follow whether there is a pattern to the different features. Perhaps an analysis using the highest/lowest scores graphically might be more helpful. It does not appear that any pitfalls were included in the questions. In the article, the authors later discuss possibility of loss of technical support, updates and possibility that the sponsor may go out of business. It is not clear that the survey included that possibility and how the respondents might have answered. I would consider this a modest weakness, but would like to hear some comment from the authors.“Obstacles and pitfalls”This is an excellent section that is most useful to the potential purchaser of a system and what are the minimum requirements that should be available should the support be terminated.This is an important section. Are there alternatives? For example, upfront costs and uncertain future of any wholly integrated platform are reasons that some labs have set up their own cloud based systems, although without all the functionality of an integrated system and without FDA certification, but the data can be easily referenced to physical lab books and as such provide some of the advantages of a whole integrated system.I have a question for the authors: Do they think that “electronic laboratory notebook manager” may become a new, sought after, position analogous to the “network manager” that is needed even though there are multiple commercial products available and standards widely accepted? Perhaps the pharma experience on how to manage a large, integrated eNL might be helpful here if the authors are aware of them, or perhaps they could describe how at the department whether the management of the eLN is the responsibility of specific individuals.“Conclusions and recommendations”This is a good summary of the authors’ thoughts. Perhaps a little harsh with the term “inertia of scientists”, there may be many reasons that scientists have not embraced eLN in their work, particularly if it is a small laboratory or one strapped for resources other than inertia.The authors only once mention the important issue of need for restrictions on alteration of data, and only in the Box 1. Perhaps some mention of this issue would be helpful in the text as well.The final list of recommendations is an excellent primer for exploring the possibility of an eLN. I commend the authors for their efforts to increase the visibility of such systems and providing their experience and perspective on this topic.",
"responses": []
},
{
"id": "11789",
"date": "06 Jan 2016",
"name": "Christoph Kleinschnitz",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDirnagl and Przesdzing provide a highly innovative and comprehensive summary for the use of electronic laboratory notebooks (eLNs) in life science research. They highlight advantages of eLNs over traditional hand written LNs, comment on possible obstacles and inform readers about distinct features/specification of different types of eLN systems, e.g. complex rights management, integration of original data, direct link to laboratory equipment etc. In addition, they offer a step-by-step instruction of how to establish eLN systems in everyday laboratory practice based on the successful implementation of eLNs in their own institution. From an internal survey of their staff (scientist, students, technicians) they deduce which features of eLN systems were considered most desirable and which ones were regarded less relevant.In total, the article is highly informative for any researcher working in the life science field and is a very well suited guide for everybody who plans to switch from LN to eLN.",
"responses": []
},
{
"id": "11785",
"date": "08 Jan 2016",
"name": "Barry W. McColl",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 persuasive argument for replacing conventional lab notebooks with electronic versions (eLNs). Crucially, they outline the current drawbacks of conventional lab books in the current age and why there is a need to consider alternatives. One comment would be that the authors could perhaps have indicated if alternatives other than the systems mentioned in the article exist? The benefits of adopting eLNs described seem clear and comprehensive but importantly the potential weak points are also considered to give some balance. A similarly balanced tone is evident in discussion of the willingness of researchers to adopt eLNs and their reaction to doing so. Thus while the article is clearly presenting the authors' favoured opinion they also reflect on alternative views. As commented on by the authors, adopting an electronic system particularly in larger departments or organisations will need the appropriate IT infrastructure and support. While the authors surveyed opinions from researchers it might also have been informative to gauge and record opinion from IT professionals as to some of the practical issues of particular relevance to lab personnel. Overall, this article is very well structured and presents a helpful and informative perspective and primer on this emerging issue.",
"responses": []
},
{
"id": "11788",
"date": "14 Jan 2016",
"name": "Ádám Dénes",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article the authors present their views on laboratory record keeping procedures and argue that electronic laboratory notebooks (eLN) would be most suitable to support modern scientific research replacing the traditional, paper based version. One major strength of this paper is the personal experience of the authors with eLN from their own department. As it comes clear from the text, researchers are often reluctant to switch to eLN despite their advantages over the old fashioned laboratory notebooks. The authors arguments for using eLN are well taken: original data generated in laboratories are mostly digital, storage of large files, protocols or images requires electronic data management anyway, whilst data sharing or searching large databases, protocols becomes much easier if using eLN. In addition, eLN run on portable devices and entries can be time stamped and changes recorded, which is required by the scientific community and regulatory pressures. Whilst the authors’ preference is obvious for eLN, they are cautious with their recommendations and clearly outline the potential pitfalls of eLN as well. In my opinion, the following issues might deserve a short explanation:What is the level of detail researchers should record in eLN? In the case of standard LN, complex data files and large figures mostly remain electronic, whilst some figures, tables, analysis results and conclusions are incorporated into the LN. However, software running on dedicated machines (flow cytometers, imaging devices, etc) generate large files that are only partially extracted in text, PDF or other forms due to their incompatibility with common word processing tools. Should eLN users attempt to incorporate some of these data into eLN considering limitations in storage space, or such files should remain stored and analysed as earlier? How safe do the authors feel keeping most of their research data in an organized manner in eLN using cloud-based systems or large databases that are available from external servers or institutions? Are there measures large research units should consider before switching to eLN to support the protection of intellectual property or their patent applications?Overall, I find this article very useful and informative. I suspect that in the near future most research labs will not be able to avoid using eLN due to a number of reasons explained clearly in the paper. Practical information from experienced users will greatly support the willingness of researchers to start using eLN and this could also contribute to dealing with reproducibility issues in the future.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/5-2
|
https://f1000research.com/articles/5-1/v1
|
04 Jan 16
|
{
"type": "Review",
"title": "Primary Hyperparathyroidism",
"authors": [
"Leonardo Bandeira",
"John Bilezikian",
"Leonardo Bandeira"
],
"abstract": "Over the past several generations, primary hyperparathyroidism (PHTP) has undergone a change in its clinical presentation in many countries from a symptomatic disease to an asymptomatic one. The reasons for this change in clinical presentation are related to the widespread use of biochemical screening tests, to the measurement of PTH more routinely in the evaluation of metabolic bone disease and to the status of vitamin D sufficiency in the population. Along with recognition of a broader clinical spectrum of disease, including a more recently recognized normocalcemic variant, has come an appreciation that the evaluation of classic target organs that can be affected in PHPT, such as the skeleton and the kidneys, require more advanced imaging technology for complete evaluation. It is clear that even in asymptomatic patients, evidence for microstructural disease in the skeleton and calcifications in the kidneys can be demonstrated often. Potential non-classical manifestations of PHPT related to neurocognition and the cardiovascular system continue to be of interest. As a result of these advances, revised guidelines for the management of asymptomatic PHPT have been recently published to help the clinician determine whether surgery is appropriate or whether a more conservative approach is acceptable.",
"keywords": [
"Primary Hyperparathyroidism",
"parathyroid glands",
"adenomas"
],
"content": "Introduction\n\nPrimary hyperparathyroidism (PHPT) is characterized by hyperactivity of one or more parathyroid glands, disordered calcium homeostasis, and a consequent increase in serum calcium and elevated or inappropriately present circulating levels of parathyroid hormone (PTH)1. In the 1940’s Fuller Albright described the classic manifestations of PHPT as a disorder of bones and stones2. Indeed, historically, PHPT was characterized best by skeletal and kidney involvement. This clinical landscape changed rather dramatically in the 1970s with the advent of the multichannel autoanalyzer that provided a serum calcium concentration whenever the biochemical screening test was ordered. The diagnosis of PHPT was made much more commonly thereafter by mild hypercalcemia, lack of any specific symptomatology, or obvious renal or bone disease3. Currently, up to 80% of patients with PHPT in parts of the world where biochemical screening is routine have “asymptomatic” PHPT1.\n\nMost patients with PHPT have a single, benign adenoma. A smaller percentage, about 15% to 20%, have multigland disease, including multiple adenomas and hyperplasia. Multigland disease is more common in familial syndromes such as multiple endocrine neoplasia (MEN) 1 or 24. Parathyroid carcinoma is rare, occurring in fewer than 1% of patients with PHPT1.\n\nGene mutations can be associated with the development of parathyroid tumors. These genes include MEN1, calcium-sensing receptor (CASR), HRPT2, RET (familial forms), and PRAD1/cyclin1 (sporadic tumors)1,5.\n\n\nEpidemiology\n\nPHPT is the leading cause of hypercalcemia6 and one of the most common endocrine disorders; the estimated prevalence is between 0.1% and 0.5% in the US3,7. It is observed mainly in postmenopausal women (female-to-male ratio of 3–4:1) over 50 years old6,8. A large Brazilian study showed a prevalence of 0.78%, and the vast majority of these patients were asymptomatic (81%)7.\n\nIn younger patients, PHPT should call attention to the possibility of MEN, especially type 1, because this genetic syndrome has rather high penetrance in the decades up to 30 years of age. By age 50, hypercalcemia will have surfaced in virtually all patients with MEN-1. Besides its earlier onset, PHPT in MEN-1 has a gender distribution more likely to be balanced and bone and renal involvement is likely to be more evident4. Autopsy studies have estimated the prevalence of MEN-1 in PHPT to be 1% to 18%9. A multicenter study conducted in Italy, involving 533 patients with PHPT, found a MEN-1 prevalence of about 13%. This study also showed that patients with MEN-1 were younger10.\n\n\nDiagnosis\n\nThe diagnosis of PHPT is very straightforward after confirmation of persistent hypercalcemia. The associated PTH level is frankly elevated or inappropriately normal. In the latter regard, PTH levels can be as low as the “20s” (with a normal range of 10 to 65 pg/mL) and still be regarded as inappropriate for a hypercalcemia state11. The total serum calcium must be adjusted for albumin. For every gram-per-deciliter reduction in the serum albumin concentration, the total calcium measurement should be adjusted upwards by 0.8 mg/dL. In regard to PTH, either the second generation (measuring the intact molecule and large inactive fragments) or third generation (measuring the intact PTH molecule exclusively) can be used for diagnosis1.\n\nAnother form of PHPT has been recognized over the past 15 years. Normocalcemic PHPT (NPHPT) is characterized by elevated PTH levels in the presence of serum and ionized calcium that are consistently normal. Secondary causes of an elevated PTH—such as renal compromise (creatinine clearance of less than 60 mL/min), 25-hydroxyvitamin D insufficiency (less than 30 ng/mL or 80 nmol/L), primary hypercalciuria, malabsorption syndromes, and medications such as lithium or thiazides—must be excluded. In patients who present with normal levels of serum calcium, high PTH concentrations, and vitamin D deficiency, treatment with vitamin D can lead to the common hypercalcemic form of PHPT3.\n\nAlso important in the evaluation of PHPT is a 24-hour urine collection. A calcium clearance-to-creatinine clearance ratio (CaCrR) will help in differentiating familial hypocalciuric hypercalcemia (FHH), a rare disorder that can be confused with PHPT. FHH is an autosomal dominant disease characterized by mild hypercalcemia, very low urine calcium excretion, and elevated serum PTH. It is caused by a mutation in the CASR gene. It is important to distinguish FHH from PHPT because FHH is a benign condition for which parathyroid surgery is virtually never indicated12. A CaCrR of less than 0.01, on a normal dietary calcium intake, is observed in 80% of patients with FHH. There is some overlap between FHH and PHPT, particularly in the CaCrR range of 0.01 to 0.02. However, it is unlikely in FHH for the CaCrR to exceed 0.02. Similar to the MEN syndromes, FHH displays high genetic penetrance and hypercalcemia becomes evident usually before the age of 30. There is also often a family history of hypercalcemia12,13.\n\nIn addition to the determination of urinary calcium excretion in PHPT, it is important to evaluate other urinary constituents that together may comprise stone risk in these patients. Whereas urinary calcium excretion alone is not helpful vis-à-vis determination of stone risk in PHPT, the complete urinary biochemical stone risk profile is. Therefore, the 24-hour urine collection in PHPT should include a full urinary stone biochemical risk profile14.\n\n\nClinical presentation\n\nPHPT presentations vary worldwide. This is particularly true now with the availability of the autoanalyzer that varies in usage throughout the world. Where biochemical screening is routinely performed, PHPT usually presents with mild hypercalcemia. Overt bone and stone diseases are unusual15. A good illustration of how the availability of biochemical screening can influence the clinical presentation of PHPT is seen in the study comparing PHPT in the US and China. In a cohort studied in New York City, 80% of subjects were asymptomatic, whereas virtually everyone in Beijing was symptomatic16. Thirteen years later, in 2013, China showed a reduction in symptomatic PHPT to 60% and this was due presumably to the wider availability of multichannel biochemical screening in that country. In the US, the disease continues to present primarily asymptomatically (80% of patients). However, a new phenotype, NPHPT, has emerged (see below)17.\n\nAlthough the trend for asymptomatic PHPT to become progressively more the dominant form of PHPT is clear worldwide, symptomatic PHPT continues to be the predominant form of the disease in some countries, such as India, Iran, and Saudi Arabia15.\n\nIn Brazil, an increase in the incidence of asymptomatic PHPT has also been observed recently. Within a decade, albeit in different cities, the incidence of asymptomatic PHPT increased from 34% in São Paulo to 82% in Recife7,8. Data from other Latin American countries are limited, but it seems that the majority of patients still present with symptomatic PHPT15.\n\nOsteitis fibrosa cystica (OFC) is the classic presentation of skeletal involvement in PHPT. Characteristic radiological features include a salt-and-pepper appearance in the skull, phalangeal subperiosteal bone resorption, cysts, and brown tumors. In addition, bone pain, skeletal deformities, and pathological fractures can be present18. Typically, bone mineral density (BMD) is very low and bone markers are elevated. Radiologically and densitometrically, cortical bone is more involved than trabecular bone. Thus, skeletal sites with a predominance of cortical bone, such as the distal third radius, will show greater bone loss. By dual-energy X-ray absorptiometry (DXA), the distal 1/3 radius site, thus, is preferentially affected14.\n\nNephrocalcinosis and nephrolithiasis are the most common complications of PHPT14. In Shanghai, where approximately 60% of the patients are symptomatic, the prevalence of kidney stones among patients with PHPT is 48%17. Hypercalciuria contributes to the formation of stones but the etiology is multifactorial14.\n\nPHPT can affect organs other than the skeleton and kidney when it presents as a symptomatic disease. Other affected organ systems include the neuromuscular (peripheral polyneuropathy and proximal muscle weakness), gastrointestinal tract (peptic ulcer and pancreatitis), cardiovascular (hypertension, atherosclerosis, left ventricular hypertrophy, valve calcification, and arrhythmias), and psychiatric (anxiety, irritability, apathy, psychosis, and loss of memory) disorders15,18,19. Also, manifestations related to symptomatic hypercalcemia can be present; these include nausea, vomiting, constipation, polyuria, polydipsia, mental confusion, coma, and a short QT interval on the electrocardiogram20. It should be emphasized that multi-organ involvement in PHPT is the classic presentation of symptomatic disease. The extent to which these organ systems may be involved in asymptomatic disease is not clear (see below).\n\nOwing to the prevalence of asymptomatic PHPT, OFC is rarely seen these days in developed countries. However, skeletal involvement continues to be readily detected by technologies such as DXA15,21. Reduced BMD is observed most often in distal 1/3 radius site, comprised mostly of cortical bone, with relative preservation of lumbar spine, a site that is endowed primarily with trabecular bone22. However, these densitometric findings are not consistent with epidemiological data that have consistently reported a greater incidence of fractures at trabecular (vertebral) as well as cortical (non-vertebral) sites in PHPT. Vignali et al. evaluated the incidence of vertebral fractures (VFs) in postmenopausal women with PHPT as compared with a control group. Patients with PHPT, regardless of whether they were symptomatic, had more VFs (24.6% versus 4.0%, P <0.0001)23. Also, there was no difference in VF risk between symptomatic and asymptomatic patients (34.1% versus 21.1%, P = 0.15) but compared with matched non-hyperparathyroid control subjects, these figures were clearly and significantly higher23.\n\nAnother study published recently compared the incidence of VF in patients with symptomatic and asymptomatic disease. Both groups had a high percentage of VFs (34.4% and 34.7%) but the difference between the two groups was not appreciated24. The availability of new technologies such as high-resolution peripheral quantitative computed tomography (HRpQCT) and trabecular bone score (TBS) has provided a greater understanding of skeletal microstructure in asymptomatic patients22.\n\nHRpQCT analyzes images from peripheral sites, such as the radius and tibia, and is capable of discerning microstructural characteristics. It can discern trabecular and cortical compartments separately with regard to volumetric bone density, bone geometry, cortical thickness, and trabeculae distribution and number25. A recent study showed, by HRpQCT, that patients with PHPT versus controls had decreased volumetric densities at trabecular and cortical compartments, thinner cortices, and more widely spaced and heterogeneously distributed trabeculae. These changes were more pronounced at the radius, suggesting a protective effect of increased mechanical load on the tibia, a weight-bearing site26. A study with a similar design found changes at cortical and trabecular bone in patients with PHPT as compared with a control group but only at the radius and not on the tibia27.\n\nTBS is a textural index that evaluates pixel gray-level variations in the lumbar spine DXA image, providing an indirect index of trabecular microarchitecture. It can be readily applied to a DXA image through the use of a specific software program that is approved by the US Food and Drug Administration. A low TBS value is associated with fewer, less well-connected, and more widely distributed trabeculae22,28,29. Studies have shown a positive correlation between TBS values and HRpQCT indices30,31. Silva et al. evaluated TBS, compared with HRpQCT findings in 22 postmenopausal women with PHPT, most of whom were asymptomatic31. TBS revealed a degraded trabecular index of 1.24 (normal is more than 1.35). Moreover, the TBS value was correlated with many HRpQCT indices of trabecular microarchitecture.\n\nThese new imaging technologies have revealed abnormalities in trabecular bone in addition to the well-known cortical involvement in PHPT. They help to resolve the clinical paradox of relatively normal lumbar spine BMD by DXA in the context of an increase in VFs in PHPT21,22.\n\nThe incidence of nephrolithiasis in patients with PHPT has declined as the percentage of patients with asymptomatic PHPT has increased21. However, when imaging studies are conducted among subjects who have no history or symptoms of renal stone disease, nephrolithiasis or nephrocalcinosis (or both) can be detected rather often32,33.\n\nNon-traditional manifestations of PHPT with particular regard to cardiovascular and neurocognitive features are the subject of interesting but inconclusive studies. At this point, we cannot reach any conclusions about either of these “off-target” systems with regard to their direct relationship to PHPT (see neurocognitive features in the “Quality of life” section). Although cardiovascular morbidity and mortality seem to be increased in patients with severe PHPT, these findings have not been confirmed in those with asymptomatic disease. Among these complications, increased vascular stiffness is the one that has been most consistently demonstrated. Other findings, such as cardiovascular mortality, hypertension, coronary artery disease, valvular calcification, ventricular hypertrophy, arrhythmias, diastolic dysfunction, and carotid atherosclerosis, are characterized by limited and conflicting data in asymptomatic patients34.\n\nMost reports on NPHPT come from referral centers in which subjects were evaluated for an osteometabolic disease35. Lowe et al. evaluated 37 patients with NPHPT and found rather high prevalence rates of osteoporosis (57%), fragility fractures (11%), and nephrolithiasis (14%)36. Amaral et al. compared patients with NPHPT and mild hypercalcemic PHPT and found no difference in prevalence of fractures and nephrolithiasis between the groups37. Charopoulos et al. examined the effects of PHPT in the skeleton by using peripheral quantitative CT and noted that the trabecular compartment is preserved in NPHPT38. In studies that have indicated more skeletal involvement than one might expect in NPHPT, the selection bias from a metabolic bone diseases center might be relevant.\n\nData are conflicting and limited about the natural history of NPHPT. Lowe et al. observed disease progression in 40% of patients36. However, other studies, with subjects randomly assigned from community populations, showed no progression to overt PHPT39,40. The prevalence of NPHPT varies widely among different reports and this is probably due to methodological differences. For example, two studies published in 2015, both performed with randomly assigned community populations in Sweden and Italy, found a prevalence of 11% and 0.44%, respectively, when using different cutoff values for PTH and 25-hydroxyvitamin D40,41.\n\nWe cannot draw many conclusions at this stage of our understanding of NPHPT, but it is fair to state that the absence of hypercalcemia does not imply that NPHPT is a mild or an asymptomatic form of PHPT. A significant proportion of patients with NPHPT can show characteristics similar to those of patients with symptomatic or asymptomatic hypercalcemic PHPT22. Screening of an unselected population can unmask subjects with NPHPT with no apparent signs or symptoms42. Thus, as is the case for the more common hypercalcemic variant of the disease, patients with NPHPT can present a spectrum of target organ involvement. Also, similar to its more established hypercalcemic counterpart, NPHPT has a variable natural history without any clear-cut factors that can predict who will or will not show disease progression35.\n\nThe “moans and groans” of PHPT, as classically described by Albright in the 1940’s, haunt us today because so many patients complain of non-specific neurocognitive issues. With the change in clinical presentation of PHPT, these neurocognitive issues have surfaced only to confound rather than to clarify a possible association between them and the disease. A central question, thus, relates to whether PHPT is associated in a causative way with these complaints. Some studies have found this association, although it remains unclear how much the events can be ascribed specifically to PHPT. Caillard et al., in a multicenter prospective study, analyzed patients with PHPT before and after a successful parathyroidectomy43. Preoperatively, non-specific symptoms were common: anxiety, body pain, abdominal distention, forgetfulness, headaches, and mood swings. Significant improvement in quality of life was found 3, 6, and 12 months after surgery. Veras et al. compared a group of patients with newly diagnosed PHPT with another group of patients with more long-standing disease and found that the latter were affected more in functional capacity, physical limitation, general health, and vitality44. Amstrup et al. found poor quality of life in patients with PHPT compared with a control group, even after parathyroidectomy45. These studies, using a generic quality-of-life questionnaire SF-36 (36-Item Short Form Health Survey), are by no means conclusive43–45.\n\nIn a randomized controlled trial with only asymptomatic patients, Rao et al. used scores to measure quality of life and psychosocial function, comparing a group that underwent parathyroidectomy and another group that was followed without surgery46. There was an increase in scores for social and emotional function in addition to declines in anxiety and phobia in the group that had surgery. Walker et al., in a case-control study that used several tests to measure cognitive function, found weaker performance in various domains such as depression, anxiety, and memory in patients with PHPT compared with a control population47. Parathyroidectomy led to an improvement in some of the cognitive parameters. However, no linear association between calcium or PTH and these abnormalities was noted.\n\n\nGuidelines for the management of primary hyperparathyroidism\n\nPatients with classic symptoms of PHPT should be referred for surgery48,49. The vexing question is who among those with asymptomatic disease should undergo parathyroidectomy. Four international workshops on the management of asymptomatic PHPT have addressed this question over the past 25 years48–50. The most recent one, in 2013, led to revised guidelines based upon advances over the previous 5 years. The newer observations have led the experts to recommend a more proactive approach to detect target bone and kidney involvement. The current indications for surgery in asymptomatic PHPT are as follows (Table 1)48:\n\n1. Serum calcium value of more than 1 mg/dL above the upper limit of normal.\n\n2. Peri- or post-menopausal women and men at least 50 years old who have a T-score of not more than −2.5 at the lumbar spine, femoral neck, total hip, or distal 1/3 radius. In premenopausal women and men younger than 50 years old, a Z-score of not more than −2.5 is recommended as the cut-point. Other approaches to skeletal evaluation (X-ray, VF assessment, TBS, and HRpQCT) are recommended to determine whether skeleton involvement is present. Substantial trabecular disease would support a decision for surgery. If a VF is present, surgery is clearly recommended.\n\n3. Creatinine clearance of less than 60 mL/minute.\n\n4. Presence of nephrolithiasis or nephrocalcinosis. Evaluation with imaging studies, such as X-ray, computed tomography (CT), or ultrasound, is now recommended.\n\n5. Presence of hypercalciuria (more than 400 mg/day) along with a complete urinary biochemical stone risk profile that places the patient at risk for nephrolithiasis or nephrocalcinosis.\n\n6. Age of less than 50 years.\n\nBMD, bone mineral density; CT, computed tomography; DXA, dual-energy X-ray absorptiometry; eGFR, estimated glomerular filtration rate48.\n\nNon-classic manifestations of PHPT are not included in the guidelines for surgery because the evidence is not yet in hand14.\n\nGuidelines are suggesting, for the first time, an approach to NPHPT. With annual follow-up, patients who become hypercalcemic should be evaluated by the guidelines for hypercalcemic disease. If there is disease-associated progression with fractures, bone loss, nephrolithiasis, or nephrocalcinosis, surgery should be considered even if the patient continues to be normocalcemic (Figure 1)48.\n\nBMD, bone mineral density; DXA, dual-energy X-ray absorptiometry; PHPT, primary hyperparathyroidism; PTH, parathyroid hormone.\n\nRevised recommendations for monitoring patients who are not to undergo parathyroid surgery are as follows:\n\n1. Annual serum calcium.\n\n2. Three-site DXA every 1 to 2 years.\n\n3. X-ray or VF assessment of spine if clinically indicated (height loss or back pain).\n\n4. Creatinine clearance and serum creatinine annually.\n\n5. If renal stones or other renal involvement is suspected: 24-hour biochemical stone profile plus renal imaging.\n\nIf, during follow-up, any criteria for surgery develop and there are no medical contraindications, surgery should be strongly recommended48.\n\n\nPreoperative localization/surgery\n\nAll patients should undergo preoperative imaging tests before parathyroidectomy, in order to locate the affected gland(s). Imaging tests are not used for diagnostic purposes. There is, thus, no indication for parathyroid imaging if surgery is not planned. The most commonly used tests are ultrasound, sestamibi imaging, and CT. The use of CT with four-dimensional techniques (4D-CT) is providing greater anatomical resolution51. A recent study that used imaging with 4D-CT only in patients whose parathyroid adenoma had not been identified with ultrasound and sestamibi showed a sensitivity of 89%52. Another study compared the cost-effectiveness of imaging tests by using a hypothetical cohort and calculating sensitivity and positive predictive value of the tests. It concluded that using more than one imaging technique is more cost-effective because it decreases the likelihood of a bilateral exploration53.\n\nThe traditional surgical approach is bilateral neck exploration, but “minimally invasive parathyroidectomy” (MIP) has gained acceptance as an attractive approach. MIP terminology refers to any operative approach, open or endoscopic, in which the goal is to remove, as non-invasively as possible, the abnormal gland. A small incision with local anesthesia is part of this concept. Although MIP has gained in popularity, the central point to remember is that the key to the successful parathyroidectomy is not so much successful localization of the parathyroid adenoma, as useful as that is, but rather identification of the expert parathyroid surgeon51.\n\nIn general, during MIP, intraoperative measurement of PTH (IOPTH) is employed to confirm that removal of the gland rectifies the problem. Because PTH has a mean half-life, measured literally in seconds in patients with normal renal function, it will normalize within minutes of removal of the parathyroid adenoma51. IOPTH improves the cure rate and is now routinely used in the setting of MIP. Different criteria for measurement and interpretation of IOPTH were suggested. One study compared different criteria (Halle, Miami, Vienna, and Rome) and noted greater accuracy when the Miami criterion was used. This criterion is defined as a PTH drop of 50% or more from the highest of either preoperative or pre-excision baseline value to the level at 10 minutes after hyperfunctioning parathyroid gland excision indicates surgical cure54. Richards et al. suggested, as a surgical cure criterion, a 50% fall into the normal range and showed a decrease in the rate of surgical failure, especially in patients with multigland disease55. The most recent guideline indicates use of the latter criterion48. Rarely (in well under 5% of cases), a patient who meets these intraoperative PTH criteria for “cure” will not be cured.\n\nImprovement in various organs affected by PHPT is noted after curative parathyroidectomy. Studies show beneficial effects in bone disease, including improvement in BMD, bone microstructure, and decrease in bone markers56–58. A reduction in the recurrence rate of nephrolithiasis has also been noted59,60. Tassone et al. recently evaluated the renal function of 109 patients before and after surgery and noted that surgical cure halts the deterioration of renal function in patients with PHPT61. Other studies suggest that curative surgery improves quality of life43,46,47,62.\n\n\nMedical management\n\nMany patients do not meet surgical criteria and elect not to pursue parathyroid surgery. There are also patients who meet surgical criteria but who opt for a non-surgical approach. There are still other patients for whom medical contraindications preclude a surgical approach1.\n\nCalcium intake should follow the guidelines established for the general population. There is no reason for calcium restriction in patients with PHPT48. In patients with 25-hydroxyvitamin D levels in the insufficient or frankly deficient range (namely, less than 30 or 20 ng/mL, respectively), vitamin D should be given cautiously. The amount of vitamin D can be titrated from 400 to 800 IU/day as the situation warrants. Many experts feel that in PHPT, a desirable goal is for the 25-hydroxyvitamin D to be maintained above 30 ng/mL48,63.\n\nMisiorowski evaluated the effect of a 2-year course of alendronate in symptomatic patients who refused surgery64. The lumbar spine and femoral neck sites improved. Khan et al. evaluated patients with asymptomatic PHPT in an experimental design that called for 2 years of continuous therapy compared with a group receiving placebo in the first year, followed by a crossover to alendronate in year 265. Lumbar spine and hip sites improved along with a reduction in bone markers. There was no improvement in BMD at the distal radius. The group that was crossed over from placebo to alendronate in year 2 showed a slope of improvement in BMD at the lumbar spine that mirrored the increase in BMD in year 1 in the group that received alendronate from the onset of the study.\n\nBisphosphonates have also been studied in NPHPT. This study matched groups receiving cholecalciferol with or without alendronate for 1 year. Similar to the study by Khan et al. with hypercalcemic PHPT, lumbar spine and hip BMD improved along with a reduction in bone turnover markers66. In virtually all studies, serum calcium, PTH, and urinary calcium excretion did not change64–66.\n\nThe calcimimetic, cinacalcet, has shown utility in PHPT. In general, cinacalcet normalizes serum calcium and modestly reduces PTH67–69. These studies have not shown any positive effects of cinacalcet on BMD or non-specific symptoms of the disease68,69. Peacock et al. evaluated patients with severe or mild disease and found normalization in serum calcium and decrease in PTH in both groups69. Faggiano et al. compared a group of patients receiving alendronate plus cinacalcet and another group receiving cinacalcet only70. In both groups, there was normalization of calcium and decrease in PTH; however, there was a significant improvement in BMD only in the group using alendronate.\n\nThese experiences give guidance as to which pharmacological agent one would select in those who are candidates for drug therapy. A cautionary note is that many patients with asymptomatic PHPT do not have to be considered for pharmacological therapy because their BMD is not low and their serum calcium is within 1 mg/dL of the upper normal range. In these subjects, there is no need to consider pharmacological therapy. For those whose bone density is low, however, a bisphosphonate would be a logical choice. For those whose serum calcium is in the range in which reduction is a desired goal, cinacalcet would be the logical choice. For those who present with both low BMD and serum calcium of greater than 1 mg/dL above the normal range, combination therapy would make sense71.\n\n\nConclusions\n\nPHPT is one of the most common endocrine diseases. Recent advances in the presentations of PHPT, with regard to both the hypercalcemic and normocalcemic forms, have led to new concepts regarding management. The revised guidelines reflect a more proactive approach to evaluation and management. We are still in need of more information regarding NPHPT as well as putative off-target manifestations of PHPT. For patients who have not met any guidelines for surgery and are not going to have surgery, conservative management with or without pharmacological intervention is an option.",
"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\nSociedade Brasileira de Endocrinologia e Metabologia, Bandeira F, Griz L, et al.: Diagnosis and management of primary hyperparathyroidism--a scientific statement from the Department of Bone Metabolism, the Brazilian Society for Endocrinology and Metabolism. Arq Bras Endocrinol Metabol. 2013; 57(6): 406–24. PubMed Abstract | Publisher Full Text\n\nAlbright F, Aub JC, Bauer W: Hyperparathyroidism: A common and polymorphic condition as illustrated by seventeen proved cases from one clinic. J Am Med Assoc. 1934; 102(16): 1276–1287. Publisher Full Text\n\nCusano NE, Silverberg SJ, Bilezikian JP: Normocalcemic primary hyperparathyroidism. J Clin Densitom. 2013; 16(1): 33–9. 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PubMed Abstract | Publisher Full Text\n\nSilva BC, Walker MD, Abraham A, et al.: Trabecular bone score is associated with volumetric bone density and microarchitecture as assessed by central QCT and HRpQCT in Chinese American and white women. J Clin Densitom. 2013; 16(4): 554–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSilva BC, Boutroy S, Zhang C, et al.: Trabecular bone score (TBS)--a novel method to evaluate bone microarchitectural texture in patients with primary hyperparathyroidism. J Clin Endocrinol Metab. 2013; 98(5): 1963–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCassibba S, Pellegrino M, Gianotti L, et al.: Silent renal stones in primary hyperparathyroidism: prevalence and clinical features. Endocr Pract. 2014; 20(11): 1137–42. PubMed Abstract | Publisher Full Text\n\nSuh JM, Cronan JJ, Monchik JM: Primary hyperparathyroidism: is there an increased prevalence of renal stone disease? AJR Am J Roentgenol. 2008; 191(3): 908–11. PubMed Abstract | Publisher Full Text\n\nWalker MD, Rubin M, Silverberg SJ: Nontraditional manifestations of primary hyperparathyroidism. J Clin Densitom. 2013; 16(1): 40–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCusano N, Silverberg SJ, Bilezikian J: Normocalcemic PHPT. In: Bilezikian J, Marcus R, Levine MA, Marcocci C, Silverberg SJ, Potts JT Jr., eds. The Parathyroids. 3rd ed. USA: Elsevier, 2015; 331–339. Publisher Full Text\n\nLowe H, McMahon DJ, Rubin MR, et al.: Normocalcemic primary hyperparathyroidism: further characterization of a new clinical phenotype. J Clin Endocrinol Metab. 2007; 92(8): 3001–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAmaral LM, Queiroz DC, Marques TF, et al.: Normocalcemic versus Hypercalcemic Primary Hyperparathyroidism: More Stone than Bone? J Osteoporos. 2012; 2012: 128352. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCharopoulos I, Tournis S, Trovas G, et al.: Effect of primary hyperparathyroidism on volumetric bone mineral density and bone geometry assessed by peripheral quantitative computed tomography in postmenopausal women. J Clin Endocrinol Metab. 2006; 91(5): 1748–53. PubMed Abstract | Publisher Full Text\n\nGarcía-Martín A, Reyes-García R, Muñoz-Torres M: Normocalcemic primary hyperparathyroidism: one-year follow-up in one hundred postmenopausal women. Endocrine. 2012; 42(3): 764–6. PubMed Abstract | Publisher Full Text\n\nKontogeorgos G, Trimpou P, Laine CM, et al.: Normocalcaemic, vitamin D-sufficient hyperparathyroidism - high prevalence and low morbidity in the general population: A long-term follow-up study, the WHO MONICA project, Gothenburg, Sweden. Clin Endocrinol (Oxf). 2015; 83(2): 277–84. PubMed Abstract | Publisher Full Text\n\nVignali E, Cetani F, Chiavistelli S, et al.: Normocalcemic primary hyperparathyroidism: a survey in a small village of Southern Italy. Endocr Connect. 2015; 4(3): 172–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCusano NE, Maalouf NM, Wang PY, et al.: Normocalcemic hyperparathyroidism and hypoparathyroidism in two community-based nonreferral populations. J Clin Endocrinol Metab. 2013; 98(7): 2734–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCaillard C, Sebag F, Mathonnet M, et al.: Prospective evaluation of quality of life (SF-36v2) and nonspecific symptoms before and after cure of primary hyperparathyroidism (1-year follow-up). Surgery. 2007; 141(2): 153–9; discussion 159–60. PubMed Abstract | Publisher Full Text\n\nVeras A, Maia J, Mesquita P, et al.: Lower quality of life in longstanding mild primary hyperparathyroidism. Arq Bras Endocrinol Metabol. 2013; 57(2): 139–43. PubMed Abstract | Publisher Full Text\n\nAmstrup AK, Rejnmark L, Mosekilde L: Patients with surgically cured primary hyperparathyroidism have a reduced quality of life compared with population-based healthy sex-, age-, and season-matched controls. Eur J Endocrinol. 2011; 165(5): 753–60. PubMed Abstract | Publisher Full Text\n\nRao DS, Phillips ER, Divine GW, et al.: Randomized controlled clinical trial of surgery versus no surgery in patients with mild asymptomatic primary hyperparathyroidism. J Clin Endocrinol Metab. 2004; 89(11): 5415–22. PubMed Abstract | Publisher Full Text\n\nWalker MD, McMahon DJ, Inabnet WB, et al.: Neuropsychological features in primary hyperparathyroidism: a prospective study. J Clin Endocrinol Metab. 2009; 94(6): 1951–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBilezikian JP, Brandi ML, Eastell R, et al.: Guidelines for the management of asymptomatic primary hyperparathyroidism: summary statement from the Fourth International Workshop. J Clin Endocrinol Metab. 2014; 99(10): 3561–9. PubMed Abstract | Publisher Full Text\n\nBilezikian JP, Khan AA, Potts JT Jr, et al.: Guidelines for the management of asymptomatic primary hyperparathyroidism: summary statement from the third international workshop. J Clin Endocrinol Metab. 2009; 94(2): 335–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBilezikian JP, Potts JT Jr, Fuleihan Gel-H, et al.: Summary statement from a workshop on asymptomatic primary hyperparathyroidism: a perspective for the 21st century. J Bone Miner Res. 2002; 17(Suppl 2): N2–11. PubMed Abstract | Publisher Full Text\n\nUdelsman R, Åkerström G, Biagini C, et al.: The surgical management of asymptomatic primary hyperparathyroidism: proceedings of the Fourth International Workshop. J Clin Endocrinol Metab. 2014; 99(10): 3595–606. PubMed Abstract | Publisher Full Text\n\nDay KM, Elsayed M, Beland MD, et al.: The utility of 4-dimensional computed tomography for preoperative localization of primary hyperparathyroidism in patients not localized by sestamibi or ultrasonography. Surgery. 2015; 157(3): 534–9. PubMed Abstract | Publisher Full Text\n\nWang TS, Cheung K, Farrokhyar F, et al.: Would scan, but which scan? A cost-utility analysis to optimize preoperative imaging for primary hyperparathyroidism. Surgery. 2011; 150(6): 1286–94. PubMed Abstract | Publisher Full Text\n\nBarczynski M, Konturek A, Hubalewska-Dydejczyk A, et al.: Evaluation of Halle, Miami, Rome, and Vienna intraoperative iPTH assay criteria in guiding minimally invasive parathyroidectomy. Langenbecks Arch Surg. 2009; 394(5): 843–9. PubMed Abstract | Publisher Full Text\n\nRichards ML, Thompson GB, Farley DR, et al.: An optimal algorithm for intraoperative parathyroid hormone monitoring. Arch Surg. 2011; 146(3): 280–5. PubMed Abstract | Publisher Full Text\n\nHansen S, Hauge EM, Rasmussen L, et al.: Parathyroidectomy improves bone geometry and microarchitecture in female patients with primary hyperparathyroidism: a one-year prospective controlled study using high-resolution peripheral quantitative computed tomography. J Bone Miner Res. 2012; 27(5): 1150–8. PubMed Abstract | Publisher Full Text\n\nTournis S, Fakidari E, Dontas I, et al.: Effect of parathyroidectomy versus risedronate on volumetric bone mineral density and bone geometry at the tibia in postmenopausal women with primary hyperparathyroidism. J Bone Miner Metab. 2014; 32(2): 151–8. PubMed Abstract | Publisher Full Text\n\nAlonso S, Ferrero E, Donat M, et al.: The usefulness of high pre-operative levels of serum type I collagen bone markers for the prediction of changes in bone mineral density after parathyroidectomy. J Endocrinol Invest. 2012; 35(7): 640–4. PubMed Abstract\n\nRowlands C, Zyada A, Zouwail S, et al.: Recurrent urolithiasis following parathyroidectomy for primary hyperparathyroidism. Ann R Coll Surg Engl. 2013; 95(7): 523–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nZha ZL, Qu F, Lian HB, et al.: [Clinical analysis of primary hyperparathyroidism with kidney stones: 23 cases report]. Zhonghua Wai Ke Za Zhi. 2013; 51(10): 887–90. PubMed Abstract\n\nTassone F, Guarnieri A, Castellano E, et al.: Parathyroidectomy Halts the Deterioration of Renal Function in Primary Hyperparathyroidism. J Clin Endocrinol Metab. 2015; 100(8): 3069–73. PubMed Abstract | Publisher Full Text\n\nMurray SE, Pathak PR, Schaefer SC, et al.: Improvement of sleep disturbance and insomnia following parathyroidectomy for primary hyperparathyroidism. World J Surg. 2014; 38(3): 542–8. PubMed Abstract | Publisher Full Text\n\nIsidro ML, Ruano B: Biochemical effects of calcifediol supplementation in mild, asymptomatic, hyperparathyroidism with concomitant vitamin D deficiency. Endocrine. 2009; 36(2): 305–10. PubMed Abstract | Publisher Full Text\n\nMisiorowski W: [Alendronate increases bone mineral density in patients with symptomatic primary hyperparathyroidism]. Endokrynol Pol. 2005; 56(6): 871–5. PubMed Abstract\n\nKhan AA, Bilezikian JP, Kung AW, et al.: Alendronate in primary hyperparathyroidism: a double-blind, randomized, placebo-controlled trial. J Clin Endocrinol Metab. 2004; 89(7): 3319–25. PubMed Abstract | Publisher Full Text\n\nCesareo R, Di Stasio E, Vescini F, et al.: Effects of alendronate and vitamin D in patients with normocalcemic primary hyperparathyroidism. Osteoporos Int. 2015; 26(4): 1295–302. PubMed Abstract | Publisher Full Text\n\nKhan A, Bilezikian J, Bone H, et al.: Cinacalcet normalizes serum calcium in a double-blind randomized, placebo-controlled study in patients with primary hyperparathyroidism with contraindications to surgery. Eur J Endocrinol. 2015; 172(5): 527–35. PubMed Abstract | Publisher Full Text\n\nNorman J, Lopez J, Politz D: Cinacalcet (Sensipar) provides no measurable clinical benefits for patients with primary hyperparathyroidism and may accelerate bone loss with prolonged use. Ann Surg Oncol. 2012; 19(5): 1466–71. PubMed Abstract | Publisher Full Text\n\nPeacock M, Bilezikian JP, Bolognese MA, et al.: Cinacalcet HCl reduces hypercalcemia in primary hyperparathyroidism across a wide spectrum of disease severity. J Clin Endocrinol Metab. 2011; 96(1): E9–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFaggiano A, Di Somma C, Ramundo V, et al.: Cinacalcet hydrochloride in combination with alendronate normalizes hypercalcemia and improves bone mineral density in patients with primary hyperparathyroidism. Endocrine. 2011; 39(3): 283–7. PubMed Abstract | Publisher Full Text\n\nMarcocci C, Bollerslev J, Khan AA, et al.: Medical management of primary hyperparathyroidism: proceedings of the fourth International Workshop on the Management of Asymptomatic Primary Hyperparathyroidism. J Clin Endocrinol Metab. 2014; 99(10): 3607–18. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11702",
"date": "04 Jan 2016",
"name": "Robert Wermers",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11701",
"date": "04 Jan 2016",
"name": "Frederick Singer",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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/5-1
|
https://f1000research.com/articles/4-1523/v1
|
30 Dec 15
|
{
"type": "Research Article",
"title": "Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcripts in the mosquito Anopheles culicifacies: an evolutionary puzzle",
"authors": [
"Punita Sharma",
"Tanwee Das De",
"Swati Sharma",
"Ashwani Kumar Mishra",
"Tina Thomas",
"Sonia Verma",
"Vandana Kumari",
"Suman Lata",
"Namita Singh",
"Neena Valecha",
"Kailash Chand Pandey",
"Rajnikant Dixit",
"Punita Sharma",
"Tanwee Das De",
"Swati Sharma",
"Ashwani Kumar Mishra",
"Tina Thomas",
"Sonia Verma",
"Vandana Kumari",
"Suman Lata",
"Namita Singh",
"Neena Valecha",
"Kailash Chand Pandey"
],
"abstract": "In prokaryotes, horizontal gene transfer (HGT) has been regarded as an important evolutionary drive to acquire and retain beneficial genes for their survival in diverse ecologies. However, in eukaryotes, the functional role of HGTs remains questionable, although current genomic tools are providing increased evidence of acquisition of novel traits within non-mating metazoan species. Here, we provide another transcriptomic evidence for the acquisition of massive plant genes in the mosquito, Anopheles culicifacies. Our multiple experimental validations including genomic PCR, RT-PCR, real-time PCR, immuno-blotting and immuno-florescence microscopy, confirmed that plant like transcripts (PLTs) are of mosquito origin and may encode functional proteins. A comprehensive molecular analysis of the PLTs and ongoing metagenomic analysis of salivary microbiome provide initial clues that mosquitoes may have survival benefits through the acquisition of nuclear as well as chloroplast encoded plant genes. Our findings of PLTs further support the similar questionable observation of HGTs in other higher organisms, which is still a controversial and debatable issue in the community of evolutionists. We believe future understanding of the underlying mechanism of the feeding associated molecular responses may shed new insights in the functional role of PLTs in the mosquito.",
"keywords": [
"Mosquito",
"feeding",
"Salivary gland",
"Plant like transcripts",
"microbial flora",
"malaria"
],
"content": "Introduction\n\nHorizontal gene transfer (HGT), an evolutionary force that modulates the movement of genetic information between distantly related organisms, is well accepted in prokaryotes1–3. However, unlike prokaryotes, uncovering the functional role of HGT from eukaryotes to eukaryotes remains challenging4. Nevertheless, the number of well-supported cases of HGT are rapidly increasing, but one fundamental question, whether observed HGT in the genome or transcriptome of higher eukaryotes is pseudogenic5–7 or plays any important role in the evolution in complex metazoans, still remains unclear8–10.\n\nCurrently, next-generation sequencing is emerging as an important tool to discover and understand the evolutionary relationship of the molecular codes identified from non-model organisms11–15. Recently, a series of good review articles have been published, where authors critically argued and discussed that HGT from symbiotic/free-living organisms1,4,16,17, can be an important mechanism to drive the acquisition of novel traits. However, documenting the role of HGTs may be more controversial for the massive gene transfer within non-mating interspecies of complex metazoans4,18–20.\n\nOne of the most debatable and contradictory HGT being argued is the massive transfer of algal nuclear and chloroplast encoded genes to mollusks18,19. In fact, it has long been documented that the herbivore sea slug Elysia chlorotica carries a unique ability to harvest plastids (absence of nuclei) from its heterokont algal prey, Vaucheria litorea, and keeps plastids for several months in the digestive tract for long-term maintenance of photosynthesis and development. To explain this complex interaction, a possible hypothesis of the HGT of algal genes in the gut of the mollusk is under extensive investigation. Although, a series of recent transcriptomic analyses provide supportive evidences of massive HGTs, however the inability to find evidence of the algal associated nuclear genes in the egg genome further leaves an open question over HGT’s role in evolution18.\n\nMosquitoes that transmit many deadly infectious diseases, e.g. malaria, dengue, chikunguniya etc. are emerging as a valuable model to understand multi-taxon interactions21. For entomologists, unraveling the molecular and evolutionary complexity associated with dual feeding behavior in adult female mosquitoes, remains one of the unresolved central questions. Acquisition of nectar sugar by adult mosquitoes (in both sexes) is essential for regular metabolic energy, while blood meal by adult female mosquitoes is needed for egg production and life cycle maintenance22. The evolution of blood feeding is believed to have arisen independently over 145–165 million years ago from herbivore insects23,24, which might have favored the evolution of the specialized feeding organ system such as proboscis25, enlarged salivary glands26, facilitating the fast acquisition of nutrient rich blood meal from vertebrate host.\n\nFor the last few years, we have investigated the salivary associated molecular factors that affect mosquito feeding behavior and Plasmodium transmission27,28. Anopheles culicifacies exists as a complex of at least five sibling species (A, B, C, D, E) with wide distribution29,30 and acts as an important rural malarial vector causing more than 65% of malaria cases in India. We believe that malaria transmission by A. culicifacies in rural areas could be attributed to its strong adaptation towards agricultural plain areas. However, there is no molecular explanation that exists in relation to feeding behavior, evolution and adaptation preference to the plain area. Therefore, to understand the complex biology and molecular genetics of this mosquito, recently we have initiated a series of multi-tissue transcriptomic studies. In our recent RNAseq analysis, we demonstrated for the first time that salivary glands are evolved with unique ability to meet and manage dual (sugar or blood) meal specific responses in the mosquito A. culicifacies31. However, unexpectedly during functional annotation of the salivary RNAseq database we also observed a cluster of plant like transcripts (PLTs) for which nature of origin remains unclear. Here, in the present investigation, we aimed to predict and examine the molecular nature, origin and evolutionary relationship of these putative PLTs. Multiple validations by PCR, real time PCR, coupled with immunoblot analysis and immuno-florescence assay (IFA) provide strong evidence that PLTs are of mosquito origin and may encode active proteins for specific functions. Phylogenomic analysis predicts that adaptation to the nectar sugar uptake might have favored the acquisition of PLTs, possibly a unique case of HGTs in the mosquito. To the contrary over HGTs role in evolution, our investigation provides another evidence of the massive transfer of genes from plant to mosquito A. culicifacies. A comprehensive molecular analysis of the PLTs and ongoing metagenomic analysis of tissue associated microbiome provide initial evidence of how the mosquito evolved and adapted for feeding over plant host. To the best of our knowledge this is the first study defining the unique relationship of mosquito-plant-microbe interactions.\n\n\nResults & discussion\n\nIn an attempt to clarify the molecular complexity associated with dual feeding behavior evolution in the mosquito, currently we are focused on sequencing, generating and annotating large scale transcriptomic databases of the mosquito feeding machinery components, e.g. salivary glands, midgut, olfactory tissues etc. In fact mosquito salivary glands are bi-lobed single epithelial layered organs that initiate biochemical communication to the plant or vertebrate hosts. Over the last decade, several investigations in adult female mosquitoes have been valuable in identifying salivary specific molecular factors that facilitate fast blood meal uptake from a vertebrate host32. But how salivary glands manage dual meal (sugar vs. blood) specific molecular responses remains unclear. Our recent RNAseq based comparative salivary transcriptomic analysis demonstrated that adult female mosquito salivary glands are evolved with a unique ability to manage and facilitate meal specific responses31.\n\nInterestingly, but unexpectedly our study31 also revealed the presence of 537 putative transcripts encoding plant like proteins associated with the sugar fed library, but absent in the blood fed salivary transcriptome database (Figure 1A; Supplementary material ST1). The surprising discovery of these transcripts, which we labeled as plant like transcripts (PLTs), raised several puzzling, but arguable questions that prompted us to clarify: (i) whether the PLTs are of mosquito origin; (ii) if they are expressed in the mosquito tissues and/or other developmental stages (iii) if expressed in mosquito, what is the possible evolutionary and functional correlation of these transcripts in feeding and (iv) whether these transcripts have any molecular relationship to plant-mosquito-microbe interactions/symbiotic associations. To uncover the molecular nature and possible functions of the putative PLTs, in the present investigation we performed a systematic and comprehensive analysis of PLTs, revealing a unique case of the massive transfer of HGTs from plant to mosquito.\n\n(A) BLAST2GO based Species distribution analysis of sugar and blood fed mosquito salivary transcriptomic database. Green star mark indicates the name of plant species, best match to the NR database in the sugar fed salivary transcriptome. Confirmation of the nature of Origin (B): RT-PCR expression of PLTs during aquatic development of the mosquitoes; (C–E): Real-Time PCR based developmental expression analysis of PLTs viz. Cysteine protease; Expansin; Aquaporin; (F): PCR based genomic DNA amplification of PLTs; S7: Ribosomal Protein S7; Aqp: Aquaporin; Cp: Cysteine protease; Dhyd/Dhy: Dehydrin; Exp: Expansin [(b): Exp1/Exp2: Experiment 1 & 2; M: Male; F: Female; E: Egg; L1–L4: Larval stages L1–L4; P: Pupae; (c) M: 100 bp Marker; NTC: No Template Control].\n\nFirst, to confirm the nature of the PLTs’ origin, we did a deep enquiry with technical staff and confirmed that under standard rearing facilities, mosquitoes are never exposed to any plant material. To further rule out the possibilities of any contamination, we separately maintained the experimental mosquitoes as detailed in the methodology section. For technical validation of the PLTs’ origin, we conducted a series of experiments: (i) in two independent experiments, we examined and verified the RT-PCR based expression of at least 10 selected PLTs (Figure 1B; Supplementary material S1A), in the salivary glands of adult male and female mosquitoes; (ii) interestingly, we also observed that PLT expression is not only restricted to the mosquito tissues, but is also expressed during the aquatic developmental stages viz. egg, larva, and pupa of the laboratory reared mosquitoes (Figure 1B). Our relative gene expression analysis revealed that PLTs are more dominantly expressed in the egg, pupa and adult than larval stages (Figure 1C–E). Although, mosquito egg and pupa stages are metabolically active, and do not take any food material, we suspected that the filter paper being used for mosquito egg laying may be a potential source of environmental contamination carry over. To clarify this doubt, we collected a small piece of moistened filter paper in RNA isolation solution (Trizol) and re-examined PLT expression along with other developmental stages. Absence of any amplification even after 35 PCR cycles, in the filter paper cDNA sample showed no sign of contamination (Supplementary material S1A); (iii) we also observed positive amplification of selected PLTs through genomic DNA PCR (Figure 1F); (iv) we further carried out the functional validation of one of the plant homolog PLTs encoding dehydrin protein, by immunoblot analysis as well as immuno-florescence assay (Figure 2A–G); (v) lastly, from ongoing annotation of another independent transcriptome sequence database originated from non-salivary tissue i.e. olfactory (OLF) tissue of adult female mosquito A. culicifacies (Das De T., Sharma P., Thomas T., Pandey KC., Dixit R. unpublished data), we were able to observe similar PLTs (Supplementary material S1B); (v) finally to test whether PLT expression is associated with feeding machinery components, we monitored the relative expression of PLTs in four tissues that included salivary glands, midgut, olfactory tissue and hemocytes, collected from 3–4 days old naïve adult female mosquitoes, by real-time PCR (Figure 5A). Interestingly, we not only observed that PLTs are dominantly expressed in the tissues associated with mosquito feeding machinery (olfactory tissue, salivary gland and midgut), but also noticed a significant down regulation in response to blood meal in the salivary glands (Figure 5A,B), evidencing that the mosquito genome may code plant like proteins.\n\n(A) Web based functional prediction of putative domain of salivary transcript encoding plant homolog dehydrin like protein; (B–C) Molecular and phylogenetic relation of mosquito encoded (Ac-Dehydrin with other plant dehydrins (dotted circle represents the conserved K/red circle and S/blue circle segments), a unique feature of plant dehydrins (see text); (D) Mosquito dehydrin alignment of K-segment sequence with wheat and synthetic Dehydrin sequence used for antibody generation; (E) Real-Time PCR based developmental expression analysis of AcDehydrin, (F) Immunoblot analysis of Ac-Dehydrin expression during the development of the mosquito: Anti-dehydrin antibody recognize three protein bands of expected size (28, 52 & 63 kDa) in the control wheat seedling samples (W). Mosquito samples included Egg, Larval stages (L1, L2, L3), Pupa (P), Male (M) and Female (F); In egg stage two major forms of dehydrin bands were seen (≥62 kDa and 26 kDa). Further in case of larva, pupa, female, the presence of 26 kDa and 22 kDa bands suggested that two isoforms of dehydrin were present. In male, it appeared that less expression of 26 kDa form of dehydrin was seen in the western blot. Lane B represents Negative reference includes bacterial protein sample. The calculated molecular weight of 206 AA long salivary protein Ac-dehydrin is 23 kDa, which was closely similar in the larval stages. In fact dehydrins are characterized by conserved K-segment comprising consensus KIKEKLPG sequence towards the C-terminus and may be repeated one to many times, encoding variable size proteins ranging from 9 -200 kDa (see text for detail); (G) Immuno-florescence assay: IFA analysis of Plant anti-dehydrin antibody binding and detection in the mosquito egg Panel-A & B and pupa Panel-C & D. The FITC labeled samples were observed and captured under bright field (BF), dark field (DF+FITC) and with FITC florescence signal only. Green florescence image (Panel-A or Panel-C) shows the presence of dehydrin expression in the mosquito. A corresponding image (Panel-B or Panel-D) of the samples represents the negative control samples processed under identical conditions, excluding primary anti-dehydrin antibody treatment only (see methods).\n\nDehydrins are a group 2 member of late embryogenesis abundant (LEA) proteins, originally identified from land plants, and known to be associated with desiccation (water stress) tolerance33. In fact LEA proteins were thought to be restricted to plants and other lower eukaryotes viz. cyanobacteria, algae, but now they have also been identified in other animals including insects34. Dehydrins are evolutionarily conserved proteins acclimated to low-temperatures (LT) that allow efficient tolerance to drought and cold stress among photosynthetic as well as some non-photosynthetic organisms such as yeast35–38. Dehydrins are characterized by lysine rich conserved K-segment comprising consensus amino acid sequence EKKGIMDKIKEKLPG, towards the C-terminus that may be repeated many times to encode 9 -200 kDa protein39–42. This unique feature renders these proteins cationic, providing cryoprotective activity towards freezing sensitive enzymes43. The biochemical characterization of a novel cryoprotective protein in freeze-tolerant Eurosta solidaginis larvae shows dehyrin like activities44, but a true homolog of dehydrin is yet to be verified.\n\nMosquito dehydrins have not been reported so far, though a putative transcript AGAP000328 has been predicted from the mosquito A. gambiae genome, carrying (PF00257 domain) a signature of dehydrin like proteins (Supplementary material S2). Finding PLT encoding proteins associated with dehydration stress e.g. dehydrin, aquaporin, expansin etc. encouraged us to further examine their possible functions in the mosquito A. culicifacies. A comprehensive molecular analysis of the identified transcript AcDehydrin showed 100% identity to the plant dehydrin, having two conserved lysine rich K-segments (Figure 2A–D). In our relative gene expression analysis, we observed a constitutive expression of AcDehydrin, throughout the aquatic developmental as well as adult stages of the mosquito (Figure 2E), indicating that AcDehydrin transcript may encode a putative functional protein.\n\nFor functional validation of AcDehydrin protein, we examined the developmental expression of the dehydrin protein through immuno-blotting assay using rabbit antiserum containing anti-dehydrin antibody, raised against conserved K-segment sequence TGEKKGIMDKIKEKLPGQH (Figure 2D) of plant dehydrin40 (kind gift from Dr. Timothy Close). In our experiments we used wheat seedling protein sample as positive reference control. The anti-dehydrin antibody not only recognized the expected (28, 53 and 62 kDa) protein band in the wheat samples45, but also identified at least two equivalent proteins (28 and 62kDa) abundantly expressing in different mosquito developmental stages viz. egg, adult male and female mosquitoes (Figure 2F). Additionally, we were also able to observe multiple isoforms ranging from (~10 ->70 kDa) expressing at low level in different developmental stages, an expected unique feature of dehydrin to form macromolecular structures39–42. Finally, immuno-florescence assay not only corroborated the abundant expression in the egg, but also suggested that mosquito encoded AcDehydrin protein may play a crucial role in the stress tolerance and survival of the embryo in the egg (Figure 2G).\n\nLike other LEAs, dehydrins accumulate to high amounts in plant embryos, but remain undetectable in other vegetative tissues until their exposure to dehydration stress. The stress exposure results in their rapid induction and binding to multiple proteins, probably through intramolecular hydrogen bonding to protect tissue damage from dehydration/cold stress46. In fact, we also find another key transcript, encoding a putative protein named expansin, a member of plant cell wall-loosening proteins. These proteins are known to be involved in cell enlargement and developmental processes requiring cell-wall modification47. Like dehydrin, a real-time PCR analysis of expansin also showed dominant expression in the egg, as compared to other developmental stages (Supplementary material S2B). Taken together, we hypothesize that the mosquito A. culicifacies may have survival benefits of cold stress tolerance as well as developmental regulation, similar to plants. Future studies involving dsRNA mediated gene silencing may unravel molecular and functional relationship of the PLTs controlling feeding and adaptation phenotypes in the mosquito48,49.\n\nNext, to understand the possible evolutionary relationship we performed an extensive phylogenomic analysis of a few selected transcripts. To do this, first we retrieved and analyzed all plant-homolog putative transcripts (537 PLTs), and performed an extensive BLASTX analysis against either the NR database or an insect specific database at NCBI (http://blast.ncbi.nlm.nih.gov/), and further characterized three categories of transcript(s) (i) one transcript: encoding highly conserved alpha-tubulin (cytoskeleton associated protein), showing highest identity to plant and insect (>95% and 85–90%, respectively); (ii) two transcripts: encoding aquaporin (water channel membrane protein)50 and active site of the cysteine protease (protein chewing enzyme)51 showing highest identity to plant (>90%) and 40–52% identity to insect (iii) two transcripts: encoding dehydrin (cold stress response protein)46 and expansin (plant cell wall loosening protein)52 only matched to plants, but remained unmatched to any insect database (Supplementary material S3 A–C).\n\nThe above results prompted to follow up the associated evolutionary consensus, favoring plant-mosquito relationship: a parallelism setting where different species from unrelated taxa faces the common selective pressure53. Initial multiple sequence alignment analysis revealed significant heterogeneity (substitution/deletion) of amino acid residues, but also indicated unique conservation of insect or plant specific residues within the mosquito A. culicifacies, result a clade formation with plant species (Figure 3A,B; Supplementary material S4). Subsequently, we also tested whether the evolution of common traits from unrelated taxa owing to similar selection pressure favors adaptive significance.\n\n(A) Molecular analysis of partial cDNA sequence encoding (100AA) Plant-like Cysteine protease active domain: Multiple sequence alignment showing molecular relationship of AcSgCp with plant (Green circle) as well as insects (Red circle) cysteine proteases: conserved residues (marked as ) as well as conserved active site residue (marked as ). Green represents conserved cysteine residues, which enables disulfide formation. Upward arrow mark represent unique plant specific amino-acids residues also conserved in the Anopheles culicifacies, while downward arrow mark represent unique insect specific residue conserved in A. culicifacies and only in Solanum lycopersicum. (B) The evolutionary history of AcSgCp inferred using the Neighbor-Joining method, favoring a clade formation with S. lycopersicum and other plant cysteine proteases. (C) Relationship between strength of convergent evolution favoring adaptive significance of feeding associated PLTs: A maximum likelihood (ML) estimation was applied to calculate and compare the sitewise likelihood (∆SSLS) values between two, species evolution (H0) and convergent adaptive evolution (H1) hypothesis, for Cysteine protease (see text for details). (D) Structural comparison between predicted 3D structure of the mosquito, and solved structure of the plants cysteine protease: Asparagine (ASN) and Histidine (HIS) indicate conserved residue of the active site.\n\nA maximum likelihood (ML) estimation was applied to calculate and compare the site-wise likelihood (∆SSLS) values between the two hypotheses, i.e. mosquito-mosquito species evolution (H0) and mosquito-plant convergent adaptive evolution (H1), for the selected PLTs. The site-wise log likelihood plot indicator, i.e. divergence towards negative (∆SSLS) was compared with LRT (likelihood ratio test), using the parametric bootstrap at 1000 replicate analysis (cut off p-value 5%). Final data analysis and comparison statistics favored the convergent hypothesis54, demonstrating that mosquito A. culicifacies PLTs followed a convergent model favoring (H1), an adaptive evolution for sugar feeding associated functional relationship with plants (Figure 3C; Supplementary material S4). Our analysis also supports the previous observations noted for the evolution of echolocating gene clusters among bats and bottlenose dolphins55. Additionally, the predicted 3D structural analysis revealed fine conservation of the active functional domains in the mosquito and plant proteins e.g. cysteine protease (Figure 3D; Supplementary material S5). From these studies, we concluded that mosquito feeding associated genes are not only evolving actively, but also acquiring new genes (e.g. dehydrin, expansin), to adapt successfully over the plant host.\n\nInsect-plant association represents one of the most dominant interactions over millions of years56–58. These interactions are thought to play an important role in the co-evolution of molecular effector arms, enabling effective adaptation over each other59. Uncovering of the molecular mechanisms of the herbivore insect-plant interaction has greatly facilitated the design of molecular strategies to save the valuable crops from insect pests60–63. However, such studies have not given special attention to mosquitoes. From the unexpected findings of the mosquito PLTs, we interpreted that either studies in relation to the sugar feeding associated biology have largely been ignored28 or the mosquito A. culicifacies may have evolved with more complex genetic architecture favoring evolution of several environmentally-guided traits viz. carbon metabolism; light mediated photo conditions for mating, feeding, survival etc. Therefore, to predict sugar metabolism associated molecular and functional relationships of salivary PLTs, initially we analyzed all the putative PLTs against three databases (Reactome, KEGG, and Biocycles) annotated for Arabidopsis thaliana, using KOBAS online software, version 2.0 (http://kobas.cbi.pku.edu.cn/home.do).\n\nNotably, we observed that 18 transcripts encoding proteins related to at least five Biocyclic pathways linked to photosynthetic organelles viz. plastid in plants (Supplementary material S6, T1). To verify the predicted ‘plastid’ related salivary transcripts, Fisher’s exact test was performed using BLAST2GO, revealing a pool of 11 transcripts differentially expressed in the sugar fed mosquitoes (Fisher test p<0.001; Supplementary material S6B) encoding important enzymes/proteins, associated with one of the key pathway “Carbon fixation in Photosynthetic Organisms” (Figure 4A). Further, we also identified four unique salivary transcripts encoding different enzymes linked to three other secondary metabolite synthesis pathways, namely: ‘Trepenoid Backbone Biosynthesis’ (4-hydroxy-3-methylbut-2-enyl diphosphate reductase/E.C.1.17.1.2, LYTB); ‘Carotenoid Biosynthesis’ (Phytoene Synthase/E.C.2.5.1.32, PS); and ‘Flavonoid Biosynthesis’ (3-dioxigenase/E.C.1.14.11.9 & 3’ beta-hydroxylase/E.C.1.14.13.88) pathways restricted to plants (Supplementary material S7). A comprehensive molecular and phylogenetic analysis of a few selected transcripts, encoding an enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase/E.C.1.17.1.2 (LYTB) and phytoene synthase/E.C.2.5.1.32 (PS) exclusively revealed a unique evolutionary relationship to the cyanobacteria, algae, plants and aphid Acyrithosiphon pisum (Figure 4B,C; Supplementary material S7). In fact, during its early development mosquito larvae start to feed on diverse micronutrients e.g. bacteria, algae, fungi etc., and switch to feed on nectar sugars in adult mosquito stage. Thus, it could be possible that a long association and regular microbe-mosquito-plant interactions64,65, might have favored insects (mosquitoes) to adapt, feed, and digest sugar as well as selective synthesis of secondary metabolites/pigments, essential for specific phenotype e.g. visual pigmentation/dark body coloration66. A recent study on light-induced ATP synthesis from the chloroplastid-like carotene pigments in Acyrithosiphon pisum, a plant sap sucking aphid, provides the first molecular evidence that the aphid genome may carry plant like photosynthetic machinery components67. A fungal mediated lateral HGT mechanism has been proposed for the evolution of the carotenoid biosynthesis gene in this aphid20.\n\n(A) KEGG prediction of salivary transcripts (differentially expressed/Fisher test p<0.001) encoding enzymes (Green) involved in “Carbon Fixation in Photosynthetic Organisms” pathway known to be restricted to the photosynthetic plants organelles e.g. plastids only (see text). (B) Phylogenetic analysis of a unique mosquito salivary transcript, encoding a Plant homolog 4-hydroxy-3-methylbut-2-enyl diphosphate reductase/E.C.1.17.1.2 linked to the “Trepenoid Backbone Biosynthesis” pathway. (C) Phylogenetic analysis of a unique mosquito salivary transcript, encoding a Plant homolog Phytoene Synthase/E.C.2.5.1.32 linked to the “Carotenoid Biosynthesis” pathway. In fact like other animals, insects are also believed to absorb carotenoid pigment (an eye pigment) from plant food. Additionally, lower microbes such as algae and cyanobacteria also carries LYTB/PS gene in their genome. Phylogenetic analysis of the salivary LYTB & PS showed unique association with the plant, as well as microbial LYTB while PS also showed evolutionary relationship to the novel PS gene recently identified from sap sucking insect Acyrithosiphon pisum, suggesting that mosquito LYTB/PS might have evolved, for independent synthesis of the carotenoid synthesis assisting feeding adaption preference over plant host. (D) Identification of symbiotically associated salivary microbial flora predominated and unique bacteria (marked green circle), probably assisting mosquito to adapt, feed and metabolize diverse carbon rich sugar sources of plant origin (see another report for detail).\n\nIn nature, mosquitoes are regularly exposed to various environmental factors which have adverse effects on their reproductive success, longevity & vector competence68. Gut bacterial endosymbionts are known to play a part in several functions including food digestion, metabolism, reproduction and immunity69. Our recent metagenomic analysis of salivary microbiome identified several unique bacterial phyla, including Chlorobium, Cyanobacteria, Nitrospira and other phototrophic bacteria associated with salivary glands (Figure 4D), but absent in the gut of laboratory reared 3–4 days old adult female mosquitoes A. culicifacies70. Indirectly, the above findings further support the hypothesis that mosquitoes may have feeding associated distinct plant like molecular machinery components, partly shared by the residing symbiotic bacterial community for diverse carbon/nitrogen rich plant sugar source metabolism. For example, finding of prominent salivary associated Acidobacteria (2.4%), may facilitate the utilization of plant polymer viz. cellulose/xylan sugars of diverse origin71, as reported in the gut of the wood feeding larvae of Huhu Beetle (Prionoplus reticulari)72.\n\nThe observation of a large pool of chloroplast and nuclear encoded plant genes in the mosquito transcriptome supported the previous finding of similar gene transfer of photosynthetic machinery components from algae to mollusk19. In addition to this, molecular analysis of PLTs also revealed a plant related class of secondary metabolites (see above) and immune genes i.e. Remorin (anti-fungal); osmotin/thaumatin (anti-fungal); and Vicilin (Antimicrobial) (Supplementary material ST-1), a similar finding of active genes in the aphid genome73. Although, in case of algal-mollusk or Aphid-plant interaction studies, the role of microbes is yet to be established, however, our metagenomic analysis provides initial evidence that tissue associated microbial flora may also share and facilitate optimal function. Thus, we believe that the accumulating data of genetic material transfer within metazoans are still at a premature stage, but emerging evidences strongly suggest that acquisition and retention of desired active functional genes for beneficial traits, may favor improved survival and adaptation values in changing ecologies4.\n\nWith the current available data, including the present investigation, we hypothesize that HGTs in metazoans may also play an important role in the evolution and acquisition of beneficial traits that facilitate feeding and survival adaptation over diverse ecologies. This hypothesis is further strengthened by our following new observations: (a) that PLTs expression seems to be restricted to the tissues, i.e. the feeding machinery components that facilitate digestion and metabolism, e.g. salivary glands, midgut olfactory tissues in case of the mosquito (Figure 5); (b) absence of PLTs from other non-digestive tissues, e.g. hemocyte (mosquito blood cells); (c) the finding of dominantly associated unique bacterial species to the mosquito digestive tissues viz. salivary gland and midgut70, e.g. Acidobacteria (sugar metabolism); Agromonas bacteria, a soil oligotroph (nitrogen fixing bacteria) that usually grow at extra low nutrient environments of the paddy field, complementing the high larval density of the mosquito A. culicifacies in paddy fields of the rural India74. Indeed, Agromonas has been previously isolated from paddy fields71; but largely remain unidentified from any insect species so far.\n\n(A) Tissue specific expression of PLTs; (B) Blood meal response of salivary glands PLTs viz. dehydrin, expansin and light harvesting complex (LHC) in the adult female mosquito. SG_SF: Salivary Gland Sugar Fed; SG_BF: Salivary Gland Blood Fed; MG: Midgut; OLF: Olfactory; HC: Hemocyte; NTC: No Template Control.\n\nFurthermore, in mosquitoes it has long been accepted and proven that a significant variation exists in the chromosomal DNA as well as genome size within Anopheline and other mosquito species75,76, but how these variations differentially affect the mosquito biology viz. behavior, physiology, immunity and vectorial capacity etc., are poorly understood at the molecular level.\n\n\nMaterial & methods\n\nMosquito rearing: A cyclic colony of A. culicifacies sibling species A, were reared and maintained at 28 ±2°C/RH 80% in the insectary fitted with an automated dawn and dusk simulator allowing a light and dark cycle for 11hrs with 1hr transition from light to dark and vice-versa, essentially required for proper mating and feeding at NIMR77. All protocols for rearing and maintenance of the mosquito culture were approved by the Institutional Animal Ethics Committee (IAEC) of the institute (Reg. No. 33ReBi/GO/S/99/CPCSEA). For our specific research work, pupa stage A. culicifacies were collected from the insectary and kept in a round plastic cage fitted with mosquito net, perfectly wiped with 70% ethanol prior to the experiment. Post emergence adult mosquitoes were fed daily on sterile sugar solution (5%) using a glass test tube supplied with a sterile cotton swab throughout the experiment, while mosquitoes were allowed to feed directly on a rabbit for blood meal acquisition. For aquatic development, gravid females were allowed to lay eggs on moistened filter paper mounted inside small plastic cups (e.g. ice cream cup), semi-filled with pre-cooled boiled water. Hatched larvae were feed on mixed dried powder of yeast and fish food (Taiyo Plus, Tetra Gmbh, Germany). The utensils viz. plastic bowl, cup or tray used to rear larvae were properly washed with soap solution, followed by multiple washing with boiled water and air dried. The waste removal and fresh nutrient supply to the larva was maintained with an interval of 24 hrs in 300–400 ml pre-cooled hot water.\n\nTissue collection: For the desired tissues viz. salivary glands, midgut, hemocytes, olfactory tissues collection, we followed essentially the same protocol as established for our recent metagenomic and transcriptomic studies31,70. Briefly, prior to dissection, 3–4 day old sugar or blood fed adult female mosquitoes were surface sterilized using 70% ethanol for 1 min followed by dissection in a sterile water drop on a microscopic slide in a sterile working area under laminar flow. Sterile entomological needles/forceps were used to manually pick up and collect the tissues in the pre-sterilized 1.5 ml Eppendorff tubes containing 50 μl Trizol solution. For the hemocyte collection, a flushing method was opted for as described previously80. Briefly 2–3 μl of Schneider’s (RPMI): FBS: citrate buffer (60:10:30) was injected into the lateral wall of the mesothorax of cold anesthetized mosquitoes, followed by flushing out the diluted hemolymph with an additional 3–5 μl of Schneider’s (RPMI), by clipping of the last abdominal segment. The diluted hemolymph was directly collected by pipette in Trizol. For the egg collection, a clean fine art paint brush was used to scrape the eggs from moistened filter paper, rinsed with sterile water and collected in the Trizol. The other aquatic developmental stages viz. larva, pupae were also manually picked up with a Pasteur pipette, washed with sterile water twice and collected in Trizol for RNA isolation.\n\nRNA isolation, cDNA preparation and PCR analysis: The desired tissues viz. salivary glands, midgut and hemocyte78 or the whole body, were collected in Trizol. Total RNA was isolated using standard Trizol method, followed by first-strand cDNA synthesis using Oligo-dT or Random Hexamer primers (Verso kit). For differential expression analysis, routine RT-PCR and agarose gel electrophoresis protocols were used. Relative gene expression was assessed by QuantiMix SYBR green dye (Biotool Biolabs, Madrid, Spain) in Eco-Real-Time PCR Machine (Illumina). PCR cycle parameters involved an initial denaturation at 95°C for 5 min, 40 cycles of 10 s at 95°C, 15 s at 55°C, and 22 s at 72°C. Fluorescence readings were taken at 72°C after each cycle. A final extension at 72°C for 5 min was completed before deriving a melting curve, to confirm the identity of the PCR product. Actin gene was used as an internal control in all qPCR measurements, where minimum two technical replicates were used in each real-time experiment. To better evaluate the relative expression, each experiment was performed in three independent biological replicates. The relative quantification results were normalized with internal control Actin gene and analyzed by 2–ΔΔCt method79.\n\nPLTs identification and phylogenomic analysis: In an attempt to clarify and improve the functional annotation of a cluster of unique sequences encoding plant like proteins, unexpectedly observed from our recently sequenced salivary transcriptomes31, we performed a comparative analysis for both the sugar fed as well as blood fed salivary transcriptomic databases. Initially, to do this we did a species distribution analysis and manually sorted and catalogued the sequences that best match to the plants from the FASTA file. The shortlisted transcripts were subjected to a similarity search against NCBI's NR database using the BLASTx algorithm80, with a cut-off E-value of ≤10−3 using BLOSUM62 matrix as well as GO annotation/Interproscan analysis using BLAS2GO81. Biocyclic pathway analysis for PLTs KOBAS online (http://kobas.cbi.pku.edu.cn/home.do) software, version 2.082. Following primary BLASTX analysis, the reference sequences from the selected top hits were retrieved and edited for subsequent analysis in the FASTA format. Multiple sequence alignment was performed using ClustaX2, version 2.083. The CLC Sequence viewer (http://www.clcbio.com) software (version 6.9.1) was used for better quality graphics. The phylogenetic relationship was inferred through MEGA5.1 (http://www.megasoftware.net/) software. The evolutionary history was inferred using the Neighbor-Joining method, and percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates). The evolutionary distances were computed using the p-distance method, presented in the units of the number of amino acid differences per site. A work flow for the Phylogenomic analysis has been presented in the Supplemental material S6. The following major steps were followed:\n\nI. Alignment of orthologous sequences for the selected genes Cysteine Protease, Aquaporin and Alphatubulin using MAFFT v6.864 at default parameters (Auto (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i) with Amino Acid substitution matrix (BLOSUM62), Gap Penalty (1.53), offset penalty (0.123) and saved in Phylip Interleaved alignment format.\n\nII. Alignment was used to generate RAxML tree, using T-REX online84, at following parameters for generating de-novo phylogeny at following parameters: PROTCATDAYHOFF substitution model, Hill Climbing Algorithm, Number of alternative runs on distinct starting trees =100, Rapid bootstrap random seed =12345, Bootstrap random seed =12345. This alternate phylogeny was called H1, as compared to commonly accepted Species phylogeny which was called H0 (the null hypothesis).\n\nIII. For Delta SSLS estimation, site wise log likelihood values were calculated using85 for both H0 and H1 phylogeny. Difference in Sitewise Log likelihood was calculated (Delta SSLS= H0-H1), where negative value supports convergent evolution and positive value supports species phylogeny.\n\nIV. For LRT test (Tree Finder), Phylogenetic reconstruction for H0 and H1 was done under WAG substitution model & Likelihood method for identifying best fit protein model with optimized frequencies with Heterogeneity models (G, GI and I). Parametric bootstrapping analysis was done to compare the two evolutionary hypotheses ‘H0’ and ‘H1’. The resulting p-value is the probability that the likelihood ratio simulated under the null hypothesis is less or equal than the observed. Given a level of significance of 5%, a p-value greater than 95% indicates that H1 is better than H0, and a p-value less than 5% indicates that H1 is worse.\n\nModeling procedure & 3D structural prediction analysis: All structures of representative protein were retrieved from the Protein Data Bank (www.rcsb.org) and aligned using the structure alignment program STAMP4.086. Models using all four structures as templates were generated using Modeller9 version 1087. 3D representation of the model was prepared in VMD version 1.9 (Visual Molecular Dynamics tool)88.\n\nGenomic DNA isolation & PCR: For the genomic DNA extraction, a total of five adult female mosquitoes, decapitated with head and wing, were collected in extraction buffer and processed as described earlier70. All the PCR amplification conditions and parameters were identical as described above for RT-PCR analysis.\n\n(a) Wheat seedling protein sample preparation: Wheat seeds were surface sterilized, imbibed for two consecutive days on moist filter pads placed in the glass petridish, under deprived light, given alternate 16h/8h light/dark cycle for 3 days and then processed as described previously89. Briefly, crude protein extract was prepared by homogenization of seeds in phosphate buffered saline (PBS) with added benzamidine hydrochloride (1 mM) and phenylmethylsulfonyl fluoride (PMSF) (1 mM) followed by centrifugation at 15,000 rpm for 30 minutes at 4°C. Supernatant was collected to quantify and optimize the protein sample concentration for SDS-PAGE with different amount of protein (viz. 20 µg, 50 µg, 100 µg, 200 µg and 400 µg). For further experiments 200 µg was selected as an optimal concentration for immunoblot analysis.\n\n(b) Mosquito developmental stage (egg, larva, pupa) samples: Different stages of mosquito viz. egg, larva, pupa were collected in PBS containing benzamidine hydrochloride (1 mM) and phenylmethylsulfonyl fluoride (PMSF) (1 mM) protease inhibitors. The collected mosquito whole body samples were homogenized on ice for 10 minutes, followed by centrifugation at 15,000 rpm for 15 min at 4°C. The clean supernatant was collected and quantified for subsequent analysis as described below.\n\n(c) Bacterial protein sample : BL21* cells of E. coli (2ml) were grown in LB media containing ampicillin (100 μg/ml) at 37°C till optical density (OD: 600) reached 0.4–0.6. Harvested cells were spinned down at 12000 rpm and re-suspended with 200 µl re-suspension buffer containing 50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM Imidazole. Cell lysate was then centrifuged at 12000 rpm for 5 min and clear supernatant was analyzed through SDS-PAGE.\n\n(d) SDS-PAGE and immunoblot analysis: Protein samples (200 µg each) were separated on SDS-polyacrylamide gel with Amersham mini vertical electrophoresis system and transferred to nitrocellulose membrane. Membranes were blocked with 1.5% (w/v) gelatin in PBST and incubated with anti-dehydrin primary antibody (affinity purified polyclonal rabbit antiserum; 1:1000 dilution). The unbound antibody was washed three times for 5 min with PBST. Membranes were then incubated with anti-rabbit HRP secondary antibody (monoclonal; 1:60,000 dilution) (Santa Cruz Biotechnology, USA) for 1 hour. Unbound secondary antibody was washed for 5 minutes three times with PBST at room temperature. The blots were visualized using Amersham ECL prime Western blotting detection reagent containing Solution A: luminol enhancer and Solution B: peroxide and developed on X-ray films by developer and bands were readily fixed in fixer solution.\n\n(e) Immuno-florescence microscopy: The collected different developmental stages of mosquito viz. egg and pupa were washed with DEPC treated water and fixed with 4% paraformaldehyde (PFA) overnight at 4°C. The PFA was removed with PBST wash followed by dehydration of the samples with a methanol series as described90 and stored at -20°C until use. Before using, the samples were rehydrated with a gradual dilution series of methanol in PBS. Final traces of methanol were removed with PBST washes followed by a final wash with 150 mM Tris HCl, pH 9. Antigen retrieval was proceeded by incubating the samples with 150 mM Tris HCl (pH 9) at 70°C for 15 minutes, which were subsequently permeabilized with chilled acetone at -20°C for 20 minutes. Later blocking was done overnight with 10% BSA in PBST at 4°C. After blocking the mosquito samples were incubated with anti-dehydrin primary antibody (affinity purified polyclonal rabbit antiserum; 1:500 in 1% BSA in PBST) for 3 days. PBST washed samples were incubated with goat anti-rabbit IgG FITC labelled secondary antibody (polyclonal; Santacruz Biotechnology, USA) at 4°C for 2 days. For each washing step with PBST i.e. before permeabilization and/or after primary and secondary antibody incubation, the samples were carefully handled. Finally samples were washed with 4% PFA before mounting with a series of glycerol wash given with 25%, 50% and 75% glycerol in PBS for 20 minutes each. The mounted samples were observed under confocal microscope (Model# A1R, Nikon). Negative control samples were processed in identical conditions, except the use of anti-dehydrin antibody and observed along with test samples.\n\n\nConclusion\n\nEvolution and adaptation of dual feeding (sugar vs. blood) behavior in adult female mosquitoes remains an unresolved question. Comparative salivary transcriptomic and metagenomic analyses provide initial evidence that A. culicifacies, may have acquired and evolved with plant like machinery components partly shared by salivary associated microbes, together facilitating feeding preference and adaptation over plants grown in the plain agricultural area of rural India.\n\n\nData availability\n\nThe sequence data has been submitted to NCBI SRA database under following accession number: AC-SG-SF: SRR1753386. All other data is included as Supplementary material.",
"appendix": "Author contributions\n\n\n\nConceived and designed the experiments: RD, PS, NV, KCP. Performed the Experiments: PS, SS, TT, TDD, RD, VK, SLR. Analyzed the data: PS, AKM, SS, RD, SV.\n\nContributed reagents/materials/analysis tools: NS, RD, NV, KCP. Wrote the paper: RD, PS, NV, KCP.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWork in the laboratory is supported by Department of Biotechnology (DBT), Government of India (BT/HRD/35/02/01/2009) and Indian Council of Medical Research (ICMR), Government of India (5/87(301)v2011ECD-II), RKD is a recipient of a DBT sponsored Ramalingaswami Fellowship. The authors thank NIMR for setting a new laboratory. Punita Sharma is recipient of DBT Research Fellowship (DBT-JRF/10-11/284).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nWe are thankful to Dr. S.K. Subbarao for expert comments on the manuscript. We thank Dr. Timothy close, for kind gift of anti-dehydrin antibody. We thank DBT and ICMR for financial support to conduct the research at NIMR. We thank Kunwarjeet Singh for technical assistance and mosquito rearing.\n\n\nSupplementary material\n\nSupplementary material for ‘Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcripts in the mosquito Anopheles culicifacies: an evolutionary puzzle’.\n\nClick here to access the data.\n\n\nReferences\n\nBoto L: Horizontal gene transfer in evolution: facts and challenges. Proc Biol Sci. 2010; 277(1683): 819–827. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoonin EV, Makarova KS, Aravind L: Horizontal gene transfer in prokaryotes: quantification and classification. Annu Rev Microbiol. 2001; 55: 709–742. PubMed Abstract | Publisher Full Text\n\nSyvanen M: Evolutionary implications of horizontal gene transfer. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharma P, Sharma S, Maurya RK, et al.: Salivary glands harbor more diverse microbial communities than gut in Anopheles culicifacies. Parasit Vectors. 2014; 7(1): 235. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEichorst SA, Kuske CR, Schmidt TM: Influence of plant polymers on the distribution and cultivation of bacteria in the phylum Acidobacteria. Appl Environ Microbiol. 2011; 77(2): 586–596. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReid NM, Addison SL, Macdonald LJ, et al.: Biodiversity of active and inactive bacteria in the gut flora of wood-feeding huhu beetle larvae (Prionoplus reticularis). Appl Environ Microbiol. 2011; 77(19): 7000–7006. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGerardo NM, Altincicek B, Anselme C, et al.: Immunity and other defenses in pea aphids, Acyrthosiphon pisum. Genome Biol. 2010; 11(2): R21. 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}
|
[
{
"id": "12793",
"date": "09 Mar 2016",
"name": "Vas Dev",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Sharma and group, is one of the first reports on mosquito-microbe-plant interaction, defining the molecular relations of how mosquitoes might have evolved from herbivores to blood feeders. In fact this is a long standing question in the insect communities, where this report supports the idea that mosquitoes may have acquired the plant genes for specific functions. Although the functions of the genes are yet to be established, observation of photosynthetic genes and microbes, appears to facilitate the photo-light meditated responses, probably in feeding and/or mating. To the best of my knowledge and experience the behavior of this mosquito species (Anopheles culicifacies) is quite complex compared to other malarial vector species.The most interesting part is that the homology search analysis of PLTs reveals the name of many local plant species which commonly grown in plain areas of rural India. I believe these observations will serve as a basis for new leads for authors to understand how mosquito-plant interactions facilitated this mosquito species to evolve to feed and adapt in plain agricultural areas.",
"responses": []
},
{
"id": "12795",
"date": "10 Mar 2016",
"name": "Luis Boto",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSharma et al. article presents convincing evidence showing that Anopheles culicifacies harbor several plant like genes, probably acquired by horizontal gene transfer, whose expression is associated to the mosquito feeding mode.Starting from 537 plant like transcripts identified in a previous transcriptomic study, authors present compelling evidence that some of these transcripts are of the mosquito origin (and no contamination) and at least in one case (dehydrin) they encode an expressed and functional protein. In addition, authors discuss the possible role of these acquired plant genes in feeding adaptations. In my opinion, the article is very interesting and provides a new piece to understand the significance and importance of horizontal gene transfer in metazoan evolution. In this sense, I think that this article deserves to be indexed.My main concern is in relation to the results presentation (and discussion) through the paper. In this sense, I consider that restructuring the paper and figures could make this interesting paper more readable.In my opinion, phylogenomic analyses, showing the possible horizontal gene transfer from plants to the mosquito, should precede the presentation of the other results. Identification of mosquito genes related to plant genes is the point that supports and justifies later research and it should be highlighted. In this sense, I suggest a new figure 1 combining the actual Figure 1A and figure 2B and 2C (dehydrin gene tree as an example).In the same vein, I suggest combining in a new figure 2 the current figure 1B, 1C, 1D, 1E, 1F and figure 5A and 5B. Together, these pieces of information support the mosquito origin of the discussed genes and their involvement in the mosquito feeding mode..A new figure 3 should show results in dehydrin gene validation encompassing the current figure 2A, 2D, 2E, 2F and 2G. Other minor points are: Authors select a few genes from the initial 537 plant like transcripts pool to demonstrate that some genes are present in the mosquito genome and they are functional. However, many of the initially identified genes are not studied. In this sense, statement in the abstract that “Our multiple experimental validation…..confirmed that plant like transcripts (PLTs) are of mosquito origin” should be softened to “……..confirmed that at least some of the plant like transcripts (PLTs) are of the mosquito origin”. In the same sense, a comment in the non-studied PTLs could be welcome through of the manuscript. In the same vein, authors (page 5, “PTLs are of mosquito origin”) say that 10 PTLs are selected for RT-PCR. However in the figure 1B only five of these amplifications are shown. I think that authors should justify why they choose these particular PTLs. On page 8, please review the last sentence in “phylogenomic analysis of plant like transcripts”. It seems that mosquito feeding associated genes acquire new genes. Authors should recognize that Fig.4D is partially modified from Figure 2C in ref.70. In this sense I suggest to change “see another report for details” to “see ref.70 for details” in the Fig. 4 caption. On page 13, authors state that:\n\nThe observation of a large pool of chloroplast and nuclear encoded plant genes in the mosquito transcriptome supported the previous finding of similar gene transfer of photosynthetic machinery components from algae to mollusk. As the authors recognize in another place in the article, transfer of algal genes to Elysia chlorotica is controversial and in any case, authors should be cautious in the use of their results to support an absolutely different case study. In this sense I also suggest to soften claims for support to other similar observations in the Abstract.",
"responses": []
},
{
"id": "11757",
"date": "18 Apr 2017",
"name": "Manuel Corpas",
"expertise": [
"Reviewer Expertise I am bioinformatician with expertise on NGS analyses. I also did some work for the characterisation of the Aphid genome."
],
"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 \"Deep sequencing revealed molecular signature of horizontal gene transfer of plant like transcript in the mosquito Anopheles culicifacies\" provides an interesting point of view regarding the controversial topic of horizontal gene transfer (HGT) from plants to mosquito.\nThe article is generally very well written and provides compelling evidence about the presence of plant-like transcripts (PLT) expressed in the tissues related to sugar feeding mechanisms.\nI am impressed by the number of validation experiments such as RT-PCR or immunoblotting. These add compelling evidence about the presence of PLT in the tissue of Anopheles culicifacies (Ac).\n\nThe bit where I am slightly puzzled about is the data deposited in SRA. It refers to transcriptome sequencing using Illumina (Illumina HiSeq 2000), (Illumina Genome Analyzer IIx) and some metagenomics analyses using (454 GS FLX+) and (Illumina Genome Analyzer IIx) . The study does not have sufficient information for me to be able to know how this data was used and contributed to the overall analyses and results. Also, in the transcriptome sequencing assay I do not see any replicates. Why did you just do 1 run of the RNA-seq transcriptome? I do not see anything related to the bioinformatics analyses related to this transcriptome analysis neither the metagenomics analyses.\nThere are also a number of questions I have:\n- Where are PLT likely to be located in the tissues they are expressed in? Plastids? Is there a possible mechanism the authors propose regarding how the HGT might have happened?\n- The presence of plant-like transcripts does not preclude that HGT happened - please could you explain why you think this might be the case? Do you see a reason why PLT tend to be restricted to tissues exposed to feeding?\n- The supplementary document appears to have the right margin skewed to the right, making it slightly uncomfortable to read the document.\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?\nYes\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/4-1523
|
https://f1000research.com/articles/4-1522/v1
|
30 Dec 15
|
{
"type": "Research Article",
"title": "Computational identification of signaling pathways in protein interaction networks",
"authors": [
"Angela U. Makolo",
"Temitayo A. Olagunju",
"Angela U. Makolo"
],
"abstract": "The knowledge of signaling pathways is central to understanding the biological mechanisms of organisms since it has been identified that in eukaryotic organisms, the number of signaling pathways determines the number of ways the organism will react to external stimuli. Signaling pathways are studied using protein interaction networks constructed from protein-protein interaction data obtained from high-throughput experiments. However, these high-throughput methods are known to produce very high rates of false positive and negative interactions. To construct a useful protein interaction network from this noisy data, computational methods are applied to validate the protein-protein interactions. In this study, a computational technique to identify signaling pathways from a protein interaction network constructed using validated protein-protein interaction data was designed.A weighted interaction graph of Saccharomyces Cerevisiae was constructed. The weights were obtained using a Bayesian probabilistic network to estimate the posterior probability of interaction between two proteins given the gene expression measurement as biological evidence. Only interactions above a threshold were accepted for the network model.We were able to identify some pathway segments, one of which is a segment of the pathway that signals the start of the process of meiosis in S. Cerevisiae.",
"keywords": [
"Bayesian Networks",
"Protein Interaction Networks",
"Saccharomyces Cerevisiae",
"Signaling Pathways"
],
"content": "Introduction\n\nFor biologists and scientists in the life sciences, the successful sequencing of the genome is only one step out of many involved in understanding organisms. This has produced a lot of information that will not be useful unless refined. Biologists are interested in understanding the intricacies of the workings of the cells of an organism – the activities and reactions of such an organism to its environment. This information is useful in designing necessary interventions in order to modify the biological mechanisms of an organism or its reactions to external stimuli.\n\nAccording to the central dogma of molecular biology, genes are composed of DNA which is transcribed into RNA and the RNA is then translated into protein. Ultimately, all organisms are composed mainly of proteins in different forms and quantity.\n\nProteomic data and protein-protein interaction data from organisms form a key component in understanding an organism due to the major role played by proteins in cellular mechanisms. Protein-protein interactions are the foundation of biological mechanisms such as signal transduction, cell cycle control, DNA replication and transcription and enzyme-mediated metabolism1,2.\n\nAs a result of these interactions, understanding of organisms is facilitated by modeling the Protein Interaction Network (PIN) with a network constructed using the protein-protein interaction data. With a model such as this, a lot can be learned of the organism from its reaction to external stimuli and the effects of interventions on the biological mechanisms of the organism. For instance, it has been shown that the phenotypic effects of the deletion of a single gene depend on the position of that gene in the complex web of protein interactions3.\n\nApart from the importance of the protein-protein interactions map in studying the machinery of the proteome and the cellular behaviour of an organism, they are also practically important in the creation of interventions aimed at producing desired phenotypic outcomes such as new drug designs or disease prevention4,5.\n\nProtein-protein interaction data from organisms are obtained on a large scale using a number of high throughput techniques such as Yeast Two-Hybrid (Y2H), Co-Immunoprecipitation (Co-IP), Mass Spectrometry etc. These high throughput techniques have however, been identified to have high rates of false positives and false negatives. False positive interactions are protein-protein interactions that are reported to exist with any of the experimental techniques but do not exist in reality, while false negative interactions are true interactions that do not get reported using an experimental technique. Rates of false positives in protein interaction data have been reported to be as high as 50%6–8. As a result of analysis based on the integration of gene expression level measurement data and protein-protein interaction data, only about 30–50% of the interactions have been suggested to be biologically relevant. Reference 9 reported 47% true protein-protein interactions where a Paralogous Verification Method (PVM) was applied. The PVM may have performed better owing to its incorporation of information on paralogs of other organism to strengthen the biological evidence.\n\nThese high rates of protein-protein interaction data inaccuracy are due to peculiarities of the techniques used to generate them. For instance, unlike other affinity-based methods that cannot detect transient interacting proteins, Tandem Affinity Purification (TAP-tag) tag methods can detect transient interacting proteins which are however lost during the purification process10.\n\nFurthermore, these new high throughput methods of detecting protein interactions have no doubt rapidly generated much more data than have been collected by traditional methods in small scale experiments. This thus makes it impractical to start verifying each of these interactions by the traditional methods used in small scale experiments11.\n\nIn order to make sense of the vast data and obtain insightful information, these data need to be subjected to analytical procedures that will extract signal from the noise. This task of analyzing genomic data takes a computational approach due to the magnitude of the information involved. In reducing this level of noise in the protein interaction data different computational techniques aimed at improving the reliability of the data are applied. To predict true interactions between protein pairs, many authors have suggested a number of methods for estimating and assigning reliabilities to the interactions in the experimental data. These methods include using a logistic regression distribution function over a number of parameters to assign confidence scores to the interactions17,18, the use of expression profile and paralogs to assign reliability scores to already observed interactions9 the use of maximum likelihood technique for the estimation of domain-domain interactions in order to infer protein-protein interactions7. For computational biologists, the challenge would be the development of methods of transforming the high-throughput data obtained from these different sources into biological insights.\n\nIn this paper, we seek to bridge the gap between protein-protein interaction data and other biological data in constructing useful signaling pathway models that will lead to insightful knowledge of biological processes. We propose a probabilistic approach using Bayesian networks to assign weights to protein-protein interactions. These weighted interactions are then used to construct the weighted PIN from which signaling pathways are predicted. Refer to Figure 1 for the schematic of the computational approach used. This work thus describes a computational means to clean up the background noise inherent in the various methods of proteomic data acquisition in order to better understand bio-molecular mechanisms.\n\n\nMaterials and methods\n\nProtein interaction data was obtained from the publicly available Saccharomyces Genome Database (SGD). The protein interaction data is an amalgamation of the interactions obtained using eight different high throughput experimental procedures - Yeast Two-Hybrid, Affinity Capture Mass Spectrometry, CO-Purification, Affinity Capture Western, Biochemical Activity, Reconstituted Complex, Protein-Peptide and Far Western. This data contained 22,650 interactions between 2554 different proteins.\n\nThe S. cerevisiae expression measurement data was obtained from the Yeast Cell Cycle Analysis Project of the Stanford University. The data is housed at a publicly available database maintained by the Saccharomyces Genome Database at the Department of Genetics, School of medicine. The Yeast Cell Cycle Analysis project aimed at identifying all the genes whose mRNA levels are regulated by the cell cycle12. This data is available at the Yeast Cell Cycle Analysis Project site. The data contained the expression profiles of 800 proteins of the S. cerevisiae organism.\n\nThe processing of the data obtained from the Yeast protein-protein interaction data and the Yeast Expression measurement was carried out by first filtering for the proteins that have expression level measurement. Only the proteins in the protein-protein interaction dataset that were also present in the gene expression measurement data were used. This was done based on the hypothesis that proteins occurring in the same complex and are known to physically interact have higher correlation than proteins that are not known to directly interact. This hypothesis is supported by13–16, where it has been observed that true protein interactions have a high mRNA expression for the proteins involved. The filtration of the dataset produced 306 protein-protein interactions that have expression level measurements from the 22,650 protein interactions and the 800 gene expression measurements. These 306 protein-protein interactions represent the intersection of the two datasets as depicted in Figure 2. With reference to the yeast protein interaction data and expression measurement data respectively, these figures correspond to 0.013% and 0.382% of the original dataset respectively.\n\nIn this work, the probability estimation of protein interactions was done using a Bayesian probabilistic model. According to Bayes' theorem, the posterior probability density is proportional to the prior probability density and the likelihood function.\n\nOur interest is in drawing inference about the parameter ϕ from a probability model p(ɣ|ϕ) to give rise to observed data ɣ. Allocating a prior probability π(ϕ) to the parameters assuming they are uncertain, we can obtain a posterior probability according to Bayes' theorem where p(ɣ) which is the marginal density for ɣ is obtained by integrating over the prior. Refer to Equation 1.\n\n\n\nEquation (1) can be rewritten as equation (2) since π(ϕ|ɣ) is a function of ϕ for observed ɣ which shows the direct proportionality between the posterior probability and the product of the likelihood and the prior probability19.\n\n\n\nIn order to make use of the Bayesian model, there must be an approximation or full specification of the prior probability distribution and the likelihood function. The first step in the determination of the likelihood function which is based on the probability of observing the data is to fix a probability distribution f(θ) where θ is the parameter defining the probability distribution.\n\nIn outcome space, for a given dataset (Y1, Y2, Y3, … ,Yn), the probability of observing the dataset given θ is written as\n\n\n\nIn parameter space, the likelihood function in terms of the probability of observing the dataset given θ is\n\n\n\nFor a Bernoulli distribution, the probability distribution is\n\n\n\nTherefore for a sample of N observations (Y1, Y2, Y3, …,Yn), the joint distribution is as Equation 6, and can be rewritten as Equation 7\n\n\n\n\n\nThe likelihood function determines what value of θ makes the dataset (Y1, Y2, Y3, …,Yn) most probable.\n\nEstimating the maximum likelihood of the parameter θ, we maximize the function with respect to θ and then set it to zero to obtain the Maximum Likelihood Estimation of the parameter θ20.\n\n\n\nWe formalize the problem of constructing a weighted graph that is instrumental in building a PIN. Let (V, E, w) be the protein-protein interaction network where V = p0, p1,...pn is the set of all proteins and E = {e = (pi, pj) | pi, pj ϵ V} is the set of interactions among these proteins in the set V, and w is the weight of each edge that belongs to E. w being the weight of the interaction between two proteins (pi, pj) is a measure of the reliability of interaction between the two proteins (pi, pj) obtained using the Bayesian probabilistic approach described above.\n\nThe vertices of the interaction graph are contained in the set of the unique proteins obtained after the computation of the reliability of interaction between the protein pairs i.e. |V| = 306, and the edges of the graph are the set of interactions between these proteins in V.\n\nThe protein-protein interaction graph is constructed with an undirected sparse graph due to the sparse nature of biological networks.\n\nThe implementation of the algorithm for this computational technique was done in Java programming language using the Java Universal Network Graphics (JUNG) framework for graphs. The JUNG framework is an open-source collection of libraries providing common language for modeling, analyzing and visualizing any data that can be represented as a graph or network. The JUNG framework is extensible in order to tailor it to specific needs and also includes implementation of a host of algorithms for network analysis, graph theory and data mining.\n\nThe graph implementation in JUNG supports the representation of the different types of graphs such as directed and undirected graph, multimodal graphs, graphs with parallel edges and hypergraphs.\n\nFor this work, we used the JUNG 2.0.1 API released in January, 2010 which can be found at http://jung.sourceforge.net.\n\nA pathway is an ordered list of distinct proteins in V such that each consecutive pair is found in E21.\n\nGiven an undirected sparse graph G = (V, E, w) and a pair of nodes {(pi, pj) | (pi, pj) ϵ V} corresponding to the starting and ending proteins respectively, we wish to find a simple path from pi to pj which will be a segment of a pathway.\n\nWith the graph constructed, which is the PIN of the S. cerevisiae organism based on the data supplied, we queried the graph with a pair of proteins (pi, pj) ϵ V which are respectively the starting protein and ending proteins of the path of interest. We are interested in having a simple path corresponding to the signal transduction path from the starting protein to the ending protein returned by the search algorithm.\n\nThe search is done using a Depth First Search (DFS) algorithm. The start protein becomes the root node for the algorithm and examines all the outgoing nodes to it, expands the first child node of the apparent tree and progressively continues the search until the target node (the ending protein) is found. If the DFS algorithm however encounters a node that has no children, it backtracks to the previous node to continue exploring the children nodes.\n\nIn order to understand and make meaning of the pathway segments that are obtained from the PIN, we compared the proteins to their functional annotation. Mapping proteins in known signaling pathways and PINs to their functional annotations has an important function. The proteins in an organism may have similar biological functions such that one protein effectively replaces another in a pathway, then such proteins should share the same set of gene annotation terms. The Gene Ontology annotation, which is a functional annotation scheme, provides this basis for the identification of functional description of proteins and their interactions with other proteins and other molecules.\n\nIn this work, we used the Gene Ontology (GO) annotations to interpret the pathway segments that have been identified from the protein-protein interaction network constructed for the S. cerevisiae organism.\n\n\nResults\n\nTo validate the protein-protein interaction data that we used in this work, we applied the method that was described in section II-B to first filter the data. As was described the filtration of the data was done by integrating the gene expression measurement of the regulated Yeast Cell-cycle in order to obtain a dataset that is an intersection of both datasets.\n\nThis step was taken based on the hypothesis that there is a high correlation between the expression levels of truly interacting proteins13–16,22 and also using the gene expression measurement as a source of biological information23–25 for the computational inference.\n\nUsing the protein-protein interaction data comprising 22,650 interactions between 2554 unique proteins and the gene expression levels of 800 genes, we applied the computational approach based on Bayesian probability described earlier. Further in the validation process, the application of the Bayesian probabilistic model on the data to estimate the posterior probability of an interaction existing between two proteins given the biological evidence produced the weight estimate for the interactions. With the estimation of the interaction weight and the rejection of interaction weights below the threshold obtained from the mean expression level measurements, we obtained a dataset containing 306 protein-protein binary interactions. This dataset was used in constructing the PIN of the S. cerevisiae organism.\n\nThe 306 protein-pairs represented the proteins that had expression profile measurement, which corresponds to the intersection set of the two datasets. With reference to the protein-protein interaction data and the gene expression level measurement, this is a mere 0.013% and 0.382% of the original dataset respectively.\n\nWe applied the method described in section 2.7 to identify pathway segments in the constructed PIN for S. cerevisiae. Given a graph G = (V, E), a pathway has been described as an ordered list of distinct proteins in V such that each consecutive pair is found in E21. With a starting protein and an ending protein of interest, a simple path between these two corresponds to a pathway. Due to the size limitation of the expression measurement dataset used and the effective reduction in the overall number of proteins used to construct the graph, we were only able to identify pathway segments. A pathway segment is a chain of interacting proteins which is a part of a larger pathway. Some of the resulting pathways identified with this technique are presented in Table 1 and Table 2. These tables elucidate the protein description, the GO function and GO process of the proteins involved in the pathway segments as obtained from the AmiGO website http://www.geneontologyproject.org/go.\n\n\nDiscussion\n\nTable 1 and Table 2 present some of the pathway segments identified using the computational approach proposed in this paper. The understanding of the paths is facilitated by using Gene Ontology associations to understand the biological processes the proteins are involved in. A signaling pathway is characterized by a starting protein that is a receptor at the membrane and ends with a transcription factor.\n\nFrom Table 1 we identified a pathway segment {YMR163C- YOR326W- YCL063W- YER150W} along with the genes coding for each of the proteins using GO annotation.\n\nThe pathway segment starts with the protein YMR163C, identified to be a receptor important for peroxisome inheritance. Signaling pathways are often characterized by an activator at the membrane of the cell binding to a receptor to initiate the chain of signal transduction. These peroxisomes are organelles that metabolize fatty acids and are numerous in the S. cerevisiae organism. By blocking peroxisome transport through point mutants in the MYO2p gene that binds to it, the levels of MYO2p gene expression increased26. The implication of this is that signal is transmitted to the mother cell to stop further peroxisome transfer by lowering INP2 gene expression. The next protein, YOR326W, in the pathway segment is coded for by the MYO2p gene whose level of expression is modified in the activation of signal that is relayed to alter the level of the INP2 gene that codes for the YMR163C receptor protein. The next protein in the chain, YCL063W, coded for by the gene VAC17 has been identified to be a vacuole-specific receptor for myosin MYO2P and is involved in vacuole inheritance, a molecular anchoring function. The last protein in the pathway segment, YER150W, coded for by the gene SPI1 contributes to transcriptional regulation induced under conditions of stress during the diauxic shift27.\n\nIt is observed that this pathway which signals the start of the process of meiosis suddenly breaks off to a gene (SPI1) that participates in catalysis at the molecular level. This is not abnormal as these are pathway segments and not the full transduction pathway activated by the receptor protein.\n\nIn a similar approach, 28 used protein-protein interaction data and expression data to model pathways. They ranked candidate signaling pathways of interacting proteins using expression data based on the rationale that proteins in the same signaling network must simultaneously exist with the activation of the pathway; the genes coding for these proteins must also under the same environmental factors required for the signaling network and about the same time, be transcribed.\n\nTheir approach to predicting pathways included specifying the starting protein, a membrane protein, and an ending protein of interest, such as a DNA-binding protein, based on a prior knowledge of genetic relationship between them. In their findings, the pathways that the algorithm identified were not complete pathways owing to incomplete maps.\n\n21 also applied a computational approach that is similar to our own by assigning scores to protein-protein interaction data, creating a PPI network from the data and mining signaling pathways from the network. The parameters for the search on the network included a starting protein and an ending protein as well as the length of the pathway segment. Although their approach involved training the algorithm using association rules mining from known pathways, they were only able to mine pathway segments too.\n\nThe incompleteness of pathways mined from using computational techniques on protein-protein interaction data can be attributed to false negative interactions that were not detected by the high throughput experiments that generated the data.\n\nFurthermore, a number of computational techniques that have been applied to cleaning the noise in the protein-protein interaction data used often entails eliminating some data presumed to be noise from the dataset. The proteins removed in this manner could be important proteins that would then be missing in the modeled PIN. Our own approach involved filtering the protein-protein interaction data with the gene expression measurement data such that only the proteins with expression level measurement were used in the construction of the protein interaction network. This resulted in a reduction of the 22,650 pair-wise interactions by the gene expression measurement for just 800 proteins to 306 pair-wise interactions. This reduction in the size of the data used to construct the protein interaction network was a constraint on the number of pathways identified using this approach.\n\n\nConclusion\n\nIn this paper, we proposed a simple computational approach to identify signaling pathways in PINs by first estimating true interactions within protein-protein interaction data obtained from high throughput experimental techniques which are susceptible to generating high rates of false positive and false negative interactions. We proposed a technique using Bayesian Probability to estimate the probability of true interactions between two proteins and assigned weights to the pair wise interaction based on this. Using the validated protein-protein interaction data, we constructed a PIN of the S. cerevisiae organism from where simple paths between two proteins of interest were mined. Using the Gene Ontology annotation to understand the biological process taking place within the pathway, we were able to identify a pathway which signals the start of the process of meiosis, albeit broken off for want of more data.\n\nKnowledge of signaling pathways are generally useful in designing biological interventions on an organism aimed at producing specific desired outcomes such as new drugs design and disease prevention and control.\n\n\nData availability\n\nF1000Research: Dataset 1. Yeast Expression Data, 10.5256/f1000research.7591.d11032541\n\nF1000Research: Dataset 2. Protein-protein interaction data, 10.5256/f1000research.7591.d11032642",
"appendix": "Author contributions\n\n\n\nAUM conceived the study and supervised it. TAO carried out the study. Both authors interpreted the results and were involved in the revision of the final draft manuscript and agreed to the content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKone BC: Protein-protein interactions controlling nitric oxide synthases. Acta Physiol Scand. 2000; 168(1): 27–31. PubMed Abstract | Publisher Full Text\n\nWang Jh: Protein recognition by cell surface receptors: physiological receptors versus virus interactions. Trends Biochem Sci. 2002; 27(3): 122–126. PubMed Abstract | Publisher Full Text\n\nJeong H, Mason SP, Barabási AL, et al.: Lethality and centrality in protein networks: The most highly connected proteins in the cell are the most important for its survival. Nature. 2001; 411(6833): 41–42. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nGaldieri L, Mehrotra S, Yu S, et al.: Transcriptional regulation in yeast during diauxic shift and stationary phase. OMICS. 2010; 14(6): 629–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteffen M, Petti A, Aach J, et al.: Automated modelling of signal transduction networks. BMC Bioinformatics. 2002; 3: 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFagarasanu A, Fagarasanu M, Eitzen GA, et al.: The peroxisomal membrane protein Inp2p is the peroxisome-specific receptor for the myosin V motor Myo2p of Saccharomyces cerevisiae. Dev Cell. 2006; 10(5): 587–600. PubMed Abstract | Publisher Full Text\n\nMortimer RK, Contopoulou CR, King JS: Genetic and physical maps of Saccharomyces cerevisiae, Edition 11. Yeast. 1992; 8(10): 817–902. PubMed Abstract | Publisher Full Text\n\nTang F, Kauffman EJ, Novak JL, et al.: Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole. Nature. 2003; 422(6927): 87–92. PubMed Abstract | Publisher Full Text\n\nIshikawa K, Catlett NL, Novak JL, et al.: Identification of an organelle-specific myosin V receptor. J Cell Biol. 2003; 160(6): 887–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPuig S, Pérez-Ortín JE: Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift. Yeast. 2000; 16(2): 139–48. PubMed Abstract | Publisher Full Text\n\nKurlandzka A, Rytka J, Rózalska B, et al.: Saccharomyces cerevisiae IRR1 protein is indirectly involved in colony formation. Yeast. 1999; 15(1): 23–33. PubMed Abstract | Publisher Full Text\n\nTóth A, Ciosk R, Uhlmann F, et al.: Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 1999; 13(3): 320–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaurent BC, Yang X, Carlson M: An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family. Mol Cell Biol. 1992; 12(4): 1893–902. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsuchiya E, Uno M, Kiguchi A, et al.: The Saccharomyces cerevisiae NPS1 gene, a novel CDC gene which encodes a 160 kDa nuclear protein involved in G2 phase control. EMBO J. 1992; 11(11): 4017–26. PubMed Abstract | Free Full Text\n\nGuacci V, Koshland D, Strunnikov A: A direct link between sister chromatid cohesion and chromosome condensation revealed through the analysis of MCD1 in S. cerevisiae. Cell. 1997; 91(1): 47–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeo SJ, Tatebayashi K, Kato J, et al.: The RHC21 gene of budding yeast, a homologue of the fission yeast rad21+ gene, is essential for chromosome segregation. Mol Gen Genet. 1998; 257(2): 149–56. PubMed Abstract | Publisher Full Text\n\nMichealis C, Ciosk R, Nasmyth K: Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell. 1997; 91(1): 35–45. PubMed Abstract | Publisher Full Text\n\nMakolo AU, Olagunju TA: Dataset 1 in: Computational identification of signaling pathways in protein interaction networks. F1000Research. 2015. Data Source\n\nMakolo AU, Olagunju TA: Dataset 2 in: Computational identification of signaling pathways in protein interaction networks. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11809",
"date": "18 Jan 2016",
"name": "Winston A. Hide",
"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 work is appropriately applied in principle with adequate application of methods. According to the criteria: \"work has been well designed, executed and discussed\" it has not quite yet been adequately designed would benefit from a more synthetic discussion that explores the results in context of existing work.Of concern is that the aim of the project appears to be to improve the delivery of signal over noise in PINs. But there is no means to judge if there has been an improvement - no tests, validation or comparison over a start state. Instead there is provision of results that show some interactions that are already known - which is promising - but no ability to judge if this is an improvement over just the use of protein-protein interaction data, or just the use of gene expression data.There could be some more reference to existing work - reference and comparison with that which is current in the field (see examples of refs below).",
"responses": []
},
{
"id": "11808",
"date": "27 Jan 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\nThis paper describes a computational method for extracting information from a large variety of inherently noisy biological data describing protein-protein interactions and purports to be able to discover signalling pathways, or at least segments of signalling pathways.Not being an expert on Bayesian modeling, I can't comment directly on the method although it seems to be predicated on well-supported hypotheses and aims to be conservative in the interests of decreasing noise and increasing biological validity. Significantly, the pathway segments suggested by the model are annotated with existing functional information from Gene Ontology annotations. The authors claim to validate their results by correlating the proposed interactions with existing gene expression relying on the hypothesis that highly co-expressed genes are true interactors. It should also be noted that this study was performed on S. cerevisiae, a highly studied model organism for which the authors had access to 8 different types of high-throughput methods aimed at inferring protein-protein interactions (PPIs).I've personally always struggled with the validity of using computational methods to amalgamate high-throughput PPI data for the purposes of pathway discovery. PPI networks are dynamic and I'm not convinced that we can measure them completely (in every condition, cell type, tissue type, etc.) or that we can always assume that PPIs observed in one cell, organism, or condition can be extrapolated to others so any attempt we make to catalog PPIs is necessarily vastly incomplete. Furthermore, given the wealth of data necessary to attempt applying a computational method I wonder how generally applicable these methods can be. For example, this paper relied on data from 8 different methods - how often can we expect to have that much information about a cell or organism of interest?Is it appropriate to validate computationally-derived PPIs with gene expression data? Would it not be more appropriate to perform an assay that directly or indirectly interrogates the actual interaction between proteins?I also wonder how circular the logic behind these computational methods is. The authors used data from SGD, a well-known public resource, in order to generate PPI networks and then bootstrapped these networks by using GO, another well-known public resource. I would be surprised if GO annotation was performed without knowledge from SGD so can we believe that the networks derived in this paper are based on solely on the Bayesian model? Or are we just re-discovering information we partially already knew?And if we believe the signalling pathway segments reported here are newly and independently discovered, how widely applicable is the proposed method? Can we use it for other organisms or for yeast under changed conditions, for example? How much PPI data do we need before a computational method is more efficient and informative than well-designed biochemical experiments? Were the two reported pathway segments the only ones that could be inferred from 22,650 interactions between 2554 proteins (roughly half of the entire proteome)? Is there anything exciting (and new) to be found if the model is allowed to be less conservative?What contribution to biology do the authors expect from this method?",
"responses": [
{
"c_id": "4519",
"date": "29 Mar 2019",
"name": "Temitayo Olagunju",
"role": "Author Response",
"response": "Thank you so much for your time and critique of this work. At the time this study was undertaken, the first part of the work is the development of the computational technique to be followed by a wet lab procedure to validate the findings. The scope of this report covers the first part of the study. Computational approaches to analyze and gain better understanding of biological data are to a large extent means of interpreting the data before further investigations in the lab. The task of validating interactions, between proteins that were produced with high throughput technologies, in the lab can be very intensive due to the sheer number of such interactions to be validated. Computational techniques thus aim at reducing the efforts and the time it would take to carry out such experiments. Although this technique was not tested on other organisms, the combination of PPI data and gene expression data will likely achieve the same results with minimal or no change to the algorithm."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1522
|
https://f1000research.com/articles/4-482/v1
|
05 Aug 15
|
{
"type": "Software Tool Article",
"title": "CyAnimator: Simple Animations of Cytoscape Networks",
"authors": [
"John H. Morris",
"Dhameliya Vijay",
"Steven Federowicz",
"Alexander R. Pico",
"Thomas E. Ferrin",
"Dhameliya Vijay",
"Steven Federowicz",
"Alexander R. Pico",
"Thomas E. Ferrin"
],
"abstract": "CyAnimator (http://apps.cytoscape.org/apps/cyanimator) is a Cytoscape app that provides a tool for simple animations of Cytoscape networks. The tool allows you to take a series of snapshots (CyAnimator calls them frames) of Cytoscape networks. For example, the first frame might be of a network shown from a ”zoomed out” viewpoint and the second frame might focus on a specific group of nodes. Once these two frames are captured by the tool, it can animate between them by interpolating the changes in location, zoom, node color, node size, edge thickness, presence or absence of annotations, etc. The animations may be saved as a series of individual frames, animated GIFs, MP4 movies, or H.264/MOVmovies. CyAnimator is available from within the Cytoscape App Manager or from the Cytoscape app store.",
"keywords": [
"Cytoscape",
"CyAnimator",
"network",
"animation"
],
"content": "Introduction\n\nBiological networks are typically represented as nodes and edges (node-link diagrams) that might represent the pathways, signaling cascades, interactions between proteins, and other relationships between biological entities. The problem with this representation is that it makes it seem like these relationships are static, but it is well known that biological networks are dynamic, adapting and changing in response to the cell cycle, environmental conditions, development, and, over longer periods of time, evolution. One of the best methods to represent these changes is to take advantage of motion, showing changes by interpolating between the two states. This use of motion is one way to address “change blindness”1, which makes it difficult to detect the difference between two images when they are shown in succession. This is important both for presentation of results to collaborators and the broader scientific community and for the exploration of data by individual researchers.\n\nCytoscape2 is one of the most common tools used to visualize and analyze biological networks. It provides very powerful visualization tools to map a variety of categorical and numeric data into visual attributes associated with the nodes and edges of node-link diagrams. Unfortunately, Cytoscape does not provide any inherent animation capabilities, making it difficult to use interpolation to detect changes between two states, however, Cytoscape does provide a rich infrastructure for extending its core functionality through “apps”3. Several Cytoscape apps provide some animation capabilities. For example, VistaClara4, 3DScape5, and clusterMaker6 provide support to animate through the columns of a heat map. DynNetwork (http://apps.cytoscape.org/apps/dynnetwork) reads specially constructed input files that specifically encodes changes to the network over time. Of these, only clusterMaker2 and DynNetwork are available in Cytoscape 3, and offer relatively limited restrictive animation capabilities (in the case of clusterMaker2) or require construction of special input files in advance (in the case of DynNetwork). None of the available tools allows for the animation of arbitrary changes to the network topology or visualization.\n\nCyAnimator attempts to fill this gap by providing a tool that supports animation by allowing the user to designate particular network views as “key frames”. These frames represent the state of the network at particular moments in time including the current list of nodes and edges and the visual attributes of those nodes and edges. The user may then arrange those frames in a desired sequence and CyAnimator will interpolate between the frames resulting in a smooth animation between the states of the network. The resulting animation may be saved as a movie.\n\n\nMethods\n\nThe main object in CyAnimator is a CyFrame, which contains all of the information about the visual attributes of the network background, annotations, nodes, and edges. This information is stored in a series of maps indexed either by the internal unique identifier (SUID) of the node or edge or by a hashCode of the annotation object, if the stored object is an annotation. CyAnimator makes heavy use of the visual property system in Cytoscape and the pseudocode for the general loop for populating a CyFrame is:\n\n\n\nThe captured image is used to show thumbnails to the user in the CyAnimator dialog (see below). In general, getting the visual properties results in a series of maps, one map for each property. These maps are used to restore the state of the network or to form the basis for the interpolations. In hindsight, this was an unfortunate way to implement the storage of the individual visual properties. In the current implementation, when we want to add support for a new visual property, we must create a new map for it, add code to populate the map, add code to explicitly set that visual property on a network view, and finally add the appropriate code to interpolate between two values of that visual property. This would have been much easier if we had instead stored a map of the form:\n\n\n\nwhere the Long key would be the SUID of the object. We could then maintain a list of VisualProperties, or even a map linking VisualProperty to Interpolator. This would greatly facilitate adding new visual properties to the system. We plan on refactoring CyAnimator in a future release to implement this new approach.\n\nWhen the user clicks on a frame in the CyAnimator dialog or during the animation, the stored visual properties must be mapped onto the current network. This is a straightforward mapping except in circumstances where the current network does not have the node, edge, or annotation that the stored CyFrame had or the CyFrame did not have a node, edge, or annotation that the current network has. In the first case, the missing object is added to the network and styled using the stored information. This approach depends on the fact that deleted nodes and edges are not removed from the CyRootNetwork, so the topology of the network at the time the CyFrame was stored may be recreated. In the second case, the CyFrame sets the visibility of the object to false, resulting in a network view that is consistent with the saved visual properties.\n\nInterpolation between CyFrames is handled by the Interpolator, which maintains lists of interpolators for node visual attributes, edge visual attributes, annotation visual attributes, and network visual attributes. The Interpolator makeFrames() method takes as input the list of CyFrames, the user saved (key frames) and returns an array of frames that includes interpolations between each pair of key frames. So, if the user has requested 30 frames between each key frame, and created 5 key frames, makeFrames(), will return 121 frames, which is the number of key frames to interpolate between (key frames - 1), multiplied by the number of interpolations between each key frame, plus the initial key frame ( (keyframes - 1)*interpolations + 1).\n\nCyAnimator currently supports 5 different interpolation types:\n\nTable 1 shows the interpolated visual attributes and the type of interpolation. In the current version of Cytoscape, there is no mechanism to get the list of node custom graphics for a particular node, so charts, gradients, and other custom graphics can not be interpolated. CyAnimator also doesn’t currently support interpolation of arrow colors. This is an oversight that will be rectified in a future version.\n\nTo implement the creation of a movie, three different mechanisms are used. In each case, an image file is created for each interpolated frame. One option the user has is to just write out the individual frames and allow them to use whatever movie making tool they desire. We have also implemented an animated GIF writer using the native Java ImageIO library. Finally, for creating MP4 or H.264 movies, we utilize the Xuggler (http://www.xuggle.com/xuggler/) Java library, which includes the necessary video codecs.\n\nTo bring up CyAnimator select Apps→CyAnimator. This will bring up an empty CyAnimator dialog (Figure 1). Note that CyAnimator is only able to animate between networks in the same network collection. Starting CyAnimator on a new network collection will create a new, empty, CyAnimator dialog. Once a CyAnimator dialog is open, the general workflow would be to manipulate the network to what you want it to look like at the start of your movie, then select Add Frame to add the frame to CyAnimator. Once the frame has been added, you can modify your network to what you want it to look like in the next frame of your movie and then again select Add Frame. Note that CyAnimator will do all of the interpolation to get from one frame to another, so the manipulations of the network can include a variety of changes, including changes in color, position, zoom, annotations, etc. Repeat this process until you are happy with your movie, then simply press the record () button. If you want more time between any two frames, use the context menu to change the Interpolations value.\n\nThe CyAnimator dialog provides the main interface to CyAnimator, including the following controls:\n\nIn addition to the menu bar controls discussed above, each frame provides a context menu that’s available through a Right-click (or Control-click on Mac). These menu items are:\n\nThe Output Options Dialog (see Figure 2) provides the controls to choose the type of video you want to produce, options about the resolution and speed of the video, and the location of the video output.\n\nFrames. This is the simplest of the video types. This will output each frame as a \".png\" file at the requested resolution (see ’Resolution’ below) into the Video location directory specified. To make a movie, you could use any of the standard video packages that accept individual frames (e.g. iMovie on Mac). Note that the Frames Per Second option doesn’t make sense for this output type since we’re only writing individual frames (no time encoding), so this option is disabled (grayed out) in the interface.\n\nGIF. This will output an animated GIF of the interpolated frames. Animated GIF files are easy to show on web sites, but are not the currently accepted standard format. On the other hand, animated GIF files are computationally very easy to produce.\n\nMP4. This will output a raw MPEG4 file of the interpolated frames. Producing an MP4 (and a MOV/H264) can be computationally expensive, and will require more time and memory to complete.\n\nMOV/H264. This will output an MPEG4-encoded video of the interpolated frames. The MPEG4 video is then encapsulated in an H.264 container. This is considered the current standard for web deployment of video content. Producing an MOV/H264 can be computationally expensive, and will require more time and memory to complete.\n\nFrames Per Second. This controls the speed of the movie in terms of the number of frames per second. Smooth animations should be set to at least 30 frames per second.\n\nResolution. This controls the resolution of the output frames. The units are % expansion, so a Resolution of 300 would result in a 300% (or 3X) expansion of the output image. This is extremely important for high-quality videos. We recommend at least a 3X (Resolution of 300) expansion for any published video.\n\nA sample movie is provided (see Supplementary_File_1.mov) that was created with CyAnimator and Cytoscape 3.3. This movie used galFiltered.cys, which may be located in the sampleData subdirectory of the Cytoscape installation directory (e.g. /Applications/Cytoscape_v3.2.1/sampleData on a Mac). The movie consisted of 4 key frames:\n\n1. the full network\n\n2. a view focused on MCM1\n\n3. the same view, but peripheral nodes were moved out of the way, and five annotations were added: three text annotations, a shape annotation around the three text strings, and an arrow pointing to MCM1\n\n4. another capture frame of the same view\n\n\nConclusions\n\nCyAnimator is an important addition to the suite of Cytoscape apps. It provides an easy tool to interpolate between different states of a network and may be used to animate changes over time, condition, or treatment. One missing feature of CyAnimator is the ability to save the key frames of an animation as part of a session. We will be adding support for this in a future release.\n\n\nSoftware availability\n\nhttp://apps.cytoscape.org/apps/cyanimator\n\nhttps://github.com/RBVI/CyAnimator\n\nhttp://dx.doi.org/10.5281/zenodo.206847\n\nhttps://www.gnu.org/licenses/lgpl.html",
"appendix": "Author contributions\n\n\n\nJHM wrote the manuscript and enhanced the app. SF developed initial version of the app for Cytoscape 2.8. VD ported the app to Cytoscape 3. AP and TF supervised app development and provided input on the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nJHM was funded by NIGMS grants P41-GM103504 and P41-GM103311. TF is funded by NIGMS grant P41-GM103311. AP is funded by NIGMS grant P41-GM103504.\n\n\nAcknowledgments\n\nThe authors wish to acknowledge the support of the NRNB Academy.\n\n\nSupplementary material\n\nSample movie created with CyAnimator and Cytoscape 3.3. This movie used galFiltered.cys, which may be located in the sampleData subdirectory of the Cytoscape installation directory (e.g. /Applications/Cytoscape_v3.2.1/sampleData on a Mac).\n\nClick here to access the data.\n\n\nReferences\n\nSimons DJ, Ambinder MS: Change blindness: theory and consequences. Curr Dir Psychol Sci. 2005; 14(1): 44–48. Publisher Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPico AR, Bader GD, Demchak B, et al.: The Cytoscape app article collection [v1; ref status: not peer reviewed, http://f1000r.es/3tv]. F1000Res. 2014; 3: 138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKincaid R, Kuchinsky A, Creech M: VistaClara: an expression browser plug-in for Cytoscape. Bioinformatics. 2008; 24(18): 2112–2114. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Q: 3DScape: three dimensional visualization plug-in for Cytoscape. Nature Precedings. 2011. Reference Source\n\nMorris JH, Apeltsin L, Newman AM, et al.: clusterMaker: a multi-algorithm clustering plugin for Cytoscape. BMC Bioinformatics. 2011; 12(1): 436. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDhameliya V, Wu A, Morris S, et al.: CyAnimator: F1000 Release. Zenodo. 2015. Data Source"
}
|
[
{
"id": "9880",
"date": "18 Aug 2015",
"name": "Giovanni Scardoni",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 App is very useful and easy to use. As the article highlights It is very important to have a tool allowing the user to create animations from static frames of a network, and there are no other Cytoscape apps with the same characteristics of CyAnimator. The paper is well written and both implementation and features are easy to understand.Minor changes:The sentence about the future implementation of the maps in the implementation section should be moved to the conclusions session or to the supplementary materials. So the reader can concentrate on the current features of the app. Similarly, the last sentence of the conclusions sounds better as \"The ability to save the key frames of an animation as part of a session will be added .....\".",
"responses": []
},
{
"id": "10174",
"date": "02 Sep 2015",
"name": "Nathan Salomonis",
"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 useful Cytoscape app that fills in an important void not provided by the parent application: dynamically transitioning between distinct network states or associated visualized datasets. The application is thus novel and provides an important function in Cytoscape that could significantly assist with biological interpretation. Major Point 1: The authors state: “Note that CyAnimator is only able to animate between networks in the same network collection.” Can a network collection be different networks that share some or all of the same nodes? If not, I understand how this could be a technical limitation of the app, but would be of particular use when curating distinct pathway states from an original network. Often, adding, subtracting, moving and re-coloring nodes in succession to different states in the same network instances is challenging, but storing these as distinct snap shots that can be animated together is extremely powerful. If not present, is this a future option? Major Point 2: The shown movie is a reasonable simple demonstration of CyAnimator, however, this does not show of the more interesting dynamic options of the tools, such as transitioning between different developmental or temporal states (node color attributes, aka gene expression fold differences) or temporally distinct interactions for a given pathway (e.g., chain of metabolic events). While I realize this would be some work to do, this would more strongly demonstrate the capabilities of the software. Minor Point: One of the powerful uses of this software would be via programmatic generation of animations, by defining network states and visual parameters. While I know that some of these capabilities are embedded in Cytoscape 3, are these possible within CyAnimator for the purpose of making animations outside of the UI? If so, can the authors provide an example set of code to do this? A link to documentation or a tutorial would also be helpful for the end-users to properly use the software.",
"responses": [
{
"c_id": "1730",
"date": "30 Dec 2015",
"name": "Scooter Morris",
"role": "Author Response",
"response": "Thank you for your review. I have just submitted version 2 of the CyAnimator paper, which corresponds to the latest release of the App on the Cytoscape App store. The new app version is a significant rewrite and offers several advantages over the previous version, which I believe address move of your concerns. To answer point-by-point:Major Point 1: The authors state: “Note that CyAnimator is only able to animate between networks in the same network collection.” Can a network collection be different networks that share some or all of the same nodes? If not, I understand how this could be a technical limitation of the app, but would be of particular use when curating distinct pathway states from an original network. Often, adding, subtracting, moving and re-coloring nodes in succession to different states in the same network instances is challenging, but storing these as distinct snap shots that can be animated together is extremely powerful. If not present, is this a future option?Yes, a network collection in Cytoscape 3 includes multiple networks that share nodes and edges. Essentially, a network collection can be thought of as a set of projections of a single network, very similar to the Cytoscape 2 model. Cytoscape 3 allows users to create multiple network collections and the nodes and edges aren't shared between them. Both versions of the App support this capability.Major Point 2: The shown movie is a reasonable simple demonstration of CyAnimator, however, this does not show of the more interesting dynamic options of the tools, such as transitioning between different developmental or temporal states (node color attributes, aka gene expression fold differences) or temporally distinct interactions for a given pathway (e.g., chain of metabolic events). While I realize this would be some work to do, this would more strongly demonstrate the capabilities of the software.The sample movie has been updated as suggested to show three different expression fold changes and a heat strip representation showing all three at once.Minor Point: One of the powerful uses of this software would be via programmatic generation of animations, by defining network states and visual parameters. While I know that some of these capabilities are embedded in Cytoscape 3, are these possible within CyAnimator for the purpose of making animations outside of the UI? If so, can the authors provide an example set of code to do this? A link to documentation or a tutorial would also be helpful for the end-users to properly use the software.This version of the App introduces some simple commands that may be used by app developers to create frames. At this point, the capability remains somewhat rudimentary and doesn't allow programmatic changes to frame ordering or transition duration. This will be added in a future version and more detailed documentation will be written at that time."
}
]
}
] | 1
|
https://f1000research.com/articles/4-482
|
https://f1000research.com/articles/4-1520/v1
|
30 Dec 15
|
{
"type": "Software Tool Article",
"title": "CausalTrail: Testing hypothesis using causal Bayesian networks",
"authors": [
"Daniel Stöckel",
"Florian Schmidt",
"Patrick Trampert",
"Hans-Peter Lenhof",
"Florian Schmidt",
"Patrick Trampert",
"Hans-Peter Lenhof"
],
"abstract": "Summary Causal Bayesian Networks are a special class of Bayesian networks in which the hierarchy directly encodes the causal relationships between the variables. This allows to compute the effect of interventions, which are external changes to the system, caused by e.g. gene knockouts or an administered drug. Whereas numerous packages for constructing causal Bayesian networks are available, hardly any program targeted at downstream analysis exists. In this paper we present CausalTrail, a tool for performing reasoning on causal Bayesian networks using the do-calculus. CausalTrail's features include multiple data import methods, a flexible query language for formulating hypotheses, as well as an intuitive graphical user interface. The program is able to account for missing data and thus can be readily applied in multi-omics settings where it is common that not all measurements are performed for all samples.Availability and Implementation CausalTrail is implemented in C++ using the Boost and Qt5 libraries. It can be obtained from https://github.com/dstoeckel/causaltrail",
"keywords": [
"software",
"Bayesian networks",
"causality",
"interventions",
"counterfactuals",
"GUI",
"expectation-maximisation",
"do-calculus"
],
"content": "Introduction\n\nAn important task in molecular biology is the experimental validation of new hypotheses. This, however, can prove to be an expensive and time-consuming endeavour. Computational methods that allow to assess hypotheses in-silico can, consequently, decrease costs and increase productivity considerably. A popular class of methods for this purpose are graphical models. Graphical models are statistical models for which the dependencies between its variables can be interpreted as a graph structure. Bayesian networks (BNs), a special class of graphical models, are frequently used in bioinformatics as they allow to model dependencies between biological entities as a directed acyclic graph. In a BN, an arc from parent to child is often assumed to model a causal relationship. This, however, is not true in general. Often, multiple equivalent BNs for one probability distribution with differing topological order exist. Hence, they encode different “causal” relationships. Pearl et al. showed under which conditions the dependencies in a BN do, in fact, model real causal effects and described a formal framework for causal reasoning1. This framework, known as do-calculus, allows to examine hypothesis on how external changes (interventions) affect a system’s behaviour. Examples for interventions in a causal BN (CBN) are to add/remove edges or to set a node to a constant value. The do-calculus allows the modeling of the effects of mutations, gene knockouts, or counter-factual questions such as “Would the patient have recovered when administered drug B, knowing that he did not recover when administered drug A?”. The ability to answer questions like this is essential for the study of gene regulation or the evaluation of treatment regimens and, therefore, should be well supported by appropriate tools.\n\nFor inferring BNs and learning their parameters various packages such as bnlearn2, BANJO3, BNFinder24, or SMILE5 exist. SMILE additionally provides the graphical user interface (GUI) GeNIe. The pcalg R package6 allows to infer the structure of CBNs. Murphy7 compiled an extensive list of available software for working with graphical models. Although many of the listed tools are able to conduct Bayesian inference, we only found one commercial tool, BayesiaLab8, supporting causal reasoning through interventions. None of the tools seem to support counterfactual queries. With CausalTrail we provide a software for conducting causal reasoning using the do-calculus with which we attempt to fill the apparent lack of free tools in this area. Given a predefined CBN structure, CausalTrail infers parameters using an expectation maximization (EM) procedure that can cope with missing data. This makes CausalTrail applicable to multi-omics datasets where some measurements may be missing or must be discarded due to quality issues. After parameter learning the user can pose, possibly counterfactual, queries containing causal interventions. For the implementation of CausalTrail we put special emphasis on the performance and reliability of the implemented methods. We additionally provide a simple, but flexible query language for formulating hypotheses as well as a user friendly GUI. CausalTrail is licensed under GPLv3 and can be obtained from https://github.com/dstoeckel/causaltrail.\n\n\nMethods\n\nCausalTrail is written in C++ and uses the Boost and Qt5 libraries, as well as the Google Test framework for unit tests. CBN topologies are read from simple interaction format (SIF) and trivial graph format (TGF) files. Experimental data must be provided as a whitespace separated matrix.\n\nAs CausalTrail does not directly support continuous variables, continuous input data must be discretised using one of the provided discretisation methods. The ceil, floor, and round methods discretise the inputs to the nearest integers. In contrast thresholding-based methods like the arithmetic or harmonic mean, median, z-score and fixed threshold methods create binary output. The bracket medians and Pearson-Tukey9 procedure create three or more output classes. Discretisation methods can be directly specified using the GUI or via a JSON-based input file.\n\nFor parameter learning the EM procedure described by Koller et al.10 is used in order to account for missing values. To avoid local minima, the EM algorithm is restarted multiple times using different initialization schemes. For Bayesian reasoning, we implemented the variable elimination algorithm (cf. Koller et al.10). Counterfactuals are computed using the twin network approach1.\n\nCausalTrail uses an intuitive query language for formulating hypotheses. Every query starts with a ’?’ followed by a list of nodes for which the posterior probability of a certain state. Alternatively it is possible detect the most likely state of a variable using the argmax function. It is possible to condition on a list of nodes using the ’|’ character. Similarly, interventions can be stated after ’!’. Possible interventions are: fixed value assignments (N = v), edge additions between nodes N and M (+N M) and edge removals (-N M). Example queries are given in Table 1.\n\nHigh phosphorylation levels for ERK increase the likelihood of AKT being phosphorylated. In contrast, no such influence is detectable for PKA. The last two rows show the effect of conditioning on ERK.\n\nMultiple network instances can be loaded and used in the same session. The session itself can be saved and restored at any point in time. Network layouts are computed using a force-directed algorithm or, if installed, using Graphviz11.\n\nWe developed and tested CausalTrail under Ubuntu Linux 14.04. Compiling the code under Windows is possible using MSVC 2015, but not officially supported.\n\nWhen invoking the command line application, a file containing the observations, a file specifying how the observed variables should be discretised, as well as the used network topology need to be specified. Once the input files are read, CausalTrail computes and prints the parameters of the Bayesian network. After the parameters have been computed, the user can enter queries in the query language.\n\nNodes represent proteins and edges phosphorylation events. Nodes for which probabilities should be computed are coloured light green. Nodes with fixed values due to an intervention are coloured light yellow. The dashed edges are not considered during evaluation due to the intervention on ERK.\n\nThe graphical user interface workflow is similar to the CLI workflow and all functionality available in the CLI is also available in the GUI. First, the user needs to load a network topology, followed by observational data. Then, the discretisation methods to be used for the variables can be selected or loaded from a file. Queries can be entered manually via a text field or built interactively by right-clicking on the network nodes and edges. In the first case, queries are automatically checked for validity while typing. The nodes and edges involved in a query are highlighted. Counterfactual queries can be generated by conditioning and creating an intervention on a variable simultaneously.\n\n\nUse-case\n\nWe demonstrate an application of CausalTrail, using the protein signaling network inferred by Sachs et al.12 (see Figure 1). The authors validated the existence of the arc between ERK and AKT by showing that an intervention on ERK has an effect on AKT, but no effect on PKA. To this end, the phosphorylation of AKT and PKA was measured with ERK being (i) unperturbed, (ii) stimulated, and (iii) knocked down using siRNAs. Whereas the stimulation of ERK had no effect on PKA, it lead to an increase in AKT phosphorylation. For the knockdown, again no change of PKA phosphorylation could be detected whilst the phosphorylation of AKT dropped slightly below the level of the unperturbed case. To test whether the inferred network models the experimental data faithfully, we used the dataset and topology provided by Sachs et al.12 to train the parameters of a CBN and examined the arc between ERK and AKT more closely. To this end, we discretised each protein’s phosphorylation level into the classes low (0), medium (1), and high (2) using the bracket medians procedure. We then computed the most likely phosphorylation state of AKT and PKA in (i) unperturbed, (ii) stimulated, and (iii) ERK knockout cells, which we modelled using interventions that fix the ERK phosphorylation level to high and low respectively. The computed queries are given in Table 1. We find that the stimulation of ERK leads to an increased AKT phosphorylation level. When ERK is knocked out AKT phosphorylation drops to low showing that the previous increase was, in fact, mediated by ERK. In contrast the activity of ERK has no effect on the phosphorylation of PKA. Note that using an intervention is essential for this observation as conditioning on ERK would render PKA dependent on ERK resulting in a different prediction (see bottom lines in Table 1).\n\n\nDiscussion\n\nCausalTrail enables its users to harness the additional expressivity offered by the do-calculus to formulate and test biological hypotheses in-silico. In addition to basic interventions, CausalTrail supports the evaluation of counterfactuals using the twin network approach. To the best of our knowledge, it is the only available tool that offers this functionality. Our software offers efficient implementations for parameter learning and query evaluation that allow examining experimental data in an interactive fashion. The showcased application of causal reasoning demonstrates that CausalTrail may be a valuable addition to a bioinformatician’s toolbox for the interpretation of Bayesian networks.\n\n\nSoftware availability\n\n1. URL link to the author’s version control system repository containing the source code (https://github.com/dstoeckel/causaltrail)\n\n2. Link to source code at time of publication (https://github.com/F1000Research/causaltrail)\n\n3. Link to archived source code at time of publication (http://dx.doi.org/10.5281/zenodo.35611)\n\n4. Software license (GNU General Public License version 3)",
"appendix": "Author contributions\n\n\n\nFS and DS implemented the software. PT advised FS’s work. DS and HPL drafted the manuscript. All authors contributed to proofreading, provided corrections and have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the [SPP 1335] (Scalable Visual Analytics) of the DFG [LE 952/3-2].\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nPearl J: Causality: models, reasoning and inference. Cambridge Univ Press, 2nd edition, 2009. Publisher Full Text\n\nMarco S: Learning Bayesian networks with the bnlearn R package. J Stat Softw. 2010; 35(3). Publisher Full Text\n\nSmith VA, Yu J, Smulders TV, et al.: Computational inference of neural information flow networks. PLoS Comput Biol. 2006; 2(11): e161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDojer N, Bednarz P, Podsiadło A, et al.: BNFinder2: Faster Bayesian network learning and Bayesian classification. Bioinformatics. 2013; 29(16): 2068–70. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDruzdzel MJ: Smile: Structural modeling, inference, and learning engine and genie: a development environment for graphical decision-theoretic models. In AAAI/IAAI, 1999; 902–903. Reference Source\n\nKalisch M, Mächler M, Colombo D, et al.: Causal inference using graphical models with the R package pcalg. J Stat Softw. 2012; 47(11): 1–26. Publisher Full Text\n\nMurphy K: Software packages for graphical models. 2014; 7. Reference Source\n\nConrady S, Jouffe L: Bayesian Networks & BayesiaLab A Practical Introduction for Researchers. Bayesia USA, 1st edition, 2015. Reference Source\n\nCraciun MD, Chis V, Bala C: Methods for discretizing continuous variables within the framework of Bayesian networks. In Proceedings of the International Conference on Theory and Applications in Mathematics and Informatics, ICTAMI. 2011; 433–443. Reference Source\n\nKoller D, Friedman N: Probabilistic graphical models: principles and techniques. MIT press, 2009. Reference Source\n\nGansner ER, North SC: An open graph visualization system and its applications to software engineering. Softw Pract Exp. 2000; 30(11): 1203–1233. Publisher Full Text\n\nSachs K, Perez O, Pe’er D, et al.: Causal protein-signaling networks derived from multiparameter single-cell data. Science. 2005; 308(5721): 523–529. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11879",
"date": "18 Jan 2016",
"name": "Maxime Gasse",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn interesting tool that provides an easy-to-use user interface to play with causal Bayesian networks. Do-calculus provides a safe and sound framework for reasoning with effects, causes and interventions. It is good see the development of such kind of software to help in the adoption of CBNs and do-calculus to express and compare the results of experimental studies.I can not judge on the protein signaling use-case presented in the article, however the methods employed to perform inference and parameter learning are standard and appropriate. Still, more details about the parameter learning implementation would be welcome: is there any kind of regularization done along with EM? Is it possible to specify some priors?The tool is young and suffers from occasional crashes (I encountered some while testing), however the code is open source which should greatly help in fixing bugs or implementing new functionalities upon it. Some ideas of such functionalities which could improve the software:being able to specify toy Bayesian network structures by hand using the GUI being able to specify and modify the CPTs by hand in the GUI being able to generate data from the network, from the joint distribution p(x), conditional distributions p(y|x), post-intervention distributions p(y|do(x)) or even mixing both p(y|y,do(z))",
"responses": []
},
{
"id": "11738",
"date": "29 Jan 2016",
"name": "Vishakh Hegde",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 fill a void in the computational biology toolkit, as I am not aware of a software package for performing inference on nodes, only packages for structure learning. This important functionality is a useful contribution.My main critique is on the usability - if this is meant to target biologists, it would be helpful to make the file formats very simple, in particular most biologists use Excel for their data, so it would be helpful if they could specify the model and the data in a csv or txt file.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1520
|
https://f1000research.com/articles/4-1519/v1
|
30 Dec 15
|
{
"type": "Study Protocol",
"title": "Risk of intracranial hemorrhage in users of oral antithrombotic drugs: Study protocol for a nationwide study",
"authors": [
"Sasha Gulati",
"Ole Solheim",
"Sven M. Carlsen",
"Lise R. Øie",
"Heidi Jensberg",
"Agnete M. Gulati",
"Charalampis Giannadakis",
"Asgeir Store Jakola",
"Øyvind Salvesen",
"Ole Solheim",
"Sven M. Carlsen",
"Lise R. Øie",
"Heidi Jensberg",
"Agnete M. Gulati",
"Charalampis Giannadakis",
"Asgeir Store Jakola",
"Øyvind Salvesen"
],
"abstract": "Background A wide range of antithrombotic medications can be used in the prevention and treatment of thrombosis. Among hemorrhagic complications of antithrombotic drugs, intracranial hemorrhage may have particularly devastating consequences with high morbidity, disability and mortality rates. The incidence and risks of intracranial hemorrhage in patients on antithrombotic treatments from regular clinical practice outside clinical trials remain largely unknown. It is not known if results from clinical trials can be extrapolated to everyday clinical practice. We will conduct a nationwide study to investigate the risks and incidence rates of intracranial hemorrhage in users oral antithrombotic drugs in Norway from 2008 through 2014. Methods and design The aim of this nationwide study is to investigate the incidence rates of intracranial hemorrhage requiring hospitalization in users of oral antithrombotic drugs. The study will be conducted within the approximately 4.7 million inhabitants of Norway from January 1st, 2008, to December 31st, 2014. Treatment and outcome data are obtained from the Norwegian patient registry and the Norwegian prescription database. Trial registration number Clinicaltrials.gov (NCT02481011)",
"keywords": [
"Stroke",
"Hematoma",
"Drug-Related Side Effects and Adverse Reactions",
"Hematoma",
"Subdural",
"Intracranial"
],
"content": "Background\n\nAccording to the World Health Organization (WHO) the disorders caused by thrombosis are collectively the most frequent cause of death and disability in the developed world1. A wide range of antithrombotic medications can be used in the prevention and treatment of thrombosis. There are two main groups of antithrombotic therapy: anticoagulants which limit activity of the coagulation cascade, and antiplatelet agents which limit activation or activity of blood platelets2. Certain disorders are best managed with antiplatelet medications, others with anticoagulants, and some with both2.\n\nThe challenge is to prevent thrombosis while maintaining hemostasis, namely the capacity to preclude hemorrhage. The most serious adverse effect of antithrombotic therapy is bleeding. Combinations of antithrombotic agents are now frequently used, and this may lead to an increased frequency of significant bleeding complications3–6. Among hemorrhagic complications of antithrombotic drugs, intracranial hemorrhage (ICH) may have particularly devastating consequences with high morbidity, disability and even mortality rates7,8. Intracerebral hemorrhage is generally associated with a higher risk for death and incurs greater loss of health over a lifetime than ischemic stroke9–11.\n\nAlthough a certain risk for bleeding may be acceptable in the context of even greater protection against ischemic events, it is important to quantify the magnitude of bleeding risk. So far the efficacy and safety profile of antithrombotic agents are generally assessed in randomized controlled trials (RCT). However, extrapolating the results from RCTs to the general patient population in this context is challenging. Patients who participate in clinical trials are frequently highly selected and may therefore not be representative of users in everyday clinical practice. Clinical follow-up and drug compliance are often better in clinical trials and polypharmacy is less common than in clinical practice. In addition, the primary endpoint of RCTs is usually not complications, and complication numbers are limited as treatment period is often much shorter than in routine management of a chronic disease or condition. In everyday practice there is a risk of drifts in indications, inclusion criteria, exclusion criteria, and stop-criteria suggested by RCTs. Specifically, in the context of antithrombotic drugs this may potentially lead to treating patients with lesser risk of thromboembolic events but with higher risk of hemorrhage. Collectively these factors may lead to other incidence rates in the general clinical population than what is frequently reported in the literature.\n\nThe incidence of intracranial hemorrhage due to antithrombotic therapy could theoretically be monitored by post-marketing surveillance by including spontaneously reported events. Unfortunately, it seems this does not provide more reliable estimates. A recent study from Finland showed that bleeding complications due to oral anticoagulation with Warfarin are underreported in daily clinical practice12. Further, it has been shown that reporting rates of side effects following medical therapy tend to decrease over time indicating that it is more likely that adverse events to a newer drug are reported compared to a drug that has been available for many years13,14. This is why we need population-based large-scale pharmaco-epidemiological studies, i.e. real world data, in which cohorts of patients exposed to antithrombotic medications are monitored to estimate a valid and reliable risk of the treatment.\n\nThe incidence rates of intracranial hemorrhage in patients on antithrombotic treatments in the general population remain unknown. We will conduct a nationwide study to investigate the incidence rates of ICH in users oral antithrombotic drugs in Norway from 2008 through 2014.\n\n\nMethods and design\n\nReporting will be consistent with the strengthening the reporting of observational studies in epidemiology (STROBE) statement.\n\nThe primary aim of this nationwide study is to investigate the incidence rates of ICH requiring hospitalization (inpatient treatment) in users of different oral antithrombotic drugs. Secondary aims are assessments of case-fatality rates following ICH and proportion of patients undergoing neurosurgical procedures in users and non-users of antithrombotic drugs.\n\nThe study will be conducted within the approximately 4.7 million inhabitants of Norway (2008 census, Statistics Norway) from January 1st, 2008, to December 31st, 2014. All residents included in the Norwegian patient registry (NPR) and/or Norwegian prescription database (NorPD) aged 18 years or older at the start of the study are eligible for inclusion. According to NPR, the expected number of eligible patients with ICH during the study period is in the range of 50,000 to 60,000. The National Registry provides information to NPR and NorPD on vital status (dead or alive). Information about public specialist health care (both inpatient and outpatient treatment) and any dispensed prescription in Norway can be linked to residents by using a unique 11-digit personal identifier.\n\n1. Hospitalization due to ICH (Table 1)\n\n2. Residential address in Norway in the entire study period\n\n3. Age ≥18 years on January 1st 2008\n\nICD-10 = 10th revision of the International Statistical Classification of Diseases and Related Health Problems\n\nICPC-2 = version 2 of the International Classification of Primary Care\n\n1. Traumatic (high-energy) intracranial injury\n\n2. Parenteral antithrombotic treatment as this information is not retained in NorPD\n\nNorway has a public health care system with quite evenly distributed resources and uniform training and licensing for medical professionals. Only public hospitals provide health care to patients with ICH. The health authorities cover all inpatient treatment for patients with intracranial hemorrhage, and costs concerning established treatment options are generally not a concern for the individual patients or their doctors. Further, the government covers a variable proportion of the costs of antithrombotic drugs prescribed by physicians. Preapproved medicines available for general reimbursement ensure that patients get part of the medicine expenses covered by the government when having a chronic, prolonged or severe illness. This ensures access to medicines regardless of financial situation. There are in general few disparities in access to health care in the Norwegian population and insurance policies do not influence the treatment of intracranial hemorrhage. Antithrombotic medicine in Norway is only available at pharmacies if the patient has a prescription from a physician.\n\nNorPD was initiated on January 1st 2004, and contains important information on prescriptions for medications dispensed from all pharmacies throughout the country. All pharmacies are required to register each drug dispensing in NorPD, ensuring complete registration. NorPD registers the unique personal identification number of the patient, type of drug according to the Anatomical Therapeutic Chemical (ATC) classification system, number of Defined Daily Doses as defined by WHO, date of dispensing, quantity dispensed, and drug strength and formulation. NorPD does not include the prescribed daily dose. In addition, diagnoses are registered for medications with reimbursement according to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) or version 2 of the International Classification of Primary Care (ICPC-2). The 11-digit personal identification number, which is encrypted in NorPD, ensures that a complete prescription history can be established for each individual. The oral antithrombotic drugs included in the present study are presented in Table 2. The number of patients on oral antithrombotic drugs, either single agent or combined regimens) during the decade-long study period will be retrieved from NorPD.\n\nATC = Anatomical Therapeutic Chemical classification system\n\nNPR automatically receives information regarding diagnoses and procedures when patients receive both inpatient and outpatient treatment by public Norwegian specialist health care services. The information required in the present study is available from January 1st 2008. The ICD-10 subgroups of ICH that will be screened for inclusion are presented in Table 1. We will determine the incidence rates of ICH in users and non-users of oral antithrombotic treatment by linking data from NPR and NorPD.\n\nWe will identify comorbidities from both NPR and NorPD, including atrial fibrillation, congestive heart failure, thromboembolism, vascular disease, hypertension, diabetes mellitus, peptic ulcer, liver disease, alcohol abuse, osteoarthritis, previous bleeding, and chronic renal failure. Dispensed prescriptions for renin–angiotensin system inhibitors, antiarrhythmic drugs (beta-blockers, digoxin, class 1C antiarrhythmic drugs, calcium-channel blockers, and amiodarone), non-steroidal anti-inflammatory drugs, antidepressants, or proton pump inhibitors dispensed are defined as concomitant medication.\n\nAll statistical analyses are performed with SPSS 21.0, MySQL (Oracle), or R version 3.1 (R Foundation for Statistical Computing). The statistical significance level is defined as P≤0.05 with no adjustments for multiple comparisons. For each patient exposure periods for antithrombotic medications will be calculated according to the World Health Organization’s recommendation for drug utilization studies using the ATC classification system and the DDD as a measuring unit15. Exposure is defined as having occurred when patients have drug available and discontinuation as when they have no more drug(s) available. For many patients, treatment regimens are expected to change during the study period, so we treat use of antithrombotic medications in the analyses as time varying exposures. Consequently, patients can change exposure group according to dispensed prescriptions during the entire span of the study period. We consider patients to be at risk only when exposed to the drug(s) (during active treatment). We calculate risk time (person years) only for the active treatment period. Patients are followed until death, emigration or end of study period. Drug exposure and registration of comorbidity are discontinued (censored) at the time of the first event after 2008 (ICH). Due to strict Norwegian data privacy regulations the exact dates for prescription dispensing, ICH, and death are not made available to the study authors, and all time measurements are from a reference date known only to NPR and NorPD. The calendar year for ICH is available. The month and calendar year for death is available. Incidence rates will be calculated and compared between users and non-users of antithrombotic drugs for overall risk of intracranial hemorrhage and for the subgroups non-traumatic intracerebral hemorrhage, acute or chronic subdural hematoma, and subarachnoid hemorrhage. We will estimate hazard ratios with 95% confidence intervals for ICH using Cox regression models with adjustments for age, sex, concomitant drugs, and comorbidity. The time variable in the Cox model is patient age. We will investigate case fatality rates at three-month and one-year timepoints in addition to an analysis of overall survival following ICH. Differences in time to event (death) will also be presented in survival curves. We will investigate the proportions of patients with ICH undergoing neurosurgical procedures for different antithrombotic drug exposures. We will analyze continuous variables using an unpaired two-tailed t test for normally distributed data and continuous data with skewed distribution using the Mann-Whitney U test. The Chi-square test is used to examine the associations between categorical variables. For all outcome measures the statistician (ØS) will be blinded to drug exposure. All tables and figures are determined before any statistical procedure is undertaken, all tables will be filled in with results before the code for actual drug exposure is broken, and no information will be deleted when results are known.\n\nThe study protocol has been approved by the Regional Committee for Medical Research in Central Norway (2014/958).\n\nNo patients are involved in setting the research question or the outcome measures; nor are they involved in the design and implementation of the study. There are no plans to involve patients in dissemination.\n\nThere will be no additional data available.\n\nThe study will give rise to a scholarly publication that will be published in an international, peer-reviewed journal.\n\n\nDiscussion\n\nIn this article, we present a protocol for a nationwide study designed to investigate incidence rates of intracranial hemorrhage requiring hospitalization in users of different oral antithrombotic drugs. Treatment and outcome data are obtained from the Norwegian patient registry and the Norwegian prescription database. Secondary end-points are case-fatality rates at 3 months and 1 year after ICH, overall survival after ICH, and proportion of patients undergoing neurosurgical procedures in users and non-users of various antithrombotic drugs. In addition we will provide incidence rates for the different antithrombotic drugs in subgroups of ICH as they are defined in this study.\n\nThe major strength of this study is the large nationwide sample size collecting real world data for antithrombotic medication and its relation to ICH. Further, the study is performed in a well-defined region, Norway, where patients have equal access to a public health care system and medications. This will assure results with high external validity. Another strength is the inclusion of subdural hematomas, which are often omitted from studies evaluating the incidence of ICH in patients on antithrombotic treatment (except when associated with high energy or penetrating trauma mechanism). We will also like to stress that all tables will be set in advance before any statistical procedure is undertaken, the statistical procedures will be performed by a statistician blinded to the actual antithrombotic treatment, all results will be filled in the tables before the codes for actual treatment are broken, and no information will be deleted when the results are known. By performing the study by these strict methodological measures we will as far as possible avoid presenting biased results.\n\nThe main limitation of our study is its observational design and lack of randomization. There is a lack of information about important clinical parameters including body mass index, blood pressure, tobacco use, lipid levels, and coagulation profile; hence the effect of unmeasured confounders cannot be excluded. Since data are partly based on diagnoses codes set in clinical practice, the results may be affected by the quality of coding practice.\n\nThere are tools available to estimate the risk of major bleeding for patients on anticoagulation treatment to help determine risk-benefit16. Unfortunately, we have no data on the necessary variables in our patient population. Antithrombotic drugs are prescribed to patients with perceived higher risks of thrombosis. Some risk factors associated with thrombosis are also risk factors for ICH. Making causal inference from observed incidence rates of ICH in various categories of users and non-users of antithrombotic medications is difficult.\n\nIn large registry- and population-based pharmacoepidemiological studies some assumptions concerning drug exposure must be made. The ATC/DDD system has been in use for more than four decades in drug utilization studies. It has been found suitable for comparisons of drug utilization between different population groups and to monitor trends in drug use. Further, the ATC/DDD system is useful for providing denominator data for drug safety assessments. In everyday clinical practice individual patient characteristics and pharmacokinetic considerations must be accounted for. Consequently, the prescribed daily dose may differ from the DDD. Other researchers have estimated a daily drug exposure for the individual patient after comparing the accumulated drug dose and the elapsed time from consecutive prescriptions6. Information about the prescribed daily dose is unfortunately not available in NorPD. However, the prescribed daily dose does not necessarily reflect actual drug doses consumed. All time measurements are from a reference date known only to NPR and NorPD. Prescriptions dispensed in the last few months of 2014 might extend beyond our study period. Some patients might therefore have had an undetected ICH in early 2015 with ongoing exposure. Moreover, only patients with ICH admitted to hospital are included in the present study. Together, these factors contribute to a more conservative risk estimate of ICH in users of antithrombotic medications. Further, there is likely to be some missing drug exposure in the first few months of the study period in 2008 as prescriptions were dispensed in 2007. This might increase the incidence rate of ICH in the control group without antithrombotic drug exposure.\n\nDue to data privacy regulations all patient data are de-identified and we are not allowed to access patients electronic hospital records or diagnostic imaging to validate diagnoses and drug exposure. Validation of diagnoses and drug exposure, at least in a representative subgroup of patients, would probably strengthen our results.\n\n\nConclusion\n\nIn this article, we present a protocol for a nationwide study designed to investigate incidence rates of ICH requiring hospitalization in users of different oral antithrombotic drugs. Treatment and outcome data are obtained from the Norwegian patient registry and the Norwegian prescription database. We want to perform this study by strict methodological measures and have discussed some of the methodological issues pertinent to the successful execution of this large-scale pharmacoepidemiological study.\n\n\nList of abbreviations\n\nATC = Anatomical Therapeutic Chemical\n\nDDD = Defined Daily Dose\n\nICD-10 = 10th revision of the International Statistical Classification of Diseases and Related Health Problems\n\nICH = Intracranial hemorrhage\n\nICPC-2 = version 2 of the International Classification of Primary Care\n\nNorPD = Norwegian prescription database\n\nNPR = Norwegian patient registry\n\nRCT = Randomized controlled trials\n\nSTROBE = strengthening the reporting of observational studies in epidemiology",
"appendix": "Author contributions\n\n\n\nAll authors read and approved the final manuscript. SG is the guarantor and gave the original concept of the study. SG and HJ were involved in the study design, collection of the data, statistics and writing of the manuscript. OS, SMC, LRØ, AMG, CG and ASJ took part in the study design and writing. ØS contributed in the study design, writing of the manuscript and statistics.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis study is funded by a grant from the Liaison Committee between the Central Norway Regional Health Authority and the Norwegian University of Science and Technology. The funders have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank the Norwegian patient registry, the Norwegian prescription database, and Ingrid I. Riphagen at the Unit for Applied Clinical Research, NTNU.\n\n\nReferences\n\nWHO: Global health risks: mortality and burden of disease attributable to selected major risks. 2009. Reference Source\n\nSchneider DJ, Sobel BE: Conundrums in the combined use of anticoagulants and antiplatelet drugs. Circulation. 2007; 116(3): 305–15. PubMed Abstract | Publisher Full Text\n\nLee M, Saver JL, Hong KS, et al.: Risk-benefit profile of long-term dual- versus single-antiplatelet therapy among patients with ischemic stroke: a systematic review and meta-analysis. Ann Intern Med. 2013; 159(7): 463–70. PubMed Abstract | Publisher Full Text\n\nCaldeira D, Canastro M, Barra M, et al.: Risk of Substantial Intraocular Bleeding With Novel Oral Anticoagulants: Systematic Review and Meta-analysis. JAMA Ophthalmol. 2015; 133(7): 834–9. PubMed Abstract | Publisher Full Text\n\nLamberts M, Lip GY, Hansen ML, et al.: Relation of nonsteroidal anti-inflammatory drugs to serious bleeding and thromboembolism risk in patients with atrial fibrillation receiving antithrombotic therapy: a nationwide cohort study. Ann Intern Med. 2014; 161(10): 690–8. PubMed Abstract | Publisher Full Text\n\nSchjerning Olsen AM, Gislason GH, McGettigan P, et al.: Association of NSAID use with risk of bleeding and cardiovascular events in patients receiving antithrombotic therapy after myocardial infarction. JAMA. 2015; 313(8): 805–14. PubMed Abstract | Publisher Full Text\n\nGonzalez-Perez A, Gaist D, Wallander MA, et al.: Mortality after hemorrhagic stroke: data from general practice (The Health Improvement Network). Neurology. 2013; 81(6): 559–65. PubMed Abstract | Publisher Full Text\n\nPurrucker JC, Haas K, Rizos T, et al.: Early Clinical and Radiological Course, Management, and Outcome of Intracerebral Hemorrhage Related to New Oral Anticoagulants. JAMA Neurol. 2015; 1–10. PubMed Abstract | Publisher Full Text\n\nCadilhac DA, Dewey HM, Vos T, et al.: The health loss from ischemic stroke and intracerebral hemorrhage: evidence from the North East Melbourne Stroke Incidence Study (NEMESIS). Health Qual Life Outcomes. 2010; 8: 49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim HC, Choi DP, Ahn SV, et al.: Six-year survival and causes of death among stroke patients in Korea. Neuroepidemiology. 2009; 32(2): 94–100. PubMed Abstract | Publisher Full Text\n\nLee HY, Hwang JS, Jeng JS, et al.: Quality-adjusted life expectancy (QALE) and loss of QALE for patients with ischemic stroke and intracerebral hemorrhage: a 13-year follow-up. Stroke. 2010; 41(4): 739–44. PubMed Abstract | Publisher Full Text\n\nNavgren M, Forsblad J, Wieloch M: Bleeding complications related to warfarin treatment: a descriptive register study from the anticoagulation clinic at Helsingborg Hospital. J Thromb Thrombolysis. 2014; 38(1): 98–104. PubMed Abstract | Publisher Full Text\n\nSouthworth MR, Reichman ME, Unger EF: Dabigatran and postmarketing reports of bleeding. N Engl J Med. 2013; 368(14): 1272–4. PubMed Abstract | Publisher Full Text\n\nHoffman KB, Dimbil M, Erdman CB, et al.: The Weber effect and the United States Food and Drug Administration's Adverse Event Reporting System (FAERS): analysis of sixty-two drugs approved from 2006 to 2010. Drug Saf. 2014; 37(4): 283–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuidelines for ATC classification and DDD assignment 2015. WHO Collaborating Centre for Drug Statistics Methodology. Oslo, 2014. Reference Source\n\nPisters R, Lane DA, Nieuwlaat R, et al.: A novel user-friendly score (HAS-BLED) to assess 1-year risk of major bleeding in patients with atrial fibrillation: the Euro Heart Survey. Chest. 2010; 138(5): 1093–100. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11731",
"date": "18 Jan 2016",
"name": "Terje Sundstrøm",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written and ambitious study proposal. Many patients are prescribed antiplatelet and anticoagulant therapies, and intracranial hemorrhage (ICH) is a particularly feared complication as it is associated with severe morbidity and mortality. These therapies are most frequently used in elderly people, and the extent of their use and adverse effects will be increasingly relevant, as the population is progressively getting older. This study aims to assess the risk of ICH in patients on different anti-thrombotic therapies in Norway. We agree with the authors that the reported incidence rates of ICH in such patients are probably lower than what is seen in regular clinical practice. Thus, it is of considerable interest to evaluate this in a real-world and heterogeneous population where these drugs are in use, and especially within a homogenous and transparent health system such as in Norway. The study is generally well designed and methodologically sound. The described analyses will involve large groups of patients from the Norwegian Patient Registry and the Norwegian Prescription Database. However, we find the inclusion and exclusion criteria somewhat unclear and not in concordance with other sections of the manuscript. The first inclusion criterion is “hospitalization due to ICH”; this will enable the analysis of incidence rates of antithrombotic use in ICH patients. The most interesting incidence rates are those of ICH among all patients on these therapies, which will require inclusion of all Norwegian citizens on these drugs. These analyses are however described repeatedly throughout other sections of the protocol, and will give sound estimates of ICH incidence rates and a foundation for comparative analyses between different drugs and patient groups. We also find the exclusion criteria slightly confusing. With the collected information, which specifically does not include detailed information from hospital journals, it will not be possible to exclude patients with “traumatic (high energy) intracranial injury “or “parenteral antithrombotic treatment”. On the other hand, it would be very interesting to evaluate the in-hospital incidence (and outcomes) of ICH in patients undergoing such treatments for stroke and myocardial infarction, but this is beyond the scope of this study. The authors also want to assess the frequency of neurosurgical procedures in users and non-users of antithrombotic drugs. It is not clear which types of procedures this analysis will involve, but we suppose that craniotomies with hematoma evacuation, burr hole evacuation, ventricular drainage and intracranial pressure measurement are relevant procedures. The NOMESCO Classification of Surgical Procedures (NCSP) codes should be clearly described. We believe that it will be difficult to differentiate between chronic and acute subdural hematomas (no specific ICD code for chronic or acute), as well as traumatic and non-traumatic hematomas (divergent practices using traumatic ICD code S06.5 and non-traumatic ICD code I62.0). One could deduce if the hematoma was chronic or acute through linkage analysis with the conducted neurosurgical procedures. However, it will be impossible to assess if it was chronic or acute in non-operated patients, or traumatic or non-traumatic, without detailed review of patient journals.Taken together, this study protocol outlines a comprehensive and interesting study that can provide clinically relevant estimates of ICH risk in patients on antithrombotic drugs. In our opinion, the authors only need to provide minor clarifications to the study protocol.",
"responses": []
},
{
"id": "12194",
"date": "15 Feb 2016",
"name": "Peter Siesjö",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study outline by Gulati et al. aims to evaluate the incidence and relative risk of ICH after parenteral anti-thrombotic drug use. The aim is meritorious since very little data of these are known1. Mostly the question of prolongation of anti-thrombotic therapy after ICH have been discussed2. The presentation and discussion of the strengths and limitations of the proposed study is adequate and well organised. The only alternative to the present design would be a prospective trial but, given the limitations, the proposed retrospective study will be able to make a reasonable estimate of incidence and relative risks.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1519
|
https://f1000research.com/articles/4-1518/v1
|
30 Dec 15
|
{
"type": "Research Article",
"title": "Transcriptome sequencing revealed differences in the response of renal cancer cells to hypoxia and CoCl2 treatment",
"authors": [
"Nadezhda Zhigalova",
"Artem Artemov",
"Alexander M. Mazur",
"Egor B. Prokhortchouk",
"Nadezhda Zhigalova",
"Alexander M. Mazur",
"Egor B. Prokhortchouk"
],
"abstract": "Human cancer cells are subjected to hypoxic conditions in many tumours. Hypoxia causes alterations in the glycolytic pathway activation through stabilization of hypoxia-inducible factor 1. Currently, two approaches are commonly used to model hypoxia: an alternative to generating low-oxygen conditions in an incubator, cells can be treated with CoCl2. We performed RNA-seq experiments to study transcriptomes of human Caki-1 cells under real hypoxia and after CoCl2 treatment. Despite causing transcriptional changes of a much higher order of magnitude for the genes in the hypoxia regulation pathway, CoCl2 treatment fails to induce alterations in the glycolysis / gluconeogenesis pathway. Moreover, CoCl2 caused aberrant activation of other oxidoreductases in glycine, serine and threonine metabolism pathways.",
"keywords": [
"Hypoxia",
"CoCl2",
"renal cancer",
"gene expression",
"metabolism pathways"
],
"content": "Introduction\n\nHypoxia is characterized by reduced oxygen supply and appears in multiple pathological conditions including tumours. However, hypoxia can also have a functional role during normal mammalian development and embryogenesis 1 . Cells respond to hypoxic conditions both on biochemical and gene expression levels by switching from aerobic metabolism to anaerobic glycolysis and by expression of stress-related genes involved in regulation of cell death, erythropoiesis, angiogenesis and survival 2–4 . The activation of many O2-regulated genes is mediated by hypoxia-inducible factor (Hif1a). Under normoxia, Hif1a is hydroxylated by specific prolyl hydroxylases (PHD1, PHD2 and PHD3). This reaction requires oxygen, 2-oxoglutarate and ascorbate 5,6 . When Hif1a is hydroxylated, it interacts with the von Hippel-Lindau tumor suppressor protein (pVHL). pVHL forms the substrate-recognition module of an E3 ubiquitin ligase complex, which directs Hif1a poly-ubiquitylation and proteasomal degradation 7,8 . Under hypoxia (less than 5% O2), PHD activity is inhibited by cytoplasmic reactive-oxygen species (ROS) which alter the oxidation state of Fe2+ (a cofactor for PHD activity) to Fe3+. This alteration inhibits PHD activity and Hif1a hydroxylation, thus Hif1a cannot interact with pVHL and promotes HIf1a stabilization 9,10 . This anaerobic condition and stabilization of Hif1a are characteristic of many tumors. The most common molecular abnormality in renal cell carcinoma is the loss of VHL, which is found in about 50–70% of sporadic cases. Consequently, renal carcinomas with mutations in VHL have high steady-state levels of Hif1a expression and are hypoxic 11 . Some divalent cations such as cobalt (Co2+), nickel (Ni2+), and the iron-chelator deferoxamine (DFX), have been applied to mimic hypoxic conditions in cultured cells as they activate hypoxic signals by stabilizing HIF1a 12 . Transition metal Co2+ could induce hypoxic response by inhibiting PHD activity via iron replacement. Therefore, treatment of a cell culture with cobalt chloride (CoCl2) is a common model of hypoxia 13 . The second classical setup to study hypoxia is hypoxia induction in a CO2 incubator with a regulated level of oxygen (less than 1% O2). In this work, we performed RNA sequencing of Caki-1 clear cell renal cancer cell lines treated with hypoxia and with CoCl2 to understand how adequate CoCl2 treatment was as a hypoxia model. We propose that CoCl2 is not a completely correct model for hypoxia, as it aberrantly induces various hydroxylases not involved in hypoxia pathways and fails to induce downstream biochemical pathways normally induced by hypoxia.\n\n\nMethods\n\nCaki-1 human clear cell renal carcinoma cells were obtained from American Type Culture Collection (ATCC). Caki-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (GIBCO). For hypoxia treatment, we placed cells into a CO2 incubator with O2 control (BINDER CO2 CB 53) with a regulated environment of 1% O2, 5% CO2 and 94% N2, or cobalt chloride (CoCl2, Sigma) 300 mkM (stock solution 100mM in water) for 24 h.\n\nTotal RNA was extracted from Caki-1 cells with Trisol reagent according to the manufacturer’s instructions (Invitrogen). Quality was checked with BioAnalyser and RNA 6000 Nano Kit (Agilent). PolyA RNA was purified with Dynabeads® mRNA Purification Kit (Ambion). An Illumina library was made from polyA RNA with NEBNext® mRNA Library Prep Reagent Set (NEB) according to the manual. Sequencing was performed on HiSeq1500 with 50 bp read length. 10 million reads were generated for each sample.\n\nReads were mapped to hg19 genome (bowtie2-indexed reference downloaded from ftp://ftp.ccb.jhu.edu/pub/data/bowtie2_indexes/hg19.zip) with tophat2 software (version 2.1.0) 14 . Gene models of non-overlapping exonic fragments (http://www-huber.embl.de/pub/DEXSeq/analysis/encode/hsa.DEXSeq.gtf) were taken from ENSEMBL 54 database (http://www.ensembl.org/). For each exonic fragment, total coverage by mapped reads in each sample was calculated with bedtools multicov tool (version 2.17.0). Total gene coverage was calculated as a sum of coverages of all non-overlapping exonic fragments of a gene. Differential expression analysis was performed by applying default read count normalization (estimateSizeFactors) and performing per-gene negative binomial tests (nbinomTest), implemented in DESeq R package (version 1.22.0), with default parameters 15 .\n\nWe considered a gene to be differentially expressed if the adjusted p-value in DESeq test was lower than 0.05 and fold-change values were higher than 2 (or lower than 12). These sets of differentially expressed genes were further used for gene category enrichment analysis. We took the subset of genes which were found differential in both hypoxia against normoxia controls and after CoCl2 treatment against normal control and only in one of each experiments. These 3 sets of genes were analyzed with DAVID web service (version 6.7) 16 to find KEGG 17 pathways enriched with the genes.\n\nTCGA data on transcriptomes of kidney tumours (KIRC cohort) was downloaded from Broad Institute FireBrowse (http://gdac.broadinstitute.org/runs/stddata__2015_11_01/data/KIRC/20151101/gdac.broadinstitute.org_KIRC.Merge_rnaseqv2__illuminahiseq_rnaseqv2__unc_edu__Level_3__RSEM_genes_normalized__data.Level_3.2015110100.0.0.tar.gz). Principal component analysis (PCA) was performed with R prcomp function.\n\nA simple transcriptome-based hypoxia signature was constructed as follows: for every sample being evaluated (e.g., TCGA cancer sample), we considered only the genes which were differentially expressed between hypoxia and untreated Caki-1 cell line (DESeq test adjusted p-value< 0.05). For these genes, we multiplied their logarithmic fold-change (hypoxia vs untreated) to their expression in the evaluated sample. The resulting values were then summed up over the genes under consideration. This yielded a per-sample hypoxia score which would be higher in samples with increased expression of hypoxia-induced genes and decreased expression of hypoxia-suppressed genes.\n\n\nResults\n\nTo compare transcriptional effects caused by hypoxia and by CoCl2 exposure, we performed transcriptome sequencing (RNA-seq) of Caki-1 clear cell renal carcinoma cell line in three conditions: untreated, treated with CoCl2, and exposed to hypoxic conditions (1% O2). We searched for differentially expressed genes in two comparisons: CoCl2-treated against untreated cells and cells under hypoxia against untreated cells.\n\nWe first asked how transcriptomic changes after both treatments fit into the established hypoxia gene signature 1,18,19 . Figure 1A shows relative expression of the genes from hypoxia signature in all sequenced samples. We observed that genes in hypoxia signature were upregulated both in hypoxia and after CoCl2 treatment, though the effect was much stronger in CoCl2-treated samples. To check if a higher magnitude of transcriptomic changes in CoCl2 compared to hypoxic conditions was characteristic to other differentially expressed genes, we compared the distributions of logarithmic fold-changes (absolute value) for gene expression in these two experiments (Figure 1B). Surprisingly, for the majority of genes, hypoxia-induced changes were higher than the ones induced by CoCl2. In other words, hypoxia resulted in broader transcriptome response than CoCl2 treatment even though specific hypoxia-related genes were more affected after CoCl2 treatment.\n\n(A) CoCl2 causes much more pronounced expression changes in the expression of key hypoxia regulators compared to real hypoxia treatment. Heatmap represents relative gene expression for key genes involved in hypoxia regulation. (B) Hypoxia results in broader transcriptome response compared to CoCl2 treatment, i.e., more genes are changing expression under hypoxia. The figure shows absolute log fold change values for gene expression between hypoxia (or CoCl2) groups relative to control group. Genes are sorted according to absolute log fold change values. P(wilcoxon) < 2.2 × 10-16.\n\nThese results suggested that CoCl2 treatment could be an incomplete model of hypoxia capturing only upstream signalling events in hypoxia pathways and not reflecting broader downstream effects. To further investigate the differences between CoCl2 model and real hypoxia, we compared the sets of differentially up- and down-regulated genes in CoCl2-treatment (against untreated) and in hypoxia-treated (against untreated) cells. Figure 2A summarizes the overlap between those gene sets. To understand what regulatory and biochemical pathways were affected in each treatment, we performed gene category enrichment analysis over KEGG 17 pathways with DAVID web service (version 6.7) 16 for genes affected in both treatments or exclusively in one treatment.\n\n(A) Summary of KEGG pathways, enriched by the genes, up- and down-regulated in CoCl2 and hypoxia treatment. No enriched pathways were discovered for the genes downregulated in both treatments. (B) Overall expression change in glycolysis/gluconeogenesis (KEGG hsa00260) genes in control, hypoxia conditions and after CoCl2 treatment. Glycolysis/Gluconeogenesis (KEGG hsa00010) is activated in hypoxia but not after CoCl2 treatment. (C) Expression change for glycine, serine and threonine metabolism genes (KEGG hsa00010). Glycine, serine and threonine metabolism (KEGG hsa00260) is activated only after CoCl2 treatment but not under hypoxia.\n\nThe genes which were significantly upregulated in hypoxic conditions (1% O2) but not after treatment with CoCl2 were significantly enriched in the glycolysis/gluconeogenesis pathway which was known to be related to hypoxia 1,15,18 . Unexpectedly, we detected no enrichment in glycolysis/gluconeogenesis pathway with the genes differentially expressed after CoCl2 treatment which was confirmed by a heatmap for glycolysis/gluconeogenesis-related genes (Figure 2B).\n\nThe genes upregulated in both hypoxia and under CoCl2 treatment were enriched in the MAPK pathway. As the MAPK pathway was known to activate hypoxic response, MAPK activation was expected in hypoxia. Even though CoCl2 affected directly HIF1a pathway, MAPK was activated in CoCl2-treated samples as well as in real hypoxia. This result supported a previous observation of MAPK-dependent activation of hypoxia response under CoCl2 treatment 12 . Surprisingly, we observed systemic lupus erythematosus-related pathway activation in both treatments. The set of genes upregulated in this pathway (histone proteins H2A, H2B, H3, H4 and MHCII antigen-presenting genes) could be unrelated to lupus erythematosus, but rather could indicate increased proliferation and inflammation.\n\nWe also explored the genes specific to CoCl2 treatment but not affected by hypoxia. Surprisingly, the genes upregulated after CoCl2 treatment but not changed in hypoxia were enriched in the glycine/serine/threonine biosynthesis pathway (Figure 2A and 2B). We hypothesized that Co2+ ion could substitute metal cofactors of several oxidoreductases in the pathway and subsequently impair their activity. This, in turn, could require greater amounts of enzyme to be synthesized.\n\nHypoxic conditions within a tumour have been shown to predict worse clinical outcome 20 . We used our data on whole-transcriptome profiling of kidney cancer cell lines in normal and hypoxic conditions to extract a wide hypoxia signature and validate it with TCGA data on transcriptomes of kidney tumours 21 . Major variation in TCGA transcriptomes (Figure 3A) is generated by the difference between tumours and adjacent normal samples. We projected our sequenced samples to the principal components derived from TCGA samples and observed that the direction of transcriptome changes between untreated and hypoxic cell lines is slightly similar to the difference between normal and tumour samples, though hypoxia-related changes couldn’t explain normal-tumour difference. We constructed a transcriptome-based hypoxia signature as described in the Methods section. To test if this hypoxia signature predicted clinical outcome for kidney cancer patients, we explored the distribution of our hypoxia scores between disease free patients and patients in which the disease had recurred or progressed. Hypoxia scores were significantly higher for recurred/progressed patients (Wilcoxon test p-value=0.0009, Figure 3B).\n\n(A) PCA plot for TCGA samples of kidney tumours, adjacent normal tissue samples, untreated Caki-1 cells, Caki-1 cells treated with CoCl2 and the cells in hypoxic environment. (B) Hypoxia signature derived from RNA-seq results predicted significantly higher hypoxia scores for recurred or progressed TCGA tumours.\n\n\nDiscussion\n\nThe current study for the first time provides RNA-seq data revealing hypoxia-induced transcriptomic changes, which allows broader understanding of the processes related to hypoxia. In our analysis, we explored the limits of applicability of CoCl2 treatment as the model of hypoxia. Briefly, we observed that CoCl2 strongly alters expression of few genes important for hypoxia signalling, but fails to influence the essential downstream consequences of hypoxia, particularly the glycolysis/gluconeogenesis pathway. This might suggest the existence of alternative regulation mechanisms which trigger the downstream events in hypoxia together with main VHL/HIF1a pathway. CoCl2 treatment also abberantly induced pathways which did not respond to hypoxia. These included the glycine, serine and threonine metabolism pathways. We hypothesized that its aberrant activation might be caused by Co2+ ion binding to the enzymes (other than PHD proteins) involved in Glycine, Serine and Threonine biosynthesis.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw per-gene expression counts for individual genes (see Methods), 10.5256/f1000research.7571.d109570 23\n\nRNA-seq data was deposited to NCBI SRA under SRP066934 study accession code. The study contained experiments under the following accession codes: untreated (SRX1459966, SRX1459967, SRX1459969), treated with CoCl2 (SRX1459974, SRX1459977, SRX1459978), exposed to hypoxia (SRX1459979, SRX1459981, SRX1459984).",
"appendix": "Author contributions\n\n\n\nNZ performed the experiments, AM constructed the libraries for RNA sequencing, AA performed data analysis and prepared the figures, AA and NZ wrote the manuscript, EP designed and supervised the experiment.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by Russian Scientific Foundation (RSF) grant #14-14-01202.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nHong SS, Lee H, Kim KW: HIF-1alpha: a valid therapeutic target for tumor therapy. Cancer Res Treat. 2004; 36(6): 343–353. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHöckel M, Vaupel P: Tumor hypoxia:definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst. 2001; 93(4): 266–76. PubMed Abstract | Publisher Full Text\n\nPouysségur J, Dayan F, Mazure NM: Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006; 441(7092): 437–443. PubMed Abstract | Publisher Full Text\n\nYee Koh M, Spivak-Kroizman TR, Powis G: HIF-1 regulation: not so easy come, easy go. Trends Biochem Sci. 2008; 33(11): 526–534. PubMed Abstract | Publisher Full Text\n\nHu CJ, Wang LY, Chodosh LA, et al.: Differential roles of hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene regulation. Mol Cell Biol. 2003; 23(24): 9361–9374. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIvan M, Kondo K, Yang H, et al.: HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science. 2001; 292(5516): 464–8. PubMed Abstract | Publisher Full Text\n\nMaxwell PH, Wiesener MS, Chang GW, et al.: The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature. 1999; 399(6733): 271–5. PubMed Abstract | Publisher Full Text\n\nTanimoto K, Makino Y, Pereira T, et al.: Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein. EMBO J. 2000; 19(16): 4298–309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHagen T, Taylor CT, Lam F, et al.: Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF1alpha. Science. 2003; 302(5652): 1975–1978. PubMed Abstract | Publisher Full Text\n\nSimon MC: Mitochondrial reactive oxygen species are required for hypoxic HIF alpha stabilization. Adv Exp Med Biol. 2006; 588: 165–170. PubMed Abstract | Publisher Full Text\n\nThomas GV, Tran C, Mellinghoff IK, et al.: Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med. 2006; 12(1): 122–7. PubMed Abstract | Publisher Full Text\n\nTriantafyllou A, Liakos P, Tsakalof A, et al.: Cobalt induces hypoxia-inducible factor-1alpha (HIF-1alpha) in HeLa cells by an iron-independent, but ROS-, PI-3K- and MAPK-dependent mechanism. Free Radic Res. 2006; 40(8): 847–56. PubMed Abstract | Publisher Full Text\n\nPiret JP, Mottet D, Raes M, et al.: CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2. Ann N Y Acad Sci. 2002; 973: 443–447. PubMed Abstract | Publisher Full Text\n\nKim D, Pertea G, Trapnell C, et al.: TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013; 14(4): R36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnders S, Huber W: Differential expression analysis for sequence count data. Genome Biol. 2010; 11(10): R106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang da W, Sherman BT, Lempicki RA: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009; 4(1): 44–57. PubMed Abstract | Publisher Full Text\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000; 28(1): 27–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBenita Y, Kikuchi H, Smith AD, et al.: An integrative genomics approach identifies Hypoxia Inducible Factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res. 2009; 37(14): 4587–602. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEustace A, Mani N, Span PN, et al.: A 26-gene hypoxia signature predicts benefit from hypoxia-modifying therapy in laryngeal cancer but not bladder cancer. Clin Cancer Res. 2013; 19(17): 4879–88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJubb AM, Buffa FM, Harris AL: Assessment of tumour hypoxia for prediction of response to therapy and cancer prognosis. J Cell Mol Med. 2010; 14(1–2): 18–29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe Cancer Genome Atlas Research Network: Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013; 499(7456): 43–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeigand JE, Boeckel JN, Gellert P, et al.: Hypoxia-Induced alternative splicing in endothelial cells. PLoS One. 2012; 7(8): e42697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhigalova N, Artemov A, Mazur A, et al.: Dataset 1 in: Transcriptome sequencing revealed differences in the response of renal cancer cells to hypoxia and CoCl2 treatment. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11723",
"date": "04 Jan 2016",
"name": "Alexey Ruzov",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors analysed the response of renal cancer cells Caki-1 to hypoxia and CoCl2 treatment using whole transcriptome sequencing. The main finding of the manuscript that the profiles of gene expression of Caki-1 cells are qualitatively different under these two conditions, and, therefore, the CoCl2 treatment may not represent an adequate model mimicking hypoxia, is interesting and merits indexation, in my opinion.Although, as far as I can ascertain, most of the experiments are technically sound and the main conclusions of the study are justified, I feel that there is a number of issues with the text/interpretation of the data, which need to be addressed to improve the quality of the manuscript:It is not sufficiently clear from the text why the authors have elected the Caki-1 cell line for their study. Is there anything peculiar about these cells? Is VHL mutated/lost/misexpressed in these cells? The last question is especially important since the authors mention that this protein is lost in 50-70% of renal cell carcinomas. According to the Dataset 1, the authors used triplicates for each condition in their analysis. This should be stated in Methods section. The authors should also comment on the nature (biological vs technical) of these replicates there. It is not clear if the numbers presented in the Venn diagram in Figure 2A represent all genes up/down regulated for each condition or only the genes enriched for the indicated KEGG pathways. It is also not clear from the annotation or legend which numbers correspond to upregulated and downregulated genes in this figure. The Discussion is very weak at the moment with no references cited in the section. The authors should rewrite it relating their findings to available literature. Other points:The abstract lacks a sentence summarizing results of the study and/or pointing at potential implications of the findings for medicine/cancer biology. It would be beneficial for readers if such statement was included in the abstract. The last sentence in Introduction (starting with \"We propose...\") may well be an overinterpretation. The conclusion that CoCl2 treatment is not a \"correct model for hypoxia\" may need to be additionally confirmed using other cell line systems. Presenting the KEGG enrichment scores for each category alongside the Venn diagram may be helpful for assessing significance of the results.",
"responses": []
},
{
"id": "11907",
"date": "12 Jan 2016",
"name": "Colyn Crane-Robinson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis project uses mRNA sequencing to monitor changes in gene expression in a human kidney cell line in response to direct hypoxia (5% O2) as compared to treatment with 300mM CoCl2, frequently used as a surrogate for oxygen depletion. The results are very clear-cut: monitoring several genes known to be key hypoxia regulators shows CoCl2 to be more effective than oxygen depletion but in contrast, monitoring a much broader gene response showed O2 depletion to have greater effects. This contrast led the authors to concentrate attention on several specific biochemical pathways: a) glycolysis/gluconeogenesis is strongly activated in hypoxia (as expected) but not following treatment with CoCl2; b) the reverse is the case for glycine, serine, threonine metabolism which is strongly induced by CoCl2 but not by hypoxia.These last examples make clear the non-equivalence of CoCl2 treatment and real hypoxia and this article therefore issues a strong caveat regarding the use of the inorganic surrogate. It is most certainly worth publishing for this reason alone.Since hypoxic conditions in tumours are taken to be predictive of a poor clinical outcome, the authors tested whether the direction of changes in the transcriptome of their untreated and hypoxic cell lines mirrors the difference between tumours and their normal cell counterparts. Hypoxia scores from mRNA expression data were somewhat (but significantly) elevated in recurrent/progressive patients.However, this difference does not (unfortunately) appear to be sufficient to use clinically (if I am understanding their intentions correctly). The upshot is that the authors do not mention this comparison in their Discussion. On practical matters: they might expand the caption to Fig 1. In A) I assume the three tracks are biological replicates: if so, write it. In B) a fuller explanation of the X-axis ‘Index’ would be helpful. Check on the printing of ‘300mM CoCl2’ (in Methods, bottom of p2).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1518
|
https://f1000research.com/articles/4-1517/v1
|
30 Dec 15
|
{
"type": "Method Article",
"title": "Detecting “protein words” through unsupervised word segmentation",
"authors": [
"Wang Liang",
"Zhao Kaiyong",
"Zhao Kaiyong"
],
"abstract": "Unsupervised word segmentation methods were applied to analyze protein sequences. Protein sequences, such as “MTMDKSELVQKA…,” were used as input to these methods. Segmented protein word sequences, such as “MTM DKSE LVQKA,” were then obtained. We compared the protein words derived via unsupervised segmentation and protein secondary structure segmentation. An interesting finding is that unsupervised word segmentation is more efficient than secondary structure segmentation in expressing information. Our experiment also suggests the presence of several “protein ruins” in current non-coding regions.",
"keywords": [
"Word segmentation",
"Protein sequence",
"Protein secondary structure",
"Unsupervised method",
"Soft counting",
"Protein word",
"Gene finding",
"Description length"
],
"content": "Introduction\n\nWord segmentation mainly refers to the process of dividing a string of written language into its component words. For some East Asian languages such as Chinese, no space or punctuation is placed between letters. The “letter” sequences must be segmented into word sequences to process the text of these languages. For example, “iloveapple” is segmented into “I love apple.”\n\nThe main concept of the segmentation method is simple. For example, the letter sequence “iloveapple” can be segmented into many forms of word sequences, including “I love apple,” “ilove apple,” “il ove apple,” “iloveapple,” “ilo vea pple,” etc. We select the “word sequences” with the highest probability as the segmentation of this letter sequence. For example:\n\nP(“I love apple”) = 0.8\n\nP(“ilove apple”) = 0.3\n\nP(“iloveapple”) = 0.03\n\nHence, we will select “I love apple” as the segmentation of letter sequence “iloveapple.”\n\nThe probability of a word sequence is calculated by the probability of the multiplication of each word.\n\nFor example:\n\nP(“I love apple”) = P(i) × P(love) × P(apple)\n\nP(“ilove apple”) = P(ilove) × P(apple)\n\nTherefore, the key to segmentation is determining the probability of each word.\n\nIf we have many possible segmented sequences such as that for “i love apple,” then we can simply calculate the probability of each word by dividing the number of its occurrences by the number of occurrences of all the words. For example, we have two sequences, namely, “i love apple” and “i love mac;” that is, we have six word occurrences. “i” appears two times; thus, its probability P(“i”) = 2/6. “apple” appears one time; thus, its probability P(“apple”) = 1/6. This method is called supervised segmentation. For words such as “ilove” that do not appear in corpus, an extremely small probability is provided.\n\nIf we have no segmented sequence but many letter sequences such as “iloveapple,” then we can still calculate the probability of all the possible words and develop the word segmentation method. The main concept of this method is described as follows.\n\nFirst, the random or equal probability of each possible word is provided. In general, we need to set a maximal word length. For example, for “iloveapple,” if we set four as the maximal word length, then possible words include “ilov,” “love,” “ovea,” etc.\n\nSecond, given that we have the word list with probability, we can segment all the letter sequences into word sequences.\n\nThird, we calculate the new word probability according to the word sequences in the second step.\n\nWe repeat the second and third steps until the probabilities of all the words reach a stable value. Then, we can use this word list with probability to segment letter sequences. This method is called unsupervised segmentation1.\n\nThe basic theory of word segmentation is depicted in Figure 1.\n\nA. Supervised word segmentation. B. Unsupervised word segmentation.\n\nFor English text, the accuracy of supervised segmentation is over 95%. Meanwhile, the unsupervised segmentation method can reach approximately 75%. Supervised methods are better than unsupervised methods; however, unsupervised approaches are highly adaptive to relatively unfamiliar “languages” for which we do not have sufficient linguistic knowledge2.\n\nUnsupervised segmentation can be described as “inputting letter sequences and outputting meaningful word sequences.” If the input is a protein sequence such as “MTMDKSELVQKA…,” then what is the output? The answer to this question is the topic of this study.\n\nIn the following sections, we will perform an unsupervised segmentation of protein sequences. Identifying the functional equivalents of words in protein sequences is a fundamental problem in bioinformatics. The elements of protein secondary structure are generally regarded as the functional building blocks of protein sequences. Therefore, we compare our “protein word” sequences and related secondary structure segmentation in Section 3. We find our “protein word segmentation” to be a better encoding method than secondary structure segmentation in terms of expressing information. We discuss this confusing result in Section 4. The last section provides a short summary of the study.\n\n\nWord segmentation experiment\n\nThe soft-counting method is a representative unsupervised segmentation method; it uses the standard expectation–maximization algorithm to estimate the probability of any possible substring1. This operation produces a vocabulary with probability. To segment a sequence, a Viterbi-style algorithm is then applied to search for the segmentations with the highest probabilities.\n\nUnsupervised segmentation methods require only raw letter sequences. In this study, we use amino acid letter sequences. Data are selected from the Protein Data Bank (PDB, http://www.rcsb.org/pdb/) structure database. The PDB database contains approximately 100,000 protein sequences. These sequences are used as input in the unsupervised segmentation method. Thus, we obtain:\n\nDefinition 1: “protein word,” the segments produced by a word segmentation method.\n\nUnsupervised segmentation only requires selecting a maximal word length. In this study, we set nine as the maximal protein word length. This value can be adjusted according to data size.\n\nApproximately 26 million possible protein words are included in our data. Frequency and border information features are used to filter these words3–6. After performing the soft-counting method for these protein sequences, we obtain a final protein vocabulary with approximately 630,598 words. We can then use this vocabulary with word probability to segment protein sequences. An example of protein word segmentation is shown as follows.\n\nOriginal protein sequence:\n\nDADLAKKNNCIACHQVETKVVGPALKDIAAKYADKDDAATYLAGKIKGGSSGVWGQI PMPPNVNVSDADAK LADWILTLK\n\nCorresponding segmented protein word sequence:\n\nD ADLAKK NNCI ACH QVET KV VGPAL KDIAAK YADK D DAATYL AGKIK GGSSGV WGQI PMPPN VNVSD ADAKA LADWI LTLK\n\n\nComparison with secondary structure segmentation\n\nIn most human language texts, the output of segmentation is a meaningful word sequence. As expected, the same is true for a protein sequence. We compare a protein word sequence with the elements of protein secondary structure, which act as the functional building blocks of a protein.\n\nProtein secondary structure mainly refers to the local conformation of the polypeptide backbone of proteins that is frequently and discretely classified into a few states. Word segmentation is highly similar to the protein secondary structure assignment process. For protein sequences, such as “MTMDKSELVQKA,” the corresponding consecutive amino acids of the same secondary structure can also be regarded as a structure word. This process can be called secondary structure segmentation (Figure 2). A total of 437,537 distinct structure words are included in our data set.\n\nA protein sequence is segmented by its secondary structure. The sequence in this figure has three secondary structure words: “MTM,” “DKSEL,” and “VQKA.”\n\nDefinition 2: “structure word,” the protein segments produced by secondary structure segmentation.\n\nSegmentation performance is generally evaluated using the boundary F-score measure, F = 2RP/(R + P). Recall R is the proportion of the correctly recognized word boundaries to all boundaries in the gold standard; precision P is an output of the word segmentation of a segmenter. In this study, we use structure segmentation as the gold standard (Figure 3).\n\nFor this example, the precision is 1/33 ≈ 3% and recall is 1/2 = 50%. The F-score is 2 × 0.33 × 0.5/(0.33 + 0.5) ≈ 39.8%.\n\nFor the soft-counting method, boundary precision is 39.9%, boundary recall is 28.0%, and the boundary F-score is 32.9%. A segmentation example is provided in Figure 4.\n\nThe boundary F-score is approximately 85% for unsegmented English text using the soft-counting method. For Chinese text, the boundary F-score is approximately 80%. For protein word segmentation, however, such value is only 32.9%. Therefore, protein word segmentation is different from secondary structure segmentation. This result does not satisfy our expectation. We will analyze the differences in the next section.\n\n\nEncoding efficiency of segmentation\n\nAs a basic statistical feature, word occurrence distribution may explain the differences between the two kinds of segmentation.\n\nIn a typical English data set, if we regard the top 10% frequency words in vocabulary as high-frequency words, then the occurrences of these words generally account for approximately 90% of all the letters in the data set. For low-frequency words, for example, 1-frequency (only appear once) words account for approximately 50% of all the words in vocabulary; however, their occurrences only account for approximately 1% of the letters of the entire corpus. This law holds true for most human languages. It is also a fundamental assumption in most unsupervised segmentation methods. The related values of soft-counting segmentation and secondary structure segmentation are provided in Table 1.\n\nBased on Table 1, secondary structure segmentation appears to be “inefficient” primarily because its vocabulary contains too many low-frequency words. We can use “description length (DL)” to describe the efficiency of segmentation7. The segmentation process can be regarded as an encoding process that replaces the letters of a word with related word symbols. A codebook, in which each word is represented by a unique string, can be used to encode a corpus of words as efficiently as possible. The total number of letters required to encode the corpus (i.e., the sum of the lengths of the codebook and the encoded corpus) that uses a well-designed codebook/vocabulary will be less than that for the original corpus. Smaller units, such as morphemes or phonemes, which require fewer code words, and thus, a shorter codebook, can be encoded further. However, efficiently encoding the corpus becomes increasingly difficult as the number of code words used decreases. Meanwhile, some words may never be used when the codebook has too many words. Thus, the length of the codebook and the length of the encoded corpus must be balanced. The DL principle states that a codebook that results in the shortest total combined length must be selected. Therefore DL can be used to describe segmentation efficiency.\n\nThe DL values of structure segmentation and soft-counting segmentation are provided in Figure 5.\n\nFrom Figure 5, we can determine that the entire DL of structure segmentation is more than that of soft-counting segmentation. In structure segmentation, approximately 45% of the DL value is used for vocabulary. In soft-counting segmentation, such value is only 16%. Structure segmentation designs a large codebook, but only a few of its capabilities are used. Thus, secondary structure segmentation may not be a good encoding method. Two opposing explanations may explain this result.\n\nFirst, current structure segmentation is really inefficient. Therefore, we can find a different structure assignment method whose structure segments can express the functions of genes better. Our data set uses the DSSP secondary structure definition. We also try other kinds of secondary structure definitions such as STRIDE8, but we obtain similar low efficiency segmentation.\n\nSecond, the structure segmentation scheme is truly efficient. Given that the segmentation method is appropriate, some problems may exist in the data set. A highly common problem in the data set construction process is the unbalanced data problem. For example, if we only select several paragraphs of a long book to build a data set, then this problem will occur.\n\nWe develop the experimental data by filtering similar protein sequences in PDB. The PDB protein data can also be regarded as randomly selected from all available protein data. No obvious problem is observed in our data construction process. Hence, if our data set is unbalanced, then all current protein data are unbalanced. If yes, then more protein words are present in current non-coding regions. For example, several short DNA regions can be translated into protein words; however, such protein words that cover a region may not comply with the strict definition of a protein sequence. Therefore, we have:\n\nDefinition 3: “protein word coverage region,” the DNA region that can be translated into protein words.\n\nWe can also detect a protein word coverage region using segmentation methods. We back-translate the protein words into DNA forms and obtain a DNA vocabulary. Then, we can use this vocabulary to segment DNA sequences using supervised segmentation methods. For example, a DNA sequence “ATGGTTTTGTGTCTTGAGGGT” is segmented as Figure 6.\n\nThe green parts can be translated into protein words. The red parts cannot be translated. (Note: This sequence is not a protein sequence. Only some regions can be covered by “protein words”).\n\nThe following is a typical example of DNA sequence segmentation:\n\nTTGTGG G TTGTCTATTGATGTT TTTGGT C TATTCTAAG A A TTGGAG A GAG A GAGGTT A A AAT C TCT G ACT ATG A TTGTGG A TTGTCT GCTGAT GTT TTTGGT C TATTGTTCT AAGAAT TGG A GAG A GAG A GAGGTT A A A A TCT C C G ACT ATG A TTGTGG A TTGTCT ATTGCTGTTTTT GGT C TATTGTTCT AAGAAT.\n\nThis sequence is part of “gi|157704448|ref|AC_000133.1| Homo sapiens chromosome 1, alternate assembly HuRef, whole genome shotgun sequence:307501-308000.” The green segments can be translated into “protein words.” Although 87% of the region of this sequence is a protein word coverage region, no distinct intron, exon, and stop codon is found. Hence, such sequence cannot be regarded as a coding sequence.\n\nWe use the human genome to judge whether the data imbalance can be improved by adding all the protein word coverage regions. The genome is divided into 500 bps equal-length sequences. We use two vocabularies, namely, soft-counting word vocabulary and secondary structure word vocabulary, to segment the aforementioned DNA sequences. Then, we calculate their DL individually. The results are presented in Figure 7.\n\nWe determine that the two kinds of segmentation methods have similar DL values. Then, we convert all the DNA protein word coverage regions into protein word sequences. These sequences are regarded as gold standard segmentation. Then, we delete the space between words and perform unsupervised segmentation test again. Boundary precision reaches 67%, boundary recall reaches 60%, and the boundary F-score reaches 63%. These results concur with our conjecture. Adding protein word coverage regions can address the data imbalance and improve structure segmentation efficiency.\n\nThe distribution of protein word coverage regions is shown in Figure 8.\n\nFor example, DNA sequences whose percentage of protein word encoding region is within [0.6, 0.7] are approximately 75%.\n\nWe determine that approximately 16% of the DNA sequences have a “protein word coverage region” percentage of over 70%. Among these sequences, only approximately 10% are actual gene areas, the remaining sequences may be regarded as protein ruins, which may be abandoned in evolution.\n\n\nConclusion\n\nWe use protein sequences as input to perform a word segmentation test. As expected, the output is several meaningful protein word sequences. Thus, we compare them with secondary structure segments, which are generally regarded as the basic functional blocks of a protein. However, we determine that the two kinds of segmentation methods are different. Subsequently, we use DL to analyze their difference and determine that word segmentation is more efficient than structure segmentation. This result is irrational. We suppose that this problem is mainly caused by the data imbalance. That is, the gene is similar to a large book and current protein sequences are only several paragraphs in this book. Our DNA segmentation experiments also support this conjecture.\n\nOverall, “protein word” and “protein word coverage region” may have no biological meaning. Word segmentation is a fundamental technology in processing Chinese and other languages without space or punctuation between words, similar to that of protein sequences. Word segmentation is the preliminary step in search engine systems, translation systems, and proofreading systems, etc. The main objective of this study is to inspire new ideas that apply these technologies in analyzing protein sequences. Word segmentation simply acts as a bridge.\n\n\nData and software availability\n\nUnsupervised segmenting methods only need raw letter sequence. We mainly use the data of PDB (http://www.rcsb.org/pdb/) as our experiment data. This dataset contains about 100,000 pieces of protein sequences. We use CD-HIT algorithms to delete the similar protein sequence. The code is available from: http://weizhong-lab.ucsd.edu/cd-hit/download.php.\n\nLatest source code and experimental instructions: https://github.com/maris205/secondary_structure_detection\n\nArchived source code as at the time of publication: http://dx.doi.org/10.5281/zenodo.346639\n\nLicense: MIT (http://opensource.org/licenses/MIT)",
"appendix": "Author contributions\n\n\n\nWang Liang designed the experiments, wrote the codes. Zhao Kaiyong prepared the experimental data and provided the biology knowledge supports.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nGe X, Prat W, Smyth P: Discovering Chinese words from unsegmented text. Proceedings on the 22 Annual International ACM SIGIR Conference On Research and Development in Information Retrieval. USA, 1999; 271–272. Publisher Full Text\n\nWang H, Zhu J, Tang S, et al.: A new unsupervised approach to word segmentation. Comput Linguist. 2011; 37(3): 421–454. Publisher Full Text\n\nCohen P, Adams N, Heeringa B: Voting Experts: An unsupervised algorithm for segmenting sequences. Intell Data Anal. 2007; 11(6): 607–625. Reference Source\n\nHewlett D, Cohen P: Word segmentation as general Chunking. Proceedings of the Fifteenth Conference on Computational Natural Language Learning, USA. 2011; 39–47. Reference Source\n\nZhao H, Kit C: Exploiting unlabeled text with different unsupervised segmentation criteria for Chinese word segmentation. Proceedings of the Conference on Empirical Methods in Natural Language Processing, Israel. 2008; 17–23. Reference Source\n\nZhao H, Kit C: An empirical comparison of goodness measures for unsupervised Chinese word segmentation with a unified framework. The Third International Joint Conference on Natural Language Processing, India. 1: 2008; 9–16. Reference Source\n\nKit C, Wilks Y: Unsupervised learning of word boundary with description length gain. CoNLL, Norway. 1999; 1–6. Reference Source\n\nZhang W, Dunker AK, Zhou Y: Assessing secondary structure assignment of protein structures by using pairwise sequence-alignment benchmarks. Proteins. 2008; 71(1): 61–7. PubMed Abstract | Publisher Full Text\n\nLiang W: Detecting “protein words” through unsupervised word segmentation: First release. Zenodo. 2015. Data Source"
}
|
[
{
"id": "12078",
"date": "01 Feb 2016",
"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 authors have applied unsupervised word segmentation methods to protein sequences which in itself is a very novel undertaking and overall the manuscript is well written. However there are some outstanding points which the authors could consider to improve the quality of the manuscript.Some justification should be provided as to why 9 was selected as the maximal protein word length. How are the results affected as a function of maximal word length. On page 3, the authors write 'In human language texts, the output of segmentation is a meaningful word sequence. As expected, the same is true for a protein sequence.'It is not clear what the authors mean by 'meaningful' in the case of protein sequences. What kind of 'meaning' do the authors expect on applying an ostensibly linguistic method to protein sequences. Again on the same page the authors note that 'word segmentation is highly similar to the protein secondary structure assignment process.' Secondary structural assignment in proteins is based on the successive values of the main chain phi,psi angles and the hydrogen bonding patterns. The authors however could mean the propensity based assignment of secondary structure in case of protein structure prediction which is known to be inaccurate, the propensities being estimated from appropriate databases. Although the authors cite back references (3-6) how the structural segmentation was performed should be discussed in much greater detail in the present manuscript. If the structural segmentation was performed on the basis of actual secondary structure present in a protein (phi,psi and hydrogen bonding pattern) then there does not appear to be any connection between the two processes of segmentation, in terms of word and structure. So they should naturally be expected to be different. On fails to understand the contrary expectations of the authors when they note on page 4 ,'Therefore, protein word segmentation is different from secondary structure segmentation. This result does not satisfy our expectation'. On should also note that identical sequences have been known to adopt variable secondary structures in proteins due to other factors such as tertiary contacts and its position with respect to the rest of the polypeptide chain. How does this fact reflect on the overall results of the manuscripts? Therefore although the work concerns a novel implementation of the word segmentation method in the context of protein sequences, some of the 'expectations' and interpretations of the authors possibly require greater clarity or stronger justification.",
"responses": []
},
{
"id": "12977",
"date": "21 Mar 2016",
"name": "Judith Klein-Seetharaman",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting article and approach, but the results presented are too preliminary to assess how useful this approach may be for biologists. The title is fine but the abstract is not sufficient and reflects the deficiencies of the paper. While the idea is interesting and could potentially lead to discovery of new “words” in protein sequences, in its current state, the paper is restricted entirely on the language aspects. There is no expert knowledge on protein sequence/structure/bioinformatics that has gone into the design of the study or the analysis of the results. There are no references at all on prior work on this topic and the methods are described in too brief format. For example, when were the 100,000 sequences downloaded from pdb and does this represent all sequences. What secondary structure elements were included from DSSP/Stride? What cut-off was used to arrive at the reduced dataset, and how many protein sequences were included in the final data analysed? While it is reasonable to compare unsupervised protein words with secondary structure elements, it does not make sense to assume that the boundaries have to be fixed. In protein structures, the exact boundaries are not precise and that contributes to difficulties in further improvement on accuracy of secondary structure prediction. Furthermore, there are differences between the types of boundaries predicted, so discovery of a coil-helix versus a coil-sheet would have different error rates. Secondary structures are only one type of segmentation, there is others such as protein interaction recognition elements, enzyme active sites, posttranslational modification indicators etc that also carry “meaning” as a “word”. Last but not least, the paper neglects the contributions of tertiary structure in the definition of meaning in biology. All of these issues are much more likely to contribute to the low performance than the speculation on data imbalance. Of course, the pdb structure dataset is extremely imbalanced, and this is noted wrongly in the text that “all current protein data are unbalanced”. The proteins in the pdb are a very biased subset of proteins that crystallize, thus highly over representing small soluble proteins.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1517
|
https://f1000research.com/articles/4-1507/v1
|
23 Dec 15
|
{
"type": "Review",
"title": "Reconciling Techno-simplicity and Eco-complexity for future food security",
"authors": [
"Santiago L. Poggio",
"Sarina Macfadyen",
"David A. Bohan",
"Sarina Macfadyen",
"David A. Bohan"
],
"abstract": "Ecological intensification has been proposed as a paradigm for ensuring global food security while preserving biodiversity and ecosystem integrity. Ecological intensification was originally coined to promote precise site-specific farming practices aimed at reducing yield gaps, while avoiding negative environmental impacts (techno-simplicity). Recently, it has been extended to stress the importance of landscape complexity to preserve biodiversity and ecosystem services (eco-complexity). While these perspectives on ecological intensification may seem distinct, they are not incompatible and should be interwoven to create more comprehensive and practical solutions. Here, we argue that designing cropping systems to be more diverse, across space and time would be an effective route to accomplish environmentally-friendly intensification of crop production. Such a novel approach will require better integration of knowledge at the landscape level for increasing agro-biodiversity (focused on interventions outside fields) with strategies diversifying cropping systems to manage weeds and pests (focused on interventions inside fields).",
"keywords": [
"Agricultural intensification",
"Agroecology",
"Biodiversity",
"Ecological intensification",
"Ecosystem services",
"Organic agriculture",
"Yield gap analysis"
],
"content": "Introduction\n\nWorldwide demand for food, fuel and fiber is expected to increase for at least the next four decades owing to continuing population growth (Godfray et al., 2010). Some potential pathways to address the future gap between supply and demand of agricultural production exist through reducing waste and altering consumer diets (Godfray et al., 2010; Tscharntke et al., 2012), but these may still be insufficient. We will therefore need to increase both land productivity and resource use efficiency of agricultural systems. This challenge for agriculture is set against global environmental changes and increasing pressure on natural ecosystems, some of which are driven by agriculture itself (e.g. forest clearance and reduced carbon sequestration capacity). The consequences of these problems of food production, along with water scarcity and declining energy resources, could produce significant global social unrest and destabilization in the coming years, which has been described as ‘The Perfect Storm’ (Beddington, 2009). Degradation of both land productivity and ecological processes, in turn, creates potentially dangerous feedbacks. Increasing desertification, soil salinization, and water eutrophication, for instance, all present long-term threats to agricultural production and the well-being of communities, significantly displace the boundaries of humans’ safe operating space (Rockström et al., 2009).\n\nTo cope with the challenge of ensuring global food security while preserving biodiversity and ecosystem integrity, the ‘ecological intensification’ of cropping systems paradigm has been proposed (Cassman, 1999). Advocates of the ecological intensification of agriculture presently propose two apparently contrasting ‘paradigms’ or perspectives of the future of farming. The first is focused on bridging yield gaps of the major commodity crops worldwide, through targeted breeding strategies of a few crop types, and precise applications of fertilizers and pesticides (Cassman, 1999; van Ittersum et al., 2013). This technology-based perspective renders management options that are simple to implement and reduce farmers’ uncertainty, but also externalize a range of environmental costs (Techno-simplicity). The second emphasizes the role of landscape complexity on biodiversity, supporting the ecological processes that underpin ecosystem services (Eco-complexity), such as pollination, pest control, and soil fertility (Bommarco et al., 2013; Doré et al., 2011). This perspective emphasizes the importance of spatial heterogeneity, especially landscape complexity, to preserve both biodiversity and the ecological functions related to ecosystem services (Chaplin-Kramer et al., 2011; Garibaldi et al., 2011).\n\nWhilst eco-complex management options may be more sustainable than techno-simple practices, they are more complicated to implement, requiring co-ordination among farmers and local authorities, and carry higher levels of uncertainty and risk for farmers. Furthermore, the broader public is keen on preserving heterogeneous rural landscapes, particularly due to their scenic, recreational and cultural values, and complain when intensive agriculture leads to countryside homogenization. Thus, there is an urgent need to conserve biodiversity and rural landscapes, driven in part by public concern, while ensuring food security, particularly in poor countries where key areas of hot-spot biodiversity and severe poverty coincide (Fisher & Christopher, 2007). However, pesticides and transgenic crops are unpopular with the public, especially in some developed countries (Costa-Font et al., 2008), while both biotechnology and precision agriculture are helping to increase food supply and reduce environmental pollution worldwide. For instance, the use of insecticides in cotton has been markedly reduced since the adoption of Bt-transgenic varieties in Argentina and Australia (Qaim, 2009). Hence, attitudes and perceptions of benefits from techno-simplicity and eco-complexity perspectives greatly vary among the different societal actors, with a marked gap between where management interventions are implemented and where the benefits are perceived.\n\nA considerable body of research has highlighted that designing more diverse agricultural landscapes will be crucial for increasing yields and simultaneously conserving biodiversity to ensure food security (Loos et al., 2014). However, the apparent dichotomy between Eco-complexity and Techno-simplicity has led to an undue focus on how small, uncropped patches in landscapes can be better managed. Fields have been left to techno-simple solutions and the constraints of landscape research have limited the scales at which eco-complex management options can be tested. As a result, management actions for increasing diversity within the field are uncommon and, where they do exist, they have not been widely adopted and have had equivocal effects.\n\nHere, we argue that integrating Techno-simplicity and Eco-complexity perspectives would deliver environmentally-friendly, intensive and food-secure agro-ecosystems, through the provision of more tractable management options for farmers. We stress that bringing about change of practices in intensified cropping systems is about making small changes to usual farming methods. In practice, each individual change may only lead to small gains in biodiversity. However, when actions are combined and implemented across large areas, marked increases in biodiversity and ecosystem services may emerge, expressed at broader spatial scales and over longer periods than a single growing season. We also claim that the knowledge needed is already available (Shennan, 2008), but needs to be properly integrated into intensively managed cropping systems to effectively design more multifunctional agricultural systems. We argue that achieving future food security through integrating eco-complex and techno-simple perspectives will require reconciling goals, costs and benefits perceived by social actors across scales, including farmers, city dwellers, practitioners and policy makers.\n\n\nSimplifying cropping systems for further increasing yields\n\nModern agriculture has succeeded in increasing global food supply. This has been achieved through targeted breeding of a few, highly productive crops, providing resources through fertilization and irrigation, protecting crops by applying pesticides and, more recently, by genetic modifications to crops. This agricultural intensification has led to declines in biodiversity due to habitat loss, farmland homogenization and environmental pollution (Tscharntke et al., 2005). To overcome these negative environmental impacts, the paradigm of ecological intensification was initially coined to promote a site-specific, precision agriculture. This would increase yields of the main cereal crops, ensuring future global food security, while simultaneously reducing both soil degradation and pollution, derived from plowing and excessive fertilization (Cassman, 1999). This techno-simple perspective increased yields of the major commodity crops worldwide through breeding a handful of crop types and being highly precise in the applications of fertilizers and pesticides, but reducing crop diversity at local, regional and global scales (Khoury et al., 2014). Similar crop yields have been achieved in GM herbicide-tolerant varieties, where herbicides applications decline significantly in comparison to counterpart conventional crops (Champion et al., 2003). Ultimately, these crops were rejected in Europe both because of public concern and measurable impacts on biodiversity (Bohan et al., 2005), despite demonstration that these biodiversity effects could be reversed through modified management, such as herbicide timing (Dewar et al., 2003). However, in the Americas, where glyphosate-resistant crops have been rapidly adopted, biotypes of some major weeds have evolved resistance to this herbicide (Powles, 2008).\n\nTechno-simplicity is, thus, directed at improving crops and their environment to bridge the gaps between actual and potential yields, through advancement in plant ecophysiology, molecular biology and soil science. As originally conceived, the ecological intensification paradigm is the latest reconceptualization of a long history of empirical research on resource use efficiency in agriculture (Cassman, 1999; de Wit, 1992; van Ittersum et al., 2013). The conceptual framework applied is based on yield gap analysis, which is aimed at identifying the key environmental and management factors defining the gaps between potential and actual yields (see Box 1). However, the techno-simple perspective of ecological intensification has not given proper attention to biodiversity and its benefits, through associated ecological processes and functions regulating the incidence of weeds, pests and diseases.\n\n\n\nCrop yields, measured as the amount of harvestable production per land area unit (kg ha-1), are determined by factors influencing crop cycle length, resource use, growth rates and reproductive allocation (van Ittersum & Rabbinge, 1997; van Ittersum et al., 2013). Yield-defining factors determine the maximum yield levels (potential yields) for a given crop grown in a particular site, with no water and nutrients limitations, and effective control of biotic factors reducing yields. Potential yields are defined, on the one hand, by environmental factors that are location-specific, such as the growing season length (frost-free period), atmospheric CO2, and the annual variability in incoming solar radiation, day length, and temperature. On the other hand, potential yields are also determined by crop plant (genotypic) characteristics, such as developmental rates (type of response to day length), growth rates (photosynthetic pathway), and plant morphology and architecture (growth, reproductive allometry). Both environmental and genotypic attributes of crops can be managed to some extent in regular farming conditions. Hence, farmers make tactical decisions to maximize the use of resources available during the growing season by choosing well-adapted crop varieties and optimum sowing dates, thus determining the growing period to maturity, planting density and spatial arrangement.\n\nYield-limiting factors include water and nutrients, which determine the water- and nutrient-limited yields (van Ittersum & Rabbinge, 1997; van Ittersum et al., 2013). Water-limited yields are usually obtained from crops grown under rain-fed conditions, where water limitations result from climate (annual rainfall, dry season, evapotranspiration, vapor pressure deficit) and soil factors (runoff, infiltration, retention capacity, soil depth). Nutrient limitations are due to soil characteristics that may affect both soil nutrient availability and crop uptake efficiency, such as the organic matter content, soil reaction (pH) and salinity. Soil biotic activity may also influence nutrient availability for crops. Yield-reducing factors comprise biotic agents, such as weeds, pests and diseases, and abiotic stressors, such as pollutants, which reduce yields by decreasing or hindering either resource use, growth rates or reproductive partitioning. Actual yields at farm level result from combinations of limiting and reducing factors, which are defined by the actual supplies of water and nutrients and protection levels against biotic adversities. Farmers can increase actual yield levels by taking measures to overcome the effects of factors limiting and reducing yields. Yield-increasing measures consist of providing resources; water through irrigation and nutrients by applying synthetic fertilizers or managing soil fertility and structure (composting, green manuring) (Table 1, van Ittersum & Rabbinge, 1997). Yield-protecting measures are aimed at preventing and reducing the incidence of biotic factors reducing crop growth and yields (Table 2). These may involve direct control with specific tools or management strategies.\n\nSupporting and regulating ecosystem services in agro-ecosystems closely interact with the measures farmers take for increasing and protecting yields. Supporting services are related to the biogeochemical processes governing organic matter decomposition, mineral and carbon cycling, soil porosity and structure. Although fertilization is the main practice against nutrient limitations, yield-increasing measures may involve strategies to sustain or enhance soil organic matter, such as diversified crop rotations (see Table 1 and Table 2, Bommarco et al., 2013; Shennan, 2008). These strategies are exclusively applied at field scale from a single growing season to several years. Regulating services have been mostly associated with the biological control of insect pests through predation and parasitism (Bommarco et al., 2013). Unlike supporting services, the influence of spatio-temporal heterogeneity (i.e. landscape complexity) is critical to ensure the provisioning of regulating services. Hence, most initiatives to promote regulating services in agro-ecosystems emphasize the role of semi-natural habitats for retaining biodiversity, thus promoting ecological processes in farmland mosaics. Moreover, abundance and activity of populations providing regulating services may closely interact with agrochemicals controlling target biotic adversities. Animal pollination, though considered a regulating ecosystem service, would actually be an essential yield-defining factor because it determines seed- and fruit-setting in many crops, and consequently crop yields.\n\nOther management practices may involve a combination of both.\n\nOther management practices may involve a combination of both.\n\nThe high crop yields currently achieved in intensively managed agro-ecosystems are the successful outcome of a long process of techno-simplification of natural ecosystems. This process imposed increasing levels of spatiotemporal homogenization through standardized management and specialization in cropping systems (Figure 1). Hence, natural vegetation was cleared to grow crops, which promoted some desirable species while many others were controlled. Then, proportions of crop harvest and fallows intervals in the growing season were gradually reduced, whereas the natural regeneration of grasslands and forests was replaced by seeding and planting. In addition, recurrent disturbances during the growing season, such as plowing, maintained early successional conditions that promoted short-lived life-forms to the detriment of perennial ones. Moreover, many ecological functions have been progressively substituted by artificial inputs, such as the use of synthetic fertilizers in place of nutrient cycling by the soil biota (Figure 1). Therefore, achieving high monoculture yields relies on breeding high-yielding varieties and using high levels of petrochemical inputs, which consequently maintain biodiversity at low levels in intensively managed agro-ecosystems (Figure 1). Accordingly, croplands alone currently account for nearly half of human appropriation of global net primary productivity (Haberl et al., 2007). A substantial part of productivity, which in the past was biodiverse, has been transferred to (low diversity) crop yield productivity by farming intensification. Biodiversity has been actively selected against in these systems and has declined (Figure 1). Species composition and trophic structure have changed, thus modifying ecosystem processes of availability, uptake and use of soil resources, such as water and nutrients, among biotic communities (Tscharntke et al., 2005).\n\nThe “zone accessible to intensive management” (light red area on the upper left hand side) corresponds to intensively managed agro-ecosystems where high productivity levels can be achieved at very low species richness (ideally only one species in monocultures), but through substituting ecosystem functions (nutrients provided by SOM, biological regulation of pests) for the use of off-farm inputs (fuels, fertilizers, pesticides, and/or water resources; see Box 1 for further details). Curves in red on the left hand side are yield-input production functions showing how crop yields increase as more inputs are applied and species diversity decreases (i.e. agricultural intensification increases as techno-simplicity progresses) in rain-fed (solid line) and irrigated (dashed line) cropping systems (modified from Cassman, 1999). There is also a “crop yield zone accessible through the design of more diverse cropping systems or complex farmland mosaics” (light green area on the upper right hand side), which rather relies on ecological processes occurring across different spatial and temporal scales (i.e. by implementing eco-complexity measures; see Table 1 and Table 2 in Box 1 for further details).\n\n\nComplexity in the agricultural context\n\nThe importance of spatial heterogeneity, particularly landscape complexity, for preserving both biodiversity and ecosystem services has been the focus of many recent studies (Chaplin-Kramer et al., 2011; Garibaldi et al., 2011; Tscharntke et al., 2012). The seminal paper by Thies & Tscharntke (1999) was one of the earliest to empirically demonstrate that landscape structure is highly influential for mobile arthropod species in farmlands. Since then, numerous studies have shown that landscape complexity, in terms of the amount and configuration of habitat patches, can influence species dynamics and ecosystem services (e.g. Gagic et al., 2012; Thies et al., 2011). However, invertebrate pest species may be much more variable than other invertebrates, such as natural enemy taxa (Chaplin-Kramer et al., 2011; Jackson & Fahrig, 2014). As a consequence of this body of research, most biodiversity management initiatives in agro-ecosystems emphasize the role of non-crop habitats for retaining biodiversity and ensuring the provision of ecosystem services. This viewpoint advocates preserving, reintroducing and restoring semi-natural habitats to increase landscape complexity and, consequently, promote ecosystem services. This requires land-use changes at farm and landscape levels and, potentially, co-operation between landholders (landscape management in Figure 2). However, there is a growing appreciation that for certain species, local management changes may also show some benefits and, theoretically, they can be implemented at much smaller spatial scales (patch and field levels in Figure 2) (Gonthier et al., 2014).\n\nThe two hypothetical relationships between management options and the benefits that may accrue are identified at the local (alpha diversity-driven) and landscape (beta diversity-driven) scales of implementation. Changes in biodiversity (species richness) at landscape (gamma, red line) and local (alpha, blue line) scales and species turnover (beta, green line) are also shown as a function of spatial scale. The range of implementation scales are shown in the light grey boxes (patch/plot – field – farm – landscape/watershed). Scales setting the upper (region/nation) and lower (individual plant/plant part-insect interactions) for implementing management options are depicted in grey boxes. Different beneficiaries are also identified. Farmers usually perceive the benefits at the same level at which they make decisions and do agriculture, field and farm levels, while society perceives the benefits from the landscape/watershed level to the regional and national levels.\n\nEcosystem functions derived from complex interactions among different species and their environment lie at the heart of ecology. However, the application of these concepts to agro-ecosystems, which are heavily influenced by intensive management, has only recently begun (Scherr & McNeely, 2008). Over the last 50 years the obvious and sustained loss of biodiversity in agricultural landscapes (Geiger et al., 2010) has given new impetus to the study of the relationship between species diversity and important ecosystem functions they support.\n\nTheoretically, greater species diversity across a range of taxa has been shown to improve the way communities capture resources and convert these to biomass, thereby increasing productivity (Cardinale et al., 2012). Empirically, crop yields can be increased by intraspecific genetic diversity and crop diversity (Li et al., 2009). Diverse mixtures of native perennial grasses can produce more biomass for biofuel production (Tilman et al., 2006). A long-term grassland study showed that primary productivity in highly diverse plant communities has greater resistance to, and recovers more fully from, a major drought (Tilman & Downing, 1994). Species diversity has also shown to be important for a range of provisioning services. For instance, we know that herbivory can be reduced at higher crop diversity levels, while pest mortality can be increased at higher trophic diversity levels (Wilby & Thomas, 2002). However, whether this relationship is due to species diversity per se, or the result of species abundance and resource partitioning is dependent upon the system or the context (Northfield et al., 2012). There is consistent evidence that maintaining wild pollinator diversity improves pollination outcomes (Garibaldi et al., 2013), but for many other ecosystem functions the link to diversity is often unclear.\n\nWhile complexity has been proved valuable at a range of spatial scales, methods for introducing complexity into intensive agricultural landscapes are still rare. Proponents of organic farming, which excludes synthetic pesticides and fertilizers, have long considered their systems to support more complex and diverse communities. Studies are starting to provide evidence supporting this theory, at least for some taxa (Fuller et al., 2005). Crowder et al. (2010) showed that organic farming promotes evenness of communities and enhances pest control. On just one organic farm, 1501 unique interactions among 560 plant and animal taxa have been shown (Pocock et al., 2012). Moreover, a few studies have demonstrated that organic farming can change the structure and function of interacting species networks (Lohaus et al., 2013; Macfadyen et al., 2009). Organic farming is typically adopted across an entire farm and consequently implements many different managements in a holistic way, going beyond the piecemeal implementation of individual managements that is typical in conventional agriculture. However, studies disentangling the influence of different organic management options suggest that some managements may be very much more influential than others (Puech et al., 2014).\n\nComplexity of agricultural systems is beginning to be embraced and measured by researchers (Pocock et al., 2012). Eco-complexity management recommendations (see Table 1 and Table 2 in Box 1), which are derived from these research studies, emphasize the complex nature of agro-ecosystems and rely on the functioning of naturally occurring species. Some recommendations are intrinsically difficult and costly for individual landholders to adopt. Their implementation should be carried out at large spatial scales to make their full benefits evident (Figure 2). Despite those difficulties for landholders, many of these management options are attractive to consumers as they tend to have less negative environmental impacts over the long-term, and they also provide a marketing niche for some farmers.\n\nMany management recommendations derived from eco-complex farming systems would be feasible in techno-simple cropping systems. Excessive emphasis has been put on agro-ecosystem complexity rather than on the simplicity of management interventions required to support complexity. For example, in many agricultural landscapes, we know that both pests and natural enemy species resort to various plant species throughout their life-cycle. Often a recommendation is made such as “greater diversity of native plant species in a landscape will increase natural enemy species diversity.” Although this may be technically correct, achieving this objective in highly altered agricultural landscapes will demand significant investment by landholders. A more manageable approach may be to identify a few key plant species supporting relatively more pest species than natural enemy species, thus targeting them for removal from semi-natural patches (Storkey et al., 2013). Similarly, in the cropping parts of landscapes, simple management recommendations can introduce significant differences in biodiversity, such as cover crops and intercropping (Shennan, 2008).\n\n\nReconciling techno-simplicity and eco-complexity perspectives for future food security\n\nDesigning policies and initiatives for conserving biodiversity in agro-ecosystems, as well as encouraging environmentally friendly farming, are strongly influenced by the social, not purely economic, attitudes of people towards biodiversity and agriculture, which may differ starkly between city dwellers and landholders. On the one hand, urban consumers usually express positive attitudes towards organically grown food, which are associated with environmental concern and a rejection of GM food (Dreezens et al., 2005). These attitudes are usually based on emotion, rather than scientific knowledge (Martín-López et al., 2007), such as the appreciation of charismatic and useful species as well as the aesthetic and cultural values of landscapes (Martín-López et al., 2007). On the other hand, farmers are keen to adopt technological innovations aimed at facilitating crop management and increasing yields, such as transgenic crops (Qaim, 2009), while their willingness to implement conservation schemes is influenced by many factors interacting in complex social and natural networks (Siebert et al., 2006).\n\nRelationships between both the goals and costs of implementing eco-complex management options, as well as the benefits that may accrue, can be described at the local (alpha diversity-driven) and landscape (beta diversity-driven) scales of implementation (Figure 2). Techno-simplicity and eco-complexity perspectives may appear distinct, from a consideration of their goals and approaches to achieve ecological intensification. However, a synthesis of both can be created because they are applied in the same systems and face trade-offs between costs and benefits across similar scales of management implementation and outcomes perception (Figure 2). Articulating goals, costs and benefits of any management intervention will be, therefore, critical for designing farming systems that synthesize both techno- and eco- perspectives for ensuring food security. The conceptual framework needed to articulate these three components can be articulated with insights that have been made from integrating different management strategies and tactics involved in crop protection systems (Cardina et al., 1999; Irwin et al., 2000). To be operational, such a synthetic framework should also properly integrate processes and contexts at different space and time scales (i.e., biological, ecological, agricultural, socioeconomic, and political domains).\n\nPractically, within our synthetic framework, the main components can be conceptually defined for any management intervention (whether agronomic, restoration or conservation). Firstly, both the space and time scales in which interventions are applied should be specified (Figure 2). For instance, herbicides to control weeds are applied to the entire field area, while grass strip schemes are installed only along field margins. Secondly, the scale at which the expected benefits from a particular intervention are effectively perceived should be identified. Hence, actions applied at a particular scale may express their beneficial effects directly or indirectly at the same or different scales. Finally, implementing any intervention has a cost, which can vary across scales, not only in monetary terms, but also in the complexity and nature of tasks involved and the time required to be implement (e.g. installing a grass strip along the margin of a particular field vs. implementing grass strip schemes in a region).\n\n\nConclusions\n\nDesigning more diverse cropping systems is crucial for increasing yields and, simultaneously, conserving biodiversity to ensure the functioning of agro-ecosystems and improvements to future food security. We argue that space and time dimensions require special attention. Doing so would allow us to synthesize a more comprehensive and practical ecological intensification from the techno-simple and eco-complex paradigms. We highlight that the knowledge needed is already available in the different disciplines of agroecology, crop ecophysiology, food-web ecology and landscape ecology, to build a more integrative and effective cropping system design. This could lead to the design of inter-connected habitats, which contain both managed and naturally-occurring species and their services and are thereby multi-functional. Moreover, we argue that developing eco-complex agricultural systems requires proper articulations of goals, costs and benefits of any management intervention. This demands the integration of processes and contexts at different space and time scales. Giving special attention to the social actors involved at different domains, such as farmers, city dwellers and policy makers is also relevant. Effective communication among these social actors about their perceptions of goals, costs and benefits is fundamental for succeeding in the implementation of any program aimed at achieving future food security through reconciling eco-complex and techno-simple perspectives.",
"appendix": "Author contributions\n\n\n\nAll authors were involved in the writing and revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nThe authors declare that there are no competing interests.\n\n\nGrant information\n\nThe authors declared that no grants were involved in supporting this work specifically. SLP research is funded by Universidad de Buenos Aires (20020130100037BA) and Agencia Nacional de Promoción Científica (PICT 2010-1650). SLP was beneficiary of a travel subsidy supported by CONICET (Argentina) and CNRS (France). SM research is funded by the Grains Research and Development Corporation. DAB is funded by two Agence Nationale de la Recherche Agrobiosphere projects, PEERLESS and AgrobioSE. SLP is a research scientist of CONICET, the National Research Council of Argentina.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBeddington J: Food, energy, water and the climate: a perfect storm of global events? In Conference presentation given to the Sustainable Development UK Annual Conference, QEII Conference Centre, London, 19 Mar 2009. Reference Source\n\nBohan DA, Boffey CW, Brooks DR, et al.: Effects on weed and invertebrate abundance and diversity of herbicide management in genetically modified herbicide-tolerant winter-sown oilseed rape. Proc Biol Sci. 2005; 272(1562): 463–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBommarco R, Kleijn D, Potts SG: Ecological intensification: harnessing ecosystem services for food security. Trends Ecol Evol. 2013; 28(4): 230–38. 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PubMed Abstract | Publisher Full Text\n\nPowles SB: Evolved glyphosate-resistant weeds around the world: lessons to be learnt. Pest Manag Sci. 2008; 64(4): 360–5. PubMed Abstract | Publisher Full Text\n\nPuech C, Baudry J, Joannon A, et al.: Organic vs. Conventional farming dichotomy: Does it make sense for natural enemies? Agric Ecosyst Environ. 2014; 194: 48–57. Publisher Full Text\n\nQaim M: The Economics of Genetically Modified Crops. Annu Rev Resour Econ. 2009; 1: 665–93. Publisher Full Text\n\nRockström J, Steffen W, Noone K, et al.: Planetary boundaries: exploring the safe operating space for humanity. Ecol Soc. 2009; 14(2): 32. Reference Source\n\nScherr SJ, McNeely JA: Biodiversity conservation and agricultural sustainability: towards a new paradigm of 'ecoagriculture' landscapes. Philos Trans R Soc Lond B Biol Sci. 2008; 363(1491): 477–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShennan C: Biotic interactions, ecological knowledge and agriculture. 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}
|
[
{
"id": "12055",
"date": "05 Feb 2016",
"name": "Joern Fischer",
"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 based on the premise of there being two kinds of ecological intensification. One focuses largely at the field scale, and emphasizes technological improvements (including agrochemicals and GM crops), whereas the other focuses beyond the field scale, and emphasizes ecological interactions (e.g. pest control, pollination). The paper attempts to reconcile these two perspectives. The rationale underpinning this paper, to my mind, is interesting and worthwhile. Indeed, there are strongly contrasting approaches, and seeing how they can be reconciled would be useful. I believe this paper can make an interesting contribution, but the current version does not live up to its promise, namely to reconcile the two paradigms presented. Below, I explain why. The paper mixes up food security and food production. The focus is, however, only on food production, and hence the word food security needs to be used far more carefully and/or not at all. Food production is a necessary but insufficient prerequisite for food security. The introduction lacks a clear outline of the structure of the paper. This makes it difficult to follow the argument. The first half of the paper (up to page 5) was clear and easy to follow. After this, however, I feel the paper needs to be carefully re-written. It is not clear to me how the two perspectives actually are to be reconciled. Figure 1 is confusing. It shows multiple production possibility frontiers (if that is what they are – it is not explained very clearly), and open and filled dots. But it’s unclear what these are. What is the present state of a system, and what needs to happen to move from one PPP to another? The explanation given in the text and the caption is insufficient to make sense of this figure. The figure is somewhat overloaded. Figure 2 is even more confusing. It contains far too many concepts and ideas in the one figure. Also, some of the details are counter-intuitive – why does biodiversity correlate with temporal scale? Why is the temporal scale associated with a patch minutes to days? What is a patch – why is it smaller than a plot? Why are there three curves for beta-diversity in the right-hand diagram? How do the small diagrams relate to the large one? Most importantly, what does this figure suggest regarding reconciling the two paradigms? Despite trying quite hard, I am afraid I simply could not follow this figure. One page 7, top, right column, the authors state that farmers are keen to adopt high-yielding technologies. This is overly simplistic, and depends very much on the context. In general, the paper does not pay attention to resilience vs. efficiency issues, which are important in the context of comparing the two paradigms. Smallholder systems in poor countries, for example, may be designed first of all for resilience (so there is enough food no matter what happens), and second for efficiency (so that there may be a surplus, for example to sell). Related to this, ideas of variability are missing. E.g. in Box 1, there are limitations to yields from water deficiency, but these also vary unpredictably in some systems. Such variability has typically been minimized by technological systems, whereas agro-ecological, diversified systems are more likely to embrace variability as something natural and not necessarily bad. On page 2, it could be mentioned that diets have been simplified by focusing on a narrow set of crops only. See deFries et al. 2015, Science1. In summary, then, I believe this paper requires major revision before I would consider it “acceptable” for a reputable international journal. To my mind, the clarity of figures, the overall argument, and the second half of the manuscript need to improve substantially. I hope my comments are useful in providing some pointers where the most important changes are needed.",
"responses": []
},
{
"id": "12054",
"date": "16 Feb 2016",
"name": "R Ford Denison",
"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\nWhatever merits this paper may have are undermined by its pejorative application of “simplicity” to “ecological intensification.” Much of industrial agriculture may indeed be “techno-simple” (few crops, uniform application of high rates of fertilizer, etc.) and “externalize a range of environmental costs.” But precision farming (sometimes called “ecological intensification”) is much less simple to implement – Cassman (1999) focuses on its challenges, rather than claiming that the approach is fully developed – and reduces those environmental costs. Yet, as written, this paper attributes simplicity and environmental costs to “ecological intensification”, rather than to the simpler but more-harmful methods this approach it is intended to replace. For example, “ecological intensification” is blamed for “reducing crop diversity” (p. 2-3). If the intent is reconciliation, as implied by the title, I would suggest “technological sophistication” and “ecological sophistication” as descriptions of these two approaches.The article raises the issue of space and time dimensions of biodiversity, making some important points, such as the challenging need for cooperation among farmers to address landscape-scale diversity. These issues aren't really explored in enough depth to be useful, however. For example, a one-sentence treatment of “cover crops and intercropping” describes them as “simple” options, vastly understating the challenges they can pose for farmers. Similarly, although Thies and Tscharntke (1999) did indeed show that “landscape structure is highly influential for mobile arthropod species in farmlands”, they also found that increasing noncrop land to 50% of total only reduced crop bud damage from 28% to 18%. With benefits this small, what would be the overall effects of taking 50% of ag land in a region out of production? Would the net reduction in production lead to price increases, undermining food security for the urban poor and stimulating clearing of forests for agriculture elsewhere? Although I endorse the stated goal of this paper, it would need to be completely rewritten to make a significant contribution. In particular, it would need quantitative data on actual outcomes (such as increased yield or reduced pollution) and not just proxies (such as more-abundant beneficials) resulting from precision farming or from increased biodiversity at different scales.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1507
|
https://f1000research.com/articles/4-1504/v1
|
23 Dec 15
|
{
"type": "Review",
"title": "Sharing the sandbox: Evolutionary mechanisms that maintain bacterial cooperation",
"authors": [
"Eric Bruger",
"Christopher Waters",
"Eric Bruger"
],
"abstract": "Microbes are now known to participate in an extensive repertoire of cooperative behaviors such as biofilm formation, production of extracellular public-goods, group motility, and higher-ordered multicellular structures. A fundamental question is how these cooperative tasks are maintained in the face of non-cooperating defector cells. Recently, a number of molecular mechanisms including facultative participation, spatial sorting, and policing have been discovered to stabilize cooperation. Often these different mechanisms work in concert to reinforce cooperation. In this review, we describe bacterial cooperation and the current understanding of the molecular mechanisms that maintain it.",
"keywords": [
"microbes",
"biofilm",
"bacterial cooperation"
],
"content": "Introduction\n\nBacteria were once thought to be solitary individuals, but it is now clear that they lead complex social lives1,2. Multicellular bacterial communities termed biofilms are now considered a normal form of bacterial growth. Bacterial chemical communication, including quorum sensing (QS), is ubiquitous3,4, and the molecular underpinnings of multicellular bacterial structures such as Myxococcus xanthus fruiting bodies and Streptomyces filaments are also being elucidated5,6. With our increased understanding of bacterial sociality comes a further appreciation of the role of cooperation in many bacterial processes. Microbial cooperative behaviors have important impacts on our own lives, including antibiotic resistance7, biofilm formation in chronic infections8, and virulence during acute infections9,10. Explaining the evolution of cooperative tasks has long challenged evolutionary biology, as these systems appear ripe for exploitation by non-cooperating defector/cheater cells that receive the benefits of cooperation without paying the cost of production11. Because of their short generation times, large population sizes, small genomes, and asexual reproduction, bacteria are now recognized as ideal model systems to understand the factors leading to the evolution and persistence of cooperative behaviors12–14. In this review, we will summarize from both a conceptual and a mechanistic perspective our understanding of how cooperation is maintained in bacteria.\n\n\nFacultative cooperation\n\nBacteria have evolved complex regulatory circuitry to respond and effectively acclimate to different environments, so it is not surprising that this flexible regulatory circuitry can also be utilized to control cooperative traits. Cooperative behaviors in bacteria, such as the production of extracellular “public good” molecules, defined as resources that can be utilized by both the producers and the non-producers in the community, are exploitable by non-producing cheater/defector cells. One approach to limit cheater invasion is facultative cooperation. Engaging in cooperation at limited times, particularly when the benefit is the greatest, or in environmental conditions where the cost of cooperation is low can limit or prevent cheater invasion15,16. In this way, bacteria may preserve cooperation in conditions that would otherwise favor its collapse17. It is notable, however, that facultative participation only partly mediates the problem of cooperation by limiting the times when a cell must maintain it. Other mechanisms, such as relatedness, are likely required in conjunction with optional participation to preserve cooperation.\n\nFor public goods to be effective, they often must exceed a threshold concentration in the extracellular environment15. Therefore, there must be a sufficient number of producing cells contributing to the public good. For this reason, production of many public goods such as exoenzymes, proteases, chitinases, and siderophores are regulated by QS (Figure 1A)4. This process relies on the secretion and detection by bacteria of small chemical signals known as autoinducers into the extracellular environment. As the cell density of a growing culture increases, so does the concentration of autoinducers. This is reinforced by the positive feedback of many QS system on autoinducer synthesis18,19. At a specific concentration of signal, receptors bind to and sense these autoinducers, allowing the bacteria to switch from a low- to high-cell density state. This is often seen as a transition from non-production to production of cooperative traits such as extracellular public goods. QS itself is an exploitable cooperative behavior, as QS-specific cheaters that do not signal, overproduce signal, or do not respond to signal can evolve20.\n\nA. Quorum Sensing. The cooperative behavior is induced only when a sufficient amount of signal has accumulated (left). B. Spatial Structure. When cells are able to assort with kin in space, particularly in the case of biofilm formation (bottom left), dispersal of cells and diffusion of public goods are limited and promote the maintenance of cooperative behavior. C. Policing. This mechanism may act through directed harm (left) or restraint of benefits (right). D. Metabolic Constraint. Producers of a cooperative behavior such as a public good also produce an individually retained private good that is beneficial or required for survival and growth in the focal environment. E. Metabolic Prudence. Cells detect nutrients and other cues in their environment to determine whether it is cost effective to cooperate.\n\nQS regulation of cooperation has been well studied in the bacterium Pseudomonas aeruginosa. This bacterium upregulates extracellular proteases in the high-cell density state21. These proteases degrade extracellular proteins, liberating smaller peptides that can be used for growth. Thus, the growth of P. aeruginosa in minimal media with the casein protein family as a carbon source is dependent upon a functional QS system. Mutants in the QS pathway do not secrete high levels of proteases and grow poorly in this environment, but they receive negative frequency-dependent fitness benefits when mixed with a cooperating strain20. In other words, QS mutants can invade the wild-type strain when rare but lose their fitness benefits when common, a feature likely to occur in most public goods scenarios20,22,23. Therefore, although QS limits maximum public good production to high-cell density, it is not sufficient in this case to completely prevent cheater invasion, although it may mediate the extent to which this occurs. Similar results in a casein growth medium were recently described for Vibrio cholerae24 and Vibrio harveyi (unpublished results from our laboratory), which also secrete proteases at high-cell density in a QS-dependent manner. The degree of resistance to cheating QS can provide likely depends upon the cost and benefit functions of the behavior, the manner in which regulation is imposed, and the genetic architecture of the QS system.\n\nAs QS itself does not appear sufficient to completely ward off cheaters, at least in these systems, additional mechanisms for cheater prevention are required. One such mechanism, referred to as metabolic constraint, found that the utilization of adenosine was also positively controlled by QS24 (Figure 1D). Unlike protease secretion, which is a public good, adenosine utilization primarily benefits the producing individual, making this function as a “private good”. The addition of adenosine to the medium inhibited the evolution of QS mutants, as these were not able to effectively use this resource. Therefore, the cooperators were able to access a benefit that was unavailable to the defectors. It was proposed that mutually regulating public and private goods under control of QS could be a mechanism to stabilize QS-controlled cooperative tasks. However, if utilization of adenosine is a private good that benefits the producer in a density-independent manner, it is not clear how this system would have evolved. An alternative hypothesis is that in its natural environment adenosine is primarily present in high-cell density situations. QS could function as an environmental cue to prime P. aeruginosa to utilize likely nutrient sources that may be encountered at different cell densities. While QS can explain varying behavioral differences with density, the underlying cooperative behaviors are still promoted when cells have unified interests, such as relatedness. Because of autoinducer specificity, QS provides not just evidence of general density but also the degree of relatedness of the surrounding community. It is therefore our view that metabolic constraint systems will only evolve when utilization of the private good in a density-dependent manner is favored. On a broader note, any condition in which co-regulation of the public and private good is most beneficial could exhibit metabolic constraint.\n\nAnother mechanism to limit cheater invasion is to only produce public goods when their cost is minimal, an idea termed metabolic prudence (Figure 1E). P. aeruginosa also produces carbon-rich molecules called rhamnolipids that allow large numbers of these organisms to “swarm” over certain surfaces, such as soft agar plates. Rhamnolipids are a public good that can be exploited by non-producers to swarm. Xavier and colleagues noticed25, however, that a non-producer did not exhibit higher fitness than a rhamnolipid producer during a swarm assay of chimeric populations, even though a significant portion of carbon was being directed towards synthesis of this public good. The authors deduced that P. aeruginosa only produced rhamnolipids when experiencing an excess of carbon in relation to nitrogen levels. Thus, the cost of production for this public good was minimized and did not lead to reduced fitness versus the non-producer. Interestingly, rhamnolipid production is also regulated by QS, indicating that production only occurs at high density and/or relatedness as well26.\n\nMetabolic prudence is thus a facultative cooperation mechanism, which illustrates that bacteria integrate the relative cost associated with cooperative traits. Though it has not yet been widely demonstrated, it is likely to occur for additional microbial cooperative behaviors. It is common for complex traits to be controlled by multiple regulatory inputs. For example, the catabolite repressor protein (CRP), which responds to the presence of phosphotransfer sugars, at least in Escherichia coli, regulates 300 genes, which is ~7% of its genome27. It is our opinion that regulatory connections between QS (or other signaling systems), central metabolism, and the control of cooperation will be common, and finding other systems that demonstrate metabolic prudence will be an exciting new avenue of research28.\n\n\nSpatial structure and assortment\n\nSpatial structuring of related cooperators is a key mechanism by which cooperation is likely evolved and maintained11,29,30 (Figure 1B). One critical example of cells actively structuring themselves in an environment that is proposed to encourage cooperation is the production of biofilms31. Biofilms are multicellular communities of bacteria encased in an extracellular matrix. A costly and potentially cheatable behavior itself32, biofilm formation provides a framework for cells to situate themselves in space and direct cooperative benefits preferentially towards clonal offspring and other related kin. Biofilms also restrict diffusion so that public goods, such as extracellular enzymes, remain near the producing cell rather than being dispersed by flow or other forces33,34. This was recently demonstrated as V. cholerae biofilms attached to chitin surfaces retain sufficient amounts of the extracellular enzymes chitinases to metabolize this nutrient33. Cells that do not form biofilms lose higher portions of these public goods due to increased loss via diffusive and advective forces, which likely reflects conditions encountered in natural environments33,34. This means that public goods benefits remain distributed over a narrower, more local range in space that favors their diversion toward neighboring kin cells. Additionally, biofilm formers may even be able to exclude non-producers from colonized nutrient source surfaces35.\n\nHowever, this structuring also comes with the potential for more competition between kin, especially if cells don't also possess the capacity to disperse and colonize new patches in their habitat. For example, experimentally evolved lineages that produce more biofilm through an evolved wrinkly spreader lifestyle are able to bind tightly to neighboring cells due to enhanced production of extracellular matrix materials, but this typically comes with a tradeoff for growth potential36,37. The tradeoff may be in part restrained by the reduced ability of cells to disperse from a cluster. A similar example of a tradeoff between colonization and dispersal is seen in V. cholerae, where biofilm producers compete and grow better on a surface but are less effective at dispersing to new locations in their habitat38, and natural populations of Vibrio cyclitrophicus demonstrate differential specialization for colonization onto and dispersal from particles, signifying that this phenomenon could be more widespread39. This suggests that multiple selective pressures are naturally acting upon microbes that can either reinforce or act against other cooperative behaviors.\n\nIn the examples described thus far, no assumptions have been made about the ability of cells to recognize the presence or identity of neighboring cells, and this is not always theoretically necessary for spatial structure to enable cooperation40,41. However, it may be important to be able to recognize neighbors and restrain competition if surrounded primarily by relatives. There are many examples of cells being capable of effectively distinguishing between self and non-self and adjusting behavior accordingly in ways that impact growth outcomes42–44. QS is one such system in bacteria, but other contact-dependent recognition systems such as the contact-dependent growth inhibition (CDI) system of Burkholderia, type VI secretion systems, and flocculation in yeast have been described45–47. This ability to correctly decipher amongst neighbors and the composition of the surrounding community could greatly encourage the success of cooperation, particularly if production of cooperative behaviors is predicated upon sensing members of a cell's own genotype.\n\nIt is worth noting that producer cells at low frequency can in some cases preferentially gain the benefit of public goods production compared with non-producers, even in the absence of higher ordered structure48. This creates a snowdrift scenario whereby the rare type has an advantage. This situation is, however, highly dependent on the parameters of the specific cooperative behavior, but it may contribute to maintenance of cooperative tasks.\n\n\nPolicing\n\nPolicing and related forms of punishment are proposed as another mechanism to stabilize cooperation in the face of potential cheaters (Figure 1C). In this scenario, an aggressive action that negatively impacts fitness targets cheaters relative to cooperators49. Policing has been commonly observed among many eukaryotic organisms50, including insect workers, birds, and social primates, but the prevalence and diversity of molecular mechanisms underlying bacterial policing are not well characterized. Punishment could be enacted by either restraining benefits directed to a non-contributing partner or by direct harm. In the second case, this behavior may be costly to the enacting individual but still stabilize other cooperative behaviors for cooperating kin in the population as long as the punishment and resulting cooperation are positively correlated51,52.\n\nOne example of policing is the enforcement of sanctions, seen in the interaction of the root-nodule forming bacteria Rhizobia with its host plant. Symbionts that do not sufficiently contribute fixed nitrogen to their associated host receive a limited flow of oxygen and nutrients in return53,54. Limiting benefits or imposing costs to less or non-cooperative partners in this manner should favor more cooperative partners in mutualisms54,55. In some systems, such as squid-Vibrio symbioses, the host is able to filter and selectively favor suitable partners in such a manner, and host-enforced bottlenecks are also likely to play a strong role in maintaining fidelity in the interaction56,57. Bottlenecks may more generally act to stabilize cooperative behavior, regardless of host association58–60. In this way, restraining a benefit in the face of non-reciprocating partners can have the effect of maintaining the interaction. These sanctions need not be restricted to inter-species interactions and could be imagined to occur, for instance, in populations where cells are exchanging metabolites61.\n\nRecently, QS in P. aeruginosa has also been shown to induce policing that targets QS defectors by regulating cyanide production and resistance62. As described above, QS induction of extracellular proteases is necessary for maximum growth in a minimal media environment with casein as the carbon source. In this case, certain classes of QS defectors are unable to produce co-regulated compounds that counteract the effects of cooperator-produced cyanide and are thus unable to completely invade a QS-proficient population cooperating via extracellular enzyme production that could otherwise be exploited by the potential cheats.\n\nIn all likelihood, these types of policing mechanisms may be difficult to maintain and unlikely to be common for maintaining bacterial cooperation11. Because policing behaviors are costly to perform and may target related kin, they may convey a fitness disadvantage under many conditions. However, this effect may be somewhat alleviated if such traits are only expressed conditionally, as shown in the QS-regulated example. For cooperative partners, from either the same or different species, sanctions may arise more naturally. Due to negative effects on the productivity caused by a poor partner, reciprocal sanctioning effects will more naturally emerge, as fewer partners will be present to repay the favor40.\n\n\nDivision of labor in bacteria\n\nA penultimate form of cooperation that is a requirement for the development of higher ordered multicellularity is “division of labor”. Division of labor can be defined as cooperating individuals that perform discrete tasks that are themselves costly to the individuals, but the sum total of this task distribution is beneficial to the larger community. Division of labor is clearly evident in complex multicellular eukaryotes. A heart cell has differentiated to perform very different tasks than a liver cell. In these organisms, development and terminal differentiation are keys to driving and maintaining phenotypic heterogeneity. Division of labor has also clearly been observed in bacteria. A classic example is spore formation by M. xanthus63. Upon starvation, this predatory bacterium aggregates into multicellular mounds, which ultimately form structures called fruiting bodies coated with environmentally resistant M. xanthus spores that rise above the local surface. These structures are thought to aid in dispersal of the spores to new environments.\n\nDivision of labor is also proposed to be a common feature of biofilms, although this is a controversial idea that as of yet has little experimental support. Indeed, five specific Bacillus subtilis cell types can be observed in a monospecies biofilm64. These subtypes localize to distinct regions of the biofilm, but the adaptive function of these cell types is not clear, as a locked matrix-producing cell type is sufficient to produce a robust biofilm in the lab. However, it is likely that a homogenous biofilm would be maladaptive in the natural life cycle of B. subtilis, as the laboratory environment is missing key aspects of the natural world. It also seems unlikely that biofilms will remain as genetically homogenous as liquid cultures due to limited dispersal leading to restriction of competition to local scales. Understanding the evolutionary factors that drive the emergence of phenotypic heterogeneity must rely on a better understanding of the ecology of these bacteria and ideally guide experiments on these organisms in more naturally relevant systems. Moreover, like all forms of cooperation, identifying the strategies and mechanisms that maintain these interactions in the face of defectors will be an intriguing area of research.\n\nThe above examples represent division of labor in monospecies systems, but interspecies division of labor can and does occur as well. In complex microbial communities, we predict that division of labor will be most evident in the cooperation of individuals through metabolic exchanges65,66. Indeed, this has been observed in clinical cystic fibrosis isolates of Staphylococcus aureus67. This phenomenon has been shown to be possible in synthetic as well as natural communities40,68–70. In mixed communities, members that are not directly involved in the exchange may still impact it providing a degree of separation or assortment of individual partners, a process labeled social insulation40,71. With the increasing importance of the human and animal microbiome in health and disease, understanding cooperative division of labor interactions in these communities, and potentially with the host, will be an increasingly important area in microbial evolution.\n\n\nConclusion\n\nAs we have seen, microbial cooperation occurs in diverse manners, and the mechanisms guiding its maintenance are likewise diverse. These mechanisms all fundamentally act to mediate the fitness costs imposed by expressing cooperative behavior or by altering the way that benefits are administered. Fitness gains may be directed to the acting party (direct), to related kin (indirect), or both, and thus act to increase the organism's overall inclusive fitness. Also evident throughout this review, these mechanisms may and often do work in concert with one another. As researchers discover novel examples of microbial cooperation, more mechanisms that direct their maintenance will likely come to light. We are particularly optimistic that many mechanisms of cooperation utilizing optional participation guided by communication will be discovered in experimental and natural communities. The surface has only been scratched in this area of research, and it will be exciting to see what new mechanisms are uncovered, how they may potentially be used for industrial and medical applications, as well as how they may inform what we know about biology at both the micro and macro scale.\n\n\nAbbreviations\n\nQS, quorum sensing.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis material is based in part upon work supported by the National Institutes of Health grant GM110444 and the National Science Foundation under Cooperative Agreement No. DBI-0939454 to C.M.W. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. We thank two reviewers for their insight and helpful comments.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nCrespi BJ: The evolution of social behavior in microorganisms. Trends Ecol Evol. 2001; 16(4): 178–83. PubMed Abstract | Publisher Full Text\n\nVelicer GJ: Social strife in the microbial world. Trends Microbiol. 2003; 11(7): 330–7. 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Proc Natl Acad Sci U S A. 2014; 111(4): 1237–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHammer ND, Cassat JE, Noto MJ, et al.: Inter- and intraspecies metabolite exchange promotes virulence of antibiotic-resistant Staphylococcus aureus. Cell Host Microbe. 2014; 16(4): 531–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMorris JJ, Lenski RE, Zinser ER: The Black Queen Hypothesis: evolution of dependencies through adaptive gene loss. MBio. 2012; 3(2): pii: e00036-12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorris JJ, Papoulis SE, Lenski RE: Coexistence of evolving bacteria stabilized by a shared Black Queen function. Evolution. 2014; 68(10): 2960–71. PubMed Abstract | Publisher Full Text\n\nPfeiffer T, Bonhoeffer S: Evolution of cross-feeding in microbial populations. Am Nat. 2004; 163(6): E126–35. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMitri S, Xavier JB, Foster KR: Social evolution in multispecies biofilms. Proc Natl Acad Sci U S A. 2011; 108(Suppl 2): 10839–46. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11697",
"date": "23 Dec 2015",
"name": "Jeff Gore",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11696",
"date": "23 Dec 2015",
"name": "Stuart A. West",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1504
|
https://f1000research.com/articles/4-481/v1
|
05 Aug 15
|
{
"type": "Software Tool Article",
"title": "Social Network: a Cytoscape app for visualizing co-publication networks",
"authors": [
"Victor Kofia",
"Ruth Isserlin",
"Alison M.J. Buchan",
"Gary D. Bader",
"Victor Kofia",
"Ruth Isserlin",
"Alison M.J. Buchan"
],
"abstract": "Networks that represent connections between individuals can be valuable analytic tools. The Social Network Cytoscape app is capable of creating a visual summary of connected individuals automatically. It does this by representing relationships as networks where each node denotes an individual and an edge linking two individuals represents a connection. The app focuses on creating visual summaries of individuals connected by co-publication links in academia, created from bibliographic databases like PubMed, Scopus and InCites. The resulting co-publication networks can be visualized and analyzed to better understand collaborative research networks or to communicate the extent of collaboration and publication productivity among a group of researchers, like in a grant application or departmental review report. It can also be useful as a research tool to identify important research topics, researchers and papers in a subject area.",
"keywords": [
"social network analysis",
"data visualization",
"collaborative networks",
"co-publication networks"
],
"content": "Introduction\n\nA scientist’s research output and collaborative tendencies - at least those that can be measured based on publications - can be visually summarized as a network where each node denotes an author and edges link authors who have co-published. Such a network facilitates determining who publishes with whom and in what topics, and identifying key individuals and organizations within collaborative research networks. It is useful to create and visualize a network showing a broad overview of collaborative research publications to communicate the extent of collaboration and impact of publications. As another example, creating a collaboration network from a set of publications for a specific topic, for example “Alzheimer’s”, can help highlight experts in the field and could be useful as a research tool to help identify important topics, researchers and papers.\n\nPreviously, creating co-publication networks required users to manually retrieve the relevant data and transform it into either a formatted text file or an excel workbook that defined all the individual nodes and connections. Users would then have to import the text file or workbook into Cytoscape or another network visualization tool. To streamline this workflow, we developed the Social Network app, a Cytoscape 3 app that is capable of automatically generating visual summaries of individuals connected in academia. In the simplest mode of interaction, the user supplies the first initial and last name of the individual whose network they would like to visualize, and a co-publication network is generated automatically from one of three currently supported bibliographic databases: PubMed, Scopus and Web of Science (via InCites). Users can also provide more complex and larger sets of publications, for example using the PubMed query system.\n\n\nMethods and implementation\n\nThe Social Network App supports both text and file-based inputs. As Figure 1 shows, co-publication networks can be created in four ways. With the search box, users can run queries against PubMed. A co-publication network is automatically generated from any results that are retrieved. Alternatively, users can go directly to PubMed, Scopus or InCites web sites, search for publications, export them to a specified file format and visualize them using the app. See the user guide for detailed instructions on how to do each of these tasks (http://baderlab.org/UserguideSocialNetworkApp);\n\nOften when running queries with common names (e.g. “Smith J”), the query returns publications that contain more than 500 authors. Visualizing networks containing these types of publications is challenging because generating n · (n - 1)/2 edges for each publication (n refers to the # of authors in the publication) is resource intensive and the large clusters created are difficult to visualize and interpret. To avoid this issue, the user panel includes a maximum author per publication field that allows users to specify the author number threshold at which publications are excluded. By default the threshold is set to 500.\n\nA co-publication network summary panel is also included in the user panel. For PubMed and Scopus networks, the panel displays the total number of publications parsed and the number of excluded publications (publications are excluded if the number of authors they have exceeds the threshold). Because InCites networks contain institutional affiliations for all the authors of a given publication, they have richer summaries. Charts that summarize the total number of publications and citations by location can be viewed by clicking on links in the panel that navigate to summary charts created with the Google Chart API (https://developers.google.com/chart/).\n\nSocial Network is written in the Java programming language as an app for Cytoscape 31 and is based on the Cytoscape 3-supported OSGi (Open Services Gateway Initiative) software architecture. To facilitate development, we developed a set of coding guidelines and defined them as Eclipse templates. The Eclipse templates and instructions on how to import them into an existing workspace are available at (http://baderlab.org/Software/SocialNetworkApp/Development). We also used the Maven project management tool (https://maven.apache.org/) to retrieve and organize the dependencies required by the app. An outline of the required dependencies is provided in a pom file that is located in the project source code (https://github.com/BaderLab/SocialNetworkApp).\n\nThe design of the app followed Object Oriented Principles (OOP), reflected in the following class hierarchy; To facilitate future support for other social network types, we defined a set of flexible data structures, namely: AbstractNode, AbstractEdge and SocialNetwork. These data structures are used to represent networks and conveniently associated data created by the app in a general form. They are also specialized for each network type (e.g. PubMed, InCites). We also implemented the PubMed search feature generally to support future social network sources. Implementation details are provided in the source code (https://github.com/BaderLab/SocialNetworkApp).\n\nNetworks built from different bibliographic databases require their own unique visual styles. We implemented a standard visual style that all other visual styles extend. The standard visual style describes styles for attributes that all social networks share (e.g. name, label) and it is used for PubMed and Scopus networks but not InCites networks. InCites networks require their own specialized visual style because they contain additional attributes (location) that PubMed and Scopus networks typically do not possess. We implemented a new InCites visual style by extending the standard visual style and adding new style descriptions for the locations of authors. See the source code for implementation details.\n\nCo-publication networks can be generated in four ways: (1) By entering a query into the PubMed search box. (2) Loading an InCites report (XLSX format). (3) Loading a PubMed XML file containing query results retrieved from the PubMed web interface. (4) Loading a Scopus CSV file. (5) Users can also set the maximum # of authors allowed for a publication to filter out very large author lists that may clutter the network.\n\nMultiple bibliographic databases were evaluated for support by the app: PubMed, Scopus, Web of Science/InCites, and Google Scholar. Evaluation was based on application program interface (API) availability, data export capabilities, coverage, citations and update frequency (see Table 1). PubMed is developed and maintained by the National Center for Biotechnology Information (NCBI) as part of the U.S. National Library of Medicine and is accessible through the Entrez query system (http://www.ncbi.nlm.nih.gov/pubmed/). Web of Science is a literature citation index created by Thomson Reuters containing over 90,000,000 records from all fields of science (http://wokinfo.com/citationconnection/realfacts/). Web of Science data is also accessible via the Thomson Reuters InCites web-based search engine, which facilitates access to additional information, such as author institution (http://researchanalytics.thomsonreuters.com/incites/). Scopus contains over 57,000,000 records, 27 million of which are patent records and 6.8 million of which are conference papers or proceedings (http://www.elsevier.com/solutions/scopus/content) and date back as far as 1823 (http://www.elsevier.com/__data/assets/pdf_file/0007/69451/sc_content-coverage-guide_july-2014.pdf).\n\nScopus temporal coverage was retrieved here http://www.elsevier.com/__data/assets/pdf_file/0007/69451/sc_content-coverage-guide_july-2014.pdf.\n\nScopus contains author profiles that include, among other things, the institutional affiliations of an author. These profiles are helpful when disambiguating authors with very similar or identical names. Web of Science, InCites and Scopus access require a paid subscription, which large academic institutions often provide. Google Scholar is a freely available and automatically updated database of citations with associated author pages (https://scholar.google.com/intl/en/scholar/about.html).\n\nDatabase content was evaluated by selecting a specific publication and comparing its citation counts among the different databases. Update frequency was determined by checking whether a newly published paper (published on January 1st 2015 or later) had been indexed by the database and by examining citation counts and verifying that newer citations had been captured. Prior to app development, data from PubMed, Web of Science and Scopus was available for an internal project. Thus, development was oriented towards supporting content from these three databases.\n\nAside from Google Scholar, every database that was examined had an API. Scopus and Web of Science APIs require paid subscriptions. This served to lower the desirability of both Scopus and Web of Science APIs and at the same time elevated the free PubMed API. Although both Scopus and Web of Science require paid subscriptions to view their data over the web, often large institutions have licenses to query this data which makes it accessible to many users. Scopus and Web of Science also both provide an intuitive web-based user interface that enables users to export the data to file formats that are recognizable by our app (CSV for Scopus and XLSX for Web of Science). Based on our evaluation, we chose to support PubMed (via file export and API), Scopus and Web of Science (via file export). We would have supported Google Scholar if a public API was available.\n\nPubMed is the default search engine used by the app because of the accessibility of its content and the straightforward nature of its associated retrieval mechanisms: its web-based interface and the Entrez Programming Utilities (eUtils) API. Eutils enables URL-based (non-RESTful) programmatic access to data contained in PubMed as well as any other databases linked to Entrez (http://www.ncbi.nlm.nih.gov/books/NBK1058/). Standard PubMed queries, for example “LastName First Initial”[Au], including recognized PubMed search tags (http://www.nlm.nih.gov/bsd/mms/medlineelements.html), can be entered into the PubMed search field in the app, which retrieves XML results using the eUtils web service. The results are parsed using the SAX (Simple API for XML) API included in the Java standard library and are transformed into a co-publication network using the Cytoscape API. Nodes in the network represent authors, edges represent co-authorship and how frequently authors collaborate is indicated by the thickness of an edge.\n\nEutils queries are limited to several hundred characters (http://www.ncbi.nlm.nih.gov/books/NBK25499/). In the case where users would like to build a co-publication network based on a longer query, for instance, finding all the papers that any two researchers in a given institution have published together (i.e. number of researchers choose 2 search parameters), users can perform queries directly on PubMed’s website (which allows complicated queries such as these) and export the results to an XML formatted file. Instructions for this workflow are provided in the app user guide: http://baderlab.org/UserguideSocialNetworkApp#PubMed. XML results obtained through eUtils differ slightly from XML results directly exported from PubMed. In particular, XML results exported from PubMed do not contain citations, whereas XML results retrieved by eUtils do. To correct this, the app retrieves this information using eUtils. Since the citation counts ultimately come from the same source regardless of how the initial data was obtained (PubMed or eUtils), networks generated via either method are equivalent. There is also a limit on the amount of data that can be retrieved at one time from eUtils. NCBI recommends that no more than 500 publications be retrieved from a single eUtils query (http://www.ncbi.nlm.nih.gov/books/NBK25498/). To circumvent this restriction, large data sets can be retrieved incrementally (i.e. 500 records at a time). However, there is a limit set on the frequency of eUtils requests. A maximum of three requests is allowed per second (http://www.ncbi.nlm.nih.gov/books/NBK25497/). Violating these suggested limits may result in NCBI blocking the IP address of the offender. The limit on the number of records is not present when exporting data from the PubMed website directly. Users can safely export up to tens of thousands of records to XML.\n\nScopus and Web of Science (via InCites) are supported via file import. A user must manually export query results via the respective web interface. Scopus CSV exports are supported by the app. InCites reports must be saved in Excel 2007 (XLSX) format to be input into the app. The app can recognize InCites spreadsheets with exactly six columns in the following order (from left to right): times cited, expected citations, publication year, subject area, all authors and document title. Instructions on how to export results from InCites to this format are provided at http://baderlab.org/UserguideSocialNetworkApp#InCites.\n\n\nResults and discussion\n\nWe demonstrate the app using an example from the Hughes et al. study6 in which social network analysis was used to determine whether Alzheimer Disease Centers (ADCs) based in the United States foster collaborative research. As part of the analysis, the study authors constructed multiple co-publication networks using publication data collected from PubMed. In the original publication, the authors created Ruby scripts to query PubMed for co-publications for a set of over 2000 researchers affiliated with ADCs.\n\nThe simplest way to interact with the app is to create either an individual researcher’s publication network or a co-publication network for an individual organization. Using an individual author from a single ADC, Rush University Medical Center, Figure 2 shows an individual’s publication network. We created the co-publication network by entering the researcher’s name (last name <space> first initial, as expected by Pubmed) into the PubMed search bar (see Figure 1) and clicking on the search button. Using results retrieved from PubMed the app created the network. For an individual author the same process can be easily performed on the Scopus or Incites websites to retrieve output files that can be loaded and parsed by the app. The main difference between networks generated by PubMed and Scopus and Incites is the number of citations attributed to each author. PubMed counts paper citations only for articles found in the freely available PubMed Central literature archive whereas Scopus and Incites use a much larger set of publications stored in their databases. Thus, Scopus and Incites provide more accurate citation counts.\n\nEach node represents a co-author of the original query author (the highlighted yellow node). The network was created by entering the author’s last name and first initial into the PubMed query bar within the Social Network App. The network was then automatically created. The yFiles Organic layout was applied to better visualize the network. Node size represents the cumulative number of the author’s publication citation counts as automatically retrieved from PubMed based on the set of publications associated with the node (the count only includes citations of publications that are in PubMed Central). Thickness of the edges connecting the nodes represents the number of publications the two authors have published together.\n\nExtending the simple use case, using all authors from a single institution, such as Rush University Medical Center, as a query, Figure 3A shows the resulting co-publication network (searching PubMed for publications that have at least two Rush University ADC researchers). Figure 3B shows the co-publication network for only the Rush University ADC researchers. The length of the query depends on the number of researchers in the query set and would have the following format:\n\n((\"LastName1 FirstInitial1\"[Au] AND \"LastName2 FirstInitial2\"[Au]) OR\n\n(\"LastName1 FirstInitial1\"[Au] AND \"LastName3 FirstInitial3\"[Au]) OR\n\n(\"LastName1 FirstInitial1\"[Au] AND \"LastName4 FirstInitial4\"[Au]) OR\n\n(\"LastName2 FirstInitial2\"[Au] AND \"LastName3 FirstInitial3\"[Au]) OR\n\n(\"LastName2 FirstInitial2\"[Au] AND \"LastName4 FirstInitial4\"[Au]) OR ... )\n\nAND \"Rush University Medical Center\"\n\nA university department, faculty or a collaborative group typically desires to visualize and analyze all publications from the organizational unit over a period of time to help evaluate research productivity and effectiveness. Also, users may be interested in visualizing all of the publications and their topics in a particular research area. To demonstrate how the app can be used for a more sophisticated use case that also highlights how Cytoscape features can be used as part of a workflow, a simple comparison to the original broad analysis Hughes et al. was performed. We queried PubMed for the same set of researchers as used in the Hughes et al. study. Each author was queried along with their institution to reduce false positives and the entire query was limited to publications containing “alzheimer”. The set of authors was large, leading to the creation of a large PubMed query, thus the PubMed web interface was used to execute the query. Both the Scopus and InCites web interfaces were unable to process the query and it was too long to pass to eUtils. Limiting the query to papers published in 2010 returned a set of 382 publications. Using the PubMed XML file downloaded from the PubMed website, we constructed a co-publication network. By using Cytoscape’s filtering capabilities, we reduced the network to just the authors used in the original query (see Figure 4). With Cytoscape’s Styles, we colored nodes by institution as specified in the original dataset. To summarize this network we used a feature in the Enrichment Map App7 that makes use of two other Cytoscape apps (clusterMaker4 and WordCloud5) to automatically cluster and annotate the network based on the word summaries of a given attribute. Each cluster was annotated using frequent words found in the titles of publications within each cluster. This automatically highlights the collaborative research topics included in the network. The network can be further reduced by creating groups associated with each cluster. By collapsing the groups to an individual node the complexity in the network would be substantially reduced and the resulting network would highlight research themes found in this set of publications.\n\n(A) Each node represents an author from the set of publications that have at least one Rush ADC researcher. Orange nodes are Rush ADC researchers and red nodes are non-Rush ADC researchers. Rush ADC researchers were selected manually and their node fill color was modified using the style bypass option and set to orange. This can also be achieved by importing a node attribute mapping only to the query authors and using the imported attribute to select the nodes. Node size represents the cumulative number of the author’s publication citation counts as automatically retrieved from PubMed based on the set of publications associated with the node (the count only includes citations of publications that are in PubMed Central). Edge thickness represents the number of publications the two authors have published together. (B) Subset of the network in (A) containing only the ADC researchers. Author names are not shown to reduce visual clutter and to protect anonymity. Large cliques represent many-author publications.\n\nEach node represents an ADC researcher and its color represents their institution (as specified in the original dataset). Node colors were automatically generated in the visual style for the ’original institution’ attribute that was available for this author set and loaded onto the co-publication network after its creation. Node size represents the cumulative number of the author’s publication citation counts as automatically retrieved from PubMed based on the set of publications associated with the node (the count only includes citations of publications that are in PubMed Central). Thickness of the edges connecting the nodes represents the number of publications the two authors have published together. Each cluster of authors, as calculated by the clusterMaker app4, is annotated with an additional circle around its members. Cluster labels aim to summarize the group with the two most frequent words in the author’s set of publication titles as computed by the WordCloud App5).\n\nThis workflow also illustrates the challenges of working with large co-publication networks and large networks in general. There are many Cytoscape features and apps that can be used to reduce complexity of the network and help summarize the results. Given the limits of searching in PubMed, Scopus and Incites, a broad global analysis similar to the one conducted by Hughes et al. likely requires multiple queries, possibly automated by scripts to retrieve different data from the databases along with a process to collate and filter the results to generate the final set of publications to be analyzed. This is currently beyond the scope of this app, but more complex and automated query functionality could be added in the future.\n\n\nConclusions\n\nUsers interested in creating visual summaries of individuals connected via co-publication links in academia will benefit from the Social Network App. The app aids users unfamiliar with Cytoscape by providing an intuitive and navigable user interface to query multiple bibliographic databases. Advanced users who wish to analyze large networks can take advantage of many powerful Cytoscape features. In the future we plan to expand the Social Network App to enable the creation of co-publication networks made from multiple PubMed queries or files as well as support for Cytoscape commands which would enable scripted access to the app. We also plan to add support to visualize connections formed by Twitter, LinkedIn and Facebook users.\n\n\nSoftware availability\n\nhttp://apps.cytoscape.org/apps/socialnetworkapp\n\nhttps://github.com/BaderLab/SocialNetworkApp\n\nhttps://github.com/F1000Research/SocialNetworkApp\n\nhttp://dx.doi.org/10.5281/zenodo.198258\n\nhttps://www.gnu.org/licenses/old-licenses/lgpl-2.1.html\n\nhttp://baderlab.org/UserguideSocialNetworkApp",
"appendix": "Author contributions\n\n\n\nVK drafted the manuscript with assistance from RI. Both VK and RI designed and developed the software. The project was initiated by GDB and AMJB. GDB supervised the project. All authors have read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFinancial support was provided by the Faculty of Medicine at the University of Toronto.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank the authors of the Hughes et al. paper for sharing their social network data for analysis.\n\n\nReferences\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhabsa M, Giles CL: The number of scholarly documents on the public web. PLoS One. 2014; 9(5): e93949. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrduña-Malea E, Ayllón JM, Martín-Martín A, et al.: About the size of Google Scholar: playing the numbers. arXiv preprint arXiv: 1407.6239, 2014. Reference Source\n\nMorris JH, Apeltsin L, Newman AM, et al.: clusterMaker: a multi-algorithm clustering plugin for Cytoscape. BMC Bioinformatics. 2011; 12(1): 436. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOesper L, Merico D, Isserlin R, et al.: WordCloud: a Cytoscape plugin to create a visual semantic summary of networks. Source Code Biol Med. 2011; 6(1): 7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHughes ME, Peeler J, Hogenesch JB, et al.: The growth and impact of alzheimer disease centers as measured by social network analysis. JAMA Neurol. 2014; 71(4): 412–420. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerico D, Isserlin R, Stueker O, et al.: Enrichment map: a network-based method for gene-set enrichment visualization and interpretation. PLoS One. 2010; 5(11): e13984. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKofia V, Isserlin R, Buchan AMJ, et al.: F1000Research/SocialNetworkApp. Zenodo. 2015. Data Source"
}
|
[
{
"id": "9859",
"date": "14 Aug 2015",
"name": "Michael Bales",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a tool that allows users to create co-authorship network diagrams within the Cytoscape application. This tool simplifies the network production process into as few as two steps: users can enter a PubMed query and then click the “search” button. A co-authorship network is generated automatically and displayed in Cytoscape; users can then use features available within Cytoscape to carry out additional tasks, such as adjusting visual properties and measuring topological measures of network structure. The system also supports searches to Scopus and Web of Science (via InCites). The authors illustrate the system’s design and functions, describe how to use it, and present a use case in which they use the tool to replicate a search carried out in a study by Hughes et al. In so doing they highlight several challenges that may occur when working with large co-authorship networks. The authors contend that co-authorship network visualization can be useful for understanding collaborative research networks or for “communicating the extent of collaboration and publication productivity among a group of researchers”. In this paper the authors do not conceive of a method to evaluate their app, for example to assess satisfaction with the app among system users, or to see whether system users are able to integrate the app into meaningful workflows. The authors report that they plan to continue developing the app, for example, by extending it so that it can display data from Facebook, Twitter, and LinkedIn. The availability of the software is a welcome addition to existing tools for co-authorship network production. Its automated features and relatively seamless integration into Cytoscape will (we expect) make it an appealing option for analysts; to the extent possible it takes care of tedious steps that analysts may be accustomed to carrying out manually. We have identified several minor points for the authors to take into consideration. First, in the sixth paragraph of the “Database evaluation and implementation” section, the authors mention that E-utilities queries are limited to several hundred characters. While this may be the case for standard E-utilities queries, it is possible by using an HTTP Post call to make significantly longer queries. From Sayers E. The E-Utilities In-Depth: Parameters, Syntax, and More, http://www.ncbi.nlm.nih.gov/books/NBK25499/: “For very long queries (more than several hundred characters long), consider using an HTTP POST call.” In the version of the Social Network that was current as of the time of this review, one of us (M.B.) attempted a query containing more than 1,000 characters and it was successful. In any case we would like to request clarification, as it appears that the current version of the system may be capable of longer queries, possibly by doing an HTTP Post request. On a related note, later in the sixth paragraph of the “Database evaluation and implementation” section, the authors state that “There is also a limit on the amount of data that can be retrieved at one time from eUtils. NCBI recommends that no more than 500 publications be retrieved from a single eUtils query”. The citation given is http://www.ncbi.nlm.nih.gov/books/NBK25498/. However, this citation does not directly support this assertion. It is true, as the authors also point out, that requests should be limited to a maximum of three per second. Additionally, large jobs are to be limited to nights and weekends Eastern time (http://www.ncbi.nlm.nih.gov/books/NBK25497/). However, if there is a stated recommendation that no more than 500 publications be retrieved from a single eUtils query, we request that the authors identify a different source in which this is indicated in writing. In the fourth paragraph of the “Database evaluation and implementation” section the authors mention that “Scopus and Web of Science APIs require paid subscriptions”. It is our understanding that Web of Science has an API that is free to subscribers, in addition to a paid API with more data fields. We would like clarification on whether the authors were referring to the API that is free to subscribers. In the second paragraph of the “Results and discussion” section the authors state, “We created the co-publication network by entering the researcher’s name (last name <space> first initial, as expected by PubMed) … and clicking on the search button”. Due to the problem of ambiguous names in PubMed, it should be noted that this approach, without an attempt at name disambiguation, may result in many false drops, leading to invalid networks. In the third paragraph of the “Methods and implementation” section the authors mention that the user panel includes a co-publication network summary panel. Later in this paragraph they mention “charts that summarize the total number of publications and citations by location can be viewed by clicking on links in the panel that navigate to summary charts created with the Google Chart API.” We have thus far been unable to locate the network summary panel or the links to the charts, so further detail would be helpful here to describe the circumstances under which these features may be used, and/or how to activate and find them. We also have some minor editorial suggestions. First, we wanted to point out that “co-authorship” networks is far more common in the literature than “co-publication” networks, so the authors may wish to switch to this term if desired. Second, in the third paragraph of the “Methods and Implementation” section the authors point out that “Because InCites networks contain institutional affiliations for all the authors of a given publication, they have richer summaries.” It may also be worth pointing out here that Scopus does this as well. Third, the sixth paragraph of the “Database evaluation and implementation” section contains a broken link (http://baderlab.org/Software/SocialNetworkApp#PubMed). We applaud the authors for making this app available for use within a freely available tool that has an active user base and community of users, and are hopeful that the authors will continue with active development of this tool, so that they may be responsive to user suggestions that may further improve the user experience and integration into workflows.",
"responses": [
{
"c_id": "1625",
"date": "08 Oct 2015",
"name": "Victor Kofia",
"role": "Author Response",
"response": "In the version of the Social Network that was current as of the time of this review, one of us (M.B.) attempted a query containing more than 1,000 characters and it was successful. In any case we would like to request clarification, as it appears that the current version of the system may be capable of longer queries, possibly by doing an HTTP Post request.Thanks for pointing this out. We have now modified the app to use POST for all queries, which will be part of the next release.On a related note, later in the sixth paragraph of the “Database evaluation and implementation” section, the authors state that “There is also a limit on the amount of data that can be retrieved at one time from eUtils. NCBI recommends that no more than 500 publications be retrieved from a single eUtils query”. The citation given is http://www.ncbi.nlm.nih.gov/books/NBK25498/. However, this citation does not directly support this assertion. Our apologies for the wrong citation. In “Building Customized Data Pipelines Using The Entrez Programming Utilities” (http://www.ncbi.nlm.nih.gov/books/NBK1058/) under the “Handling Large Datasets” subheading, it is stated that large lists should be split into smaller batches of around 500 records. We have updated the manuscript to reflect this change.In the fourth paragraph of the “Database evaluation and implementation” section the authors mention that “Scopus and Web of Science APIs require paid subscriptions”. It is our understanding that Web of Science has an API that is free to subscribers, in addition to a paid API with more data fields. We would like clarification on whether the authors were referring to the API that is free to subscribers.We were referring to both APIs. Since users have to be subscribed to Web of Science to access the ‘free’ API we classified it as ‘requiring a paid subscription’. We have clarified this point in the revision.In the second paragraph of the “Results and discussion” section the authors state, “We created the co-publication network by entering the researcher’s name (last name <space> first initial, as expected by PubMed) … and clicking on the search button”. Due to the problem of ambiguous names in PubMed, it should be noted that this approach, without an attempt at name disambiguation, may result in many false drops, leading to invalid networks.We agree with the reviewer. Name disambiguation is definitely a problem when constructing co-authorship networks in this way. And it is a problem that affects all databases. We have updated the text to reflect this as it is something that all readers should be aware of. Until such time that databases become cleaner or reliable automatic name disambiguation services become available, we recommend that users manually clean their data to resolve errors and name ambiguities before relying on co-authorship network results to support important decisions.In the third paragraph of the “Methods and implementation” section the authors mention that the user panel includes a co-publication network summary panel. Later in this paragraph they mention “charts that summarize the total number of publications and citations by location can be viewed by clicking on links in the panel that navigate to summary charts created with the Google Chart API.” We have thus far been unable to locate the network summary panel or the links to the charts, so further detail would be helpful here to describe the circumstances under which these features may be used, and/or how to activate and find them.The network summary panel is located at the bottom of the user panel. Instead of a figure that only shows the top half of the panel we have included a new figure that contains the entire panel as this will enable readers to view the sections we are referring to. If no network summary panel exists in your setup and the problem persists then it may be a bug, in which case we would appreciate it being filed as a bug report on GitHub (https://github.com/BaderLab/SocialNetworkApp/issues) or emailed to us so we can fix it.Also note that even with the network summary panel being visible, links to the charts will only appear after an InCites network has been created. No charts are available for Scopus and PubMed networks at the moment. So please verify that you are using an InCites document to build the network. An example InCites document is provided in the user guide: http://baderlab.org/Software/SocialNetworkApp. We have made this clearer in the text.We also have some minor editorial suggestions. First, we wanted to point out that “co-authorship” networks is far more common in the literature than “co-publication” networks, so the authors may wish to switch to this term if desired.Thank you for pointing this out. We have switched to using the more common “co-authorship” term throughout the manuscript.Second, in the third paragraph of the “Methods and Implementation” section the authors point out that “Because InCites networks contain institutional affiliations for all the authors of a given publication, they have richer summaries.” It may also be worth pointing out here that Scopus does this as well.It is indeed true that Scopus provides institutional affiliations. The default setting for exporting files from Scopus is “Citation Information Only” which does not include the institutional affiliations of the co-authors. We have extended the functionality so that the app is capable of parsing Scopus reports that contain additional information (like institutional affiliations). We have made a note of this in the online manual.Third, the sixth paragraph of the “Database evaluation and implementation” section contains a broken link (http://baderlab.org/Software/SocialNetworkApp#PubMed).Thanks for noticing this. We have updated the manuscript to include the correct link: http://baderlab.org/UserguideSocialNetworkApp#PubMedWe applaud the authors for making this app available for use within a freely available tool that has an active user base and community of users, and are hopeful that the authors will continue with active development of this tool, so that they may be responsive to user suggestions that may further improve the user experience and integration into workflows.Thank you for taking the time to review our app."
}
]
},
{
"id": "9866",
"date": "21 Aug 2015",
"name": "Jiang Bian",
"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 describes a software as an extension of the Cytoscape that can automatically query popular citation databases (PubMed only) and derive co-authorship networks based on the query results. It's a laudable goal, and will be a welcomed tool for researches on collaboration networks. Especially, the tool is disseminated as a open-source tool. However, there are a number of concerns.The software does not have any process for disambiguation. This is problematic. In general, citation databases do not provide disambiguation services. Google Scholar attempts to \"learn\" which publications belong to a specific author when creating the author profile. However, it is not very accurate either at the beginning for common names. For studying social networks, getting accurate information is important, especially for studying Ego networks that focus on a specific person. It is not very user friendly in terms of gathering data. Only for Pubmed, users can enter queries through the tool. With other citation databases (InCites and Scopus), users will need to query the databases directly and then export the results. It is understandable that this is due to InCites and Scopus only provide paid API services. However, it is unclear whether the authors have implemented such integration for users who have paid those API services. Further, even for Pubmed search, the software relies on the users to understand Pubmed query syntax. It would be useful if the authors could provide (in addition to text Pubmed query) guided search (see Pubmed Advanced search) for common use cases in studying collaboration networks. It would be very useful, if the authors could allow users to use the same guided search interface to three all citation databases.",
"responses": [
{
"c_id": "1624",
"date": "08 Oct 2015",
"name": "Victor Kofia",
"role": "Author Response",
"response": "The software does not have any process for disambiguation. This is problematic. In general, citation databases do not provide disambiguation services. Google Scholar attempts to \"learn\" which publications belong to a specific author when creating the author profile. However, it is not very accurate either at the beginning for common names. For studying social networks, getting accurate information is important, especially for studying Ego networks that focus on a specific person.Thank you for the comment. We agree that name disambiguation is a key concern with co-authorship networks. This is something we hope to address in the app in the future. In the meantime we have added to the paper some tips to minimize this issue. As noted in response to referee 1, until such time that databases become cleaner or reliable automatic name disambiguation services become available, we recommend that users manually clean their data to resolve errors and name ambiguities before relying on co-authorship network results to support important decisions.It is not very user friendly in terms of gathering data. Only for Pubmed, users can enter queries through the tool. With other citation databases (InCites and Scopus), users will need to query the databases directly and then export the results. It is understandable that this is due to InCites and Scopus only provide paid API services. However, it is unclear whether the authors have implemented such integration for users who have paid those API services.Our apologies for the lack of clarity. Right now the app does not support API querying for users who have paid services, but we have added this to a list of planned features in our github issue tracker at https://github.com/BaderLab/SocialNetworkApp/issuesFurther, even for Pubmed search, the software relies on the users to understand Pubmed query syntax. It would be useful if the authors could provide (in addition to text Pubmed query) guided search (see Pubmed Advanced search) for common use cases in studying collaboration networks. It would be very useful, if the authors could allow users to use the same guided search interface to three all citation databases.Thanks for the comment. We have improved the user interface of the app and included a link to the PubMed query syntax. We have added a feature request to our issue tracker to develop a user friendly interface to assist users who may not be familiar with PubMed query syntax. For now we have included in the manuscript links to the relevant PubMed tutorials."
}
]
}
] | 1
|
https://f1000research.com/articles/4-481
|
https://f1000research.com/articles/4-1502/v1
|
23 Dec 15
|
{
"type": "Opinion Article",
"title": "New tools for a new age: An evolution or revolution in higher education?",
"authors": [
"Roslyn Gleadow",
"Melissa Honeydew",
"Allie Ford",
"Bronwyn Isaac",
"Kirsti Abbott",
"Melissa Honeydew",
"Allie Ford",
"Bronwyn Isaac",
"Kirsti Abbott"
],
"abstract": "In this paper we describe how digital technologies can be used to enhance collaboration and student engagement in a large, multicampus undergraduate science unit. Four innovations developed and implemented over a period of eight years are described: use of electronic whiteboards, on-line discussion forums, social media and blogs. In showing the intermediate steps in the evolution of the use of digital and communication technologies, we demonstrate that to be effective, good educational principles are paramount.",
"keywords": [
"higher education",
"science communication",
"communication",
"collaboration",
"social media"
],
"content": "Introduction and rationale\n\nIn the past decade we have witnessed a revolution in the use of digital media. This is epitomised by an image that circulated various social media platforms, and originally posted on Instagram by NBC news comparing a photo from the inauguration of Pope Benedict in 2005 with one from the inauguration of Pope Francis in 2013. In the first, everyone is looking up and around, with only one small flip phone visible; in the second, there is a sea of iPads and smart phones lighting people’s faces and most people are looking at the Pope through a screen. The ability to easily share information with others across and space and time is unprecedented. The fact that the first photo may have been taken shortly after John Paul II’s death (also in 2005) rather than Pope Benedict’s inauguration also shows how easy it is for misleading information to “go viral” and impact the global perception of an event. This revolution in information access and sharing has huge implications for higher education when knowledge, not just perception, is at stake. It means not only that students can take seemingly credible courses from anywhere in the world through Massive Open Online Courses (MOOCs ) or enrolment in virtual classrooms, but that they also can have more control over the content they access and their schedules.\n\nLearning primarily using digital technology tends to emphasise the acquisition of knowledge, and suits some students’ study styles. Unless this is understood, there may be reduced opportunities for social engagement and networking – factors that are an important aspect of student engagement, the sharing of ideas, and creating meaning for the learner (Conrad & Donaldson, 2004). Social interactions typically happen during on-campus learning in traditional face-to-face lessons. Despite this, more and more students studying on campus prefer to listen online rather than attend lectures in person, to save travel time, schedule education around paid work, and fit in with other commitments (Gleadow 2009, unpublished conference report), but these choices often reduce opportunities for collaboration.\n\nWe teach a course on scientific practice and communication. This unit is offered at a traditional research university where blended learning models are encouraged. We combine face-to-face teaching - using a mixture of lectures and tutorials - with online activities and value social engagement, networking and collaboration between students from different disciplines and backgrounds. The course is compulsory for all science students and runs throughout the academic year. Approximately 700 students are enrolled each semester, spread across the main campus in Melbourne, at a campus in Kuala Lumpur, Malaysia and, until recently at a regional campus, including a cohort of distance education students. An advanced form of the same unit is also delivered to a small group of 30–40 talented students each semester. The content of the unit deals with the nature and origins of science, ethical practice, science literacy, and various aspects of science communication.\n\nEngagement of students studying at all levels is generally higher when they can work with their peers, work with technology, have control over their own learning and see that the teachers themselves are engaged (Wolpert-Gawron, 2012; Zepke & Leach, 2010). We have endeavoured to improve student engagement and teamwork for those students choosing to learn primarily in the online environment using a combination of social media, collaborative document tools and, for in class use, both tablet computers and electronic whiteboards. This has evolved over a period of eight years (Table 1). In this paper we outline 1) a chronology of when and how these digital technologies were introduced to students, 2) ways in which these technologies have changed, and are continuing to change, the way we teach and 3) their effectiveness, because not everything we trialled has been effective. In doing so, we show that the introduction of new teaching tools has been an evolution of earlier styles of learning, but compared to ‘then’ what we do ‘now’, looks like a revolution. The paper is based on the Keynote lecture given by one of us (RG) at the SEB Digital tools for teaching and communicating science, in London, December 2014, described by (Scott, 2015).\n\n\nSocial media as a platform for instruction and student discussions\n\nStudent work is centred on the learning management system (LMS), beginning with Blackboard in 2005, and moved to Moodle in 2010. LMSs, including ours, have traditionally functioned as a kind of information clearance house rather than educational space (see Gleadow et al., 2015). There are discussion groups but these tend to be simple question and answer sites, where the teacher responds to specific questions. Such officially sanctioned forums are important, and their use can dramatically reduce the number of emails, but much of the discussion now takes place on social media, currently Facebook – there is one exception to this, the blogging that takes place in the advanced cohort (to be discussed later). Initially Facebook use was limited to students creating their own groups for working on joint assignments, but then in 2013, a proactive student created a group for the whole class and invited others to join via LMS. It was not until 2014, however, that students started approving membership of the group by lecturers and staff members, and since then Facebook has become an important medium for teaching and learning, further reducing the reliance on email. In the semester when we first used Facebook, 27 questions were posted on the LMS during the pre-examination study week and 148 questions were asked on Facebook. In the most recent semester there were over 300 posts over the whole semester on Facebook. Students answering each other’s questions is not only good for student learning (Driver et al., 1994), but also reduces the number of personal emails to the teachers.\n\nStudents are mostly highly connected and comfortable on social media platforms. Informal surveys of students indicate that students consider Facebook “a given”. A show of hands in class indicated that over 90% of students are registered to use it. One attraction is that they can access it easily from a smart phone or other mobile device, and so the reach is much greater than with dedicated learning management systems – particularly when the LMS is not optimised for use on a variety of devices. This lowers the barriers to communication, and promotes informal language (Figure 1). This switch to informal language has been a great help to our many international students who have been reluctant to post questions on the official forums because they report feeling embarrassed about making grammatical and spelling errors: over a third of students within the cohort do not speak English as their home language. Indeed, Yu et al. (2010) reported that students felt an enhanced social acceptance, and speedy adaptation to university culture through the use of social media in educational units.\n\n(a) Students here show engagement with the subject matter (pseudoscience) and extend beyond and apply what was presented in lectures. (b) Discussion of examinable material from the lectures (on research ethics). Not only do students answer each other’s questions but go beyond, to express their own position on this ethical dilemma.\n\nPlatforms such as the Facebook group give the students some control over the content and the way in which it is delivered. Educationally, this level of autonomy is a good thing, as it allows students to drive their own learning (Gleadow et al., 1994). Students are also very good at answering each other’s questions using examples relevant to their cohort and/or sourcing relevant and entertaining videos that explain a point covered in lectures such as a video by Gentleman Thinker on critical thinking on the ‘Philosophy’ Youtube channel.\n\nMany students appreciate the almost instantaneous nature of receiving the answer to a query via Facebook because often one or two of the tutors are online too and it is simple for us to write a quick response. We are not advocating that teaching become a 24-hour day activity, but it certainly is convenient to deal with enquiries as they arise.\n\nI really felt you went above and beyond in teaching the unit. Especially answering last minute frazzled assignment questions on Facebook. (Student feedback, 2015)\n\nThere are some drawbacks to using a public forum such as Facebook rather than relying on the functionality and accessibility built into the LMS. Not all students (or teachers) want to use mainstream social media platforms, nor should they be forced to. There may be privacy concerns, or philosophical opposition to them. Students may want their social spaces to remain separate from their online study resources, and make deliberate choices to disconnect from their studies when engaged in other activities. In our unit, we attempt to ensure that such students are not disadvantaged if they are not Facebook users by making all official announcements via LMS, and cross post on social media.\n\nThere is the danger, common to all social media platforms, that some students will use public forums inappropriately. It is important to bring the Facebook group under the umbrella of the University’s code of conduct. The administrator must set ground rules for the scope of the Facebook group in the group description, stating what topics are appropriate, defining what unacceptable behaviour is, and linking it to university policies regarding appropriate use of social media and IT. In our unit, these policies are discussed further as part of the ethics and communication components of workshops. Importantly, the course coordinator and the teaching team need to monitor the site, and have the power to remove posts that are not consistent with university policy. In the four semesters that this program has been operating we have only needed to remove posts by one student that could have been interpreted as derogatory. We have also observed a small number of cases of religious and political advocacy. Our strategies for dealing with these issues have so far been effective – the perpetrator is counselled via email and invited to a face-to-face meeting. At this point a simple reminder that they can be expelled from the course or even from the university for unacceptable behaviour is all that has been needed to ensure compliance. Thus, while using Facebook has numerous strengths, we need to learn from it, rather than rely on it. It is important to carefully consider the reasons students like and use specific platforms, so that we can incorporate these preferences into other things we do.\n\n\nEvolution from tablet PCs to electronic whiteboards\n\nIn 2010 and 2011, we participated in a limited trial of tablet PCs run by the university (Lindsay, 2012). Tablet PCs are normal laptop computers with screens that can be written on with a stylus, allowing annotation of existing documents, as well as creation of new ones. At this time, many students did not bring their personal computers to university, and collaborative software was not commonplace. The university was also developing its own collaborative software that allowed students (and their teachers) to work on the same document at the same time. Prior to this trial, any use of PCs required pre-booking of computer labs elsewhere on campus. The labs were available to the whole of our faculty, so it was often difficult to access them at the most appropriate times for specific course activities. When the tablet trial took place, we were able to access computers in our tutorial rooms every week for the first time. We made a deliberate choice to have one tablet PC per two students (rather than one each), to encourage collaboration.\n\nThe trial allowed us to make significant changes to our workshop structure and activities, for example students could search academic databases in class, rather than just watching a tutor demonstrate the process and then needing to try to remember it later. Shared brainstorming activities, collectively annotating journal articles and posters, and sharing appropriate items that each pair had found on the internet were new activities that added depth, and importantly 21st century skills, to the existing curriculum. In one particular activity, working together to identify grammatical and stylistic errors in writing samples, students were enjoying themselves so much that they had to be asked to leave so the next class could begin; never before have we had to kick students out of a lesson on academic English.\n\nInformal feedback from the students covered a number of themes. Many reported that the use of computers in their classes added more fun. Others liked that they could participate or look up information, as they needed it, rather than having to wait until they could get to the library. An important message came from some of the students from backgrounds other than English: they felt that they had a voice in the classroom. Some came from cultures where interrupting or contradicting someone else was rude. Many were concerned about their level of spoken English, or found that by the time they had worked out how to express their ideas in English, activities had moved on. Because of the change of pace brought about by the tablets, as students needed to research or develop ideas more, and the option to contribute in writing, these students reported feeling more confident, and more involved in their classes. This is consistent with the recommendations of Tanner (2013) for engaging students in learning. Gradually the tablet trial wound up, as other collaborative hardware and software began to appear on the market, so we took the lessons (and activities) from the small screen to larger screens.\n\nThe use of electronic interactive whiteboards has revolutionised the way that we educate students within the workshop setting. These screens, incorporating both electronic displays and traditional whiteboard capabilities allow for a more blended mode of teaching. Used appropriately, they can foster collaborative exchanges of ideas in small group work, and allows the students to become the teachers. Typically up to six students share a table and whiteboard, and we have four electronic interactive whiteboards in each tutorial room (Figure 2). This setting creates an environment where anecdotally it seems to be easier for students to alternate between whole class activities with up to 25 students, and discussions with their smaller groups. It is often in these small groups where students are more confident to voice opinions and work effectively to develop content together.\n\nHere students are accessing on-line databases to search for primary literature. There are approximately 600 students enrolled at this campus, with students broken into tutorial groups of 20–25. There are two rooms side-by-side to accommodate the large number of students.\n\nElectronic whiteboards are highly versatile for use in a dynamic student-driven classroom environment. Collaborative small-group tasks range from interactive brainstorming of a concept relevant to lecture material, through to proof reading and editing a scientific poster before presenting it to the class, and even to the collaborative preparation of written documents, such as a cover letter addressing key selection criteria for a real science job. If necessary, the work can then be saved for future reference and emailed directly to group members from the classroom. Where students are given an open-ended group task, they use the technology available to them in a variety of ways that suit their learning style and expertise. For example, a small cohort of advanced students can quickly and collaboratively create visuals to communicate ideas for impromptu talks. They are able to conduct research and access materials from a variety of online sources and used the whiteboards in a variety of other ways as a platform to showcase the results of their work to the rest of the class. Each student brings with them knowledge of different tools, so that together their power to create and innovate is far greater than them working alone.\n\nThe new Interactive whiteboards [we used in] the unit aid learning and add stimulation for visual learners. (Student feedback, 2014)\n\nAccess to a range of online resources, including the LMS and other suitable websites, is also easy from the electronic whiteboards. We encourage students to work together to develop skills in searching online databases, as well as identifying and effectively reading primary articles. Because the members of each group share a screen, rather than working on their own individual devices, there is increased transparency, collaboration and discussion. Teaching staff are also better able to observe how the group approaches each activity, monitor progress, and offer timely guidance and feedback, rather than needing to wait for students to self-identify difficulties or concerns. Teachers, thus, move from didactic teaching and the dispensing information to working alongside students and facilitating learning, similar to that described by Gleadow et al. (1993).\n\n\nCollaboration tools\n\nAn important breakthrough for education is in the development of new tools that when used online allow collaboration across time and space. Our university moved to using Google-administered email accounts for all students and staff in 2010, fundamentally changing the way we were able to work with these collaborative tools. As part of a module on conceptualisation, we introduced brainstorming strategies, and as an example of this, mind-mapping. To facilitate discussion and exploration and to highlight the power of this method we built a tool to allow collaborative brainstorming both in real-time in classrooms and later via the unit’s LMS. The tool is a simple template built in Google Drawings, as shown in Figure 3. The workshop effectively introduces the student to the idea and benefit of mind mapping and also allows students to gain familiarity with the tool itself. Links to copies of the template are made available for the tutors to share with their class via the LMS. During the workshop the students use both electronic whiteboards and their personal laptops to access the template via their university Google account. Each student in the class then has an opportunity to collaborate in real-time simultaneously. (A video showing how this looks in practice can be viewed in the supplementary information.) This fast-paced activity has proven to be highly successful across the entire cohort with strong engagement from students and reports from tutors that the tool was easy set up and use in class. After the workshop, students are then able to access, again via the LMS, a copy of the mind-mapping template for their own use. Examination of the LMS records suggests that the majority of the students take advantage of this tool. This demonstrates the power and flexibility of adapting emerging tools in the rapidly changing digital space to enhance our students’ learning experience.\n\nHere students have worked on a practice, plus topic about insects. Student can download a clean copy for personal use. A video showing how the ideas develop and the map builds is given in the supplementary information.\n\nReflective learning has always been an important part of the program for off-campus students helping the tutor to identify gaps in understanding and promote discussion within the group. This was done by using threaded discussion boards on the LMS, and while extremely valuable, the threads could become very unwieldy as people commented in posts within posts, and so on. As blogging had previously been reported to be an effective way to promote learning and understanding (Farmer et al., 2008), we explored this as an alternative to the discussion boards. Fortunately, the LMS that we use contains a blogging module that we could implement simply to both to encourage reflection on the lecture content, and to allow students to create their own knowledge platform. We now incorporate the blogging activity into the assessment of small group of students (typically 40 per semester) who are enrolled in an advanced version of the unit (by this stage we were phasing out our off-campus program). This activity requires students to make blog posts about aspects of the theoretical content of interest to them, and to comment on other students’ posts and discussions. When the blog was first implemented, the students were asked to make four posts during the semester and at least one comment.\n\nInitially not all students could see the value of the blogs, as demonstrated by the following comments in the feedback from the 2013 cohort:\n\nA better explanation as to how the blogs are to be done would be great. … I had no idea what I was doing when I wrote my first one.\n\n… blogs were kind of annoying rather than helpful\n\nThis year, to encourage discussion, and to thereby assist the students in seeing the “point” of blogging, comments were also rewarded with marks. This led to much greater engagement with the blog, and many students seemed to not only understand the purpose of the blog, but to enjoy the lively discussions online. The students have had extensive discussions on lecture content ranging from logic to pseudoscience, to ethics in research. The students initiate these discussions themselves, commenting on posts to add information, ask questions or challenge points. The blog posts themselves, and the discussions that followed, gave many students an opportunity to identify concepts they were interested in, share their enthusiasm for a topic with an audience, develop their critical thinking and research skills in order to make meaningful comments, find points to challenge or logical responses and practice their scientific writing skills at the same time. The discussions have had an added benefit: content which would not normally be covered in the unit (through lack of time or lack of knowledge about specific examples) has been presented, by the students themselves, drawing on a wide range of sources.\n\nFeedback continues to show that students find it hard to navigate around the blogs. The most important request made by students is for notification when a peer comments on a post they have made. This feature, common in most social media applications, suggests that these digital resident students expect their LMS experience to match that of other platforms, and also that they value comments on their posts – although whether this is out of a desire to further the discussions, or merely as trigger for reward mechanisms is not clear as there is an addictive element to the desire to endlessly check comments on Facebook (Holmes et al., 2014).\n\nReflective learning has been important for many years, often through writing reflective journal entries or diaries, or specific reflective essays. The difference between these activities and blogs is the openness of the latter, in terms of the posts being accessible by someone other than the student and their teacher. Whereas in an individual reflection, the focus is on the learner producing something that shows the teacher they have developed new knowledge or understanding, with blogging, there is a stronger emphasis on sharing; ideas written in a blog are meant to be seen by others, and this awareness of audience can be enough to encourage deeper learning because the final result will be on display and open to ‘public’ critique. The blogs also share some benefits with the tablet computer trial mentioned above: students from culturally and diverse linguistic backgrounds, and those who are less confident to speak up in a group, have the time and access to resources they need to understand material, and to express themselves with some degree of anonymity.\n\n\nConcluding remarks\n\nMany different developing platforms and tools have applications in teaching over the years as technology use in education keeps pace with the rapidly changing digital world (e.g. Gleadow et al., 1993). Information and communication technologies are constantly changing and in order to identify new, different or better options, horizon scanning is important, to keep up-to date with how students are interacting with the world academically and socially. We also have a responsibility to make sure that our teaching does not become locked into some early 21st century paradigm. The manner with which we engage in education needs to match and be continuous with engagement with the rest of the digital world. To this end, we have also experimented with Wikis, Twitter and citizen science platforms, with varying degrees of success. For our purposes, the current formula of in-class electronic white boards, collaborative tools built using Google Docs, Facebook, and online blogs works mainly because the tools are readily accessible, familiar to a wide range of students, and enhance student connection.\n\nUltimately, the focus should be on what the students need to learn. We view the medium we use as secondary, and acknowledge that these tools are rapidly changing. Lectures did not end with the advent of the printing press (Figure 4), but they did change from reading of texts to presenting knowledge with the assistance of blackboards, to overhead transparencies and then to projectors with slide slows. While digital technologies, that will themselves come and go, do enable greater control of content and information access by students, the students still need to guidance, and assistance develop the skills to use such tools safely, appropriately and confidently and we must ensure our learning objectives are not lost as we take on the challenge of keeping pace with the changing digital world.\n\nIn the 14th Century, lectures often consisted of the lecturer reading from a book at a lecturn. Before printing, books were rare and very valuable and this made the contents more widely available. Today lectures are important in defining the area for learning and to inspire students. Note the students in the audience behave has a lot of similarities to the way to students behave in lectures today. Citation: Laurentius de Voltolina 001\" by Laurentius de Voltolina - The Yorck Project: 10.000 Meisterwerke der Malerei. DVD-ROM, 2002. ISBN 3936122202. Distributed by DIRECTMEDIA Publishing GmbH. Licensed under Public Domain via Commons - https://commons.wikimedia.org/wiki/File:Laurentius_de_Voltolina_001.jpg#/media/File:Laurentius_de_Voltolina_001.jpg",
"appendix": "Author contributions\n\n\n\nGleadow is the course coordinator, leader is the innovation projects and wrote the first draft of the manuscript. Honeydew, Ford, Isaac and Abbott drove particular innovations, oversaw their implementation and wrote relevant sections of the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding came from a series of small grants from the Faculty of Science Teaching Initiatives Fund, Monash University.\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 many tutors, particularly the senior tutors from each campus (Jo-ann Larkins, Joash Ban Tan Lee, Mary Lush and Trisha Wevill) for their enthusiasm in embracing and implementing the new programs, providing feedback and making them work. We also acknowledge the support of the Faculty of Science, eEducation, eSolutions and the Faculty of Science at the Clayton campus, and the School of Science in Malaysia for renovating our teaching spaces and making the use of the digital technologies in class possible.\n\n\nSupplementary materials\n\nSupplementary video.\n\nDevelopment of a mindmap\n\nClick here to access the data.\n\n\nReferences\n\nAtwell BJ, Kriedemann PE, Turnbull CGN: Plants in action. 1st Edition. Macmillan Education Australia Pty Ltd, Melbourne, Australia. 1999. Reference Source\n\nConrad RM, Donaldson JA: Engaging the online learner: Activities and resources for creative instruction. San Francisco, California. John Wiley & Sons Inc. 2004. Reference Source\n\nDriver R, Asoko H, Leach J, et al.: Constructing Scientific Knowledge in the Classroom. Educ Res. 1994; 23(7): 5–12. Publisher Full Text\n\nFarmer B, Yue A, Brooks C: Using blogging for higher order learning in large cohort university teaching: a case study. Australasian J Educ Tech. 2008; 24(2): 123–136. Reference Source\n\nGleadow R, Macfarlan B, Honeydew M: Design for learning - a case study of blended learning in a science unit [version 2; referees: 2 approved]. F1000Res. 2015; 4: 898. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGleadow RM, Ladiges PY, Handasyde K, et al.: Innovative teaching methods in Biology incorporating self-study and multimedia programs. In: Promoting Teaching in Higher Education. Reports from the National Teaching Workshop (eds. J Bain, E Lietzow and B Ross). Griffith University Press: Brisbane. 1993; 305–318. Reference Source\n\nHolmes JM, Kerne B, Timko CA: Craving Facebook? Behavioral addiction to online social networking and its association with emotion regulation deficits. Addiction. 2014; 109(12): 2079–88. PubMed Abstract | Publisher Full Text\n\nLindsay S: Top tips on Tablet PCs and MeTL. eEducation Centre, Monash University. 2012. Accessed August 2015. Reference Source\n\nMunns R, Schmidt S, Beveridge C: Plants in Action. 2nd Edition. Australian Society of Plant Scientists. 2014. Reference Source\n\nScott G: Teaching and communicating science in a digital age [version 1; referees: not peer reviewed]. F1000Res. 2015; 4: 83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWolpert-Gawron H: Kids Speak Out on Student Engagement. Edutopia. 2012. Accessed Nov 20, 2015. Reference Source\n\nTanner KD: Structure matters: twenty-one teaching strategies to promote student engagement and cultivate classroom equity. CBE Life Sci Educ. 2013; 12(3): 322–331. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYu AY, Tian SW, Vogel D, et al.: Can learning be virtually boosted? An investigation of online social networking impacts. Comput Educ. 2010; 55(4): 1494–1503. Publisher Full Text\n\nZepke N, Leach L: Improving student engagement: Ten proposals for action. Active Learning in Higher Education. 2010; 11(3): 167–177. Publisher Full Text"
}
|
[
{
"id": "11749",
"date": "08 Jan 2016",
"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 reflection on the evolution of a programme of study through the introduction of emerging digital technologies. Although in themselves adoption of none of the technologies discussed would today be considered innovative this paper presents a useful commentary on the pros and cons of adopting each through the experiences of a teaching team who have themselves clearly been innovators in their adoption. My only concern is that in reading the paper I was left wanting more details about the technologies involved and about the evaluation of their effectiveness. I acknowledge however that this paper is an opinion piece/commentary rather than an empirical study and that the reader can access the information required if they follow the citations that are included. Should the authors feel it necessary to provide more information in a second version of the paper there are a few minor typographical errors that could be corrected (but these do not currently impact negatively upon the paper).",
"responses": []
},
{
"id": "11752",
"date": "11 Jan 2016",
"name": "Mary Williams",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading this and approve it. This is a very useful article for many instructors, particularly those who remain sceptical of the value that can be added from digital tools, and those who are interested in adopting them but unsure of how to introduce them and use them effectively. The timeline presented is interesting as it shows that, as the title suggests, big changes can take place through a series of small steps; this should be reassuring to those who hope to update their approach to teaching. The article provides a narrative account of ten years of innovation in a large course, with explanations of what was tried, its success or not, and whether it has been continued as part of the course. I particularly appreciate the indications that tools and approaches must change as technologies and students’ experiences change – instructors must keep adopting and adapting. A few key points:Facebook, unlike many institutional LMSs, is optimized for mobile devices and user-friendly – even those most sceptical of social network sites should recognize that their design and technology is superior. I appreciate that the question of “What about students/ instructors who for some reason refuse to use Facebook?” is addressed. By providing the same announcements via LMS, the students who chose to exclude themselves from the Facebook discussions are not disserviced, other than by their own choosing. I feel that it is essential for students to develop their professional skills in a structured environment, and incorporating group work on tablet PCs and whiteboards is an excellent approach. I particularly agree with this statements, “Where students are given an open-ended group task, they use the technology available to them in a variety of ways that suit their learning style and expertise……Each student brings with them knowledge of different tools, so that together their power to create and innovate is far greater than them working alone.” This is exactly the type of student-centered, problem-based learning that needs to be supported and that digital tools can support. I’m curious, what is the topic / question they are addressing in the mindmap? I also appreciate the distinction that is made between blogs and the reflective essays written for the instructor that are a common component of a course. It is easy to believe that the quality of the thought and presentation would be enhanced through the more open platform. I look forward to sharing this article, as it demystifies the uses and benefits of the digital tools that ought to be a part of every instructional environment.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1502
|
https://f1000research.com/articles/4-810/v1
|
15 Sep 15
|
{
"type": "Opinion Article",
"title": "The Antibody Two-Step Solution",
"authors": [
"Mike Browning"
],
"abstract": "Problems with antibody quality have been described in numerous recent publications. In the present commentary it is argued that these quality problems are due primarily to issues of antibody variability and antibody validation. Further it is argued that the problem of antibody variability must be solved before validation can be useful. A two-step solution to the antibody problem is thus proposed.",
"keywords": [
"Polyclonal",
"Monoclonal",
"Antibody",
"Variability",
"Validation",
"Pooled Serum"
],
"content": "\n\nIn the past year there have been a number of articles in Nature and other major journals that discuss the antibody problem in somewhat apocalyptic terms1. In my mind there are only two main issues in the antibody problem: antibody validation, and antibody variability. Both of these issues have straightforward solutions that do not require any massive influx of cash or massive restructuring of antibody production.\n\nAntibody validation is the hardest nut to crack and causes the most confusion. There is no consensus on what constitutes suitable validation and this is complicated by the different methods for antibody use. However, antibody validation is a process like all science where knowledge increases as more and more work is done with the antibody. Many journals now insist that antibody validation data be provided and this is a key part of the solution. As long as the data are clear and the methods used adequately described, progress in validation will occur with time. However, none of this progress will matter unless we deal with the antibody variability problem. What difference does it make if an antibody is validated if it is not possible to obtain the same antibody for future work?\n\nThere are two main reasons for the variability in an antibody’s performance. The first is that once an antibody is found to have a high demand, many different antibody manufacturers will try to make their own version of the antibody so they can sell it. But all these new antibodies will differ in unknown and unpredictable ways from the original antibody. Thus validation done on the original antibody may or may not be true for the new antibodies. One way to deal with this problem was recently suggested by Andrew Chalmers and his colleagues2. They argue that all publications using commercial antibodies should report the name of the supplier and the catalog number of the antibody used. That way even if a supplier sells many varieties of the antibody a researcher will be able to order the same antibody that was used in the publication. This suggestion is being incorporated into the instructions to authors in more and more journals.\n\nEven though this action would greatly improve the value of antibody validation, an additional source of antibody variability would remain, namely lot-to-lot variability. This variability occurs because even if one buys the same antibody with the same catalog number, one still often encounters large variability in different lots of the same antibody obtained from different bleeds of the same animal or bleeds from different animals. There is a very straightforward fix to this type of variability. The solution is to pool all the positively screened serum collected from the animals. Virtually all lot-to-lot variability can be eliminated for polyclonal antibodies if this procedure is used. The antibody manufacturer could simply label the antibody as “pooled serum” to denote this fact. If this procedure is followed, it will no longer be necessary to reinvent the antibody validation wheel each time an antibody is used. Thus science can build upon itself as it is supposed to do.\n\nSome may argue that one should use monoclonal antibodies to eliminate variability. This is unnecessary and also unwise. It is unnecessary because for most antibodies a single rabbit can produce a stable 20–30 year supply of antibody. Only a small percentage of all antibodies sold ever sell more than can be produced by a single rabbit. It is unwise because monoclonals cost at least 3X what polyclonals cost and we are unlikely to see a time in the near future when cost will be irrelevant. Moreover, polyclonal antibodies have been shown to be superior to monoclonal antibodies in a number of different applications3.\n\nScientists and journals can fix the validation problem if antibody manufacturers will first fix the variability problem. This is called the Antibody Two-Step Solution.",
"appendix": "Competing interests\n\n\n\nMDB is majority owner and CEO of PhosphoSolutions LLC, an antibody manufacturer.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBaker M: Reproducibility crisis: Blame it on the antibodies. Nature. 2015; 521(7552): 274–276. PubMed Abstract | Publisher Full Text\n\nHelsby MA, Fenn JR, Chalmers AD: Reporting research antibody use: how to increase experimental reproducibility. F1000Res. 2013; 2: 153. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLipman NS, Jackson LR, Trudel LJ, et al.: Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J. 2005; 46(3): 258–268. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "10333",
"date": "25 Sep 2015",
"name": "C. Fernando Valenzuela",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of Browning proposes a two-step solution to solve problems related to antibody performance in biomedical research. The author proposes that antibody variability must be fixed first (for instance, by pooling positive serum from multiple animals). Once this issue has been addressed, validation will become more straightforward and will progress with time. This well-written opinion article is timely and addresses an important issue that has been the focus of intense discussion in recent years. There are only a few issues for the author’s consideration: Although it is clear what the author means by the two-step solution, relatively little detail is given regarding the issue antibody validation. A more detailed discussion of this issue is warranted. For instance, what is the author’s take on the approach proposed by Bordeaux et al. (Biotechniques. 2010 Mar;48(3):197-209. doi: 10.2144/000113382). It is stated that antibody validation will essentially be accomplished as more and more work is done with the antibody. However, there are many factors that limit the access of this information to both suppliers and users. If the antibody does not work on a particular application, the researcher often discards it without reporting any problems in any form. Perhaps being more aggressive about requesting feedback and making sure that all validation information is included in technical data sheets could contribute to solve the problem; the quality of the information included in some web sites and technical sheets is not particularly impressive. Encouraging users to submit information to sites such as antibodypedia.com or histoneantibodies.com could also be a step forward. Several companies provide trial size antibodies that allow the users to test them in a particular application in exchange for feedback. What are the author’s thoughts on how the task of antibody validation be distributed between vendors and users? Regarding reporting of antibody use, in addition to reporting catalog numbers, it seems as if it would be crucial to report lot numbers as well. What is the author’s take on the proposal that an independent body be recruited (e.g., National Institute of Standards) to oversee an antibody certification program? The abstract does not explicitly define the two-step solution.",
"responses": [
{
"c_id": "1652",
"date": "13 Oct 2015",
"name": "Mike Browning",
"role": "Author Response",
"response": "Many thanks for your comments Dr. Valenzuela. I share your frustration with the issue of antibody validation. However, if variability is not dealt with first, validation has little value to subsequent studies. Moreover the issue is very complex and beyond the scope of this opinion article. I have discussed my perspectives of this issue in my blog. You offer a number of suggestions about validation that I strongly support. These include the proposal you cite by Bordeaux et al., the need for publishing negative data and contribution to sites that catalog validation data.One issue you raise that I’d like to expand on is the use of lot numbers. Lot numbers can be both a blessing and a curse. Unfortunately, the use of lot numbers sometimes masks a tendency among suppliers to fail to follow best practices that will eliminate most antibody variability. The pooling of serum is the first best practice that must be followed to eliminate variability. Unfortunately very few suppliers do this. This is due primarily to an increase in cost and effort needed for pooling serum and also possibly to ignorance of best practices among some suppliers. The increase in cost is due to the fact that sales of the antibody must be delayed until all bleeds of the rabbits have been collected. The increase in effort is due to the need to screen all bleeds to determine which bleeds should be pooled. Many suppliers start selling an antibody as soon as they get a positive bleed. Then when that bleed is exhausted they switch to the next etc. However there can be huge differences in the quality of the antibody in subsequent bleeds. Serum pooling must be used to avoid this form of lot to lot variability.There is a second even more serious form of antibody variability that is often obscured by simply changing the lot number. This results when an antibody supplier runs out of all the bleeds from the original animal. Some antibody suppliers then use new animals to make the antibody and simply denote this fact by changing the lot number. The potential differences between bleeds from the same animal pale beside the differences seen in different animals. It is not enough to simply change the lot number. The best practice is to modify the catalog number to reflect to new animal source.Lastly, with regard to antibody certification by some independent body we wholeheartedly support such efforts to develop a set of best practices. One key issue in such an effort would need to be communication between all the stakeholders including researchers, the journals AND the suppliers."
},
{
"c_id": "1655",
"date": "14 Oct 2015",
"name": "Fernando Valenzuela",
"role": "Reader Comment",
"response": "I have no further comments. I recommend final approval of this commentary."
}
]
},
{
"id": "10334",
"date": "29 Sep 2015",
"name": "Andrew D. Chalmers",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 piece by Mike Browning makes a clear proposal to reduce the problems associated with using research antibodies. He proposes a two-step process, where step 1 is to reduce variability and the second step is to improve validation. The main contribution seems to me to be highlighting the importance of variability- step 1.Overall it is nicely written, concise and makes a very useful contribution to the ongoing discussion about how to reduce the problems with research antibodies, something which is crucial to support progress and reproducibility in life sciences. I feel it is suitable for indexation.I had a couple of comments/questions about the commentary:A reasonable conclusion from the argument about variability seems to me that we should be working towards encouraging researchers to record and report lot numbers of the antibodies they use. Do you feel this would be beneficial? The suggestion of pooling positive serum seems very sensible. I would be interested to know how frequently is this done and if it is not done very often why is this?",
"responses": [
{
"c_id": "1659",
"date": "19 Oct 2015",
"name": "Mike Browning",
"role": "Reader Comment",
"response": "Many thanks for your comments Dr. Chalmers. You raise the issue of lot numbers and suggest that these numbers should also be recorded and reported. As I indicated in my comment to Dr. Valenzuela, lot numbers can be both a blessing and a curse. Unfortunately, the use of lot numbers sometimes masks a tendency among suppliers to fail to follow best practices that will eliminate most antibody variability. The pooling of serum is the first best practice that must be followed to eliminate variability. In my experience few suppliers or researchers do this. See for example Dr. Nairn’s discussion of serum pooling is his comment and also Vivien Marx’s article in Nature Methods (2013). This is due primarily to an increase in time and effort needed for pooling serum and also possibly to ignorance of best practices among some suppliers. The increase in time is due to the fact that either the research use or sale of the antibody must be delayed until all bleeds of the rabbits have been collected. The increase in effort is due to the need to screen all bleeds to determine which bleeds should be pooled. Many suppliers and researchers start using an antibody as soon as they get a positive bleed. Then when that bleed is exhausted they switch to the next etc. However there can be huge differences in the quality of the antibody in subsequent bleeds. Serum pooling must be used to avoid this form of lot-to-lot variability.There is a second even more serious form of antibody variability that is often obscured by simply changing the lot number. This results when an antibody supplier runs out of all the bleeds from the original animal(s). Some antibody suppliers then use new animals to make the antibody and simply denote this fact by changing the lot number. The potential differences between bleeds from the same animal pale beside the differences seen in different animals. It is not enough to simply change the lot number. The best practice is to modify the catalog number to reflect the new animal source."
}
]
},
{
"id": "10697",
"date": "06 Oct 2015",
"name": "Angus C. Nairn",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short commentary article presents an opinion on the ongoing discussion of quality control of commercial antibodies. This is an important topic given the importance of these reagents to progress in a wide range of biomedicine, and of the large economic costs involved in their successful use. But more importantly of the costs when they fail, are subject to variable success, or when they introduce error and artifact. The comments come from the CEO of PhosphoSolutions, one of many companies who provide and generate general and state-specific antibodies to a wide variety of proteins and epitopes, and who have a vested interest in providing a high quality product. Two general issues are discussed, namely antibody variability and antibody validation. These are closely interconnected aspects of quality control, and two reasonable solutions are presented. In terms of variability, which arises for a number of different reasons, including batch-to-batch differences in a single source reagent, to multiple variants of protein or epitope-specific reagents generated by different individuals or companies, the suggestion to provide a catalog number and supplier is a reasonable start. These sorts of detail should be a minimal requirement, and have been discussed in other recent initiatives (see for example Helsby et al cited; Bandrowski et al http://dx.doi.org/10.12688/f1000research.6555.1). But why not be more specific and provide full details of the characterization of the antigen, epitope if known, and precise lineage related to the origins of the reagent. Importantly, there needs to be specific information that indicates which particular batch is being sold. One could even consider some sort of unique bar code that would enable investigators to keep track electronically of the reagent they receive. Greater transparency and greater detail would benefit all parties involved. In terms of variability, the reasonable suggestion is made to pool batches to avoid batch to batch variation. The context of the suggestion is that the discussion here is most relevant to the generation of rabbit polyclonal antisera and it makes sense. But in logistical terms, typically initial studies of a particular rabbit serum is done on a pilot basis to establish quality, and antibodies are often affinity purified in batches. Decision points may vary depending on whether the best strategy is to pool sera, or pool batches of purified antibody. A key missing element is the establishment of robust criteria to establish a quantitative threshold of an acceptable quality that will consistently ensure success of whatever pools of serum or purified antibody are used eventually in the desired application, be it immunoblotting, immunohistochemistry or ELISA.\n\nA third short ending deals with the strengths and weaknesses of monoclonal versus polyclonal antibodies. This is really a different issue from the rest of the discussion of validation and variability which is most relevant to rabbit polyclonal antiserum/antibodies. I suggest that the material in the last paragraph be used as part of a slightly revised introduction and used to set up the context for the later discussion. Monoclonal antibodies clearly have the capacity to overcome the variability issue and are more routine to validate once an assay is standardized. The second sentence “This is unnecessary and unwise” would be best removed, and the strengths of polyclonal antibodies (price and amount per rabbit, superior use in different applications) still made. Another suggestion would be to remove the ambiguous comparison that “validation is the hardest nut to crack” while also saying “the variability problem” is most important. Better to say that variability and validation are two inter-connected issues, both of which need to have improved quality control.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-810
|
https://f1000research.com/articles/4-1492/v1
|
22 Dec 15
|
{
"type": "Review",
"title": "Management and Prevention of Anaphylaxis",
"authors": [
"Anne-Marie Irani",
"Elias G. Akl",
"Elias G. Akl"
],
"abstract": "Anaphylaxis prevalence has increased within the last few years. This may be due to a marked increase in allergic sensitization to foods especially in the pediatric population, as well as to an increase in outdoor recreational habits and the availability of new biologic medications. Furthermore, guidelines for the diagnosis of anaphylaxis have been published, thus facilitating the recognition of this disorder. Diagnosis of anaphylaxis is mainly based on history and clinical criteria of organ system involvement. The serum tryptase assay is now commercially available and may be a helpful diagnostic tool in certain clinical situations involving hypotension, but not in the context of food-induced anaphylaxis. Treatment of anaphylaxis mainly involves the use of epinephrine as a first line medication for severe manifestations followed by symptomatic management of specific symptoms, such as antihistamines for urticaria and albuterol for wheezing. Although commonly practiced, treatment with systemic corticosteroids is not supported by evidence-based literature. Observation in a medical facility for 4-6 hours is recommended to monitor for late phase reactions, although these rarely occur. Education is an essential component of management of a patient with a previous history of anaphylaxis, emphasizing early use of epinephrine and providing a written action plan. Referral to a board-certified allergist/immunologist is recommended to determine the cause of the anaphylaxis as well as to rule out other potential conditions. In this review, our main focus will be on the treatment and prevention of anaphylaxis while providing our readers with a brief introduction to the diagnosis of anaphylaxis, its prevalence and its most common causes.",
"keywords": [
"Anaphylaxis",
"management",
"prevention",
"epinephrine",
"hypersensitivity",
"food",
"exercise",
"asthma"
],
"content": "Introduction: Definition, Prevalence, and Common Triggers\n\nThere are several accepted definitions of the term anaphylaxis in the medical literature, all of which share the common characteristic of a severe, life-threatening, generalized hypersensitivity reaction1–3. The term anaphylactoid reaction is no longer used based on the World Allergy Organization’s recommendation to further define anaphylaxis into immunologic, immunoglobulin E (IgE) mediated, and non-immunologic reactions2. The lifetime prevalence of anaphylaxis is estimated at 1.6–5% based on a recent national telephone survey4,5. Food remains the most common overall outpatient cause of anaphylaxis across all ages combined6, accounting for 30% of cases of fatal anaphylaxis6. The most common foods that trigger anaphylaxis include peanuts, tree nuts, fish, and shellfish, with the addition of cow’s milk in children7,8. There has been a recent increase in patients with sesame seed anaphylaxis6. Among medications, antibiotics and primarily penicillin are most commonly involved in subjects aged 18 years and above5 followed by non-steroidal anti-inflammatory drugs (NSAIDs)9. Other drugs implicated include biologics and monoclonal antibodies10,11. Anaphylaxis to insect stings has occurred in 3% of adults and 1% of children who have been stung12,13. Other less common causes of anaphylaxis include seminal fluid and vaccines or vaccine components6. Exercise-induced anaphylaxis (EIAn) usually occurs with a co-trigger14–16 such as food ingestion. Patients with systemic mastocytosis are at increased risk of anaphylaxis from all causes, given the increase in mast cell burden. Anaphylaxis secondary to latex remains a concern in patients with spina bifida as well as in healthcare workers, but this has become less common, especially with hospitals using latex-free products throughout the country6. Patients on allergen immunotherapy (AIT) also carry a small risk of 0.25%–1.3% of anaphylaxis6,17,18, especially in the context of poorly controlled asthma6. The risk of a fatal reaction from AIT is estimated at around 1 in 2.5 million injections6,19,20. Idiopathic anaphylaxis remains a diagnosis of exclusion when extensive trigger identification fails.\n\n\nDiagnosis\n\nHistory: the diagnosis of anaphylaxis relies principally on the history, including the time course of the event, such as history of exposure to a particular trigger, the time course between exposure and development of symptoms, and the evolution of symptoms and signs over minutes to hours.\n\nDiagnostic criteria: the diagnostic criteria set forth by the National Institutes of Health (NIH) in 2006 were based on three clinical scenarios:\n\nFirst, in the absence of an allergen, anaphylaxis is diagnosed by a rapid onset (minutes to hours) of a reaction that involves the skin, mucosal tissue, or both, alongside at least one of the following symptoms: respiratory compromise, reduced blood pressure, or symptoms of end organ dysfunction.\n\nSecond, after a likely allergen exposure, two or more of the following occur: involvement of the skin or mucosal tissue, respiratory symptoms, decreased blood pressure, and/or gastrointestinal involvement.\n\nThird, in the case of a known allergen, reduced blood pressure alone is sufficient for the diagnosis of anaphylaxis1.\n\nLaboratory test: elevated serum tryptase levels can be detected within 15 minutes and up to 3 hours after the anaphylactic episode21–23. Levels greater than 11.5 ng/mL are considered elevated. Serum tryptase levels are rarely increased in the absence of shock or when food is the trigger6. Baseline elevations of serum tryptase levels should prompt consideration of the diagnosis of systemic mastocytosis24. A recent consensus document defined a significant acute elevation of serum tryptase to be equal to or greater than 1.2 times the baseline +2 ng/mL, indicating likely mast cell activation25.\n\n\nManagement\n\nThe management of anaphylaxis includes treatment of acute episodes and preventive measures including management of comorbidities, identification and avoidance of specific triggers, and select instances of immunomodulation.\n\nThe recommendations for acute treatment of anaphylaxis are largely based on expert opinion and consensus, as there are no randomized controlled studies for any of the pharmacologic therapies used.\n\nAll published guidelines clearly identify epinephrine as the first-line medication for the treatment of anaphylaxis1,6. Epinephrine 1:1000 (1 mg/mL) at a dose of 0.2–0.5 mg in adults and 0.01 mg/kg in children up to a maximum of 0.3 mg dosage should be used6. Injection in a large muscle, usually the lateral thigh, results in better absorption of the medication26. There are currently two commercially available doses of epinephrine autoinjectors in the United States: 0.15 mg (ideal for a 15 kg body weight) and 0.3 mg (ideal for a 30 kg body weight). In Europe, a third dose of 0.5 mg has been marketed but is not available for use in the US. It is common practice to prescribe the 0.15 mg dose to children weighing as low as 10 kg and the 0.3 mg dose to children after they reach a body weight of 24 kg. The practice parameters allow physicians to use epinephrine every 5–10 minutes and even at shorter intervals if deemed necessary6. It is important to remember that patients on oral or even ophthalmic beta-blockers might not adequately respond to epinephrine27,28. In these patients, isotonic saline and intravenous glucagon given at a dose of 1–5 mg in adults and 20–30 µg/kg in children, up to a maximum of 1 mg, should be given, followed by an infusion at a rate of 5–15 µg/minute titrated to clinical response29–31. Depending on the setting (healthcare versus at home), intravenous fluids should be initiated to maintain adequate circulation32,33.\n\nAnother important consideration, which is often ignored, is to position the patient in the Trendelenburg position (lying flat on the back with legs elevated) in order to allow blood flow to the heart and to prevent the \"empty ventricle syndrome\" described by Pumphrey34.\n\nOther supportive measures could be considered as second-line therapy. These include oxygen use, H1 and H2 antihistamines for the treatment of hives, and albuterol for the treatment of bronchospasm. We recommend using a non-sedating antihistamine as opposed to the common practice of prescribing diphenhydramine, as the sedative effect might obscure possible central nervous system symptoms. Corticosteroids are not useful for the acute treatment of anaphylaxis but may be effective in preventing biphasic or protracted anaphylaxis. As a result, many centers will administer a single dose of systemic corticosteroids (orally or intravenously) after the patient has been stabilized35,36. A prolonged 3 or 5 day course is not indicated. The frequency of occurrence of biphasic reaction has been reported to be from as low as 1% to as high as 23%6,37,38. These different estimates are likely due to varying definitions of anaphylaxis and the criteria used to identify a biphasic reaction. Using the NIH definitions for anaphylaxis in a retrospective chart review of two urban academic hospitals in Canada, Grunau et al. reported the incidence of a biphasic clinically important reaction to be 0.18%39. Currently, expert consensus recommends observation in the emergency room for a period of at least 6 hours after stabilization6,38,40–43. Patients should be discharged home with a prescription for an epinephrine autoinjector (EpiPen), along with instructions for self-administration and a referral to an allergy/immunology specialist for diagnosis and prevention.\n\nLong-term preventive measures include the recognition and management of risk factors for anaphylaxis in general, as well as measures directed to the specific triggers in particular.\n\nIt is important to identify and manage comorbid conditions that increase the risk of a severe anaphylactic reaction when poorly controlled. These include asthma, cardiovascular disease, and mastocytosis or mast cell activation syndrome. Furthermore, administration of certain medications such as beta-blockers may interfere with the therapeutic response to epinephrine as previously mentioned. Young children may not be able to recognize and report early symptoms of anaphylaxis, leading to a delay in administration of epinephrine. Adolescents and young adults often display risky behavior with regards to food avoidance and poor compliance in carrying the epinephrine autoinjector.\n\nThe next section will review preventive measures specific to the various diagnostic categories of anaphylaxis.\n\nFood-induced anaphylaxis. Avoidance of the confirmed food trigger requires lifelong vigilance, including education on reading food labels, informing family and friends, and caution while eating in public establishments. Given the difficulty in implementing complete food avoidance and the resultant negative effect on quality of life, clear and consistent information should be provided regarding the specific food triggers. In some patients, food challenges performed in a clinical setting may be necessary to assess the clinical significance of positive skin tests or serum IgE levels. Various forms of immunotherapy for food desensitization are currently being investigated, including oral, sublingual, and patch application44–51. Primary prevention of peanut allergy in high-risk infants with severe eczema and/or egg allergy was recently reported in a landmark study where early introduction of peanut between the ages of 4 and 11 months in infants with negative oral peanut challenge resulted in a rate of peanut allergy of 3% at 5 years of age compared to 17% in the group of infants who practiced peanut avoidance, an 86% relative risk reduction in infants with negative peanut skin tests52.\n\nMedication-induced anaphylaxis. As with foods, an accurate determination of the offending medication is needed. In situations when the patient is receiving multiple medications simultaneously a detailed history is crucial. Standardized skin testing is available for penicillin only53, although many protocols have been reported for other antibiotics and miscellaneous drugs54,55. Once identified, the offending medication should be avoided and alternative therapies used. In that respect, it is important to identify medications with potential cross-reactivity to the offending agent. If no alternative medication is adequate to treat the underlying condition, careful desensitization by administering incremental doses of the offending drug can be performed, often in an intensive care unit setting56,57. This procedure does not confer long-term tolerance to the drug, so future administration of the drug would once again require a desensitization procedure. Omalizumab, a monoclonal antibody against IgE, given subcutaneously for the treatment of difficult-to-control asthma as well as chronic spontaneous urticaria carries a 1 in 1000 risk of anaphylaxis, especially after the first three doses. Current expert opinion recommends injections to be administered at a medical facility, together with monitoring for 2 hours following the first three injections and for 30 minutes after subsequent injections. Patients are also advised to carry an epinephrine autoinjector for a period of 24 hours following the injections11.\n\nInsect sting anaphylaxis. Anaphylaxis to insects occurs in 3% of adults and 0.4–0.8% of children who are stung6,58. History, as always, is key in identifying the insect, correlating the onset of symptoms to the sting event and helping in avoidance of future stings. Different insects build nests in different places: hornets build large nests in trees, yellow jackets in the ground, and wasps under houses or barns. Honeybees usually leave a stinger and build nests in tree hollows. Wasps, yellow jackets, and hornets are scavengers and are likely to be encountered in picnic areas where food is available. Fire ants build their nests in soil and often sting in a circular pattern multiple times. Patients with a history of insect sting hypersensitivity should be educated on avoidance of stings, carry an epinephrine autoinjector, and obtain a consultation with an allergist/immunologist in order to undergo specific serum IgE testing and skin testing to identify the culprit insects. Randomized controlled trials have demonstrated the development of long-lasting protection against anaphylaxis in most patients who are treated with subcutaneous venom immunotherapy for a period of 3–5 years6,58–61. Venom extracts are available for honeybee, yellow jacket, white-faced hornet, yellow hornet, and wasp, and whole body extract is available for fire ant. Patients with mastocytosis and mast cell activation syndrome have an increased risk of anaphylaxis with insect sting, whereby the anaphylactic episode could be the presenting sign of the disorder62,63.\n\nExercise-induced anaphylaxis. EIAn, as the name implies, is anaphylaxis induced by physical activity. The mechanism behind it is still not entirely clear. Symptoms usually start within a few minutes after exercise and include fatigue, flushing, itching, and urticaria. If exercise continues, symptoms may progress in severity with angioedema of the airways and death64,65. Often, a co-trigger is required for symptoms to develop, such as a specific (or any solid) food, NSAIDs, menstruation, alcohol, or even pollen exposure in sensitized individuals14,66. The risk of anaphylaxis with exercise may occur within 4–6 hours of food and alcohol ingestion and within 24 hours after administration of NSAIDs. The most common food trigger in the USA is wheat, followed by grains and seafood14,15. Potential co-triggers could be identified through skin testing and exercise challenge testing despite its low sensitivity. It is imperative to identify the co-triggers in order to provide education on avoidance. H2 antagonists should be avoided, as preliminary data show that they might interfere with the normal digestion of food and potentially lead to a more severe reaction16,67. Therefore, prevention is individualized to the patient and to the co-triggers. These patients can exercise regularly once the co-trigger is avoided for a period of time prior to exercise. They should be counseled to exercise with a partner at all times and should carry epinephrine for autoinjection. If early signs or symptoms develop, the patient should stop exercising in order to avoid progression6.\n\nAllergy to galactose-alpha-1,3-galactose, also known as “alpha-gal”. More recently, a new cause of anaphylaxis has been linked to red meat consumption with a delayed onset of 3–5 hours or more after ingestion68,69. Patients usually report a history of a lone star tick bite 1–3 months prior to anaphylaxis70. The pathogenesis is due to the development of an IgE response to a mammalian oligosaccharide epitope, galactose-alpha-1,3-galactose, known as alpha-gal, present in the tick and conserved in mammalian meat70. A typical presentation would be a patient waking up in the middle of the night and collapsing on the way to the bathroom after ingestion of mammalian products for dinner. Episodes are sporadic70. There is a commercially available serum test to detect IgE against alpha-gal. Avoidance of mammalian meat is recommended as well as availability of an epinephrine autoinjector.\n\nIdiopathic anaphylaxis. Idiopathic anaphylaxis remains a diagnosis of exclusion after extensive history and testing to rule out specific triggers, including foods, exercise, medications, and insect hypersensitivity. Laboratory workup to look for evidence of mast cell activation is indicated. Serum tryptase levels obtained at baseline, as well as within 3–4 hours of an acute episode, can be helpful in demonstrating acute mast cell activation23–25. Other mast cell mediator measurements of urinary metabolites include N-methylhistamine, leukotriene E4, and prostaglandin F2 alpha (PGF2alpha). An elevated basal serum tryptase level suggests the diagnosis of systemic mastocytosis. A bone marrow biopsy can also be considered70,71. In patients with elevated serum PGD2 (or its urinary metabolite, PGF2alpha), treatment with 650 mg aspirin twice a day is recommended72,73. Treatment with high-dose prednisone, 60–100 mg daily for 1–2 weeks along with non-sedating H1 and H2 antihistamines, followed by tapering the dose of prednisone on alternate days over a period of 3 months has also been shown to decrease the frequency and severity of anaphylactic episodes, but results in high toxicity74,75. Multiple reports76–78, as well as our own experience, have shown that treatment with omalizumab, a monoclonal anti-IgE antibody, can lead to decreased frequency of episodes and is very well tolerated. An epinephrine autoinjector should be carried at all times.\n\n\nConclusions\n\nAnaphylaxis is a potentially life-threatening condition. Given its high prevalence, 2–5% of the population, physicians of all specialties are likely to be tasked with the recognition and management of anaphylactic episodes. In this regard, several consensus guidelines, including the American, European and World Allergy Organization guidelines, have been published to facilitate this task1–3. A careful history and specialized testing to identify potential triggers are paramount in preventing future events. Measurements of mast cell mediators in biologic fluids can improve the diagnostic accuracy of anaphylaxis. Epinephrine remains the mainstay of treatment for acute episodes. Emerging therapies include the use of omalizumab as well as allergen-specific immunotherapy.",
"appendix": "Competing interests\n\n\n\nDr Elias Akl declares no competing interests.\n\nDr Anne-Marie Irani: Virginia Commonwealth Institute receives royalties from ThermoFisher for the tryptase assay, which are shared with the author’s spouse.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nSampson HA, Muñoz-Furlong A, Campbell RL, et al.: Second symposium on the definition and management of anaphylaxis: summary report--Second National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network symposium. J Allergy Clin Immunol. 2006; 117(2): 391–7. PubMed Abstract | Publisher Full Text\n\nJohansson SG, Bieber T, Dahl R, et al.: Revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol. 2004; 113(5): 832–6. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nRavi A, Butterfield J, Weiler CR: Mast cell activation syndrome: improved identification by combined determinations of serum tryptase and 24-hour urine 11β-prostaglandin2α. J Allergy Clin Immunol Pract. 2014; 2(6): 775–8. PubMed Abstract | Publisher Full Text\n\nWilkin JK, Wilkin O, Kapp R, et al.: Aspirin blocks nicotinic acid-induced flushing. Clin Pharmacol Ther. 1982; 31(4): 478–82. PubMed Abstract | Publisher Full Text\n\nDitto AM, Harris KE, Krasnick J, et al.: Idiopathic anaphylaxis: a series of 335 cases. Ann Allergy Asthma Immunol. 1996; 77(4): 285–91. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWong S, Yarnold PR, Yango C, et al.: Outcome of prophylactic therapy for idiopathic anaphylaxis. Ann Intern Med. 1991; 114(2): 133–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJones JD, Marney SR Jr, Fahrenholz JM: Idiopathic anaphylaxis successfully treated with omalizumab. Ann Allergy Asthma Immunol. 2008; 101(5): 550–1. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWarrier P, Casale TB: Omalizumab in idiopathic anaphylaxis. Ann Allergy Asthma Immunol. 2009; 102(3): 257–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDemirtürk M, Gelincik A, Colakoğlu B, et al.: Promising option in the prevention of idiopathic anaphylaxis: omalizumab. J Dermatol. 2012; 39(6): 552–4. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11522",
"date": "22 Dec 2015",
"name": "Victòria Cardona",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11523",
"date": "22 Dec 2015",
"name": "Sally E. Wenzel",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1492
|
https://f1000research.com/articles/4-1490/v1
|
22 Dec 15
|
{
"type": "Review",
"title": "Recent advances in understanding synaptic abnormalities in Rett syndrome",
"authors": [
"Michael V. Johnston",
"Mary E. Blue",
"Sakkubai Naidu",
"Mary E. Blue",
"Sakkubai Naidu"
],
"abstract": "Rett syndrome is an extremely disabling X-linked nervous system disorder that mainly affects girls in early childhood and causes autism-like behavior, severe intellectual disability, seizures, sleep disturbances, autonomic instability, and other disorders due to mutations in the MeCP2 (methyl CpG-binding protein 2) transcription factor. The disorder targets synapses and synaptic plasticity and has been shown to disrupt the balance between glutamate excitatory synapses and GABAergic inhibitory synapses. In fact, it can be argued that Rett syndrome is primarily a disorder of synaptic plasticity and that agents that can correct this imbalance may have beneficial effects on brain development. This review briefly summarizes the link between disrupted synaptic plasticity mechanisms and Rett syndrome and early clinical trials that aim to target these abnormalities to improve the outcome for these severely disabled children.",
"keywords": [
"X-linked",
"Glutamate",
"Rett Syndrome"
],
"content": "Introduction\n\nRett syndrome (RTT) is characterized by severe neurodevelopmental delay in psychomotor development that occurs predominantly in girls and is due to mutations in the gene on the X chromosome that codes for the transcription factor methyl CpG-binding protein 2 (MECP2)1,2. MECP2 is a transcription factor and pleiotropic protein that regulates the expression of numerous genes, including brain-derived neurotrophic factor (BDNF)3, and can mediate transcriptional activation, mRNA splicing, and post-translational processing of microRNAs4,5. A phenotype similar to RTT has also been described in children with mutations in the CDKL5 and FOXG1 genes6,7. Girls with RTT often present with features similar to autism with little or no speech development, poor contact with others, and stereotyped movements, including characteristic repetitive hand-wringing movements8. Girls with RTT typically undergo a postnatal regression phase during the first 3 years after birth in which hand wringing, seizures, loss of hand use, and loss of expressive language emerge9,10. Clinicians who have seen patients with RTT are usually able to identify others because of their unique combination of physical and neurologic features. In addition to autistic features and hand-wringing movements, the characteristic behavioral abnormalities in RTT include severely erratic breathing while awake with periods of alternating hyperventilation, cyanosis and apnea, severe seizures and autonomic instability in heart rate and blood pressure, and changes in the color of the extremities consistent with sympathetic instability11. The incidence of sudden death is increased in girls with RTT12. Developing synapses appear to be a special target of the disorder, and head circumference is usually normal at birth but then falls behind over the first year of life so that it falls into the microcephalic range9,13–16. Neuropathologic examination of brain tissue from older girls with RTT who have died has revealed that neurons are typically too close together, suggesting a stunting of axonodendritic development17,18. This hypothesis is supported by studies of nasal epithelium from girls with RTT compared with controls, which showed that the neurogenesis of olfactory receptor neurons is normal but their maturation is blocked at the point of formation of synapses with the olfactory bulb19.\n\n\nRole of excitatory synapse abnormalities in Rett syndrome\n\nEarly studies of patients with RTT, including some who had not been tested for mutations in the MECP2 gene, suggested an abnormality of glutamate excitatory synapses18. For example, Hamberger et al.20 and Lappalainen and Riikonen21 in the 1990s reported that cerebrospinal fluid from girls with RTT had concentrations of glutamate but not other amino acids that were more than twice as high as those of children without RTT. Blue et al. in 1999 reported the first autoradiographic study of glutamate and GABA receptors in postmortem brain tissue from nine girls with RTT and 10 female controls of different ages22. The authors reported that densities of N-methyl-D-aspartate (NMDA) were elevated in the frontal lobe cortex in girls younger than 8 years of age but were significantly lower in girls older than 8 years without RTT23. Similar age-related changes in glutamate receptors were observed in the basal ganglia. Horská et al.24 used magnetic resonance spectroscopy to demonstrate that glutamate was elevated in the cerebral cortex of younger girls (less than 8 years of age) with RTT, especially those with seizures, but reduced in older girls. These changes in levels of glutamate between younger and older girls correlate with the bi-phasic changes in the clinical condition of girls with RTT who have more clinical signs of excitatory activity, including seizures early in early childhood, but have reduced signs of excessive central nervous system activity later in childhood9. More recently, Blue et al. demonstrated similar age-related changes in the NMDA1 subtype in the Bird model of RTT in mice25. Taken together, the elevations in glutamate as well as in glutamate receptors in younger girls with RTT suggest that there is an abnormality of the normal homeostatic synaptic scaling by which post-synaptic receptors adjust upwards or downwards to maintain a stable level of neurotransmission26. This abnormality in synaptic function probably contributes to deficits in excitatory synaptic plasticity and enhanced excitability observed in mouse models of RTT27–30.\n\nThe pathophysiology of elevated glutamate in the face of elevated NMDA glutamate receptors in RTT is not clear. The major regulator of glutamate levels in and outside the synapse is the activity of glial glutamate transporters, which normally are activated when glutamate is released into the synapse31. Intra-synaptic levels of glutamate are thought to provide trophic influences on post-synaptic neurons, but glutamate flooding outside the synapse can activate excitotoxicity mediated by extra-synaptic NMDA receptors32. Maezawa and Jin33 reported that conditioned media obtained from Mecp2-null microglia from Mecp2tm1.1Bird/+ mice34 contain toxic levels of glutamate that is damaging to neural dendrites in vitro and this effect can be blocked by inhibiting glutaminase or glutamate receptors. Jin et al., using the same model of Mecp2 deficiency, reported that abnormal function of the SNAT1 glutamine transporter in microglia is associated with NMDA receptor-mediated neurotoxicity, mitochondrial dysfunction, and decreased viability of microglia35. Okabe et al.36 reported that astroglia from Mecp2-null mice (Mecp2tm1.1Bird/+) had downregulation of glutamate transporters that could increase extracellular glutamate to toxic levels. Lioy et al.37 also reported that the restoration of wild-type MeCP2 in astrocytes of MeCP2-deficient mice (Mecp2Stop) restored normal dendritic morphology and increased levels of the excitatory glutamate transporter. We also found that cerebellar granule cells from Mecp2-null mice (Mecp2tm1.1Bird/+) are more sensitive to cell death from hypoxia-ischemia and glutamate excitotoxicity compared with neurons from wild-type mice, suggesting that they also have intrinsic vulnerability to cell death38. These findings are consistent with the above-cited evidence of abnormal homeostatic synaptic scaling, which is expected to degrade normal excitatory neurotransmission and activity-dependent neuronal plasticity needed for learning and memory in RTT.\n\n\nAbnormal diurnal regulation of sleep and brain glutamate levels in MeCP2 deficiency\n\nRecently, the circadian sleep cycle in rodents has been associated with alterations in brain glutamate levels, with higher glutamate levels associated with wakefulness and sleep associated with a reduction in glutamate39. We applied the relatively new technology that can measure 24-hour continuous electroencephalogram (EEG) markers of sleep cycles and correlated them with biosensor measurement of in vivo glutamate concentrations40. We found that mice with a Mecp2 knockout mutation (Mecp2tm1.1Bird/+) had abnormal behaviors and remarkably abnormal sleep cycles with long periods of sleeplessness associated with very high relative concentrations of brain glutamate. These Mecp2-deficient mice also had elevated baseline levels of cortical glutamate measured with a separate colorimetric method in postmortem tissue. These data suggest that periods of sleep disturbance that are common in girls with RTT could be associated with enhanced glutamate neurotoxicity.\n\n\nRole of GABA-containing interneurons in Rett syndrome\n\nGABA (gamma-amino butyric acid) is the predominant inhibitory neurotransmitter in the brain and is used as a transmitter at approximately 15% of synapses, and there is ample evidence of its altered metabolism in the mice with Mecp2 deficiency. Chao et al.41 reported that the selective deletion of MeCP2 in GABA interneurons (Viaat-Mecp22/y mice) led to a reduction in the pre-synaptic release of GABA as well as autism-like stereotypies and RTT phenotypes in mice. Kang et al.42 reported that there is a significant location-specific downregulation of synaptic GABA transporters in Mecp2 knockout mice (Mecp2tm1.1Bird/+), and El-Khoury et al.43 reported age- and region-specific reductions in function in GABAergic pathways in MeCP2-deficient mice (B6.129P2(c)-Mecp2.tm1–1Bird). These observations along with those from the glutamate system indicate that there is a shift in the balance between excitation and inhibition in favor of excitation, as has been suggested to occur in other autism spectrum disorders8,44.\n\nOne of the most interesting recent observations on the role of altered development in GABA and glutamate-containing neurons in mice with MeCP2 mutations relates to the link between MeCP2 and visual cortical plasticity45. Visual cortical plasticity associated with the occlusion of vision in one eye can be measured precisely in the laboratory in immature mice, and GABAergic interneurons play a critical role in this process46. Durand et al. reported that MeCP2-null mice (Mecp2tm1.1Bird/+) have normal visual function early in the postnatal period but that visual acuity regresses after postnatal day 35–40 and the cortex fell silent by postnatal day 55–6047. Remarkably, this effect could be prevented by genetic deletion of the NMDA glutamate receptor subunit NR2A. He et al. also reported that the genetic conditional deletion of MeCP2 in GABAergic parvalbumin-expressing neurons prevented local circuit inhibitory functions required for experience-dependent visual cortical plasticity48. Krishnan et al.45 reported that the MeCP2 regulates the timing of the critical period of plasticity in the primary visual cortex. Kron et al.49 have also examined brain activity maps by using activation of the immediate early gene Fos and electrophysiology and found evidence of synaptic hyperexcitability in the cortical default mode network that was reduced by the administration of the NMDA channel blocking anesthetic ketamine. The results of these studies support the themes raised earlier in this discussion of RTT, namely that MeCP2 deficiency leads to excessive excitation versus inhibition and defective activity-dependent synaptic plasticity. Although ketamine can cause damage to developing neurons by excessively blocking the activity of NMDA receptors50, its use in RTT is intended to reduce excessive activity of NMDA receptors found in RTT to normal levels. Both too little and too much activity at NMDA receptors can damage the developing brain51.\n\n\nPotential new therapies based on advances in understanding Rett syndrome\n\nSeveral clinical studies are examining potential therapies for girls with RTT on the basis of the basic science insights from experiments on mice with MeCP2 deficiency. Naidu et al. launched a randomized open-label study of oral dextromethorphan, a competitive NMDA antagonist, for seizures and cognitive function in girls with RTT from 2008 to 2014 (ClinicalTrials.gov: NCT00593957). Results of this study suggest a significant improvement in a secondary outcome of receptive speech by using the Mullen Scales of Early Learning over the course of’ 6 months but no change in seizures. Based on these promising preliminary data on cognition, Naidu et al. are carrying out a randomized double-blind study of dextromethorphan for girls with RTT which was started in January 2012 (ClinicalTrials.gov: NCT01520363) with support from the US Food and Drug Administration, and this study is still seeking subjects for enrollment. Other blockers of the NMDA receptor, including memantine and ketamine as mentioned above, are under discussion but have not been registered in ClinicalTrials.gov.\n\nPublished data are available on the administration of an active peptide of insulin-like growth factor 1 (IGF-1), which has been reported to be deficient in mice with Mecp2 deficiency52. IGF-1 activates tropomyosin receptor kinase B (TrkB) receptors and stimulates downstream signaling through the PI3K, AKT, and mTOR (mechanistic target of rapamycin) signaling pathway to stimulate protein synthesis53. Recent evidence also indicates that the positive effect of Mecp2 on mTOR is mediated by post-transcriptional processing of microRNAs that enhance mTOR activity so that Mecp2 depletion leads to a reduction in mTOR activity4.\n\nIGF-1 administration has been reported to improve behavioral functional recovery in Mecp2-deficient mice (Mecp21lox/+ females54) as well as spine density, synaptic amplitude, and PSD-95, a major constituent of the synaptic post-synaptic density in excitatory synapses55,56. Khwaja et al.52 recently reported a preliminary assessment of the use of recombinant human IGF-1 (mecasermin) in girls with RTT. Some behavioral parameters, including anxiety and mood and an EEG parameter, seemed to improve, and the medication appeared to be safe and well tolerated52. Recent preclinical data suggest that fingolimod, a sphingosine-1 phosphate receptor modulator that can increase levels of BDNF, can also reduce signs of disease in a mouse model of RTT57. Valproic acid has also recently been reported to have a positive behavioral effect on mice with Rett mutations and phenotype58.\n\n\nConclusions\n\nMice with Mecp2 deficiency display a variety of aberrant behaviors and synaptic abnormalities that correlate very well with those in human RTT. Girls with RTT and mice with Mecp2 deficiency display elevations in both glutamate and NMDA glutamate receptors and this is likely to produce abnormal homeostatic synaptic scaling and probably contributes to impaired activity-dependent synaptic plasticity. RTT appears to involve an imbalance between elevated glutamate levels and reduced GABA levels, similar to models of autism spectrum disorders. Chaotic sleep in mice with Mecp2 deficiency has been shown to correlate with elevated baseline glutamate levels in the brain and very high elevations in glutamate associated with prolonged wakening. Several clinical studies are under way to attempt to normalize synaptic abnormalities in RTT, including the studies by Naidu et al. with dextromethorphan, a clinically approved competitive NMDA receptor blocker. A randomized un-blinded trial of dextromethorphan showed improvement in receptive language in girls with RTT, and a randomized trial of this drug is under way. Preclinical studies suggest that low-dose ketamine, a non-competitive NMDA blocker, might be useful for improving the connectivity of brain circuits affected in RTT and improving function. Human recombinant IGF-1 has also shown benefit in mice with Mecp2 deficiency, and a preliminary study in girls with RTT showed that it was well tolerated and may have some benefits.",
"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 FDA Grant 5RO1FD004247 and NIH-NICHD U54 HD079123.\n\n\nReferences\n\nChen L, Chen K, Lavery LA, et al.: MeCP2 binds to non-CG methylated DNA as neurons mature, influencing transcription and the timing of onset for Rett syndrome. Proc Natl Acad Sci U S A. 2015; 112(17): 5509–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaidu S, Johnston MV: Neurodevelopmental disorders: Clinical criteria for Rett syndrome. Nat Rev Neurol. 2011; 7(6): 312–4. PubMed Abstract | Publisher Full Text\n\nChapleau CA, Larimore JL, Theibert A, et al.: Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism. J Neurodev Disord. 2009; 1(3): 185–96. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nFagiolini M, Fritschy JM, Löw K, et al.: Specific GABAA circuits for visual cortical plasticity. Science. 2004; 303(5664): 1681–3. PubMed Abstract | Publisher Full Text\n\nDurand S, Patrizi A, Quast KB, et al.: NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2. Neuron. 2012; 76(6): 1078–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe LJ, Liu N, Cheng TL, et al.: Conditional deletion of Mecp2 in parvalbumin-expressing GABAergic cells results in the absence of critical period plasticity. Nat Commun. 2014; 5: 5036. PubMed Abstract | Publisher Full Text\n\nKron M, Howell CJ, Adams IT, et al.: Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment. J Neurosci. 2012; 32(40): 13860–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSlikker W Jr, Liu F, Rainosek SW, et al.: Ketamine-Induced Toxicity in Neurons Differentiated from Neural Stem Cells. Mol Neurobiol. 2015; 52(2): 959–69. PubMed Abstract | Publisher Full Text\n\nMcDonald JW, Johnston MV: Physiological and pathophysiological roles of excitatory amino acids during central nervous system development. Brain Res Brain Res Rev. 1990; 15(1): 41–70. PubMed Abstract | Publisher Full Text\n\nKhwaja OS, Ho E, Barnes KV, et al.: Safety, pharmacokinetics, and preliminary assessment of efficacy of mecasermin (recombinant human IGF-1) for the treatment of Rett syndrome. Proc Natl Acad Sci U S A. 2014; 111(12): 4596–601. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBanerjee A, Castro J, Sur M: Rett syndrome: genes, synapses, circuits, and therapeutics. Front Psychiatry. 2012; 3: 34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen RZ, Akbarian S, Tudor M, et al.: Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice. Nat Genet. 2001; 27(3): 327–31. PubMed Abstract | Publisher Full Text\n\nTropea D, Giacometti E, Wilson NR, et al.: Partial reversal of Rett Syndrome-like symptoms in MeCP2 mutant mice. Proc Natl Acad Sci U S A. 2009; 106(6): 2029–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastro J, Garcia RI, Kwok S, et al.: Functional recovery with recombinant human IGF1 treatment in a mouse model of Rett Syndrome. Proc Natl Acad Sci U S A. 2014; 111(27): 9941–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeogracias R, Yazdani M, Dekkers MP, et al.: Fingolimod, a sphingosine-1 phosphate receptor modulator, increases BDNF levels and improves symptoms of a mouse model of Rett syndrome. Proc Natl Acad Sci U S A. 2012; 109(35): 14230–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuo W, Tsujimura K, Otsuka IM, et al.: VPA alleviates neurological deficits and restores gene expression in a mouse model of Rett syndrome. PLoS One. 2014; 9(6): e100215. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11698",
"date": "22 Dec 2015",
"name": "Zhi-Qi Xiong",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11699",
"date": "22 Dec 2015",
"name": "Thierry Bienvenu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1490
|
https://f1000research.com/articles/4-1484/v1
|
21 Dec 15
|
{
"type": "Software Tool Article",
"title": "FlyOde - a platform for community curation and interactive visualization of dynamic gene regulatory networks in Drosophila eye development",
"authors": [
"Stefan A. Koestler",
"Begum Alaybeyoglu",
"Christian X. Weichenberger",
"Arzu Celik",
"Begum Alaybeyoglu",
"Christian X. Weichenberger",
"Arzu Celik"
],
"abstract": "Motivation: Understanding the regulatory mechanisms governing eye development of the model organism Drosophila melanogaster (D. m.) requires structured knowledge of the involved genes and proteins, their interactions, and dynamic expression patterns. Especially the latter information is however to a large extent scattered throughout the literature.Results: FlyOde is an online platform for the systematic assembly of data on D. m. eye development. It consists of data on eye development obtained from the literature, and a web interface for users to interactively display these data as a gene regulatory network. Our manual curation process provides high standard structured data, following a specifically designed ontology. Visualization of gene interactions provides an overview of network topology, and filtering according to user-defined expression patterns makes it a versatile tool for daily tasks, as demonstrated by usage examples. Users are encouraged to submit additional data via a simple online form.",
"keywords": [
"Drosophila development",
"gene regulatory network",
"protein interaction network",
"visualization",
"web-tool",
"database",
"community annotation",
"ontology"
],
"content": "Introduction\n\nDevelopmental biology is the study of processes that generate an entire organism from a single cell. A central question in this field is how differentiation produces specific cell types from pluripotent precursors. Drosophila melanogaster (D. m.) serves as a suitable and well-established model organism to address this question for numerous reasons including a short generation time, the multitude of available genetic methods, and its orthology shared with vertebrates (Reiter et al., 2001). The D. m. eye allows the study of morphological rearrangements as well as differentiation of non-neuronal and neuronal cell types like photoreceptors (PRs) on the single cell level (Thomas & Wassarman, 1999).\n\nUnderstanding cell differentiation requires knowledge of the involved genes, their temporally varying (dynamic) expression patterns, and interactions. Interaction data from different sources are accessible through e.g. Biogrid, Intact, String, and REDfly (Chatr-Aryamontri et al., 2015; Gallo et al., 2011; Orchard et al., 2014; Szklarczyk et al., 2015) and database collections, e.g. iRefindex or mentha (Calderone et al., 2013) (Razick et al., 2008). Expression data is mostly provided on embryonic development or organ systems, e.g. by FlyBase (dos Santos et al., 2015) and the Berkeley Drosophila Genome Project (BDGP) (Hammonds et al., 2013), but the coverage and precision of expression pattern annotation on the cellular level and the temporal resolution at later stages, e.g. larva and pupa, are limited (see Supplementary Material). On the other hand, a wealth of expression pattern data on these levels is contained in publications (e.g. (Potier et al., 2014)). Systematic use of these data requires their structured assembly through an extensive curation effort. Since automated curation is prone to errors (Mao et al., 2014), information must be extracted manually from the literature by experts, who can critically interpret the respective types of data like micrographs or expression profiles (Tomancak et al., 2007) and convert these to machine-readable data for further computer-based analyses.\n\nWe have thus developed FlyOde, an online hub for the community-driven systematic assembly of data on D. m. eye development. FlyOde is a web interface for interactive exploration of gene and protein relationships by combining visualization of the curated gene regulatory network with filters specific to fly development. FlyOde is built on an ontology-driven curation process that stores data in a specifically formatted text file, which can easily be enriched and extended upon arrival of new data.\n\n\nImplementation\n\nA directed gene interaction network representing eye development of D. m. from the third instar larva to the adult with focus on PR differentiation was constructed using Cytoscape (Shannon et al., 2003), based on data extracted manually from 77 publications (Figure 1). Currently, the network contains 146 nodes representing genes/proteins, and 284 edges representing activating or inhibiting genetic, protein-protein, or protein-DNA interactions. The layout is generated manually and organized to approximately represent developmental time along the horizontal axis, beginning with early third instar larval stage on the left, and network hierarchy along the vertical axis with master regulators (as defined by (Chan & Kyba, 2013)) placed towards the top. Nodes are associated with their FlyBase symbol, name, alternative names, FlyBase link, dynamic expression pattern, phenotypes, the terms for each of the three Gene Ontologies (GO) (Ashburner et al., 2000), and the literature references. GO annotations were added using Cytoscape. Expression pattern annotation follows a specifically developed ontology which links developmental stage and cell type (Figure 2). We support and encourage annotation from the community to continually extend the dataset.\n\nThe red circle indicates the eyeless gene (see Example 1). The inset shows selected interactions containing network motifs: negative autoregulation and feedback loop (left) and an incoherent feed forward loop (right).\n\nHierarchical representation of controlled vocabulary for the gene annotation with dynamic expression patterns is shown. In the annotation syntax terms for cell type and developmental stage are directly linked. Each term-combination represents expression in a specific cell type and developmental stage. As many comma-separated term combinations as needed to describe the expression pattern are given. During retinal development of Drosophila, cells at consecutive stages of differentiation coexist at the same developmental stage of the organism. The morphogenetic furrow marks the initiation of differentiation and moves across the eye precursor organ, the eye imaginal disc. Thus, the expression pattern is also defined by the localization relative to the morphogenetic furrow. This and additional terms that are common in the field are assigned to what we call “differentiation level”.\n\nThe interactive JavaScript based web application renders the manually curated network, which is embedded using Cytoscape Web (Lopes et al., 2010). Genes are represented as nodes and gene (or protein) interactions are visualized as directed edges. Nodes and edges have been annotated during the curation process (see previous section), and this information is displayed when an item is selected. For the selection of nodes a module for searching gene symbols, names, alternative names, and GO-terms is provided. Additionally, the network can be filtered according to gene expression patterns using dropdown menus whose structure follows the FlyOde ontology (Figure 2). Filters can be combined with the Boolean operators AND, OR, and NOT. Extensions to the network can be submitted to us via a community curation form.\n\n\nCharacterization\n\nTo characterize the content of the current FlyOde network GO enrichment analysis was performed with the ClueGO app in Cytoscape (Bindea et al., 2009). It shows that terms correlated with development and morphogenesis, pattern formation and polarity, apoptosis, mitosis, and regulation of transcription are highly over-represented, as expected (see Supplementary Materials and Supplementary Figure 1 and Supplementary Figure 2).\n\nTo evaluate the information that can be obtained from FlyOde the FlyOde filter function was compared with the FlyBase QueryBuilder. When queried for general terms like “larva and PR”, FlyBase gave more gene hits than FlyOde, but when increasingly specifying the developmental stage and cell type, like “pupa and dorsal rim area R8 (photoreceptor cell 8)”, more genes were found with FlyOde as compared to FlyBase. This shows that FlyOde already stands out in defining gene expression in a specific cell type and during a specific developmental stage of PR differentiation (see Supplementary Materials and Supplementary Figure 3).\n\nTo get an idea of the basic mechanisms represented in FlyOde, the occurrence of network motifs was analysed manually in Cytoscape. Motifs commonly found during development of organisms, like autoregulation, feedback and feed forward loops were observed. This indicates that FlyOde at least partially displays the connectivity and level of detail to qualify for representation and detailed analysis of developmental processes (see Figure 1 and Supplementary Materials, Supplementary Figure 4 and Supplementary Table) (Alon, 2007; Davidson, 2010).\n\n\nUse cases\n\nIn this example we use the search functionality to explore a specific gene in the dataset. In order to do so, Pax6 is entered into the search box. This gene is known under that name as an important regulator of development in many organisms (van Heyningen, 2002). However, the official name in D. m. is eyeless. Due to its annotation with alternative names it is found despite the search for the unofficial name Pax6 and highlighted in the network (Figure 1). Its position on the left side of the graph indicates that it is mainly expressed early during eye development. In the web interface additional information is displayed in a content related text field below the graph, which we designate “report panel”. A closer look at the expression pattern displayed there shows that it is expressed anterior to the morphogenetic furrow, in all photoreceptors in the early third instar larva, and in outer photoreceptors in the late pupa and adult. The top position in the hierarchy indicates that it is a master regulator. This is supported by its high number of interactions, and by the phenotypes, which are given in the report panel (Chan & Kyba, 2013). We also find that GO annotates eyeless as a transcription factor, and that it is involved in developmental processes of other organs. Finally, the literature references and the FlyBase link can be followed for further information.\n\nHere we apply filter combinations to display genes that are expressed in R8, but not in any other PR in the intermediate pupa to obtain candidate markers for R8 in immunostaining and cell sorting experiments. In the dropdown menus we choose “pupa”, “intermediate pupa”, “photoreceptor cell”, and “R8”, respectively. We add another filter line with the Boolean “NOT”, and in the dropdown menus select “intermediate pupa”, “photoreceptor cell”, “R7”, and another filter line with the Boolean “NOT”, “intermediate pupa”, and “at least one outer PR”, which in combination with “NOT” means “no outer PR”. All nodes are removed except for scabrous and senseless, which are therefore candidates for being R8 markers. The literature references for these two markers provide a starting point for future experimental studies.\n\nMore examples are provided in the tutorial at http://flyode.boun.edu.tr/quickguide.html.\n\n\nDiscussion\n\nHere we have presented FlyOde, which provides a platform for combining published data on gene regulatory networks (GRNs) of Drosophila organ development. As a starting point, we have equipped FlyOde with extensive GRN data for D. m. eye development, such that the web interface serves as a versatile tool for everyday tasks a fly researcher encounters.\n\nFlyOde delivers high quality data standards by manual curation. We expect to achieve efficient data collection by distributing the annotation workload among community members with minimal effort for the individual contributors, who only need to submit a simple form to add new nodes, interactions, or annotation. In return, they directly profit from the improved tool by linking their data of interest with the shared knowledge.\n\nWe are constantly improving the ontology and web application, in parallel to ongoing data curation and dataset extension. FlyOde will be expanded to include other organs with the ultimate goal to compare their GRNs.\n\nFuture work will profit from the obtained data by constraining network inference from gene expression data (Hecker et al., 2009). Another anticipated approach is to assign quantitative expression data to the established network to facilitate mathematical modelling (Graham et al., 2010).\n\nWe envision FlyOde as a companion that guides researchers through developmental processes, for example while studying a paper, and believe that the community-curated dataset and its analysis will add significant knowledge to developmental biology.\n\n\nData availability\n\nFlyOde, including all network data, annotations and their corresponding references can be freely accessed via the web-application at http://flyode.boun.edu.tr/.\n\n\nSoftware availability\n\nThe source code for the web application can be downloaded from Github (https://github.com/begum-alaybeyoglu/FlyOde).\n\nhttps://github.com/F1000Research/FlyOde\n\nhttp://dx.doi.org/10.5281/zenodo.35227\n\nThe FlyOde web application is released under the MIT License.",
"appendix": "Author contributions\n\n\n\nS.A.K. established the concept, network, annotation and ontology, performed analysis and wrote the manuscript. B.A. generated the web application. C.X.W. performed analysis and provided input during manuscript preparation. A.C. coordinated the project. All authors contributed to proofreading the manuscript and have agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nS.A.K. was supported by TÜBITAK 2236 Co-Funded Brain Circulation Scheme.\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 Stefan Fuss and the Bogazici University Computer Centre for help with the server and Elif Özkirimli (all Bogazici University, Istanbul) for proofreading the paper.\n\n\nSupplementary Material\n\nSupplementary Figure 1.\n\nGO-term enrichment analysis with ClueGO.\n\nClick here to access the data.\n\nAnalysis and characterization of the FlyOde network.\n\nIncludes Supplementary Figures 2, 3, 4.\n\nClick here to access the data.\n\n\nReferences\n\nAlon U: Network motifs: theory and experimental approaches. Nat Rev Genet. 2007; 8(6): 450–461. PubMed Abstract | Publisher Full Text\n\nAshburner M, Ball CA, Blake JA, et al.: Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25(1): 25–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBindea G, Mlecnik B, Hackl H, et al.: ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics. 2009; 25(8): 1091–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalderone A, Castagnoli L, Cesareni G: mentha: a resource for browsing integrated protein-interaction networks. Nat Methods. 2013; 10(8): 690–1. PubMed Abstract | Publisher Full Text\n\nChan SS, Kyba M: What is a Master Regulator? J Stem Cell Res Ther. 2013; 3: pii: 114. 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\nDavidson EH: Gene Regulatory Networks. In Development And Evolution. Academic Press, 2010.\n\nGallo SM, Gerrard DT, Miner D, et al.: REDfly v3.0: toward a comprehensive database of transcriptional regulatory elements in Drosophila. Nucleic Acids Res. 2011; 39(Database issue): D118–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGraham TG, Tabei SM, Dinner AR, et al.: Modeling bistable cell-fate choices in the Drosophila eye: qualitative and quantitative perspectives. Development. 2010; 137(14): 2265–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHammonds AS, Bristow CA, Fisher WW, et al.: Spatial expression of transcription factors in Drosophila embryonic organ development. Genome Biol. 2013; 14(12): R140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHecker M, Lambeck S, Toepfer S, et al.: Gene regulatory network inference: data integration in dynamic models-a review. Biosystems. 2009; 96(1): 86–103. PubMed Abstract | Publisher Full Text\n\nvan Heyningen V, Williamson KA: PAX6 in sensory development. Hum Mol Genet. 2002; 11(10): 1161–1167. PubMed Abstract | Publisher Full Text\n\nLopes CT, Franz M, Kazi F, et al.: Cytoscape Web: an interactive web-based network browser. Bioinformatics. 2010; 26(18): 2347–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMao Y, Van Auken K, Li D, et al.: Overview of the gene ontology task at BioCreative IV. Database (Oxford). 2014; 2014: pii: bau086. PubMed Abstract | Publisher Full Text | Free Full Text\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\nPinoli P, Chicco D, Masseroli M: Computational algorithms to predict Gene Ontology annotations. BMC Bioinformatics. 2015; 16(Suppl 6): S4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPotier D, Davie K, Hulselmans G, et al.: Mapping gene regulatory networks in Drosophila eye development by large-scale transcriptome perturbations and motif inference. Cell Rep. 2014; 9(6): 2290–2303. PubMed Abstract | Publisher Full Text\n\nRazick S, Magklaras G, Donaldson IM: iRefIndex: a consolidated protein interaction database with provenance. BMC Bioinformatics. 2008; 9: 405. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReiter LT, Potocki L, Chien S, et al.: A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res. 2001; 11(6): 1114–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\ndos Santos G, Schroeder AJ, Goodman JL, et al.: FlyBase: introduction of the Drosophila melanogaster Release 6 reference genome assembly and large-scale migration of genome annotations. Nucleic Acids Res. 2015; 43(Database issue): D690–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–504. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzklarczyk D, Franceschini A, Wyder S, et al.: STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2015; 43(Database issue): D447–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThomas BJ, Wassarman DA: A fly’s eye view of biology. Trends Genet. 1999; 15(5): 184–190. PubMed Abstract | Publisher Full Text\n\nTomancak P, Berman BP, Beaton A, et al.: Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 2007; 8(7): R145. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "12186",
"date": "11 Feb 2016",
"name": "Pelin Cayirlioglu Volkan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFlyOde is a very nice resource for labs, which are interested in visualizing developmental dynamics of gene regulatory networks required for the development for many neuronal types in the Drosophila eye, in addition to finding lineage specific markers. It will be valuable in the future to expand this to other sensory systems such as the olfactory system. The title and the abstract are appropriate. The video presentation is very useful.",
"responses": []
},
{
"id": "12188",
"date": "12 Feb 2016",
"name": "Bassem A Hassan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 nice and useful tool to quickly and efficiently access information about fly retina development. The fact that it can be extended to any tissue and is an open platform for community-based annotation makes it all the more useful. I applaud the authors for their efforts and thank them for making such a cool and intuitive tool available. The video tutorial is very nice and helpful as well.",
"responses": []
},
{
"id": "11677",
"date": "15 Feb 2016",
"name": "Fernando Casares",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Drosophila eye gene regulatory network ranks among the best textbook examples of organogenetic GRNs. FlyODE, by Koestler and colleagues is a welcomed attempt at integrating the many bits of expression, regulation, molecular interactions and functional analyses obtained to date, by providing a cytoscape-like tool in the form of a curated, directed graph network. FlyODE’s also allows the expansion of this network through community-based contributions –which is a great philosophy. Therefore FlyODE is a valuable tool for those working on gene networks in general and on fly eyes in particular, and will help in expanding the network (and our knowledge of the system) further. FlyODE includes references to spatial gene expression patterns. It would be good if the authors tried to unify the somehow ambiguous terms used by different labs, such as “preproneural/precursors/anterior to the MF” or “proneural/immediately anterior to the MF”, or perhaps provide some “equivalence” terms. When sorting genes according to their expression along third instar, I found that the network includes more genes at early L3 than at mid-L3, which shouldn’t be the case –as the process unfolds more genes are called into action.",
"responses": [
{
"c_id": "1813",
"date": "15 Feb 2016",
"name": "Stefan A. Koestler",
"role": "Author Response",
"response": "Thank you very much for your feedback!We are working on improving the ontology, e.g. by adjusting it to the Gene Ontology, and will incorporate your suggestions.The layout is currently done manually and is certainly to some extent biased depending on the references used for annotation. This can be expected to improve with an increasing number of annotations/references. In addition, we are planning an automatic layout to calculate the position from the annotations. In any case one has to keep in mind that it will only indicate a single (mean) value, while a gene might display a more complex expression pattern. Thus, currently one has to look at the report panel below the network for more detailed information. We are thinking about adding a graph displaying the expression profile when hovering over a node to make this information more intuitively accessible."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1484
|
https://f1000research.com/articles/4-1483/v1
|
21 Dec 15
|
{
"type": "Case Report",
"title": "Case Report: Postpartum hemorrhage associated with Dengue with warning signs in a term pregnancy and delivery",
"authors": [
"Le Phi Hung",
"Tran Diem Nghi",
"Nguyen Hoang Anh",
"Mai Van Hieu",
"Nguyen Thien Luan",
"Nguyen Phuoc Long",
"Than Trong Thach",
"Le Phi Hung",
"Tran Diem Nghi",
"Nguyen Hoang Anh",
"Mai Van Hieu",
"Nguyen Thien Luan"
],
"abstract": "Background: Dengue infection during peripartum period, although rare in endemic regions, has challenged clinicians regarding its management, especially if a parturient woman experiences postpartum hemorrhage due to a classical risk factor of maternal bleeding.Case: A full-term pregnant Vietnamese woman was diagnosed with polyhydramnios and Dengue with warning signs (DWS). She was administered platelet transfusion prior to delivery and then gave birth to a healthy newborn. After active management of the third stage of labor, the patient suffered a postpartum hemorrhage which was caused by uterine atony and accompanied with thrombocytopenia. Therefore, we decided to administer uterotonic drugs and additionally transfuse platelets.Conclusion: We describe a case of postpartum hemorrhage caused by uterine atony and coinciding with Dengue infection during delivery period, which is a rare clinical entity. With timely detection and management, the patient was finally discharged without complications.",
"keywords": [
"Dengue with warning signs",
"pregnancy",
"uterine atony",
"postpartum hemorrhage",
"polyhydramnios"
],
"content": "Introduction\n\nDengue infection in parturient women living in endemic regions only accounts for 2.5% of all pregnancies1. Besides the possibility of vertical transmission to newborns, Dengue infection may act as one of the potential risk factors of massive postpartum hemorrhage2 as well as many other serious complications for mothers. Particularly, during the peripartum period, if the mother simultaneously experiences any bleeding conditions such as cesarean section, the hemorrhage might be exacerbated and life-threatening. Even though postpartum hemorrhage is one of the most aggressive obstetric complications, especially when it coexists with Dengue infection, there have been few papers in the medical literature reporting the management of such cases.\n\nWe hereby present a pregnant Vietnamese woman who neither respected the antenatal appointment schedule nor fulfilled prenatal tests. Prior to hospitalization in our obstetric hospital, she was diagnosed with Dengue with warning signs. The parturient woman, at the same time, suffered from polyhydramnios and uterine atony. After her delivery of a completely healthy infant, she suddenly had a severe postpartum hemorrhage. Owing to effective resuscitation, the patient and her neonate were finally discharged in stable condition. The one-week follow-up did not show any abnormalities.\n\n\nThe case\n\nA 34-year-old gravida III woman presented to our hospital at 39.5 weeks of gestation with no significant medical history. During her pregnancy period, she did not regularly follow the consultant schedule nor fulfill prenatal tests. At 39 weeks of gestation, the parturient woman was reported to have polyhydramnios by ultrasound and then admitted to a provincial hospital for observation. Unfortunately, we could not obtain the images confirming her polyhydramnios state.\n\nOn her fourth inpatient day in the provincial hospital, she suddenly had a continuous high-grade fever (39°C) associated with myalgia and arthralgia. The first complete blood count result showed a platelet count of 69.000/mm3 and hematocrit of 38.8%. The patient was suspected to have Dengue and received symptomatic and supportive treatment with acetaminophen and intravenous fluids for 3 days. Vital signs and bleeding manifestations were also closely monitored. On the seventh inpatient day, the body temperature declined and then remained stable at 37°C. In the same day, the patient noted small petechiae sized 1–2 mm in diameter, around her arms and forearms. The platelet count at this time was 50.000/mm3 and hematocrit was 40.8%. One day later, the patient abruptly experienced an intermittent hypogastric pain and had vaginal bleeding. She was transferred to our tertiary obstetric hospital where she was confirmed to have Dengue with warning signs.\n\nOn the day of admission, the patient was alert and the vital signs were unremarkable (blood pressure of 120/80 mmHg, temperature of 37°C, pulse of 100 beats/min and respiration rate of 20 breaths/min). On obstetric examination, the clinician recorded a 33 cm fundal height, fetal heart rate of 142 beats/min. The cervix was 2 cm dilated and 60% effaced. Regarding laboratory analysis, the rapid test for Dengue diagnosis revealed the following results: NS1 antigen (+), IgM Dengue (+) and IgG Dengue (-). Hematocrit increased to 45.2% and the platelet count showed a continuously downward trend to 32.000/mm3. Liver and renal function tests as well as coagulation workup revealed no abnormalities. In the delivery room, the patient was administered 2 units of packed platelet concentrate in around 30 minutes before giving birth. The patient successfully delivered a healthy female infant weighing 3.500 grams by vaginal route with APGAR score of 8 and 9 after 1 and 5 minute(s), respectively. A uterine atony and a first-degree vaginal tear were recorded, leading to the loss of about 200 ml of blood. The third stage of labor was actively managed, then the perineal laceration was sutured and the retained products of conception were excluded. After that, the uterus was well contracted and a blood pressure of 120/72 mmHg and pulse of 86 beats/min were noted. The mother and newborn were transferred to the waiting room for observation. After 10 hours, the uterine contractions decreased suddenly and became boggy; the total amount of blood loss was about 1000 ml. The patient was diagnosed with postpartum hemorrhage due to uterine atony. Intravenous fluid resuscitation with 500 ml of 0.9% NaCl in two hours and a half was implemented. 20 IU of oxytocin diluted with 500 ml of Lactated Ringer’s solution was also administered intravenously with the infusion rate of 3 ml/min. In addition, the patient was given a dose of 200 mcg Methylergometrine by intramuscular injection. After 2 hours of intensive treatment, there was no further bleeding episodes and the normal range of vital signs were recorded. Repeated tests after childbirth showed moderate thrombocytopenia with a platelet count of 47000/mm3 but no abnormal coagulation was detected. After bleeding was well-controlled with oxytocin, the clinicians decided to transfuse one more unit of platelets in order to increase the patient’s platelet concentration to a safe range. The platelet count and hematocrit were 52.900/mm3 and 35.9% respectively, after transfusion. The patient was transferred to the department of postpartum care. The patient gradually recovered without any abnormality. The laboratory findings of the newborn were unremarkable, especially the negative serological test for detecting Dengue infection. Consequently, the patient and her neonate were discharged in a stable state after 7 inpatient days in our hospital. The one-week follow-up also did not show any abnormal conditions.\n\n\nDiscussion\n\nSeveral studies have suggested that Dengue infection can predispose full-term pregnant women to postpartum hemorrhage, even massive bleeding. Chotigeat et al. reported one case of a patient suffering from Dengue shock syndrome (DSS) which later developed postpartum hemorrhage4. Thaithumyanon et al. described a of heavy bleeding following a C-section3. The report of Chye JK. et al. described a parturient woman with substantial thrombocytopenia and abnormal coagulation which required intensive treatment5. Our case report, however, may be the first detailed description of a severe postpartum hemorrhagic case involving uterine atony6. Postpartum hemorrhage arose after vaginal delivery and was complicated by thrombocytopenia secondary to DWS. Diagnosis of DWS in late pregnancy requires a high degree of clinical suspicion because signs and symptoms frequently seen in Dengue may be confused with preeclampsia complicated by HELLP syndrome5,7. Moreover, physiological hemodilution in late pregnancy8 may obscure the degree of vascular leakage and challenge clinicians to evaluate it as well as to treat the patient appropriately. In our case, the patient experienced high fever, marked thrombocytopenia, hematocrit above the upper limit of the normal value and particularly, no clinical or laboratory findings suggesting preeclampsia with HELLP syndrome; this made the diagnosis of Dengue more probable. The diagnosis was confirmed by positive results from NS1 antigen and IgM/IgG rapid tests. We chose the rapid diagnosis test due to the fact that our laboratory’s resources were limited and the patient needed emergency management.\n\nVaginal delivery may be allowed without prophylactic platelet transfusion even though platelet count falls below 50000/mm36. Concern about the risk of profound bleeding due to uterine atony, however, led to our decision to raise the platelet count. Optimal timing for transfusion remains controversial, but it should not be too far away from the time of delivery9, since a study demonstrated that in DSS patients, platelet count declined quickly and returned to pre-transfusion level only in several hours10. In our case, platelet transfusion was initially performed 30 minutes before giving birth and once again 8 hours after delivery. Repeated post-transfusion blood analyses, however, indicated significant decrement of platelet count, which was in the same pattern with the findings of the study mentioned above10. Regardless of prophylactic platelet transfusion, according to a previous retrospective study, clinical bleeding in an adult patient with Dengue infection may still occur11, partially due to the intricate affect of Dengue infection on the hemostatic system12. Therefore, in such cases, platelet transfusion might not be overemphasized and management of other risk factors of bleeding in the obstetric background, such as polyhydramnios in this case, must also be considered. In spite of successful active management of the third stage of labor, the lack of close follow-up thereafter, which was partly due to hospital overload, made us unable to detect earlier delayed postpartum hemorrhage. Recurrence of uterine atony might be the major cause owing to the finding of boggy, hypotonic uterus. The fact that uterotonic drugs controlled bleeding effectively before platelet transfusion was performed also indicated the diagnosis of uterine atony. Treatment with uterotonic drugs was essential because myometrial contraction acts as the major factor preventing blood loss after natural delivery13, even if there are abnormalities in the hemostatic system.\n\nMedical literature recorded many cases of vertical transmission of Dengue virus3–5. Dengue infection in mothers at or near the time of delivery may predispose neonates to suffer from many dangerous complications because the vast majority of antibodies which are transferred across placenta do not have a protective effect14 but actually enhance the entry of virus into the host cells15. As the result of this, careful neonatal follow-up in the hospital must be performed. Time to observation remains elusive, but several studies have reported that time of febrile onset varied between 1 and 11 days of life16. In our case, a 14 day observation was not feasible given the background of hospital overload. As a result, we kept the baby in the hospital during the first 7 days of age, and then discharged mother and child after counselling the family to visit for a check-up one week later.\n\n\nConclusion\n\nA case of postpartum hemorrhage resulting from uterine atony and accompanied with Dengue infection in term pregnancy and delivery has rarely been reported in obstetric medical literature. Until now, no standard treatment has been proposed. Our current case, fortunately, reported successful management with conservative treatment and timely uterotonic administration.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.",
"appendix": "Author contributions\n\n\n\nTTT, LPH and NPL conceived the case report. LPH, TDN, NHA, MVH, NTL, and NPL collected, classified, and analyzed the contents of the case report. LPH, TDN, NHA, and MVH prepared the first draft of the manuscript. All authors have read, revised critically, and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing of interests was disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful for all medical staff who took care of the patient. TDN, NHA and MVH have been awarded scholarships from the Vietnam Student Development Fund (VNSDF; www.vnsdf.org). The organization had no role in any part of the case report or decision to publish.\n\n\nReferences\n\nTan PC, Rajasingam G, Devi S, et al.: Dengue infection in pregnancy: prevalence, vertical transmission, and pregnancy outcome. Obstet Gynecol. 2008; 111(5): 1111–7. PubMed Abstract | Publisher Full Text\n\nJanjindamai W, Pruekprasert P: Perinatal dengue infection: a case report and review of literature. Southeast Asian J Trop Med Public Health. 2003; 34(4): 793–6. PubMed Abstract\n\nThaithumyanon P, Thisyakorn U, Deerojnawong J, et al.: Dengue infection complicated by severe hemorrhage and vertical transmission in a parturient woman. Clin Infect Dis. 1994; 18(2): 248–9. PubMed Abstract | Publisher Full Text\n\nChotigeat U, Kalayanarooj S, Nisalak A: Vertical transmission of dengue infection in Thai infants: two case reports. J Med Assoc Thai. 2003; 86(Suppl 3): S628–32. PubMed Abstract\n\nChye JK, Lim CT, Ng KB, et al.: Vertical transmission of dengue. Clin Infect Dis. 1997; 25(6): 1374–7. PubMed Abstract | Publisher Full Text\n\nAmerican College of Obstetricians and Gynecologists: ACOG Practice Bulletin: Clinical Management Guidelines for Obstetrician-Gynecologists Number 76, October 2006: postpartum hemorrhage. Obstet Gynecol. 2006; 108(4): 1039–47. PubMed Abstract\n\nKerdpanich A, Watanaveeradej V, Samakoses R, et al.: Perinatal dengue infection. Southeast Asian J Trop Med Public Health. 2001; 32(3): 488–93. PubMed Abstract\n\nCostantine MM: Physiologic and pharmacokinetic changes in pregnancy. Front Pharmacol. 2014; 5: 65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO/TDR: Handbook for clinical management of dengue. Geneva, Switzerland: World Health Organization; 2012; 124. Reference Source\n\nLum LC, Abdel-Latif Mel-A, Goh AY, et al.: Preventive transfusion in Dengue shock syndrome-is it necessary? J Pediatr. 2003; 143(5): 682–4. PubMed Abstract | Publisher Full Text\n\nLye DC, Lee VJ, Sun Y, et al.: Lack of efficacy of prophylactic platelet transfusion for severe thrombocytopenia in adults with acute uncomplicated dengue infection. Clin Infect Dis. 2009; 48(9): 1262–5. PubMed Abstract | Publisher Full Text\n\nde Azeredo EL, Monteiro RQ, de-Oliveira Pinto LM: Thrombocytopenia in Dengue: Interrelationship between Virus and the Imbalance between Coagulation and Fibrinolysis and Inflammatory Mediators. Mediators Inflamm. 2015; 2015: 313842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnderson JM, Etches D: Prevention and management of postpartum hemorrhage. Am Fam Physician. 2007; 75(6): 875–82. PubMed Abstract\n\nCarroll ID, Toovey S, Van Gompel A: Dengue fever and pregnancy - a review and comment. Travel Med Infect Dis. 2007; 5(3): 183–8. PubMed Abstract | Publisher Full Text\n\nRothman AL: Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nat Rev Immunol. 2011; 11(8): 532–43. PubMed Abstract | Publisher Full Text\n\nSirinavin S, Nuntnarumit P, Supapannachart S, et al.: Vertical dengue infection: case reports and review. Pediatr Infect Dis J. 2004; 23(11): 1042–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11676",
"date": "04 Jan 2016",
"name": "Michael Paech",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis was an interesting case report for those not familiar with clinical cases of Dengue fever, although postpartum hemorrhage is not unexpected in a woman with multiple risk factors including multiparity, polyhydramnios, absent antenatal care and Dengue-induced thrombocytopenia. The report is well written apart from a few English phrases that are not quite correct (check first couple of sentences of the Discussion for example). I think it would be helpful to mention other diagnostic features (neutropenia, bradycardia, positive tourniquet test and full laboratory assessment). Perhaps also some more detail about the time course / natural history of Dengue with respect to pathologies. The decision to use a precious resource such as platelets after postpartum hemorrhage had resolved with oxytocic therapy may not have been appropriate and a caveat should be included.",
"responses": []
},
{
"id": "11878",
"date": "11 Jan 2016",
"name": "Gamal Serour",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very much for asking me to referee this interesting paper. It is a good paper which will be interesting to the readers. I recommend its acceptance for indexation",
"responses": []
},
{
"id": "11675",
"date": "14 Jan 2016",
"name": "Carolyn F Weiniger",
"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\nGeneralThis is an unusual topic, and the literature available on the topic is sparse.There is a large review by Basurko et al. (2009) on over 50 women from 2009 that has not been referenced.SpecificThere are unclear sentences that require more detail e.g.:“hospital where she was confirmed to have Dengue with warning signs.” – what specific warning signs did she present with? “The mother and newborn were transferred to the waiting room for observation.” – this is likely a translation confusion, do the authors mean that she was moved to a monitored bed? “A uterine atony and a first-degree vaginal tear were recorded, leading to the loss of about 200 ml of blood.” How were they managed? In the discussion – please provide more detail about Dengue fever and how PPH may be related, identified, managed.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1483
|
https://f1000research.com/articles/4-1482/v1
|
21 Dec 15
|
{
"type": "Review",
"title": "Recent advances in treatment for Benign Prostatic Hyperplasia",
"authors": [
"Simon van Rij",
"Peter Gilling",
"Simon van Rij"
],
"abstract": "Clinical benign prostatic hyperplasia (BPH), often identified as a worsening ability of a male to pass urine, is a significant problem for men in our society. In 2015, the use of personalised medicine is tailoring treatment to individual patient needs and to genetic characteristics. Technological advances in surgical treatment are changing the way BPH is treated and are resulting in less morbidity. The future of BPH treatments is exciting, and a number of novel techniques are currently under clinical trial.",
"keywords": [
"benign prostatic hyperplasia",
"personalised medicine",
"prostate obstruction",
"aquablation",
"mirabegron",
"Holmium laser enucleation of the prostate",
"HOLEP"
],
"content": "\n\nClinical benign prostatic hyperplasia (BPH), often identified as the worsening ability of a male to pass urine, is a multi-billion-dollar industry1. BPH increases with age and therefore with an aging society the incidence will continue to increase significantly. BPH is both extremely common and can cause significant harm2. Treatment options for men with prostate obstruction have existed for centuries. But what is new in 2015? What is making this branch of medicine such an exciting area to be working and researching in? Much of it is to do with the personalised medicine mantra and the medical technology revolution. Using these two key principles, we will show the changing nature of the treatment of BPH.\n\nBPH is often used as a colloquial term to describe urinating difficulties and bother in men. However, within the literature, a number of different terms and acronyms are used to describe the symptoms and conditions associated with urinating difficulties3. Strictly speaking, BPH is a pathological change that occurs in the prostate, leading to enlargement. This enlargement can lead to obstruction now termed benign prostatic obstruction (BPO). BPO can lead to urinary symptoms; however, one must remember that a number of other conditions can cause urinary symptoms in men. Both the European Association of Urology and the American Urology Association publish clear evidence-based guidelines on these subjects4,5. To keep this review focused, we will stick to advances in BPH and BPO.\n\n‘Personalised medicine’ has become a buzzword in the management of many medical conditions6. It involves moving away from a one-size-fits-all treatment. Instead, it is a process of weighing up the benefits of different treatments for an individual on the basis of their own specific characteristics. Much of this has grown from our increased understanding of genetics and the human genome. To apply this to BPH, we must look at recent advances in the pathogenesis of this condition.\n\nThe effect of androgens on the development of BPH has been well studied7. However, as our understanding of the topic has increased, so too has the complexity of this condition. Inflammation, growth factors, stromal interactions, and genetic factors have all been shown to contribute to the hyperplasia of the prostate glands and stroma. Much of the current research has focused on the gene expression that differs between individuals, particularly around cellular pathways and receptors. The enzyme 5-alpha reductase 2 (5AR2) plays a key role in the conversion of androgens in the prostate, leading to hyperplasia6. Epigenetic studies have shown that the expressions of these 5AR2 proteins are varied amongst subjects and appear to be linked to the development of BPH8. The exciting prospect for the future is developing commercial tests to identify these proteins in individuals to tailor medication to that individual.\n\nManagement of BPH has often been divided into medical and surgical options. However, in 2015, the lines between the two are now more blurred. This has been driven by patients’ expectations of their treatments and new technology making treatments less invasive. In the last year, no new medications that specifically act on the prostate have been brought to commercial market. Alpha-blockers, which cause relaxation of the smooth muscle fibers within the prostate, continue to be the first-line treatment5, although the drugs have no impact on the progression of BPH or on the potential to avoid surgical treatment9. Also, recent research shows that only 40% of patients commenced on these medications remain on treatment 6 months later10. An alternative medication group are the 5-alpha reductase inhibitors. Large multi-centre randomised controlled trials have shown the benefit of these medications in improving urinary symptoms9. However, evidence in relation to the side effects of these drugs has surfaced over the last few years. The most publicised came from the Prostate Cancer Prevention Trial, in which men on finasteride showed a possible increase in high-risk prostate cancer compared with those on placebo11. The absolute difference in cancer rates was extremely low, and subsequent commentaries and analysis have aimed to disprove this12. The other concerning side effect of finasteride has been a reported worsening sexual function, which in some men can be longstanding13. Finasteride given at a lower dose is a common medication used for male hair loss, and reports have shown these same concerns on sexual function when used for this purpose14. The only new medications on the market for BPH in the last few years have been the phosphodiesterase type 5 (PDE5) inhibitors, most notably tadalafil. The most common indication for this medication is the treatment of erectile dysfunction, but trials have shown improvement in BPH symptoms without adverse sexual side effects15. The exact mechanism for the effect of tadalafil for symptomatic BPH still has not been elucidated16. It is possible that it has little effect on BPH but that because men have improved sexual function they feel better!\n\nApart from traditional oral medication, attempts have been made to inject drugs directly into the prostate. This has been spurred on by the increased use and efficacy of Botox (Allergan, Dublin, Ireland) treatment in the bladder17. Initial trials in intraprostatic injection have been promising. However, the only long-term randomised controlled trial of intraprostatic Botox did not show significant benefit18. Other novel agents are being explored but still require further evidence before they can be used clinically. As definitive trials have not yet been published, it is possible that they have not resulted in adequate clinical effectiveness19.\n\nTechnology has played a significant role in the latest advances in BPH management, no more so than in the increase in interventions that do not require general anaesthetic or lengthy stays in hospital. A number techniques, known as ‘office procedures’, have involved different forms of energy, including heat and water. The most promising new technique has been the prostatic urethral lift. This is a novel mechanical implant placed into the prostate that pulls the encroaching lobes of the prostate out of the way to improve men’s flow20. As discussed previously, the effect of treatment on sexual function can be a key determinant for a patient deciding what treatment option to have. From a surgical point of view, most procedures result in retrograde ejaculation, which can affect both fertility and sexual performance/satisfaction. The UroLift device has been shown to cause minimal disruption to ejaculation and, when compared with the traditional standard form of surgery—transurethral resection of the prostate (TURP)—in a recent trial, had similar outcomes, including patient satisfaction with minimal complications21. Many of these novel techniques are introduced early without long-term data to show that they remain efficacious. It remains to be seen whether the UroLift will became a mainstay of treatment or fall by the wayside like other techniques before it.\n\nA more controversial new technique with only relatively recent published data is prostatic artery embolisation. Performed by an interventional radiologist rather than a urologist, this technique is performed under local anaesthetic and involves a groin artery puncture with super-selective vascular embolisation of the arteries to the prostate. This is postulated to cause shrinkage of the prostate and an improvement in urinary function. To date, this technique has been hampered by poor study design with no comparative randomised controlled trials published22. An article published this year from China shows that, in men with enlarged prostates, a significant decrease in the size of prostates along with symptom improvement is obtained23. The jury remains out on this technique, and professional societies have published guidelines cautioning mainstream use until proper rigorous data are published24. A number of ongoing trials will be reported in the next two years, and so we will wait and see.\n\nTURP has long been the standard of care for the surgery of BPH unless the prostate was very large and in that case an open operation was performed. The goal with technology has been to improve upon these techniques to provide better outcomes with less morbidity. In 2015, the surgical options open to a patient are many, including traditional surgery, laser surgery, and in some cases robotic surgery. The key questions are whether the technology is actually better and the role of device companies as a driving force.\n\nLasers have been used in endoscopic BPH surgery for over 20 years25. The unique properties of each laser and its individual wavelength allow precise cutting and vaporisation of tissue with excellent haemostasis. Techniques to remove prostate BPH tissue fall broadly into ablative techniques with destruction of the tissue versus enucleation techniques, whereby tissue is shelled out in large anatomic lobes for removal. Although these techniques have been around for a long time, only recently have they become the standard of care when compared with traditional surgery26. This has come about from well-designed trials comparing laser techniques with traditional surgery. The GOLIATH trial compared the 180-W version of the 532-nm laser (often called the ‘greenlight’ laser) with traditional surgery within a randomised control trial. The results at two years showed non-inferiority of the laser with lower rates of complications, particularly in blood transfusion27. Vaporisation techniques—in particular, the greenlight laser—have evolved over time by increasing the power delivered by the laser to allow faster treatment28. However, the limitations of the vaporisation technique include the lack of tissue retrieved for analysis to ensure that no cancer is present and a higher rate of needing to convert the technique to an alternative in men with large prostate glands. Holmium laser enucleation of the prostate (HOLEP) remains the most rigorously researched technique suitable to all situations and to men with varying degrees of enlargement. Further randomised controlled trials comparing HOLEP with traditional surgery have been performed with clearly decreased morbidity and similar outcomes at one-year follow-up29. The issue of longevity of the HOLEP technique has also been put to rest with articles publishing long-term outcomes out to 10 years showing low rates of re-intervention and continued relief of symptoms30. The unique property of enucleation is the ability to treat any size of gland, in particular in extremely large prostates, which have traditionally required an open incision operation. This is a morbid procedure and avoiding this is a true advantage of the HOLEP procedure31. With the success of HOLEP and in particular the enucleation technique, a number of new lasers and energy sources based on mimicking this technique have been brought to market. The thulium laser has been the most popular of these along with the diode lasers. Limited data are currently available and it remains to be seen whether these lasers will provide an improvement on the established HOLEP technique32.\n\nSo which laser technique is best? There are few trials that compare lasers against each other, as up until this point they have been competing against traditional techniques. A recent trial comparing photoselective vaporisation of the prostate and HOLEP in men with small prostate glands showed similar efficacy and is an example of what is required33. In 2015, we do not have the answer as to which is best. This is due to the variation in surgeons’ training, the equipment available, and the individual patient characteristics such as size of prostate and severity of symptoms. Regardless of which is best, it is clear that lasers are here to stay in the management of BPH.\n\nRobotic surgery in urology is well established for treatment of other conditions unrelated to BPH. A recent trend shows increased use of robotic techniques for treatment of large prostates. These techniques are currently restricted to high-volume robotic centres, but a recent published meta-analysis has shown comparability to open surgery with less morbidity34. However, HOLEP has already established itself as the minimally invasive technique of choice for men with large prostates and therefore any future comparative study would need to compare the robotic approach with this modality. Robotics already has a major hurdle when compared with HOLEP, as a recent cost analysis shows that this procedure is significantly more expensive35.\n\nSo what does the future hold for the management of BPH? From a pharmacological point of view, it is using the evidence that male urinary symptoms are not based solely on prostate obstruction. Complex interactions between multiple factors, including bladder receptors, neural pathways, and structural changes, are the targets for future medications. Using combinations of drugs, including anticholinergics and agents such as mirabegron, and the synergies between them will bring increased benefit to men’s urinary symptoms, particularly those that are refractory to standard medication. Novel surgical techniques remain experimental and chase the goal of optimal patient outcomes. Currently, also in clinical trials, water jet ablation is an exciting prospect for the future36. Using the efficient properties or water for tissue ablation and the precision of robotic technology, this procedure is termed aquablation. A large randomised study is under way.\n\nThe management of BPH in 2015 is an exciting area to be involved in. Despite all the new medical and surgical options available to the treating doctor, the key message remains that there is not a one-size-fits-all approach. An elderly man with a very large prostate who ends up in urinary retention with a catheter will have different requirements than a young sexually active man with worsening of his symptoms over time causing significant social impact. Both are affected by clinical BPH, but it is up to the physician, in discussion with the patient, to come up with an individual treatment plan suited to their needs. With new technology, this will allow us to do this more efficiently with fewer side effects.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nPeter Gilling is a meeting participant and has received research funding from Procept Biorobotics Ltd.\n\n\nReferences\n\nSaigal CS, Joyce G: Economic costs of benign prostatic hyperplasia in the private sector. J Urol. 2005; 173(4): 1309–13. PubMed Abstract | Publisher Full Text\n\nParsons JK: Benign Prostatic Hyperplasia and Male Lower Urinary Tract Symptoms: Epidemiology and Risk Factors. Curr Bladder Dysfunct Rep. 2010; 5(4): 212–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAbrams P, Cardozo L, Fall M, et al.: The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Neurourol Urodyn. 2002; 21(2): 167–78. PubMed Abstract | Publisher Full Text\n\nGratzke C, Bachmann A, Descazeaud A, et al.: EAU Guidelines on the Assessment of Non-neurogenic Male Lower Urinary Tract Symptoms including Benign Prostatic Obstruction. Eur Urol. 2015; 67(6): 1099–109. PubMed Abstract | Publisher Full Text\n\nMcVary KT, Roehrborn CG, Avins AL, et al.: Update on AUA guideline on the management of benign prostatic hyperplasia. J Urol. 2011; 185(5): 1793–803. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBechis SK, Otsetov AG, Ge R, et al.: Personalized medicine for the management of benign prostatic hyperplasia. J Urol. 2014; 192(1): 16–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcNeal JE: Origin and evolution of benign prostatic enlargement. Invest Urol. 1978; 15(4): 340–5. PubMed Abstract\n\nNiu Y, Ge R, Hu L, et al.: Reduced levels of 5-α reductase 2 in adult prostate tissue and implications for BPH therapy. Prostate. 2011; 71(12): 1317–24. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMcConnell JD, Roehrborn CG, Bautista OM, et al.: The long-term effect of doxazosin, finasteride, and combination therapy on the clinical progression of benign prostatic hyperplasia. N Engl J Med. 2003; 349(25): 2387–98. PubMed Abstract | Publisher Full Text\n\nSchoenfeld MJ, Shortridge EF, Gelwicks SC, et al.: Treatment patterns in alpha-blocker therapy for benign prostatic hyperplasia. Am J Mens Health. 2014; 8(3): 267–72. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nThompson IM, Goodman PJ, Tangen CM, et al.: The influence of finasteride on the development of prostate cancer. N Engl J Med. 2003; 349(3): 215–24. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLebdai S, Bigot P, Azzouzi A: High-grade prostate cancer and finasteride. BJU Int. 2010; 105(4): 456–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFwu C, Eggers PW, Kirkali Z, et al.: Change in sexual function in men with lower urinary tract symptoms/benign prostatic hyperplasia associated with long-term treatment with doxazosin, finasteride and combined therapy. J Urol. 2014; 191(6): 1828–34. PubMed Abstract | Publisher Full Text\n\nSingh MK, Avram M: Persistent sexual dysfunction and depression in finasteride users for male pattern hair loss: a serious concern or red herring? J Clin Aesthet Dermatol. 2014; 7(12): 51–5. PubMed Abstract | Free Full Text | F1000 Recommendation\n\nOelke M, Shinghal R, Sontag A, et al.: Time to onset of clinically meaningful improvement with tadalafil 5 mg once daily for lower urinary tract symptoms secondary to benign prostatic hyperplasia: analysis of data pooled from 4 pivotal, double-blind, placebo controlled studies. J Urol. 2015; 193(5): 1581–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVignozzi L, Gacci M, Cellai I, et al.: PDE5 inhibitors blunt inflammation in human BPH: a potential mechanism of action for PDE5 inhibitors in LUTS. Prostate. 2013; 73(13): 1391–402. PubMed Abstract | Publisher Full Text\n\nKarsenty G, Denys P, Amarenco G, et al.: Botulinum toxin A (Botox®) intradetrusor injections in adults with neurogenic detrusor overactivity/neurogenic overactive bladder: a systematic literature review. Eur Urol. 2008; 53(2): 275–87. PubMed Abstract | Publisher Full Text\n\nMcVary KT, Roehrborn CG, Chartier-Kastler E, et al.: A multicenter, randomized, double-blind, placebo controlled study of onabotulinumtoxinA 200 U to treat lower urinary tract symptoms in men with benign prostatic hyperplasia. J Urol. 2014; 192(1): 150–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAndersson K: Intraprostatic injections for lower urinary tract symptoms treatment. Curr Opin Urol. 2015; 25(1): 12–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGarcia C, Chin P, Rashid P, et al.: Prostatic urethral lift: A minimally invasive treatment for benign prostatic hyperplasia. Prostate Int. 2015; 3(1): 1–5. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSønksen J, Barber NJ, Speakman MJ, et al.: Prospective, randomized, multinational study of prostatic urethral lift versus transurethral resection of the prostate: 12-month results from the BPH6 study. Eur Urol. 2015; 68(4): 643–52. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMcVary KT: Prostatic arterial embolization for LUTS/BPH-an opportunity for collaborative research or next prostatic gizmo candidate? J Urol. 2014; 191(3): 577–8. PubMed Abstract | Publisher Full Text\n\nWang M, Guo L, Duan F, et al.: Prostatic arterial embolization for the treatment of lower urinary tract symptoms caused by benign prostatic hyperplasia: a comparative study of medium- and large-volume prostates. BJU Int. 2015. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPosition Statement on Prostatic Arterial Embolization for the Treatment of Lower Urinary Tract Symptoms due to Benign Prostatic Hyperplasia - Urological society of Australia and New Zealand. 2003. Reference Source\n\nGilling PJ, Cass CB, Malcolm AR, et al.: Combination holmium and Nd:YAG laser ablation of the prostate: initial clinical experience. J Endourol. 1995; 9(2): 151–3. PubMed Abstract | Publisher Full Text\n\nvan Rij S, Gilling PJ: In 2013, holmium laser enucleation of the prostate (HoLEP) may be the new 'gold standard'. Curr Urol Rep. 2012; 13(6): 427–32. PubMed Abstract | Publisher Full Text\n\nThomas JA, Tubaro A, Barber N, et al.: A Multicenter Randomized Noninferiority Trial Comparing GreenLight-XPS Laser Vaporization of the Prostate and Transurethral Resection of the Prostate for the Treatment of Benign Prostatic Obstruction: Two-yr Outcomes of the GOLIATH Study. Eur Urol. 2015; pii: S0302-2838(15)00713-7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEken A, Soyupak B, Acil M, et al.: Safety, efficacy and outcomes of the new GreenLight XPS 180W laser system compared to the GreenLight HPS 120W system for the treatment of benign prostatic hyperplasia in a prospective nonrandomized single-centre study. Can Urol Assoc J. 2015; 9(1–2): e56–60. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSun N, Fu Y, Tian T, et al.: Holmium laser enucleation of the prostate versus transurethral resection of the prostate: a randomized clinical trial. Int Urol Nephrol. 2014; 46(7): 1277–82. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGilling PJ, Wilson LC, King CJ, et al.: Long-term results of a randomized trial comparing holmium laser enucleation of the prostate and transurethral resection of the prostate: results at 7 years. BJU Int. 2012; 109(3): 408–11. PubMed Abstract | Publisher Full Text\n\nKuntz RM, Lehrich K, Ahyai SA: Holmium laser enucleation of the prostate versus open prostatectomy for prostates greater than 100 grams: 5-year follow-up results of a randomised clinical trial. Eur Urol. 2008; 53(1): 160–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZhang F, Shao Q, Herrmann TR, et al.: Thulium laser versus holmium laser transurethral enucleation of the prostate: 18-month follow-up data of a single center. Urology. 2012; 79(4): 869–74. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nElshal AM, Elkoushy MA, Elmansy HM, et al.: Holmium:YAG transurethral incision versus laser photoselective vaporization for benign prostatic hyperplasia in a small prostate. J Urol. 2014; 191(1): 148–54. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAutorino R, Zargar H, Mariano MB, et al.: Perioperative Outcomes of Robotic and Laparoscopic Simple Prostatectomy: A European-American Multi-institutional Analysis. Eur Urol. 2015; 68(1): 86–94. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMatei DV, Brescia A, Mazzoleni F, et al.: Robot-assisted simple prostatectomy (RASP): does it make sense? BJU Int. 2012; 110(11 Pt C): E972–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFaber K, de Abreu AL, Ramos P, et al.: Image-guided robot-assisted prostate ablation using water jet-hydrodissection: initial study of a novel technology for benign prostatic hyperplasia. J Endourol. 2015; 29(1): 63–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11665",
"date": "21 Dec 2015",
"name": "Tevita 'Aho",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11666",
"date": "21 Dec 2015",
"name": "Kevin Zorn",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1482
|
https://f1000research.com/articles/4-1480/v1
|
21 Dec 15
|
{
"type": "Research Article",
"title": "Pilot experience with opebacan/rBPI21 in myeloablative hematopoietic cell transplantation",
"authors": [
"Eva Guinan",
"David E Avigan",
"Robert J Soiffer",
"Nancy J Bunin",
"Lisa L Brennan",
"Ilana Bergelson",
"Spencer Brightman",
"Al Ozonoff",
"Patrick J Scannon",
"Ofer Levy",
"David E Avigan",
"Robert J Soiffer",
"Nancy J Bunin",
"Lisa L Brennan",
"Ilana Bergelson",
"Spencer Brightman",
"Al Ozonoff",
"Patrick J Scannon",
"Ofer Levy"
],
"abstract": "Bacterial infection and inflammation contribute significantly to the morbidity and mortality of myeloablative allogeneic hematopoietic cell transplantation (HCT). Endotoxin, a component of the outer membrane of Gram-negative bacteria, is a potent inflammatory stimulus in humans. Bactericidal/permeability increasing protein (BPI), a constituent of human neutrophil granules, binds endotoxin thereby precluding endotoxin-induced inflammation and also has direct anti-infective properties against bacteria. As a consequence of myeloablative therapy used in preparation for hematopoietic cell infusion, patients experience gastrointestinal leak of bacteria and bacterial toxins into the systemic circulation and a period of inflammatory cytokine elevation associated with subsequent regimen-related toxicities. Patients frequently become endotoxemic and febrile as well as BPI-deficient due to sustained neutropenia. To examine whether enhancing endotoxin-neutralizing and anti-infective activity by exogenous administration of a recombinant N-terminal fragment of BPI (rBPI21, generic name opebacan) might ameliorate regimen-related toxicities including infection, we recruited patients scheduled to undergo myeloablative HCT to participate in a proof-of-concept prospective phase I/II trial. After the HCT preparative regimen was completed, opebacan was initiated 18-36 hours prior to administration of allogeneic hematopoietic stem cells (defined as Day 0) and continued for 72 hours. The trial was to have included escalation of rBPI21 dose and duration but was stopped prematurely due to lack of further drug availability. Therefore, to better understand the clinical course of opebacan-treated patients (n=6), we compared their outcomes with a comparable cohort meeting the same eligibility criteria and enrolled in a non-interventional myeloablative HCT observational study (n = 35). Opebacan-treated participants had earlier platelet engraftment (p=0.005), mirroring beneficial effects of rBPI21 previously observed in irradiated mice, fewer documented infections (p=0.03) and appeared less likely to experience significant regimen-related toxicities (p=0.05). This small pilot experience supports the potential utility of rBPI21 in ameliorating HCT-related morbidity and merits further exploration.",
"keywords": [
"endotoxin",
"innate immunity",
"allogeneic hematopoietic cell transplantation",
"regimen-related toxicity",
"infection",
"engraftment"
],
"content": "Introduction\n\nRegimen-related toxicities, including infection, organ damage, and acute graft-versus-host disease (aGvHD), remain significant barriers to successful allogeneic hematopoietic cell transplantation (HCT). The “cytokine storm” hypothesis - that regimen-related injury to host cells creates a pro-inflammatory environment contributing to aGVHD and other toxicities – has been well substantiated in various experimental models1,2. One consequence of such host cell injury, specifically gastrointestinal (GI) damage from myeloablative therapy, results in leakage of bacterial lipopolysaccharide (LPS), also referred to as endotoxin, into the systemic circulation in both mice and humans undergoing HCT3,4. LPS, which is a constituent of the outer membrane of Gram-negative bacteria5 is one of the most potent inflammatory stimuli in humans, and LPS-induced production of pro-inflammatory and Th1-polarizing cytokines has been linked to subsequent aGvHD in model systems6–8. Moreover, administration of a synthetic LPS antagonist for 6 days starting from the day of transplantation reduced TNFα production, intestinal damage, aGvHD and mortality after murine myeloablative HCT while preserving the graft vs. leukemia (GvL) effect7,9.\n\nUpon entry into the systemic circulation, LPS is recognized by proteins that enhance its activity by shepherding LPS to its major tripartite pro-inflammatory cell surface receptor composed of Toll-like receptor 4 (TLR4), CD14 and MD-210–13. This multi-step delivery system amplifies the effect of small amounts of LPS. Given the resulting potency of LPS, there are also numerous mechanisms, including clearance, detoxification and neutralization, that decrease LPS-mediated inflammation10–13. Antimicrobial proteins and peptides (APPs) associated with neutrophil granules provide a potent source of LPS neutralizing activity14. One of these granule constituents, BPI, is a cationic 55 kDa protein with high affinity for the lipid A region common to most LPS variants15,16. Among APPs, including defensins, BPI is a particularly potent LPS inhibitor active at nanomolar concentrations17. Binding of BPI to LPS precludes LPS binding to both lipopolysaccharide-binding protein (LBP) and LPS receptors such as TLR4, thus inhibiting LPS-induced inflammation, including TNFα production18.\n\nRecombinant N-terminal fragments of BPI (including rBPI23 and rBPI21 [opebacan, NEUPREX®]) possessing the LPS-binding and LPS-neutralizing activity of native BPI were developed by XOMA (US) LLC (Berkeley, CA) as anti-infective agents for use in sepsis and other infectious disease indications. These BPI congeners have potent in vitro endotoxin-neutralizing activity15 and have demonstrated efficacy in multiple animal models of endotoxemia19–23. In human trials, intravenous (IV) administration of either rBPI23 or rBPI21 has appeared safe and non-immunogenic24–27 and ameliorated LPS-induced changes in parameters including cardiac index, cytokine release and coagulation. rBPI21 and rBPI23 have bactericidal activity and exhibit synergy with conventional antibiotics, including activity against antibiotic-resistant bacteria18,28.\n\nWe have proposed that opebacan may be most beneficial for individuals deficient in endogenous BPI29. Myeloablative HCT, during which recipients experience simultaneous endotoxemia and neutropenia3,4,30, represents a condition where the LPS:BPI ratio is high. To further pursue the hypothesis that providing additional LPS-neutralizing activity would abrogate LPS-related toxicity when systemic LPS is present and endogenous BPI is inadequate, we undertook a Phase I/II study of opebacan during myeloablative HCT to investigate safety and preliminary correlative clinical and laboratory data strategic decisions related to inflammation and regimen-related toxicity. The sponsor prematurely discontinued the study because of strategic decisions related to unanticipated insufficient drug supply. In order to generate hypotheses for future work and to begin to describe the effects of rBPI21 in this setting, we therefore compared the outcomes of the completed cohort of opebacan-treated participants with those of individuals meeting the same eligibility criteria and enrolled in a non-interventional study of innate immunity after myeloablative HCT.\n\n\nMaterial and methods\n\nStudy design and eligibility. From 9/2007-7/2008, sequential eligible patients scheduled to undergo allogeneic HCT were offered participation (SM Figure 1) in an open-label, dose-finding study of opebacan (NCT00454155) at the Children’s Hospital of Philadelphia (protocol CHP 871), the Beth Israel Deaconess Medical Center (BIDMC) and the Dana-Farber Cancer Institute/Brigham and Women’s Hospital (DFCI, BWH) (all protocol 06155). Eligibility criteria included: use of a myeloablative regimen (total body irradiation [TBI] ≥1000 cGy or busulfan ≥14 mg/kg PO or IV equivalent); Lansky or Karnofsky performance score >80%; first HCT; no active infection. Additional criteria included: room air oxygen saturation >95%; serum creatinine <1.5× upper limit of normal (ULN); AST and ALT ≤3×ULN and total bilirubin ≤1.5×ULN; normal cardiac shortening or ejection fraction; no history of congestive heart failure; normal cardiac troponin T level; cumulative anthracycline exposure <300 mg/m2. Exclusion criteria included: use of cord blood cells; T-cell depletion regimen; prophylactic antibiotics beyond standard practice; or planned heparin anticoagulation or dextran sulfate use (both antagonize opebacan activity) during opebacan infusion. The study was originally open to those <60 years and modified to have a lower limit of >18 years. The study was approved by the DFCI and the Children’s Hospital of Philadelphia Institutional Review Boards (IRBs). All participants and/or legal guardians gave written consent and/or assent.\n\nThe original interventional study design included 5 cohorts (each n=6) with sequential escalation of opebacan dose and duration (Supplementary material: Figure 2 and Opebacan trial protocol 06155). Opebacan was administered via central IV catheter after completion of the myeloablative regimen and ≥18 and ≤36 hours prior to donor cell infusion. In order to reach a steady-state plasma level rapidly, an initial bolus was followed by continuous IV infusion.\n\nThe protocol anticipated comparisons between 5 cohorts. However, the study was discontinued after the first cohort based on a strategic manufacturing decision by the sponsor that precluded sufficient drug availability to complete planned accrual. To better understand the outcomes of cohort 1, we therefore identified a comparison group (COMP, n=35) enrolled on a non-interventional, sample collection study of endotoxin-related innate immunity after HCT who met eligibility criteria for the opebacan trial. COMP participants had been recruited prospectively from 8/2005-7/2009 at Boston Children’s Hospital (BCH), DFCI and BWH onto protocol 05127 (Supplementary material: Comparison group protocol 05127), approved by the DFCI IRB. Comparison of the two data sets was IRB approved. All participants and/or legal guardians gave written consent and/or assent.\n\nPatient characteristics are shown in Table 1. Endpoints were defined according to the opebacan protocol (Supplementary material: Opebacan trial protocol 06155). All tests were conducted in clinical laboratories per routine. Day 0 was defined as the day of cell infusion. Engraftment was defined as the first of 3 consecutive days with absolute neutrophil count (ANC) of ≥500/µL. Platelet recovery was defined as the first of 7 consecutive days with untransfused platelet count ≥20,000/µL. The maximal temperature ± 1 day of sample acquisition was recorded. Supportive care included acyclovir prophylaxis in herpes simplex sero-positive individuals, quantitative polymerase chain reaction (PCR) screening for cytomegalovirus (CMV) reactivation followed by treatment if indicated, and antifungal prophylaxis. Participants received “gut decontamination” consisting either of oral non-absorbable antibiotics or oral or intravenous fluoroquinolone, per the treating medical team. aGVHD was graded according to modified Glucksberg31. Per FDA practice, toxicity was recorded for 30 days after drug completion (rounded to Day 35 post-HCT) on case report forms. Per institutional routine, toxicity data were collected through Day 100. Maximal severity of adverse events (AEs) was reported per NCI Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. Infections were defined by positive blood culture; focal complaint or finding with positive culture from a normally sterile or clinically indicated site; or other confirmatory laboratory evaluation (e.g., PCR). Absent infection, a fever occurring during neutropenia was classified as febrile neutropenia. CMV viremia on routine screening absent a clinical complaint or CMV-relatable organ disease was denoted CMV reactivation. Biopsy-confirmed CMV was denoted CMV infection. Focal physical and/or radiologic findings without microbiological confirmation were scored culture-negative (i.e., possible) infections. Clostridium difficile in stool, as it is frequently present on HCT admission, was excluded from the infection analysis. Research staff with HCT expertise abstracted data.\n\nAbbrev. MDS, myelodysplasia; CML, chronic myelogenous leukemia; CLL, chronic lymphocytic leukemia; TBI/CY, total body irradiation/Cyclophosphamide; BU/CY, Busulfan/Cyclophosphamide;\n\nPBSC, peripheral blood stem cells; BM, bone marrow; GVHD, graft vs. host disease;\n\nCI, calcineurin inhibitors; MTX, methotrexate;\n\n*All p-values are Fisher's exact test except for age, which is derived by Wilcoxon Rank Sum.\n\n\nStatistical analysis\n\nWe generated summary statistics of sample characteristics and outcomes for both cohorts. For between-group comparisons of proportion of subjects with toxicity, we used Fisher’s exact test with exact p-values calculated directly from the hypergeometric distribution. For comparisons of toxicity rates we used Poisson regression with a generalized linear model with log (patient days) as offset and a single binary predictor for cohort. We present the p-value for the cohort effect i.e. the estimate divided by the standard error and fit to the standard normal. We used the Mantel-Cox log rank test for event rates and the Wilcoxon rank sum test for the age comparison. The software was the R statistical package v3.1.0 (R Institute, Vienna, 2014). In Table 1, the diagnosis of acute leukemia was compared to all other diagnoses and for stem cell source, PBSC were compared to BM and BM plus PBSC.\n\n\nResults\n\nParticipant characteristics. Thirty sequential HCT candidates were screened for the opebacan interventional study. Of these, 24 were ineligible [non-myeloablative regimen (n=13), excessive anthracycline exposure (n=8) and enrollment on another trial (n=3)]. Six eligible patients enrolled. All completed the planned treatment. Of 54 individuals enrolled on the innate immunity observational study, 19 were ineligible for the opebacan trial and were excluded from the COMP cohort. Reasons for exclusion included: age (n=10), non-myeloablative regimen (n=5), relapse prior to HCT (n=2), and HCT cancellation (n=2). The remaining 35 constituted the COMP group. Both groups had complete follow-up to death or Day 100. Participant characteristics in both groups were similar with respect to age, gender, diagnosis, stem cell source and GVHD prophylaxis (Table 1). Conditioning in both groups favored TBI-containing regimens. All but one participant had an HLA matched donor (100% opebacan vs 97% COMP).\n\nOpebacan treatment trial. Six participants were enrolled from 3 institutions into cohort 1. All received opebacan per protocol with 2 deviations [drug interrupted inadvertently for 30 minutes on treatment day 1 (1 patient) and drug discontinued 1 hour prematurely (1 patient)]. There were no infusional toxicities or serious adverse events (SAEs) related to study drug. Per the Investigator’s Brochure, cardiotoxicity had rarely been observed in animal studies examining higher doses and longer durations of rBPI21 than prescribed here. This had not been observed in prior clinical trials of rBPI congeners, including opebacan24–27,32. Here, all participants had normal shortening or ejection fractions prior to study entry, and none developed clinical evidence of cardiotoxicity during infusion or thereafter. Participants had echocardiograms weekly through Day 30 or hospital discharge and again at Day 100. One participant (aged 17) had a shortening fraction decrease at Day 100 read by the echocardiographer as “mild left ventricular dysfunction”. This participant had no cardiac findings at last follow-up. The remaining 5 participants had normal ejection (measured in 5 of 5) and shortening (measured in 4 of 5) fractions at all time-points.\n\nClinical course. All opebacan and COMP participants had neutrophil engraftment (median Day 17 [range 14–28] vs Day 14 [range 10–30], respectively; p=0.35; Table 2). All participants met platelet engraftment criteria, with the exception of one COMP participant who died prior to achieving this endpoint. Opebacan-treated individuals had significantly more rapid platelet engraftment (median 12 days, range 10–26) than observed in COMP participants (median 19 days, range 13–109; p=0.005; Figure 1). There was no severe (Grade III/IV) aGVHD observed in opebacan participants. Grade III/IV aGVHD was observed in 4/35 (11%) of COMP participants.\n\nAbbr: ANC, absolute neutrophil count; PLT, platelet, F/N= febrile neutropenia; DVT, deep vein thrombosis; RF, renal failure; VOD, veno-occlusive disease of liver; ARDS, acute respiratory distress syndrome; DAH, diffuse alveolar hemorrhage.\n\n* All p-values determined by Fisher's exact test except for ANC and PLT which are by Mantel-Cox log rank.\n\n**No grade 5 toxicity in either group ≤D 35.\n\nDepicted is a box and whisker plot of the median (horizontal bar) and quartiles (bottom and top of boxes; 25th to 75th) for days to platelet engraftment (as defined in Methods) in opebacan treated and COMP participants. The opebacan treated group demonstrated more rapid platelet engraftment by Mantel-Cox log rank test; p=0.005.\n\nRegimen-related toxicity. During the first 35 days, 4 opebacan-treated participants experienced at least one grade 3 toxicity for 10 aggregate grade 3 toxicities (4.8 toxicities/100 participant days at risk, Table 2). In the COMP group, we observed at least one grade 3 toxicity in 33 participants for 75 aggregate grade 3 toxicities (6.1 toxicities/100 participant days at risk). No grade 4 (life-threatening) toxicities were observed in the opebacan group in contrast to at least one grade 4 toxicity in 6 COMP participants (17%) for 8 aggregate grade 4 toxicities (0.7 toxicities/100 participant days at risk). No grade 5 toxicities were observed in either group. Thus, opebacan-treated participants experienced a lower rate of grade 3–5 toxicity through Day 35 than the COMP group (4/6 participants vs 34/35, respectively; p=0.05). In total, 10 (or 4.8/100 participant days at risk) and 83 (or 6.8/100 participant days at risk) grade 3–5 toxicities were observed in the opebacan and COMP groups, respectively (p=0.29).\n\nTo characterize the complete period for which acute HCT toxicities are commonly reported, we also analyzed toxicities during Day 36–100. During this period, we observed 2 and 7 grade 3 toxicities in the treatment and COMP groups, respectively (Table 2). Opebacan-treated participants experienced no grade 4–5 toxicities. Three COMP participants (9%) each experienced one grade 4 toxicity and 4 experienced a fatal (grade 5) toxicity. Thus, we observed 0.51 and 0.73 grade 3–5 toxicities/100 participant days at risk in the opebacan and COMP groups, respectively (p=0.65).\n\nInfection. We first examined the incidence of possible or proven infection during the period of opebacan administration. One participant (not neutropenic) in the opebacan-treated group had fever (Day -1; 16.7%). Five of 35 COMP participants developed fever (median day +1; 14.3%) during this time, of whom 3 were not neutropenic and one had bacteremia meeting sepsis criteria. No other sources for fever were identified. There was no statistically significant difference in occurrence of early fever.\n\nWe next examined all regimen-related toxicities classified as infections (Table 3). As opebacan was administered for only 72 hours peri-transplant, we again examined the early and later post-HCT intervals separately. Through Day 35 post-HCT, COMP participants were more likely to have positive cultures than those treated with opebacan (p=0.03). One opebacan participant had possible infection based on CVL-insertion site erythema and tenderness, but cultures were negative. In contrast, 6 possible (culture-negative) and 18 documented infections occurred in the COMP group. Of these, 14 were bacterial, including 10 bacteremias of which 4 fulfilled criteria for sepsis, 1 fungal and 3 viral.\n\nAbbrev. CMV, Cytomegalovirus; HSV-Herpes simplex virus; E.coli, Escherichia coli; S. aureus, Staphylococcus aureus; S.epidermidis, Staphylococcus epidermidis.\n\n* blood; # urine; + broncheolar lavage, ^ oral swab; $ colon biopsy, % nasopharynx swab\n\nIn the Day 36–100 interval (Table 3), one opebacan participant developed concurrent bacteremia and urinary tract infection as well as oral HSV. COMP participants experienced 9 confirmed infections. Five were bacterial, including 4 bacteremias of which 2 fulfilled criteria for sepsis. There were also 4 viral infections. There was no statistically significant difference in infection incidence (p=0.65).\n\n\nDiscussion\n\nHere we report the first experience of rBPI21/opebacan administration to humans undergoing myeloablative allogeneic HCT. The drug appeared well tolerated, without attributable SAEs. Time to engraftment, incidence of regimen-related toxicities, including infection, and aGVHD appeared equivalent to or better than a cotemporaneous comparison cohort meeting the opebacan trial eligibility criteria.\n\nWe have published that administration of rBPI21 and daily enrofloxacin (a veterinary ciprofloxacin equivalent) is associated with significantly increased survival and accelerated hematopoietic recovery in a murine model of myeloablative TBI mimicking unintended radiation exposure (e.g. after a nuclear event)30. In the murine model, single-fraction TBI was given, subcutaneous rBPI21 treatment was initiated 24 hours after TBI, and no stem cells were administered to restore hematopoiesis. While there are significant differences between the murine TBI model and human HCT, the rapid hematopoietic reconstitution observed in rBPI21-treated irradiated mice prompted our interest in the potential effects of rBPI21 on engraftment. Consistent with the effects observed in the murine TBI model, we found the median time to platelet engraftment was significantly decreased (by one week) in opebacan-treated participants. Time to neutrophil engraftment was similar despite greater use of peripheral blood stem cells, which are associated with more rapid neutrophil and platelet engraftment33,34, in COMP participants (86% vs 67% in opebacan participants). However, these are small groups and these results require confirmation.\n\nInfection is one of the most common toxicities experienced by patients undergoing myeloablative HCT and one of, if not the major, contributors to non-relapse mortality35,36. Documented infections in patients undergoing myeloablative HCT are largely Gram-positive35,36 as was observed here (Table 3). While both native BPI and rBPI21 have well-recognized anti-infective activities toward Gram-negative bacteria37, they also bind to and contribute anti-infective activities against Gram-positive organisms38,39. Relevant mechanisms of action in both settings include membrane permeabilization, bacterial-toxin binding, facilitation of phagocytic opsonization and effects on membrane polarization37–39. Cell wall-deficient (e.g. L-form) bacteria, which may contribute to culture-negative fevers in HCT patients, are also susceptible to BPI-mediated killing32,40. Overall, the lower infection incidence observed in opebacan-treated participants appears consistent with BPI’s antimicrobial properties as well as associations of BPI gene single nucleotide polymorphisms with infection risk after myeloablative HCT41.\n\nOpebacan treatment was associated with less early, significant regiment-related toxicity (p=0.05). In addition, the opebacan group experienced only common regimen-related toxicities, such as febrile neutropenia and mucositis. In contrast, the toxicities noted in the COMP group included well-known but less frequent and more serious post-HCT complications such as hepatic veno-occlusive disease, pneumonitis and pleural effusions. Interestingly, low level endotoxemia has been associated with greater degrees of organ dysfunction in several other settings, including congestive heart failure, renal failure, and HIV infection42–44. Moreover, lower levels of BPI in neutrophils have been associated with atherosclerotic disease severity45, and BPI polymorphisms have been associated with bronchiolitis obliterans, a devastating HCT complication46. In murine allogeneic HCT models, endotoxin has been shown to play a significant role in the incidence of aGVHD and regimen-related toxicity, and therapies mitigating endotoxin-mediated effects have decreased such effects7–9. Our preliminary observations coupled with the literature thus support the hypothesis that rBPI21-mediated endotoxin neutralization may limit the occurrence and severity of toxicities in human myeloablative HCT.\n\nAlthough the findings reported here are novel and support the study hypotheses, several substantial limitations pertain. The number of subjects is small. In addition, while the groups compared above met the same eligibility criteria and underwent HCT during a similar timeframe, this was not a prospective randomized trial and there were no formal power calculations performed for the newly constituted comparison of outcomes. Finally, we were unable to complete the original trial of increasing opebacan dose and duration due to long-term disruption of drug supply. Given that rBPI21 has concentration-dependent effects28, it is possible and perhaps likely that greater benefit would be observed with prolonged and/or higher dosing. These results suggest that opebacan can be safely administered to individuals undergoing myeloablative HCT. In addition, positive trends or significant findings with respect to time to platelet engraftment, and the incidence of regimen-related toxicity and infection are consistent with beneficial anti-infective, systemic and hematopoietic effects of BPI described previously. This pilot experience suggests that further investigation of the potential role of opebacan in mitigating toxicity and infection in this and other clinical settings characterized by reduced neutrophil quantity or quality is warranted.\n\n\nData availability\n\nF1000Research: Dataset 1. Opebacan-treated participant demographic, and transplant information as well as transplant-related toxicity, 10.5256/f1000research.7558.d10917447\n\nF1000Research: Dataset 2. COMP cohort participant demographic, and transplant information as well as transplant-related toxicity, 10.5256/f1000research.7558.d10917548\n\n\nConsent\n\nWritten informed consent for participation including publication of deidentified information was obtained from all participants.",
"appendix": "Author contributions\n\n\n\nECG and OL conceived the study and with PJS designed the experiments. ECG, DEA, RJS, NJB, LLB, IB, SB, AO and OL carried out the research. ECG, LLB, SB, AO, and OL 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\nXOMA held the study IND. XOMA personnel, ECG, OL and LLB collaboratively designed the opebacan study and data collection forms. XOMA had no role in the current comparative study design, data collection and analysis, decision to publish, or manuscript preparation other than thoughtful review by Dr. Scannon. XOMA has supplied research support for the OL laboratory.\n\n\nGrant information\n\nThis work was supported by the Dana Foundation (Guinan and Levy PIs) and NIH NHLBI R21 HL089659 (Guinan PI). OL is also supported by an interal BCH award to the Precision Vaccines Program. As the treatment study IND (Investigational New Drug) was held by the sponsor, XOMA (US) LLC, they were active participants in preparation of the opebacan treatment protocol. As stated in the Competing interest, XOMA provided the drug but no funding. They have had no role in the conception, initiation or implementation of the observational study or the comparative data reported here.\n\n\nAcknowledgements\n\nWe thank the individuals who generously consented to participate in these studies as well as medical and nursing staff for their able assistance.\n\n\nSupplementary material\n\nRecruitment, enrollment and subsequent flow through the study for the clinical trial cohort (n=6) is shown.\n\nThe study design included an IV bolus dose of 4mg/kg followed by daily IV administration of escalating doses (Cohorts 1–3) and extension of duration (Cohorts 4 and 5). Due to discontinuation of drug availability, only cohort 1 (i.e., 4 mg/kg IV bolus followed by 6mg/kg/day IV) was enrolled and completed.\n\nOpebacan trial protocol 06155.\n\nComparison group protocol 05127.\n\n\nReferences\n\nAntin JH, Ferrara JL: Cytokine dysregulation and acute graft-versus-host disease. Blood. 1992; 80(12): 2964–8. PubMed Abstract\n\nBlazar BR, Murphy WJ: Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD). Philos Trans R Soc Lond B Biol Sci. 2005; 360(1461): 1747–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevy O, Teixeira-Pinto A, White ML, et al.: Endotoxemia and elevation of lipopolysaccharide-binding protein after hematopoietic stem cell transplantation. Pediatr Infect Dis J. 2003; 22(11): 978–81. PubMed Abstract | Publisher Full Text\n\nSauer M, Tiede K, Fuchs D, et al.: Procalcitonin, C-reactive protein, and endotoxin after bone marrow transplantation: identification of children at high risk of morbidity and mortality from sepsis. Bone Marrow Transplant. 2003; 31(12): 1137–42. PubMed Abstract | Publisher Full Text\n\nUlevitch RJ, Tobias PS: Recognition of gram-negative bacteria and endotoxin by the innate immune system. Curr Opin Immunol. 1999; 11(1): 19–22. PubMed Abstract | Publisher Full Text\n\nCooke KR, Hill GR, Crawford JM, et al.: Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J Clin Invest. 1998; 102(10): 1882–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCooke KR, Olkiewicz K, Erickson N, et al.: The role of endotoxin and the innate immune response in the pathophysiology of acute graft versus host disease. J Endotoxin Res. 2002; 8(6): 441–8. PubMed Abstract | Publisher Full Text\n\nCooke KR, Hill GR, Gerbitz A, et al.: Tumor necrosis factor-alpha neutralization reduces lung injury after experimental allogeneic bone marrow transplantation. Transplantation. 2000; 70(2): 272–9. PubMed Abstract | Publisher Full Text\n\nCooke KR, Gerbitz A, Crawford JM, et al.: LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation. J Clin Invest. 2001; 107(12): 1581–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGioannini TL, Weiss JP: Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells. Immunol Res. 2007; 39(1–3): 249–60. PubMed Abstract | Publisher Full Text\n\nBeutler B, Rietschel ET: Innate immune sensing and its roots: the story of endotoxin. Nat Rev Immunol. 2003; 3(2): 169–76. PubMed Abstract | Publisher Full Text\n\nMunford RS: Sensing gram-negative bacterial lipopolysaccharides: a human disease determinant? Infect Immun. 2008; 76(2): 454–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDiks SH, van Deventer SJ, Peppelenbosch MP: Lipopolysaccharide recognition, internalisation, signalling and other cellular effects. J Endotoxin Res. 2001; 7(5): 335–48. PubMed Abstract | Publisher Full Text\n\nLevy O: Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes. J Leukoc Biol. 2004; 76(5): 909–25. PubMed Abstract | Publisher Full Text\n\nGazzano-Santoro H, Parent JB, Grinna L, et al.: High-affinity binding of the bactericidal/permeability-increasing protein and a recombinant amino-terminal fragment to the lipid A region of lipopolysaccharide. Infect Immun. 1992; 60(11): 4754–61. PubMed Abstract | Free Full Text\n\nLevy O: A neutrophil-derived anti-infective molecule: bactericidal/permeability-increasing protein. Antimicrob Agents Chemother. 2000; 44(11): 2925–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLevy O, Ooi CE, Elsbach P, et al.: Antibacterial proteins of granulocytes differ in interaction with endotoxin. Comparison of bactericidal/permeability-increasing protein, p15s, and defensins. J Immunol. 1995; 154(10): 5403–10. PubMed Abstract\n\nWeiss J, Elsbach P, Shu C, et al.: Human bactericidal/permeability-increasing protein and a recombinant NH2-terminal fragment cause killing of serum-resistant gram-negative bacteria in whole blood and inhibit tumor necrosis factor release induced by the bacteria. J Clin Invest. 1992; 90(3): 1122–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLin Y, Kohn FR, Kung AH, et al.: Protective effect of a recombinant fragment of bactericidal/permeability increasing protein against carbohydrate dyshomeostasis and tumor necrosis factor-alpha elevation in rat endotoxemia. Biochem Pharmacol. 1994; 47(9): 1553–9. PubMed Abstract | Publisher Full Text\n\nRogy MA, Moldawer LL, Oldenburg HS, et al.: Anti-endotoxin therapy in primate bacteremia with HA-1A and BPI. Ann Surg. 1994; 220(1): 77–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJin H, Yang R, Marsters S, et al.: Protection against endotoxic shock by bactericidal/permeability-increasing protein in rats. J Clin Invest. 1995; 95(4): 1947–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDahlberg PS, Acton RD, Battafarano RJ, et al.: A novel endotoxin antagonist attenuates tumor necrosis factor-alpha secretion. J Surg Res. 1996; 63(1): 44–8. PubMed Abstract | Publisher Full Text\n\nLin Y, Leach WJ, Ammons WS: Synergistic effect of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein and cefamandole in treatment of rabbit gram-negative sepsis. Antimicrob Agents Chemother. 1996; 40(1): 65–9. PubMed Abstract | Free Full Text\n\nvon der Möhlen MA, van Deventer SJ, Levi M, et al.: Inhibition of endotoxin-induced activation of the coagulation and fibrinolytic pathways using a recombinant endotoxin-binding protein (rBPI23). Blood. 1995; 85(12): 3437–43. PubMed Abstract\n\nBauer RJ, White ML, Wedel N, et al.: A phase I safety and pharmacokinetic study of a recombinant amino terminal fragment of bactericidal/permeability-increasing protein in healthy male volunteers. Shock. 1996; 5(2): 91–6. PubMed Abstract | Publisher Full Text\n\nWiezer MJ, Langendoen SI, Meijer C, et al.: Pharmacokinetics of a recombinant amino terminal fragment of bactericidal/permeability increasing protein (rBPI21) after liver surgery in rats and humans. Shock. 1998; 10(3): 161–6; discussion 167–8. PubMed Abstract | Publisher Full Text\n\nLevin M, Quint PA, Goldstein B, et al.: Recombinant bactericidal/permeability-increasing protein (rBPI21) as adjunctive treatment for children with severe meningococcal sepsis: a randomised trial. rBPI21 Meningococcal Sepsis Study Group. Lancet. 2000; 356(9234): 961–7. PubMed Abstract | Publisher Full Text\n\nWeitz A, Spotnitz R, Collins J, et al.: Log reduction of multidrug-resistant Gram-negative bacteria by the neutrophil-derived recombinant bactericidal/permeability-increasing protein. Int J Antimicrob Agents. 2013; 42(6): 571–4. PubMed Abstract | Publisher Full Text\n\nPalmer CD, Guinan EC, Levy O: Deficient expression of bactericidal/permeability-increasing protein in immunocompromised hosts: translational potential of replacement therapy. Biochem Soc Trans. 2011; 39(4): 994–9. PubMed Abstract | Publisher Full Text\n\nGuinan EC, Barbon CM, Kalish LA, et al.: Bactericidal/permeability-increasing protein (rBPI21) and fluoroquinolone mitigate radiation-induced bone marrow aplasia and death. Sci Transl Med. 2011; 3(110): 110ra118. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGlucksberg H, Storb R, Fefer A, et al.: Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation. 1974; 18(4): 295–304. PubMed Abstract | Publisher Full Text\n\nWoo PC, Wong SS, Lum PN, et al.: Cell-wall-deficient bacteria and culture-negative febrile episodes in bone-marrow-transplant recipients. Lancet. 2001; 357(9257): 675–9. PubMed Abstract | Publisher Full Text\n\nChamplin RE, Schmitz N, Horowitz MM, et al.: Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. IBMTR Histocompatibility and Stem Cell Sources Working Committee and the European Group for Blood and Marrow Transplantation (EBMT). Blood. 2000; 95(12): 3702–9. PubMed Abstract\n\nBensinger WI, Martin PJ, Storer B, et al.: Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers. N Engl J Med. 2001; 344(3): 175–81. PubMed Abstract | Publisher Full Text\n\nMaschmeyer G, Ljungman P: Infections in Hematopoietic Stem Cell Transplant Recipients. In Principles and Practice of cancer Infectious Diseases, A Safdar, Editor. Springer Science + Business media, LLC. 2011; 17–25. Publisher Full Text\n\nPoutsiaka DD, Price LL, Ucuzian A, et al.: Blood stream infection after hematopoietic stem cell transplantation is associated with increased mortality. Bone Marrow Transplant. 2007; 40(1): 63–70. PubMed Abstract | Publisher Full Text\n\nMannion BA, Weiss J, Elsbach P: Separation of sublethal and lethal effects of the bactericidal/permeability increasing protein on Escherichia coli. J Clin Invest. 1990; 85(3): 853–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSrivastava A, Casey H, Johnson N, et al.: Recombinant bactericidal/permeability-increasing protein rBPI21 protects against pneumococcal disease. Infect Immun. 2007; 75(1): 342–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDomingues MM, Silva PM, Franquelim HG, et al.: Antimicrobial protein rBPI21-induced surface changes on Gram-negative and Gram-positive bacteria. Nanomedicine. 2014; 10(3): 543–51. PubMed Abstract | Publisher Full Text\n\nHorwitz AH, Williams RE, Liu PS, et al.: Bactericidal/permeability-increasing protein inhibits growth of a strain of Acholeplasma laidlawii and L forms of the gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes. Antimicrob Agents Chemother. 1999; 43(9): 2314–6. PubMed Abstract | Free Full Text\n\nGuinan EC, Palmer CD, Mancuso CJ, et al.: Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin. Innate Immun. 2014; 20(7): 697–711. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzeto CC, Kwan BC, Chow KM, et al.: Endotoxemia is related to systemic inflammation and atherosclerosis in peritoneal dialysis patients. Clin J Am Soc Nephrol. 2008; 3(2): 431–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrenchley JM, Price DA, Schacker TW, et al.: Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006; 12(12): 1365–71. PubMed Abstract | Publisher Full Text\n\nWiedermann CJ, Kiechl S, Dunzendorfer S, et al.: Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: prospective results from the Bruneck Study. J Am Coll Cardiol. 1999; 34(7): 1975–81. PubMed Abstract | Publisher Full Text\n\nBleijerveld OB, Wijten P, Cappadona S, et al.: Deep proteome profiling of circulating granulocytes reveals bactericidal/permeability-increasing protein as a biomarker for severe atherosclerotic coronary stenosis. J Proteome Res. 2012; 11(11): 5235–44. PubMed Abstract | Publisher Full Text\n\nChien JW, Zhao LP, Hansen JA, et al.: Genetic variation in bactericidal/permeability-increasing protein influences the risk of developing rapid airflow decline after hematopoietic cell transplantation. Blood. 2006; 107(5): 2200–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuinan E, Avigan DE, Soiffer RJ, et al.: Dataset 1 in: Pilot experience with opebacan/rBPI21 in myeloablative hematopoietic cell transplantation. F1000Research. 2015. Data Source\n\nGuinan E, Avigan DE, Soiffer RJ, et al.: Dataset 2 in: Pilot experience with opebacan/rBPI21 in myeloablative hematopoietic cell transplantation. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11664",
"date": "05 Jan 2016",
"name": "Alan Cross",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis proof-of-concept clinical study reports that the peri-HCT administration of a neutrophil-derived protein, BPI, that has endotoxin-neutralizing and antibacterial properties, is well-tolerated and may hasten platelet engraftment and reduce regimen-related toxicities. They did not measure circulating endotoxin levels or inflammation markers (e.g. sCD14 or CRP) to support the rationale for this study, although they do cite a publication by one of the co-authors (Ref 3) that supports this hypothesis. They may want to cite earlier studies by Jonathan Cohen that shows anti-endotoxin antibodies prevent acute GVHD in a murine model.",
"responses": [
{
"c_id": "1797",
"date": "05 Feb 2016",
"name": "Eva Guinan",
"role": "Author Response",
"response": "We thank the reviewer for his thoughtful reading of the manuscript and giving us the opportunity to add additional information for readers. We agree that measurements such as endotoxin levels and levels of endotoxin or endotoxin interactive proteins would enrich the manuscript. However, because the report uses a comparator cohort which was not part of the treatment study design, we have no cohort data to report for comparative purposes. Moreover, the treatment of this small cohort was only 3 days, and given the demographic and clinical heterogeneity of patients at the time of entry it would be difficult to demonstrate trends within patients. We had hoped to do so when the treatment dose and duration extended. Because this was an early phase study, we were focused on safety and PK issues, and the blood volumes for these studies were not permissive of additional samples for frequent monitoring of efficacy biomarkers. The data reported in references 3 and 30 helped us to formulate the rationale for the study as well as the rich literature regarding inflammation and GVHD. We agree that in addition to refs 1-9 (some of which are helpful reviews containing references to additional relevant literature) Bayston et al. (1991) and Abdul-Hai et al. (2006) are helpful additional references demonstrating the effects of antibody-mediated strategies to limit endotoxin bioactivity on GVHD in murine models."
}
]
},
{
"id": "11661",
"date": "20 Jan 2016",
"name": "Brett P. Giroir",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 details the first human experience utilizing rBPI21 during myeloablative HSC. This work follows several earlier publications indicating a role for LPS and induced cytokines in GVHD or other adverse outcomes after transplant in preclinical models. Also, the authors have previously publicized a very important manuscript detailing the positive effects of rBPI21 in a mouse model of ablative radiation.\n\nThe primary findings of this current study are that rBPI21 was safely tolerated in the small group of patients, and that there are suggestive findings of benefit when compared to a non-randomized but matched control population at the same institutions during the same time period. The authors adequately explain the limitations of the study design but also discuss the important implications of this study. The importance of this study is very high, since rBPI21 has a very clean safety profile in humans even during infusion into pediatric patients with septic shock, and could therefore represent a potential easily administered drug with a high therapeutic index in this population.The authors could provide clarifying statements about the time course/kinetics of endotoxemia and LBP elevation in these patients. Are LPS levels high immediately post the ablation, or only become elevated upon infusion of cells, or later in the course? This would help explain the dosing regimen in the study. In the next study, would there be consideration of a second rBPI infusion if there is severe acute GVHD, or is the mechanism of action only prophylactic in nature and not therapeutic during early GVHD? Finally, although it is understood that there is concern over rBPI potential cardiotoxicity based on a high dose preclinical model, there was absolutely NO cardiotoxicity during human infusions, including into patients with septic shock - many of whom had pulmonary artery catheters and precise documentation of cardiac safety (no decrease in CI, SVRI, PVRI, etc.). To exclude a large group of BMT patients exposed to high dose anthracyclines (such as done in this study) may unnecessarily limit access to patients who might highly benefit from rBPI since GVHD is itself documented in BMT patients to induce significant diastolic dysfunction and cardiomyopathy - which could be even worse in patients with pre-existing low ejection fractions or high exposure to cardiotoxic chemotherapy.",
"responses": [
{
"c_id": "1796",
"date": "05 Feb 2016",
"name": "Eva Guinan",
"role": "Author Response",
"response": "We thank the reviewer for his thoughtful reading of the manuscript and giving us the opportunity to add additional information for readers. We agree that measurements such as levels of endotoxin or endotoxin interactive proteins would enrich the manuscript. However, because the report uses a comparator cohort which was not part of the treatment study design, we have no cohort data to report for comparative purposes. Moreover, the treatment of this small cohort was only 3 days, and given the demographic and clinical heterogeneity of patients at the time of entry it would be difficult to demonstrate trends within patients. We had hoped to do so when the treatment dose and duration extended. Because this was an early phase study, we were focused on safety and PK issues, and the blood volumes for these studies were not permissive of frequent, additional samples for monitoring of efficacy biomarkers such as would have been needed with only a short treatment window. The dosing regimen for this pilot study was chosen based on the original concept of the study which was intended to develop hypothesis-generating data on GVHD incidence, and there was therefore a desire to have rBPI21 present and exerting its effects on the host immune and microenvironment prior to infusion of donor cells. As the reviewer suggests, we quite uniformly observed elevation of endotoxin on day 0 (prior to the infusion of donor cells) in our prior descriptive work (ref 30 in this report). However, that study had weekly observations, and therefore there is little granularity about the exact course of endotoxemia in the days immediately prior to or shortly thereafter infusion. In a prior report using a different endotoxin detection assay (ref 3), endotoxemia was present only in 4 of 25 patients on day 0 although half of patients overall developed detectable endotoxemia during the first weeks post transplant. The intent of the study was to look at increasing dose and duration of rBPI21 and thereby establish a safety and feasibility profile that would permit an improved and more focused therapeutic design in future work. The questions of GVHD mitigation and treatment remain theoretical at this time, as outlined in refs 1-9. We agree with the reviewer‘s concern that issues related to potential for cardiac toxicity could be viewed as overblown. The manuscript does state that no cardiac toxicity had been seen in human studies and that the animal data was very circumscribed in this regard. Because patients undergoing hematopoietic stem cell transplantation frequently have a history of exposure to cardiotoxic agents prior to and often during transplant, we nonetheless thought it prudent in this Phase 1 safety study to exclude patients with potential for increased risk and observe treated patients carefully. We did not intend to infer that such patients should be excluded from treatment in subsequent clinical trials."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1480
|
https://f1000research.com/articles/3-91/v1
|
17 Apr 14
|
{
"type": "Research Article",
"title": "Ten things to get right for marine conservation planning in the Coral Triangle",
"authors": [
"Rebecca Weeks",
"Robert L. Pressey",
"Joanne R. Wilson",
"Maurice Knight",
"Vera Horigue",
"Rene A. Abesamis",
"Renerio Acosta",
"Jamaluddin Jompa",
"Robert L. Pressey",
"Joanne R. Wilson",
"Maurice Knight",
"Vera Horigue",
"Rene A. Abesamis",
"Renerio Acosta",
"Jamaluddin Jompa"
],
"abstract": "Systematic conservation planning increasingly underpins the conservation and management of marine and coastal ecosystems worldwide. Amongst other benefits, conservation planning provides transparency in decision-making, efficiency in the use of limited resources, the ability to minimise conflict between diverse objectives, and to guide strategic expansion of local actions to maximise their cumulative impact. The Coral Triangle has long been recognised as a global marine conservation priority, and has been the subject of huge investment in conservation during the last five years through the Coral Triangle Initiative on Coral Reefs, Fisheries and Food Security. Yet conservation planning has had relatively little influence in this region. To explore why this is the case, we identify and discuss 10 challenges that must be resolved if conservation planning is to effectively inform management actions in the Coral Triangle. These are: making conservation planning accessible; integrating with other planning processes; building local capacity for conservation planning; institutionalising conservation planning within governments; integrating plans across governance levels; planning across governance boundaries; planning for multiple tools and objectives; understanding limitations of data; developing better measures of progress and effectiveness; and making a long term commitment. Most important is a conceptual shift from conservation planning undertaken as a project, to planning undertaken as a process, with dedicated financial and human resources committed to long-term engagement.",
"keywords": [
"The Coral Triangle",
"which encompasses the marine waters of Indonesia",
"Malaysia",
"Papua New Guinea",
"Philippines",
"Solomon Islands",
"and Timor-Leste",
"is the epicentre of marine biodiversity and widely recognised as a global conservation priority1. In addition to their conservation value",
"the Coral Triangle’s marine resources are a cornerstone of the region’s economies and societies",
"with millions of people dependent upon them as a daily source of food and income2. The health of these ecosystems is at severe risk due to destructive and over-fishing",
"coastal development",
"poor water quality",
"and climate change3."
],
"content": "Introduction\n\nThe Coral Triangle, which encompasses the marine waters of Indonesia, Malaysia, Papua New Guinea, Philippines, Solomon Islands, and Timor-Leste, is the epicentre of marine biodiversity and widely recognised as a global conservation priority1. In addition to their conservation value, the Coral Triangle’s marine resources are a cornerstone of the region’s economies and societies, with millions of people dependent upon them as a daily source of food and income2. The health of these ecosystems is at severe risk due to destructive and over-fishing, coastal development, poor water quality, and climate change3.\n\nIn 2009, the six Coral Triangle countries, supported by USAID and other external funders, embarked upon the Coral Triangle Initiative on Coral Reefs, Fisheries and Food Security (CTI-CFF), an unprecedented multilateral partnership to address threats to the region’s marine and coastal resources through accelerated and collaborative action. The CTI-CFF goals include the designation of priority seascapes, establishment of a Coral Triangle marine protected area (MPA) system, the protection of threatened species, coordinated action on climate adaptation, and implementation of an ecosystem approach to fisheries management4. As the initial five-year phase of the CTI-CFF came to a close in 2013, much has been accomplished, but much work remains if these goals are to be achieved5.\n\nMarine conservation planning is a systematic approach to developing spatial plans, primarily focused on conservation of biodiversity, habitats and ecological processes, while facilitating multiple uses of the marine environment and promoting, where possible, goals related to climate change, fisheries, and livelihoods. In the last three decades, conservation planning has evolved from an academic discipline to have considerable influence on conservation action around the world6. Yet, systematic approaches have had relatively little influence on conservation in the Coral Triangle – one region where they are needed most.\n\nConservation planning provides benefits at both regional and local scales. Regional-scale planning is critical for achieving objectives that require broad perspectives and emergent properties7 that mean the whole (e.g. a system of MPAs) is greater than the sum of the parts (individual MPAs). Emergent properties are achieved through, for example, complementarity of ecosystems and species and connectivity between individual MPAs8,9. Systematic planning provides a transparent framework to ensure efficient use of limited resources, and offers a proactive alternative to reactive actions in the face of increasing threats to natural resources10,11. Importantly, systematic planning can be used to minimise conflict between conservation goals and the diverse aspirations of users of the marine environment12,13.\n\nThe Coral Triangle has experienced rapid growth in the number of individual MPAs designated or initiated primarily by communities and local governments. Yet, despite the many benefits of local and community-led actions, few MPAs are effectively managed14 and, without coordination, they often fail to form functional conservation networks that achieve regional-scale objectives15–17. Systematic planning can inform strategic expansion of local actions to maximise their cumulative contribution towards regional-scale goals18,19, including the broad goals of the CTI-CFF.\n\nSystematic conservation planning in the Coral Triangle faces particular challenges. With the first phase of the CTI-CFF now complete, it is timely to explore these challenges and how they might be overcome. To do so, we convened a focus group of conservation biologists, practitioners, policy makers, and donors working in the region (the authors). We first outlined a vision: of conservation planning applied throughout the Coral Triangle, at spatial scales ranging from local to region-wide, to effectively inform management actions implemented to achieve objectives for biodiversity, fisheries, and food security. We then considered constraints on this vision being realised, and sought to identify strategies to overcome the constraints.\n\nFrom an initial list of “things to get right”, we consolidated related topics, and excluded those that we considered either trivial or overly specific, to arrive at the final 10 (Table 1). The topics that we discuss here are deliberately ambitious, and we do not claim either a complete analysis of these challenges or to provide solutions. Our aim is to highlight issues that have not been widely approached or discussed in the literature, which has focused primarily on technical aspects of MPA network design e.g.20. We acknowledge that the context for, and approaches to, conservation planning vary widely throughout the Coral Triangle. While some of the strategies we propose are being applied to some extent in parts of the region, and we highlight examples of these, without exception they are not being addressed effectively or extensively enough in the Coral Triangle, or indeed in many other regions. The overall goal of this paper is to contribute to the evolving agenda for marine conservation planning in the Coral Triangle by stimulating dialogue about these important and neglected topics.\n\nEach of these topics is discussed further in the text.\n\n\nThings to get right\n\nFor each of our 10 topics (summarised in Table 1), we first outline a problem statement, and then suggest potential ways forward.\n\nHistorically, marine conservation planning in the Coral Triangle has typically been initiated and led by non-government organisations (NGOs) or academia, often through collaboration with local communities or governments18,21–23. These planning initiatives, although valuable in demonstrating concepts, analyses and, at limited scales, applications to on-ground actions, have inevitably focused on specific areas within the Coral Triangle that constitute a very small proportion of the entire region. To achieve wider application of marine conservation planning, the established and emerging approaches and tools need to be made more accessible to a much wider range of practitioners, including those in government agencies responsible for spatial planning at levels from local to national.\n\nBeyond a lack of local capacity to implement conservation planning methods (see section 3), there exists a lack of awareness of what conservation planning is and why it is needed, among people responsible for managing coastal and marine resources. In the past, the dominance of developed countries in generating research on conservation planning biased approaches toward extensive prioritizations and, where these are actually applied, toward simple governance contexts7. These biases led to earlier misconceptions amongst policy-makers and potential practitioners in developing countries that conservation planning is only relevant to top-down, centralised planning24, that it is incapable of dealing with the social, economic, and cultural complexities of the Coral Triangle25, and that planning processes depend upon the use of decision-support software and data of a quality and quantity that are generally unavailable in the region. The CTI-CFF has helped to reduce these misconceptions and recognise the importance of combining bottom-up and top-down engagement to achieve goals for conservation planning. For example, the Solomon Islands and Timor-Leste have embraced and allocated funding for community-based planning as the foundation of their national approaches (personal communication: R. Pinto, Conservation International Timor-Leste; A. Vavekaramui, Solomon Islands Ministry of Environment, Meteorology, Disaster Response and Climate Change). However, systematic conservation planning is still largely a peripheral and under-valued activity in the overall operations of government organisations.\n\nMaking conservation planning accessible requires increasing the exposure of those responsible for spatial planning and coastal resource management to conservation planning concepts and processes, and at the same time dispelling misconceptions that might have developed from limited information.\n\nMuch that is written about conservation planning appears in literature that is inaccessible to potential users in the Coral Triangle. A learning study conducted at the end of the five-year USAID Coral Triangle Support Partnership program26 revealed that, among those surveyed, 54% never or rarely used the 265 separate knowledge products produced, and only 20% frequently or often used them. These products included position papers, books, training manuals, field guidance manuals and other materials produce by the USAID Coral Triangle Support Partnership, the US National Oceanic and Atmospheric Administration and the US CTI Support Program Integrator based in Bangkok. Poor uptake is likely due to lack of awareness of recently produced materials, and the need for more simplified materials for some applications. This clearly shows that coastal resource management practitioners in the Coral Triangle are in a nascent stage with respect to accessing literature and knowledge products supporting conservation planning.\n\nA broader, more accurate awareness of conservation planning will be achieved by encouraging researchers to publish in open-access journals27, translating technical documents and case studies into local languages, and distributing presentations and documents through peer-learning networks (e.g. the Philippine MPA Support Network). Knowledge products need to be more effectively distributed, including in local languages, and materials that are made available need to be in forms that match the needs and capacity of their target audience. Simplified and demystified information products are required to dispel misconceptions about conservation planning and highlight the balance between top-down and bottom-up planning.\n\nMisconceptions that planning is necessarily a “top down” process can generate reluctance amongst stakeholders to engage. Case studies such as those presented in Game et al.18 and Weeks and Jupiter28 demonstrate that conservation planning tools and methods can be used as inputs for community-based decision making. Further examples that emphasise entire, participatory planning processes and place less emphasis on decision-support tools are required. NGO-led initiatives tend to be supported by expertise and funding rarely available to government agencies e.g.22. Case studies that demonstrate how conservation planning can be undertaken within the financial, technical, and resource constraints typical of government agencies within the Coral Triangle are needed. These are now emerging; an example is the recently approved and budgeted community-based conservation program in Timor-Leste, piloted under the USAID CTSP program29.\n\nFinally, it will be necessary to demonstrate the benefits of planning, and costs of not planning, compared to counterfactual scenarios of unplanned expansion of MPAs or alternative management strategies19. For example, it can be demonstrated that objectives for biodiversity conservation can be achieved at a lower cost to resource-users under planned than unplanned scenarios19, and that opportunity costs to different users can be explicitly and transparently identified to minimise conflict30.\n\nCoastal areas and inshore waters are subject to many potentially competing planning processes, such as those for maritime transport, environmental protection, energy, fisheries, and tourism. Frameworks for marine spatial planning and integrated coastal zone management have been proposed to integrate the spatial aspects of sectoral policies in these diverse areas31. These frameworks aim to meet ecological, economic, and social objectives32, facilitate explicit trade-offs between competing uses, improve transparency in decision-making, and help to avoid unnecessary conflicts33,34. Yet coastal resource management in the Coral Triangle remains highly sectoral, with overlapping and incompatible jurisdictions, and unclear, and sometimes conflicting, mandates for different government agencies35,36. Aside from avoiding areas obviously incompatible with conservation (e.g. ports, shipping lanes), there are few examples of fully integrated spatial plans.\n\nSpatial and non-spatial planning strategies relating to production and development sectors are likely to be better funded, more widely understood, and more strongly institutionalised within government (see section 4) than conservation planning. Consequently, to have influence, conservation plans must interface with and inject a conservation perspective into these planning processes. This integration has been referred to as mainstreaming conservation plans37,38. Failure of conservation planners to engage with the larger enterprise of marine spatial planning involves several risks: marginalisation of conservation objectives; unnecessary conflict between conservation plans and more influential, development initiatives; and adverse, avoidable impacts on local communities reliant on marine resources for subsistence or small cash economies.\n\nSeveral advances are needed to better integrate conservation planning with the diverse aspects of marine spatial planning. Influence of conservation thinking on development planning requires anticipating commencement or reviews of development plans and policies, sectoral integration between agencies responsible for conservation and development planning, strong liaison of conservation scientists and NGOs with agencies responsible for development planning, and appropriately formatted information to provide inputs to processes for planning development37. Disparity between objectives can result in differences in structure and content between the outputs of conservation planning processes and those required for spatial planning more generally37. Integration can be facilitated if decision-support software tools developed to address the problem of prioritising areas for biodiversity conservation (e.g. Marxan, Zonation, C-Plan) are refocused to guide strategic expansion of development or extractive activities to have minimal impact on high-value sites for biodiversity39–42. An example would be to delineate shipping channels to minimise impacts on marine megafauna.\n\nSome progress toward sectoral integration is evident in the Coral Triangle. In Indonesia, prior to laws relating to spatial planning and management of coastal areas and small islands passed in 2007, coastal resources were governed by a vast array of statutes and laws with dozens of implementing agencies35. Indonesia is now moving towards a more integrated approach43. However, local government agencies previously mandated to zone terrestrial and urban areas lack capacity in marine conservation planning44 (see “Building local capacity for conservation planning”).\n\nUltimately, effective integration of conservation objectives and priorities into marine spatial planning requires explicit analysis of trade-offs: specifically, identifying the extent to which diverse objectives for conservation, development, and livelihoods are mutually exclusive, and providing a decision-making framework to resolve conflicts with a proper understanding of the implications of some objectives not being fully achieved45,46. The required methods are being devised, but have seldom been applied for real-world decisions, and remain inaccessible to emerging leaders in Coral Triangle countries.\n\nSince the CTI-CFF was conceived, building capacity has been a priority for all six Coral Triangle countries, which are presently under-resourced to support the >1500 existing MPAs, let alone achieve ambitious goals of protecting 20% of marine and coastal habitats by 202012. Even countries with relatively well-developed capacity for marine conservation planning - Malaysia, Philippines, and Indonesia47 - have identified lack of in-country capacity as a key hurdle to achieving CTI-CFF goals36.\n\nAlthough training on MPA management is available (from NGOs, government, and universities), lack of communication and coordination between training providers has led to delivery of non-standard modules, duplication, and omission of key competencies36. Furthermore, capacity building is often delivered as one-off training and, without follow-up assistance and mentoring, skills and knowledge acquired during training can be quickly lost. To undertake conservation planning, individuals, or at least planning teams, need to have a broad range of skills and knowledge that extend beyond those typically covered by existing training. Skills are needed in ecology, social science, the use of specialist software or GIS (geographic information systems), stakeholder engagement, communication, and negotiation, to name a few.\n\nAs a consequence of these limitations, managers of MPA networks depend heavily upon assisting organisations (e.g. NGOs, academe, development partners, or donors) for technical support with planning18,48. There are few organisations in the Coral Triangle with sufficient capacity to develop and implement effective conservation plans, and those that have the capacity are not sufficiently staffed or resourced to extend their support to all who request it. Broadening the base of technical experts will be crucial, especially in smaller countries such as the Solomon Islands, Timor-Leste, and Papua New Guinea, where existing experts are stretched to deliver support across the many problems requiring their attention49.\n\nA common approach to develop capacity in the region is through peer-learning networks, such as the Philippine MPA Support Network, Papua New Guinea Centre for Locally Managed Areas, the regionally focused Coral Triangle Center in Bali, Indonesia, and the Locally Managed Marine Areas network, active in Solomon Islands and Indonesia. These learning networks facilitate cross-site visits and similar events that allow members to share experiences and lessons learned, and provide access to training modules or events. Although training often focuses on specific aspects of MPA management, learning networks could provide a venue through which information on conservation planning might also be disseminated.\n\nA crucial solution to develop capacity in the long-term will be to create a new cohort of conservation planners from the region, through development of specific courses, qualifications, and competency standards that recognise marine conservation planning as a profession50. Targeting students in related disciplines with university short-courses and curricula51 that focus specifically on marine conservation planning would be a short-term step in this direction. The University of the Philippines’ Marine Science Institute is currently developing a Masters program on Tropical Marine Ecosystem Management aimed at local government employees and MPA managers, which includes specialisations on MPAs and spatial planning. We envision that conservation planners will eventually be represented within the relevant national government agencies and local governments in the Coral Triangle region (see “Institutionalising conservation planning within governments”).\n\nCapacity might also be built through improved and sustained collaboration between scientists from developed nations and local research communities. For example, a Partnerships in International Research and Education (PIRE) project funded by the US National Science Foundation placed US graduate students and postdoctoral scholars in research institutions in Indonesia and Philippines for a year, providing improved laboratory infrastructure, research funding, and new educational opportunities for Filipino and Indonesian scientists and students27. USAID has also funded partnerships between US and Indonesian universities, fostering strong connections between Indonesian scientists and international collaborators27.\n\nAt present, conservation plans for regions within the Coral Triangle are frequently developed and implemented as projects led by NGOs or academic institutions with restricted time frames and limited budgets for engagement. Project-based conservation planning is undesirable for two reasons. First, supporting organisations have their own motivations for involvement in planning initiatives, which are manifest in the regions selected for planning effort: typically those with extraordinary biodiversity value or particular research interest12,52. Thus, under the project model, conservation planning is spatially biased and will be undertaken only in few parts of the region. Second, conservation plans quickly become out-dated as ecological and socioeconomic conditions change. In the common case of protracted implementation, continuity of resources and expertise is required over extended time periods7. If plans are conceived and developed as finite projects, funds might not be secured for the ongoing implementation, adaptation, and revision required to keep them relevant, and personnel with capacity to interpret and update plans might be lost to lead organisations or redeployed to other roles. Institutionalising conservation planning within government will ensure that planning effort is invested much more widely, and is necessary if resources for protracted implementation and adaptation are to be maintained.\n\nInstitutionalizing conservation planning will present substantial challenges, but none appear to be intractable. Each of the six countries of the Coral Triangle has distinct governance arrangements with respect to spatial planning, biodiversity conservation, and management of coastal resources14. Approaches to institutionalise conservation planning will therefore need to be sensitive to these differences. Within-country differences in approach will also be necessary47. Governmental reform is seldom rapid, although the need to embed conservation planning in government at all levels is urgent. Still, the groundbreaking nature of the CTI-CFF itself, and the consequent progress toward multi-jurisdictional vertical (see “Integrating plans across governance levels”) and horizontal (see “Planning across governance boundaries”) cooperation, demonstrates that high-level reform for marine conservation is possible.\n\nA practical first step towards institutionalizing conservation planning would be to review the current legislative and institutional environments, at different levels of government (including customary governance) in each country, to identify appropriate entry points at which authority, legitimacy, and willingness to undertake conservation planning overlap. For example, Indonesia has comprehensive legislation that requires district governments to prepare spatial plans. These same government units are responsible for implementing MPAs, and thus offer an entry point to integrate conservation planning perspectives (see section 1). In contrast, in Papua New Guinea, there is no formal legislation supporting declaration of MPAs or spatial planning. However, strong systems of traditional resource ownership and customary law53 provide an alternative route by which conservation plans can be developed and implemented by communities with customary tenure18, e.g.22. Here, conservation planning might better be institutionalised within customary, rather than formal, governance structures.\n\nLevels of governance in the Coral Triangle range from international to national, sub-national (provinces, states), and local (e.g. municipalities, districts, communities). Decisions made at one level of governance influence the suite of actions available to, or mandated by, decision-makers at other levels54. Thus, spatial plans must be carefully integrated across spatial scales and levels of organisation55,56 to avoid plans and policies that conflict, or are difficult to interpret or enforce.\n\nUse of marine and coastal resources in the Coral Triangle is frequently subject to overlapping legislation and unclear jurisdictions, often with multiple implementing government agencies at different levels35,57. For example, in Indonesia, the enactment of a series of laws in 1999 shifted responsibility for spatial planning and coastal resource management from the national to the district level, leading to conflict with pre-existing laws and ambiguity regarding the roles and responsibilities of national, sub-national, and local government authorities35. National, sub-national, and local governments’ roles typically address different public needs and consequently can have different, sometimes conflicting, perspectives58. In many parts of the Coral Triangle, governance is further complicated by overlap of authority between formal and customary government systems: whilst customary tenure is recognised in national constitutions, traditional systems of natural resource management tend to be poorly integrated with national policies and legislation59.\n\nIntegration of conservation plans across governance levels should operate in two directions. First, region-wide initiatives such as the CTI-CFF need to be supported by actions at national, sub-national, and local levels by translating broad policy directives and planning principles into guidelines for identifying spatial priorities at progressively lower levels of governance. As seascape-scale planning initiatives become more common, there will be a need to ensure that these effectively inform local actions. This requires larger plans to be seen, not as static products, but as starting points for ongoing adaptation to changes in local circumstances, including unforeseen errors in seascape-scale data7. Similarly, national policies must be reflected in local plans. For example, in Malaysia, national regulations spatially demarcate a “commercial fishing zone” beyond three nautical miles from the coastline and a “traditional fishing zone” within that limit; these regulations provided a foundation for the process of zoning the Tun Mustapha Park, which subdivided the “traditional fishing zone”12.\n\nSecond, local marine management actions must be legally recognised and reinforced by higher levels of governance60. This is necessary both for local-level legislation, and customary governance. Otherwise, rules conceived and implemented locally might not be enforceable to outsiders who do not respect local customs and are beyond the reach of community-imposed punitive actions61,62. A further challenge is to anticipate and keep track of local actions not planned for at higher governance levels and the contribution that these make towards wider objectives. The Coral Triangle Marine Protected Area System (CTMPAS) framework, supported by the Coral Triangle Atlas, will play an important role in facilitating this63.\n\nIn some contexts, scale-bridging organisations and networks, such as the Solomon Islands’ Locally Managed Marine Areas Network and Philippines’ MPA Support Network, can play a critical role in facilitating interactions between levels of governance48,64. Other contexts might require legal reform, to ensure that plans consider existing laws and regulations at different scales65. Later revisions of Indonesia’s decentralization framework, for example, sought to clarify jurisdictional roles by emphasizing relationships between national and district governments, rather than local autonomy35. Efforts to align policy across governance levels must be undertaken with care. In Papua New Guinea, efforts to strengthen coordination between national and provincial fisheries authorities had the unintended consequence of weakening links with local governments: provincial priorities became aligned with national interests (commercial fisheries) at the expense of local concerns58.\n\nThe boundaries of natural resources rarely match those of the governance institutions responsible for managing them56,66. This is certainly true for marine resources in the Coral Triangle, where ecological connectivity processes can operate across spatial scales of tens to thousands of kilometres67,68, but where management is, for the most part, decentralised to local governments and communities69. Where the scale of ecological processes exceeds that of management jurisdictions, transboundary coordination is essential to avoid management efforts being insufficient to adequately protect the features and processes concerned. Furthermore, some benefits from management, such as enhanced recruitment arising from protection of spawning aggregations, might be realised beyond the boundaries of managing jurisdictions, undermining support for management70.\n\nTo achieve management outcomes across ecologically meaningful scales will require coordination of planning across governance boundaries66,71, as well as arrangements for equitable sharing of the costs and benefits of management72. For example, if a fish spawning-aggregation site is protected in one jurisdiction, complementary seasonal restrictions on catch of that species in neighbouring jurisdictions can provide increased ecological and fisheries benefits in all jurisdictions73. However, inequitable distribution of the costs and benefits of conservation among stakeholders or jurisdictions might result in social or political conflict, failure during implementation, or poor compliance with management regulations74,75. Plans that span multiple jurisdictions also need to incorporate multiple (sometimes divergent) objectives identified within different governance units76.\n\nTransboundary planning might be most easily approached at local scales. This has been achieved to some extent in the Philippines, through the formation of local government alliances for coastal resource management48. Motivation for collaboration typically comes from recognition of a common resource base and shared threats, such as the intrusion of commercial fishing vessels into coastal waters77. Where such a shared vision is absent, neutral assisting organisations can act as brokers, helping to overcome social or political obstacles to coordination78,79. Alternatively, more innovative approaches to transboundary coordination, such as payments for transboundary ecosystem services80, might be required.\n\nPlanning across international boundaries is likely to present the greatest challenge. For example, achieving CTI-CFF goals on managing priority seascapes and ecosystem approaches to fisheries management will additionally require negotiating access to high-value shared stocks (e.g. tuna), issues of national sovereignty, and financing79.\n\nMarine conservation planning has, to date, focused largely on the design and implementation of ‘no-take’ MPAs and MPA networks, although approaches that consider multiple actions are emerging in the literature e.g.81. The establishment of a region-wide, comprehensive, ecologically representative, and well-managed CTMPAS is one of six strategic goals of the CTI-CFF4, and guidelines for the size and location of no-take MPAs in the Coral Triangle have recently been developed20. However, aside from the fact that few MPAs are presently well managed or adequately enforced14, there are two important limitations of no-take MPAs as tools for biodiversity conservation in this region. First, where local dependence on resources is high, and spatial or occupational mobility is limited (as in much of the Coral Triangle), no-take zones are necessarily small. The median size of no-take areas in the Philippines, for example, is just 0.12 km216. Furthermore, in some areas of the Coral Triangle, tradition or preference for alternative management strategies means that permanent no-take areas are rarely supported by stakeholders70. Second, whilst no-take MPAs have proven benefits for biodiversity, fisheries and food security, they cannot manage many threats to marine and coastal ecosystems, such as land-based sources of nutrients and sediment or coral bleaching events related to climate change, and they offer only limited protection for migratory and wide-ranging species but see23,82.\n\nFurthermore, if conservation planning is to be relevant to the CTI-CFF, it must address not only MPAs but also cross-sectoral integration of goals related to biodiversity, fisheries, and food security, and help to resolve inevitable trade-offs between these e.g.2. Part of this challenge is for conservation planning to move out of its comfort zone in designing networks of no-take MPAs to consider a wider range of coordinated management tools that can address all major threats at relevant scales83. The need for conservation planning to address a broad suite of actions is underlined by some simple facts: 90% of coral reefs in the Coral Triangle are under threat3, while >80% of the region’s coral reefs are likely to remain outside of the CTMPAS, and a large proportion of inshore reefs, whether inside or outside MPAs, are adversely affected by terrestrial runoff3.\n\nThe Coral Triangle has a long history of employing traditional and customary management practices other than no-take MPAs. Examples are temporary or periodically harvested fisheries closures variously known as sasi, tabu, or taboo,62,84. Conservation plans that employ familiar strategies such as these will likely be better supported locally70, and will fit within existing governance frameworks. Multiple-use zoning offers a more flexible approach to resource management that can help to resolve trade-offs between multiple objectives82. For example, in Indonesia’s Nusa Penida MPA, multiple-use zoning was used to resolve conflict between marine tourism, seaweed farming, and fisheries activities, ensuring that the interests of all stakeholder groups were clearly represented in the plan12.\n\nPlanning for multiple tools, zones, or objectives is more complex than designing no-take MPA networks for biodiversity conservation. It requires more parameters to be estimated (with inevitable errors), increasing the need for plans to be adjusted when errors become apparent during implementation7, and requiring further iterations of planning and stakeholder consultations. For example, planning for multiple tools requires an understanding of the contribution of different management actions towards different objectives19.\n\nIdeally, conservation planning would extend from inland watersheds to offshore waters, with integrated management of coasts and near-shore marine ecosystems85. Among the impediments to designing and implementing fully integrated land-sea planning is the need to work at multiple levels of governance (see “Integrating plans across governance levels”) and across governance boundaries (see “Planning across governance boundaries”). Although planning methods are extending into this complexity of geography and governance86, practical applications of such integration in the Coral Triangle are rare.\n\nLimitations of data are unavoidable in conservation planning87. These limitations apply not only to data on biodiversity, but also to data on costs, opportunities, threats, and other spatial variables that are increasingly being used to make spatial decisions7. This is especially true in the Coral Triangle, where data are generally sparser than in some other regions69,88,89. Whilst paucity of data should not necessarily be seen as an obstacle to initiating conservation planning processes, conservation decisions can be more effective in promoting the persistence of biodiversity and livelihoods if some important limitations of data are understood or avoided. We focus here on two aspects of mapped data: spatial resolution and surrogacy.\n\nSpatial resolution refers to the size of the smallest homogeneous area that describes biodiversity, cost, opportunities, or threats. In general, the more extensive the coverage, the coarser is the spatial resolution of consistent data e.g.90. This also means that fine-resolution data tend to be available only in small parts of many planning regions, if at all. One implication is that priorities based on coarse-resolution data can be poorly aligned to those based on fine-resolution data available over smaller extents91. A related issue is that more extensive assessments tend to use larger planning units, sometimes even whole bioregions92, thereby blurring spatial variation between management units (e.g. traditional fishing grounds), which are generally very small in the Coral Triangle69, while also increasing estimates of overall conservation costs93,94. Discordance between the resolution at which priorities are identified and that required for decisions about on-ground management mean that extensive, coarse-resolution analyses have little to offer local managers54. Importantly, there is no reason to assume that conservation priorities are spatially nested; very large planning units identified as priorities will not necessarily contain all the priority areas that would later be identified with smaller planning units7.\n\nAlmost all data in conservation planning are surrogates, meaning that they approximate the variables of actual interest but for which spatial data are impossible to collect with available resources. Familiar examples are maps of ecosystems as surrogates for poorly mapped or still undescribed species8. For threats, distance to population centres might be a surrogate for exposure of marine waters to destructive fishing practices, even though actual threats vary with types of fishing gear used, attitudes of local fishing communities, dependence on types of marine resources, and links to markets66,95,96. With assessments that are more extensive and in regions with poorer data, conservation planning will rely on surrogates that are more remote from variables of primary importance, making priorities for conservation less reliable. In the Philippines, for example, coastal population density is strongly correlated with fishing pressure at the provincial scale but, at finer spatial resolutions, greater occupational diversity in more urbanised areas makes this a poor surrogate97.\n\nThe most obvious solution to problems related to resolution and surrogacy of data is to collect more accurate information on variables of interest at the resolution of management units throughout the Coral Triangle. This is more easily said than done, of course, with about 800 coastal municipalities in the Philippines48, and many thousands of management units across the Coral Triangle. Nonetheless, whilst recognising that investment in data might compromise investment in conservation actions, better data will eventually lead to better planning. Demonstrations of the prospects for improved data in the Coral Triangle include the increasing quality and availability of remote-sensing imagery on coral reefs e.g.98, the potential to adjust collection of census information to improve socio-economic data for planning99, and participatory mapping of resource use and features such as spawning aggregation sites, which has the added advantage of engaging local stakeholders in decisions about conservation.\n\nIn some cases, data and conservation assessments might simply have to be ignored because their use would be counterproductive. Data at very coarse resolution and based on unreliable surrogates will not only fail to resolve spatial variation relevant to applying actions, but can also pre-emptively divert attention from areas that would be identified as important, had better data been used. Similarly, very extensive conservation assessments that use large planning units can be counterproductive because two key (though generally implicit) assumptions are unreliable7: uniformity (that priority is uniform within planning units); and nestedness (that high priorities at coarse resolution will contain all high-priority areas at fine resolution). These limitations mean that extensive prioritisations should be replaced with bottom-up assessments that build toward flexible regional designs.\n\nConservation, whether for biodiversity or livelihoods, receives much attention globally through policy and legislation and large amounts of funding through diverse initiatives from governments, NGOs, and private donors. The objectives and performance of conservation initiatives, in the Coral Triangle and elsewhere, are measured mainly in terms of inputs (e.g. dollars invested), outputs (e.g. protected area extent), or, less commonly, outcomes (e.g. representation of marine ecosystems in protected areas). The widespread emphasis on outputs of marine conservation efforts is illustrated in the ongoing preoccupation with one of the internationally endorsed Aichi targets (Target 11, 10% of marine and coastal areas under protection). Similarly, the CTI-CFF Regional Plan of Action “ultimate target” is to include 20% of each major marine and coastal habitat type in strictly no-take replenishment zones4.\n\nThe problem with these goals and measures is that outputs can be unrelated to progress for biodiversity conservation or livelihoods. For example, the extent of marine protected areas globally and in Australia reflects efforts made to establish them where they are most expedient politically and least required to protect biodiversity100. In terms of livelihoods, there is little evidence that the extent of protected areas is related to benefits to people101. Even outcomes can be poor measures of actual progress. For example, increases in representativeness, the number of ecosystems covered by protected areas, can mask simultaneous increases in the bias of protection away from those ecosystems most in need of protection102,103.\n\nMeasuring conservation progress in terms of inputs, outputs, and outcomes results in means (establishing protected areas) being confused with ends (making a positive difference for biodiversity or livelihoods). Fundamentally, marine protected areas and related management actions are intended to make a positive difference, yet this difference is almost never measured.\n\nThe emerging field of conservation impact evaluation104 promises to enable funders and policy-makers to extend measures of progress and effectiveness to assess directly how much difference existing conservation actions make to biodiversity and livelihoods, or how much difference future actions could make. Impact evaluation measures the effects of an intervention by comparing what happened with the intervention compared with what would have happened without the intervention (i.e. the counterfactual;105). It is important to note that impact evaluation of conservation initiatives is very distinct from environmental impact assessment of development projects.\n\nOver and above measures of inputs, outputs, and outcomes, impact evaluation offers two critical improvements. The first is attribution – ensuring that the observed changes flow from the intervention being assessed, not from unrelated contextual changes106. It is important, for example, to understand whether livelihoods improve in response to a conservation initiative, as distinct from increased living standards across a region related to, say, macroeconomic changes. The second improvement provided by impact over other measures is the distinction between means and ends6. If the ultimate goal of a program is to reduce the loss of biodiversity, then impact is the amount of loss avoided, relative to the amount had the program had not been implemented. Approaches to measuring the impact of protected areas retrospectively, to provide lessons for the future, are now well developed107. Approaches to predicting where future protected areas could have greatest positive impact are also available108.\n\nThe existing work on impact evaluation of protected areas, although mostly focused on terrestrial ecosystems, can now be adapted and applied to marine conservation in the Coral Triangle. Following the lead of the health and energy sectors106, impact evaluation can also be extended to diverse on-ground interventions, such as partial fisheries closures, and strategic interventions including legislation, policy, and education. For these changes to happen, one requirement is applied research to adapt and extend existing methods for impact evaluation to the Coral Triangle, accounting for available data, capacity and the diversity of social and governance contexts. The other need is for impact evaluation theory and methods to be made more accessible to policy makers and practitioners in the Coral Triangle (see “Making conservation planning accessible”). A first step towards this is to ensure that established monitoring and evaluation programs produce data that can be used to assess impacts.\n\nThe long-term commitment required for effective conservation planning is generally under-appreciated7,109. Temporal-scale mismatches arise where short funding or electoral cycles conflict with long-term planning needs56, and there has been a tendency towards funding models that value short-term project outputs, such as the development of conservation prioritisations or plans on paper, over long-term, effectively implemented outcomes (see “Developing better measures of progress and effectiveness”). This is likely driven in part by the ease of demonstrating fulfilment of project goals linked to outputs, as opposed to less tangible outcomes, such as increased capacity of communities to undertake adaptive management. Another crucial factor is the time taken for the conservation impact of investments to become manifest and the general lack of methods for measuring impact6. Focusing on short-term outputs fails to recognise that spatial prioritisation is merely the first, and arguably the easiest, phase of conservation planning, and must be followed by protracted processes of application7, monitoring, and ongoing adaptive management and planning. Failure to conceive, and adequately fund, conservation planning as a complete planning – implementation package is a major reason why plans have failed to find traction in many parts of the world109. Approaches that acknowledge the need for application but allow insufficient time or funds might attempt to expedite implementation but, in doing so, risk losing the support of stakeholders, leading to poor compliance and failure.\n\nMaking a long-term commitment to conservation planning requires a single organisation with responsibility for steering planning outputs towards sustained outcomes. This will be realised through a shift from project-based conservation planning, towards planning processes institutionalised within government or NGOs (see “Institutionalising conservation planning within governments”). This change in approach also requires a move away from project-oriented funding models by governments and donors towards institutionalised allocations for conservation planning that are increasingly embedded within government structures.\n\nWhilst short-term political cycles are unlikely to change, opportunities might exist to safeguard conservation plans and actions against changes in political leadership or environmental orientation. At local governance levels, leadership and legislative processes tend to move more quickly than at higher levels, facilitating rapid implementation, but also allowing laws to be quickly revoked. One way to buffer against potential setbacks at the local level is to reinforce conservation plans through legislation at higher levels of government (see “Integrating plans across governance levels”). This strategy was adopted for the Sumilon Marine Reserve in the Philippines after a newly-elected local mayor with links to commercial fishing operations actively sought to degazette the MPA110. Another example is the new Solomon Islands National Protected Areas Act, which establishes a legal process for national recognition of sub-nationally established protected areas. There is a risk that formalising local conservation plans under national legislation can negate other benefits of localised governance, such as ownership and adaptive capacity e.g.111, but this risk can be offset by transparency and participative processes.\n\nUntil long-term commitments to planning are accepted and adequately supported, planning teams dependent upon short-term funding cycles must learn to work more effectively within these constraints. For example, planning teams could communicate long-term objectives to donors and package constituent parts of the planning-implementation process as a sequence of stand-alone projects that appeal to donors, rather than focusing only on outputs or promising rapid progress to outcomes. Likewise, donors must understand that quick fixes and simplistic measures of success (see “Making a long-term commitment”) can be counterproductive; conservation success needs long-term funding, or more modest short-term objectives as part of a longer sequence from plans to actions. Two critical needs are longer-term visions and realistic expectations of outcomes. These expectations might include capacity building, consolidating the effectiveness of existing conservation actions (not just establishing new ones), and other such activities that have less concrete or prestigious outputs, yet contribute towards meaningful outcomes.\n\nMuch was made of the huge scale of investment by international donors and NGOs at the inception of the CTI-CFF (http://www.usaid.gov/global-waters/november-2010/coral-triangle). Yet achieving the Initiative's goals will take decades, and it is likely that the resources required to do this have been seriously underestimated. It was difficult for the architects of such an ambitious initiative to appreciate the full implications of its geographic and political scale, the complexity of resource-management challenges to be resolved, and the required building of capacity to ensure local ownership of plans and sustainability of management actions into the future. It was even more difficult for governments and private donors to commit funds for what was always to be a decades-long enterprise.\n\nOnly time will tell whether the CTI-CFF itself will secure the long-term commitment required at all scales and levels of governance to achieve lasting outcomes. There are the seeds of a single organisation to provide oversight and coordination in the CTI-CFF Regional Secretariat, currently hosted by Indonesia. Still in early stages, the Regional Secretariat has the potential to guide a shift from project-based conservation planning, towards planning processes institutionalised within the six CTI-CFF governments (see “Institutionalising conservation planning within governments”). This will require leadership and organisations with conservation planning capacity at all scales and levels of governance.\n\n\nConclusions\n\nThe challenges to successful implementation of conservation planning in the Coral Triangle are primarily related to issues of governance, capacity, knowledge flow, and communication. Although understanding of biodiversity patterns, processes, threats, and how to manage them continues to develop, current scientific knowledge is generally sufficient to develop effective conservation plans. Addressing the challenges discussed above will open the way for more sophisticated planning approaches, such as explicit incorporation of ecological connectivity.\n\nGetting our ten things right for marine conservation planning will be difficult, and might seem overwhelming. But the first five years of the CTI-CFF have seen progress on multiple fronts that, for many observers, would have been unimaginable beforehand. In the right-hand column of Table 1, we highlight some immediate ways forward in resolving the challenges that we have reviewed. These ways forward require action, from researchers, governments, donors, and practitioners.\n\nPresent shortcomings in the application of marine conservation planning, such as the incompatible spatial scale of many conservation prioritisations, have contributed to misconceptions about the suitability of conservation planning generally and, specifically, its appropriateness in the Coral Triangle. Resolving these shortcomings conceptually, and demonstrably through contextually-relevant case studies, will help to overcome barriers to adoption of conservation planning approaches.\n\nNevertheless, whilst case-study prototypes and “best-practice” guidelines can be useful to encourage uptake of a new approach, planners working in the Coral Triangle must have the flexibility to develop strategies that are responsive to local needs and conditions, without needing to comply with standard approaches112. Governance, capacity, planning cultures, and traditions of management of natural resources vary widely within and among the Coral Triangle countries, so there will not be a “one size fits all” approach to conservation planning. Likewise, each of the challenges discussed above will play out differently, and assume different relative importance and urgency, in different geographies and contexts.\n\nPerhaps the most important thing to get right, if conservation planning is to have real impact in the Coral Triangle, is a conceptual shift from conservation planning undertaken as a project, to planning undertaken as a process. Process-oriented planning commits agencies and stakeholders to long-term engagement, which is essential to transform conservation plans on paper into successful outcomes in the long-term. Increasingly, Coral Triangle governments are adopting leadership roles at different levels and scales, as reflected in increasing national and sub-national budget allocations for conservation planning. These leaders need direct support to ensure that emerging approaches and tools become institutionalised. Finally, conservation planning should not be considered as a new paradigm for the Coral Triangle, adding to the workload of conservation practitioners and government agencies charged with natural resource management. Instead, conservation planning can be correctly seen as a way of integrating the multiple goals of the CTI-CFF and diverse additional goals to which governments are already committed.",
"appendix": "Author contributions\n\n\n\nRW and RLP conceived the study. All authors participated in discussions to identify and explore the ten topics. RW, RLP and JRW prepared the first draft. VH, MK, RA, and RAA revised subsequent drafts of the manuscript and all authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe focus group from which this manuscript originated was made possible by funding support from the Australian Research Council provided to RLP. RW, RLP and VH acknowledge the Australian Research Council for funding support. The participation of MK and RA was made possible by the generous support of the American people through the United States Agency for International Development (USAID). 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}
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[
{
"id": "4520",
"date": "22 Apr 2014",
"name": "Helen Yap",
"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 well-written paper. It summarizes valuable lessons learned from an extensive, not to mention expensive, conservation effort in a critical biodiversity-rich region of the world, the so-called \"coral triangle\". My main comments addressed to the authors would pertain to the major obstacles that conservation initiatives have been confronted with over the decades, leading to very few success stories even to this day. I'm rather surprised they are not mentioned at all in the review. The real issues, in my own experience, are poverty, human population growth, and corruption (both in the government level, but also in the NGO community). Regarding the first issue, grinding poverty in the rural areas that abut coastal resources – not only in the countries of the coral triangle – appears to continue unabated, and is only exacerbated by dwindling natural resources, poor or negligent government, and the negative impacts of climate change (sea surface warming, drought, excessive precipitation, more violent storms). A vicious cycle ensues, as all informed individuals should know; namely, poverty begets more natural resource destruction, which begets more poverty… A desperate family (and there are millions of them) needing to put food on the table will catch the last fish, or fell the last tree, regardless of the best disseminated and best articulated conservation schemes at any level or spatial scale. If the status of biodiversity is to improve in the future, shouldn't this issue be addressed? Which will probably take the authors to territory where they have little expertise, such as the local and global economic orders with their built-in inequities in terms of trade, fiscal policy, human migration, employment opportunities, etc. Though the term \"macroeconomic\" was mentioned at least once in the manuscript. The second issue, that of human population growth, does not need much elaboration because it is a glaring one. However, it does have implications for the success of conservation campaigns, and at least deserves mention. Finally, the issue of corruption. It’s a sensitive one, and I'm not sure the authors wish to tackle it head-on in case there are repercussions from national government or local officials they need to deal with in the course of their work. The matter of corruption within the NGO community I've heard about first-hand, such as the overcharging for boat use that is then passed on to the donors. Should such facts be taken into consideration when voicing concern about the success of conservation efforts in the long-term and, with them, the well-being of local human populations (e.g. \"livelihoods\") that they are supposed to help ensure? As a minor comment, the paper might benefit from some graphs and other figures.",
"responses": [
{
"c_id": "816",
"date": "13 May 2014",
"name": "Rebecca Weeks",
"role": "Author Response",
"response": "Many thanks for your review Helen.The issues that you raise - poverty, human population growth, and corruption – are certainly major obstacles to effective conservation of biodiversity, in the Coral Triangle and elsewhere.Our aim with this manuscript was to highlight challenges specific to conservation planning, as opposed to those facing conservation initiatives more generally. This narrower focus led to our identification of ten challenges that are more directly related to the uptake and application of conservation planning and, we hope, more easily resolved through action by researchers and conservation practitioners, than the ones you mention. Of course, the context within which conservation planning is undertaken would be improved through efforts to resolve wider issues relating to poverty, population growth and corruption, and this warrants mention here.In revision, we will both clarify the scope of the manuscript better, and include mention of the overarching challenges of population, poverty, and corruption. Please note though that we will await a second peer review report before revising our manuscript."
}
]
},
{
"id": "4516",
"date": "15 May 2014",
"name": "Pedro Fidelman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, based mostly on expert opinion, identifies and discusses challenges to marine conservation planning in the Coral Triangle – a region where, despite pressing conservation issues, conservation planning has had relatively limited influence. The paper provides insights to inform management actions in the Coral Triangle that may also prove useful to other regions facing similar challenges.Though the paper is of an acceptable standard, the authors may want to consider the following points:IntroductionIt would be beneficial to provide more details about what conservation planning is and why it is needed, and the complex setting (environmental, socio-economic and political) that characterise the Coral Triangle (see Fidelman et al., 20121). These would help contextualise the challenges identified in the following sections; many (if not most) of the challenges result, to some extent, from the complex nature of large-scale marine areas such as the Coral Triangle.Planning across governance boundariesThis section addresses the problem of fit and scale; and would benefit from drawing on the corresponding scholarship (see e.g., Young, O.R., 20022). The authors may want to consider replacing the heading “Planning across governance boundaries” with “Addressing the problem of fit and scale” or something along these lines.ConclusionsIt would beneficial to generalise the discussion beyond the Coral Triangle; that is, most of the challenges identified are also true to other regions, particularly large-scale marine regions. Insights from the paper would also be applicable to those regions.Other commentsSome of the suggested actions to address the challenges identified are challenges themselves (e.g., legal reform, integration across governance levels, institutionalising conservation planning etc.). This is because we live in an increasingly complex world where (borrowing from Dietz et al., 20033) ideal conditions for governance (and for that matter conservation planning) are increasingly rare. That is, many of the challenges identified in the paper are facts of life; accordingly, the real challenge is to find innovative ways to navigate such challenges.\n\nLast, we address related challenges to the Coral Triangle in a recent article – Fidelman et al., 20144 – which the authors may find relevant to this paper.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/3-91
|
https://f1000research.com/articles/4-1475/v1
|
18 Dec 15
|
{
"type": "Review",
"title": "Recent advances in treatment of heart failure",
"authors": [
"Takeshi Kitai",
"WH Wilson Tang",
"Takeshi Kitai"
],
"abstract": "With the total cases and economic burden of heart failure continuing to rise, there is an overwhelming need for novel therapies. Several drugs for heart failure have succeeded in preclinical and early-phase clinical trials, but most of them failed to show the real benefit in pivotal clinical trials. Meanwhile, the US Food and Drug Administration recently approved two promising new drugs to treat heart failure: ivabradine and sacubitril/valsartan. Furthermore, some of the newer agents in testing offer the potential for significant progress in addition to these drugs. Patiromer and zirconium cyclosilicate are attractive agents that are expected to prevent hyperkalemia during renin-angiotensin-aldosterone system inhibition, and serelaxin and urodilatin are promising drugs in the treatment of acute heart failure. Future clinical trials with more appropriate study designs, optimal clinical endpoints, and proper patient selection are mandatory to assess the true efficacy of these attractive compounds in clinical practice.",
"keywords": [
"Heart failure",
"clinical trials",
"angiotensin-converting enzyme"
],
"content": "Introduction and context\n\nHeart failure (HF) is a major public health concern that affects as many as 23 million people worldwide1. Furthermore, hospitalization rate and costs of care for HF are enormous, and recent years have provided few indications of improvement in these trends2. There has been substantial progress in the management of chronic HF with the availability of drugs such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta-blockers, and mineralocorticoid receptor antagonist (MRA). However, community-based outcomes for patients with HF remain suboptimal. One ongoing challenge is to ensure that proven HF therapies are used at tolerated target doses in appropriate patient populations. Because of high morbidity and mortality, there is an overwhelming need for new therapies that are safe and that can improve outcomes in patients with HF.\n\nIn 2015, the US Food and Drug Administration (FDA) approved two promising new drugs to treat HF: ivabradine and sacubitril/valsartan. In addition, some of the newer agents in testing offer the potential for significant progress. In this article, we provide a brief description of novel agents in acute and chronic HF, highlighting their mechanism of action and the clinical experience, where applicable.\n\n\nLCZ696 (sacubitril/valsartan)\n\nCurrently, blockade of the renin-angiotensin-aldosterone system (RAAS) is the cornerstone of treatment of HF. However, the combination of RAAS blockade with inhibition of neprilysin, an enzyme that degrades natriuretic peptides (NPs), has recently emerged as a potentially superior treatment strategy3. In July 2015, the FDA approved sacubitril/valsartan (previously known as LCZ696) for use in patients who have chronic and stable but symptomatic HF and who have a left ventricular ejection fraction (LVEF) of less than 40%. The labeling states that the agent should be used in conjunction with other HF therapies but in place of ACE inhibitors or ARBs and is contraindicated in patients with a history of ACE inhibitor or ARB-induced angioedema.\n\nLCZ696 combines a neprilysin inhibitor (sacubitril) and an ARB (valsartan). Neprilysin is a zinc-dependent neutral endopeptidase that is responsible for the degradation of several vasoactive peptides such as NPs, bradykinin, and adrenomedullin and contributes to the breakdown of angiotensin II4. As NPs act to promote natriuresis, diuresis, and vasodilation, neprilysin inhibition is thought to be the therapeutic target for counteracting the neurohormonal activation and complementary inhibiting the RAAS.\n\nThe PARAMOUNT trial. The PARAMOUNT (Prospective Comparison of ARNi [angiotensin receptor-neprilysin inhibitor] with ARB on Management of Heart Failure with Preserved Ejection Fraction) trial was the first randomized controlled trial (RCT) that compared LCZ696 with valsartan in patients (n = 301) that have HF with preserved ejection fraction (HFpEF)5. There was a significant decrease in NT-proBNP (N-terminal of the prohormone brain natriuretic peptide) levels in the LCZ696 group at 12 weeks; however, the difference was no longer significant at 36 weeks. Furthermore, there was no change in LV size, function, or mass; diastolic function; New York Heart Association (NYHA) class; or quality-of-life scores at 12 weeks5. The trial was not designed or powered to detect clinical outcomes but has provided the rationale for the larger ongoing PARAGON-HF (Efficacy and Safety of LCZ696 Compared to Valsartan, on Morbidity and Mortality in Heart Failure Patients With Preserved Ejection Fraction) trial (ClinicalTrials.gov identifier: NCT01920711), examining the long-term outcome of LCZ696 compared with valsartan in patients with HFpEF.\n\nThe PARADIGM trial. The PARADIGM-HF (Prospective Comparison of ARNi with ACE Inhibitor to Determine Impact on Global Mortality and Morbidity in Heart Failure) Trial was conducted in 8,399 patients who had NYHA class II–IV HF and an LVEF of not more than 40% and who were randomly assigned to LCZ696 or enalapril3. The trial was stopped early because of an overwhelming benefit with LCZ696 therapy. The composite primary endpoint, including cardiovascular mortality and hospitalization for HF, occurred significantly more often in patients receiving LCZ696 compared with those receiving enalapril (hazard ratio 0.80, 95% confidence interval [CI] 0.73–0.87, P <0.001). LCZ696 was also associated with significant reductions in all-cause mortality, cardiovascular mortality, and hospitalization for worsening HF. Furthermore, those patients who received LCZ696 had lower levels of the biomarkers NT-proBNP and troponin compared with those receiving enalapril. These differences were apparent within 4 weeks of treatment and were maintained when patients were assessed again 8 months later. Interestingly, levels of B-type natriuretic peptide (BNP) actually increased and this is consistent with the mechanisms of action of neprilysin inhibition6. This trial provided strong evidence for superiority of the ARNi in patients with HF with reduced ejection fraction (HFrEF)3.\n\n\nMineralocorticoid receptor antagonist\n\nIn the activity of RAAS, aldosterone is one of the most important neurohormones in the pathophysiology of HF affecting salt and water retention, endothelial dysfunction, ventricular hypertrophy, and myocardial fibrosis7. Based on the results of RALES (Randomized Aldactone Evaluation Study)8 and EPHESUS (Epleronone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study)9, the guidelines recommended that the addition of low-dose MRA to optimal therapy be considered in all patients with moderate to severe chronic HF in the absence of hyperkalemia or significant renal dysfunction or both10,11. Therefore, inhibition of RAAS by MRAs, such as spironolactone and eplerenone, has become a milestone in the current HF treatment in symptomatic (NYHA class III and IV) patients with HFrEF in addition to ACE inhibitors or ARBs.\n\nThe EMPHASIS trial. The EMPHASIS-HF (Eplerenone in Mild Patients Hospitalization and Survival Study in Heart Failure)12 was a randomized, placebo-controlled study that enrolled 2,737 patients with NYHA class II with decreased LVEF under optimal recommended therapy. Patients with serum potassium of more than 5.0 mEq/l were excluded. In this study, eplerenone reduced significantly (by 37%) the primary composite outcome of risk of death from cardiovascular causes and first hospitalization for HF in comparison with placebo. The most frequent adverse event in patients receiving eplerenone was hyperkalemia.\n\nThe TOPCAT trial. Thus, MRAs are highly efficacious in patients with HFrEF8,12. However, the management of HFpEF represents an ongoing challenge because therapies of proven benefit in HFrEF have repeatedly been shown to add little benefit in HFpEF13–17. The TOPCAT (Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist) trial was designed to test the clinical benefit of spironolactone in patients with HFpEF18. In all, 3,445 patients with HFpEF were randomly assigned to receive spironolactone or placebo. In this trial, spironolactone failed to reduce the primary composite outcome of death from cardiovascular causes, aborted cardiac arrest, or hospitalization for HF compared with placebo (hazard ratio 0.89, 95% CI 0.77 to 1.04, P = 0.14). However, it did reduce the rate of HF hospitalizations (hazard ratio 0.83, 95% CI 0.69 to 0.99, P = 0.042)19. Of interest, those enrolled in the Americas have higher event rates and followed the NP entry criteria more closely than those from Russia/Georgia and seemed to have more consistent benefits20. Meanwhile, hyperkalemia was again more common in patients receiving spironolactone versus placebo (18.7% versus 9.1%, P <0.001). Thus, further research with a more efficient protocol is warranted to assess the efficacy of this agent in patients with HFpEF. In addition, adequate monitoring for potential side effects (mainly hyperkalemia and worsening of renal function) is needed in the addition of eplerenone to standard therapy as the current guidelines stated.\n\n\nPatiromer and zirconium cyclosilicate\n\nAs the use of RAAS inhibitors and MRAs in patients with HF increases, hyperkalemia has become a more common electrolyte disturbance in clinical practice, especially in patients with chronic kidney disease (CKD). Moreover, hyperkalemia is a major limiting factor to fully titrate these drugs in these patients who are most likely to benefit from treatment. In fact, recent clinical trials that tested the efficacy of intensive RAAS blockade had to be stopped prematurely or showed unexpected outcomes. One of the frequent adverse events was hyperkalemia21–23. Currently, non-invasive treatment of hyperkalemia is limited by a lack of safety, efficacy, and tolerability. Thus, agents to control reliably the plasma concentration of potassium while maintaining the use of RAAS inhibitors or MRAs are needed. Now, there are two novel potassium absorbents, patiromer calcium and zirconium silicate (ZS-9), that are designed to increase potassium loss via the gastrointestinal tract. Although they have not yet been approved by the FDA, both have demonstrated efficacy and safety in recent trials.\n\nMechanism of action. Patiromer is a non-absorbable polymer that binds potassium in exchange for calcium throughout the gastrointestinal tract. This agent, which is an orally administered drug, increases fecal excretion of potassium and consequently decreases plasma potassium levels24. Prior patiromer clinical trials have also demonstrated the drug’s utility in treating hyperkalemia in at-risk populations for periods ranging from a few days to up to 12 weeks24.\n\nClinical trials. The PEARL-HF study: The PEARL-HF study tested the combined use of patiromer with spironolactone in 105 HF patients receiving standard care but with previous documented hyperkalemia or CKD. Patiromer significantly lowered serum potassium levels from baseline relative to placebo and prevented the development of hyperkalemia for more than 4 weeks in normokalemic patients with HF25.\n\nThe OPAL-HK trial: The OPAL-HK (A Two-Part, Single-Blind, Phase 3 Study Evaluating the Efficacy and Safety of Patiromer for the Treatment of Hyperkalemia) assessed the efficacy and safety of patiromer in 243 patients with CKD on RAAS inhibitors with high levels of serum potassium. In this study, a mean reduction in plasma potassium levels was 1.0 mEq/l after the initial 4 weeks of active treatment. When patiromer treatment was stopped at the end of the active treatment period, hyperkalemia rapidly recurred over 8 weeks. The recurrence of hyperkalemia during this period was significantly higher in the placebo group than in the patiromer group (60% versus 15%, P <0.001), indicating the need for persistent treatment to maintain normokalemia24. The most common adverse effect of patiromer therapy was constipation.\n\nMechanism of action. ZS-9 is a high-specificity inorganic crystal that entraps potassium in the intestinal tract26. Instead of exchanging calcium, ZS-9 exchanges sodium and hydrogen ions for potassium. Dose-dependent excretion of potassium occurs in the feces, whereas urinary excretion decreased with dose27.\n\nClinical trials. The efficacy of ZS-9 was assessed in a multicenter RCT including 753 patients with hyperkalemia associated with a variety of diseases, including CKD, HF, and diabetes. Patients were randomly assigned to one of four doses of ZS-9 (1.25, 2.5, 5, or 10 g) or placebo for 2 days28. The reduction of serum potassium with ZS-9 started acutely, and there was a dose-dependent reduction in serum potassium from baseline to 2 days, with absolute mean reductions of 0.73 and 0.53 mEq/l in the 10- and 5-g dose groups, respectively (P <0.001). Reductions in serum potassium were significantly greater with ZS-9 than placebo at all time points on study day 2. Notably, 98% of patients were normalized on the 10-g dose within 2 days. The most frequent adverse effect of ZS-9 was diarrhea28,29.\n\nThe HARMONIZE study: The HARMONIZE study was an RCT evaluating long-term efficacy and safety of ZS-9 in 258 patients with hyperkalemia29,30. Patients achieving normokalemia (3.5 to 5.0 mEq/l) were randomly assigned to different doses of ZS-9 (5, 10, or 15 g) or placebo for 28 days in the maintenance phase. Mean baseline potassium was 5.6 mEq/l and declined to 4.5 mEq/l after 48 hours of 10-g ZS-9 treatment in the acute phase. Significant reduction in potassium was observed within 1 hour of ZS-9 administration, and 84% of patients achieved normokalemia at 24 hours and 98% at 48 hours30. Furthermore, studies assessing the long-term efficacy and safety profile of this novel drug are ongoing (ClinicalTrials.gov identifier: NCT02163499).\n\nThese recent trials of patiromer and ZS-9 represented short-term safety and efficacy of these attractive therapeutic strategies in patients who develop hyperkalemia during RAAS inhibition. However, the durability of the beneficial effects and the long-term safety of these agents still have to be elucidated. In addition, there are no prospective data answering whether intensive RAAS inhibition with the use of patiromer or ZS-9 would improve the efficacy of RAAS inhibition and cardiovascular outcomes. Further study is needed to address these issues.\n\n\nIvabradine\n\nOne novel potential therapeutic option for HF is heart rate (HR) control. An elevated HR, probably reflecting activation of the sympathetic nervous system, is associated with worse cardiovascular outcomes. Although beta-blockers are used mainly for reducing HR in HF treatment31, up-titration of beta-blockers can be associated with an increased risk of adverse reactions32–34. Ivabradine, which acts by directly and selectively inhibiting the If current in the sino-atrial node, has potential benefits of pharmacologic modification of HR in HF.\n\nIvabradinelowers HR by inhibiting a specific sinus node pacemaker If current without affecting the myocardial contractility or relaxation, ventricular repolarization, or intracardiac conduction35–40. This is rather different from the mechanism induced by beta-blockers, which acts wherever beta-adrenergic receptors are present, causing negative inotropism and vasoconstriction in the bronchi; and calcium channel blockers act on the calcium channels of the heart and smooth muscle, causing negative inotropism, hypotension, and constipation.\n\nThe BEAUTIFUL trial. The BEAUTIFUL (Morbidity-Mortality Evaluation of the If inhibitor Ivabradine in Patients with Coronary Artery Disease and Left Ventricular Systolic Dysfunction) trial was an RCT to test the efficacy of ivabradine in 10,917 patients with stable coronary disease and an LVEF of less than 40% and an HR of more than 60 beats per minute (bpm)41. In this trial, ivabradine reduced HR but had no effect on the primary endpoint of cardiovascular death or admission to a hospital for new-onset or worsening HF. However, in a subgroup of patients with an HR of at least 70 bpm, ivabradine revealed a clear benefit with respect to the secondary endpoints of admission to a hospital for a fatal or non-fatal myocardial infarction and coronary revascularization41.\n\nThe SHIFT trial. The SHIFT (Systolic HF Treatment with If Inhibitor Ivabradine) trial was an RCT in 6,558 patients with stable symptomatic HF and an LVEF of not more than 35% in sinus rhythm with an HR of at least 70 bpm42,43. In this trial, ivabradine significantly reduced the primary endpoint of a composite of cardiovascular death or hospital admission for worsening HF and deaths due to HF43. The effect was consistent across all pre-specified subgroups, including the elderly43. Further analyses proved that high HR as a risk factor in HF and lowering HR improves outcomes43. Other analyses showed that ivabradine reduces the risk of rehospitalization for HF44 and is associated with an improvement of quality of life45. HR targeted below a threshold rather than HR reduction itself has demonstrated potential benefits. One problem with interpreting the results of the SHIFT trial is that many patients were not on target doses of beta-blockers. If indeed these patients were intolerant of higher doses of beta-blockers, then these results are quite important for clinical care. Given its promising therapeutic value, ivabradine is clearly desirable in patients with symptomatic LV systolic dysfunction, elevated HR, and intolerance to beta-blockers.\n\n\nRelaxin\n\nSerelaxin is a recombinant form of the human hormone relaxin, which is a naturally occurring hormone that is produced by the corpus luteum and placenta in pregnancy46. Recent studies have shown that relaxin is also produced by the vasculature and failing myocardium47,48.\n\nRelaxin interacts with a G protein-coupled receptor, leading to increased cyclic adenosine monophosphate (cAMP). As a result, nitric oxide production is increased by the increased activity of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression46,49,50. Additionally, relaxin upregulates the activity of vascular matrix metalloproteinase-2 (MMP-2), which can activate endothelin-151, leading to endothelin-B receptor activation and subsequent nitric oxide production49. Activation of the endothelin-B receptor is likely involved in the relaxin-mediated increases in renal blood flow51. Thus, relaxin increases cardiac output, arterial compliance, and renal blood flow, supporting important physiological changes during pregnancy52. Given its potent vasodilator properties as well as its ability to increase renal perfusion, relaxin became of interest as a potential therapy for acute HF.\n\nThe Pre-RELAX-AHF study. The Pre-RELAX-AHF (Relaxin in Acute Heart Failure) study evaluated the effects of relaxin in 234 patients with acute decompensated heart failure (ADHF) within 16 hours from presentation53. Patients were randomly assigned to receive four doses of relaxin or placebo for 48 hours. The key findings were that dyspnea relief and safety were optimal at 30 μg/kg per day and sustained results for dyspnea improvement. This dose also led to a substantial reduction in the composite endpoint of cardiovascular mortality or readmission due to HF or renal failure as well as a decrease in cardiovascular mortality at 180 days. However, several subjects (14%) had to discontinue relaxin therapy because of the significant fall in blood pressure53.\n\nThe RELAX-AHF study. The RELAX-AHF was an RCT enrolling 1,161 ADHF patients who have a systolic blood pressure of more than 125 mmHg and renal dysfunction. Patients were randomly assigned to receive serelaxin 30 μg/kg per day or placebo as a continuous 48-hour infusion within 16 hours from presentation54. In this study, serelaxin significantly improved dyspnea, shortened the length of hospital stay, and decreased the incidence of worsening HF as compared with placebo. There was also an improvement in the 6-month mortality outcomes and no evidence of adverse effects of this agent on kidney function55. Although relaxin has shown success in improving the clinical course of patients with ADHF during the initial hospitalization with an acceptable safety profile, a larger trial (RELAX-AHF2, n = 2,685) is ongoing to hopefully validate whether this drug could indeed provide long-term mortality benefit.\n\n\nUlaritide\n\nDecongestion is an important part of managing both acute and chronic HF, and retention of fluid and sodium metabolism play a fundamental role in this. NPs are activated in HF and exert compensatory effects by inhibiting the RAAS and inducing vasodilation and natriuresis56. Therefore, NPs have received much interest as a potential therapy in ADHF. NPs consist of atrial NP (ANP), BNP, C-type NP (CNP), D-type NP (DNP), and urodilatin57.\n\nUrodilatin was first isolated from human urine in 1988 as a modified version of pro-ANP58. It is produced mainly by distal renal tubule cells and is secreted into urine and is involved in renal sodium handling59. Synthetic NPs such as carperitide (a recombinant form of ANP) and nesiritide (a recombinant form of BNP) are currently used to treat congestive HF (carperitide is available only in Japan). When it is administered to patients with ADHF, a rapid reduction of pulmonary capillary pressure and consequent relief of dyspnea often result because of natriuresis, diuresis, and venous and arterial dilation. However, NP-induced vasodilatation and reductions in renal perfusion pressures and the potential for reflex sympathetic responses can cause clinically significant systemic hypotension and worsening of renal function in some patients60. In contrast to ANP and BNP, urodilatin is effective in more distal parts of the renal tubular system because of its slower elimination rate61.\n\nThe SIRIUS II study. The Prospective Double-blind Study in Patients with Symptomatic, Decompensated Chronic Heart Failure (SIRIUS) II study was aimed to assess the clinical effects of ularitide in 221 patients with ADHF59. The primary endpoint was a significant decrease in pulmonary capillary wedge pressure (PCWP) as well as improvement in dyspnea at 6 hours after completion of the 24-hour infusion. Ularitide demonstrated a significant reduction of PCWP for all three dosage groups (7.5, 15, and 30 ng/kg per min). At higher doses, the agent reduced systemic vascular resistance and increased cardiac index. Besides the beneficial hemodynamic effects, improvement in dyspnea was reported. The most frequently reported drug-related adverse events were dose-dependent blood pressure decrease. Currently, a randomized, placebo-controlled, phase 3 study—Efficacy and Safety of Ularitide for the Treatment of Acute Decompensated Heart Failure (TRUE-AHF), n = 2,152—is ongoing to measure the effect of 48-hour infusion of ularitide.\n\nNPs offer us a unique and attractive strategy for HF treatment, acting as diuretic, natriuretic, vasoactive agents without any inotropic or chronotropic effects. However, the future role of NPs in ADHF therapy is still not yet clear, especially following the rise and fall of nesiritide use. Initial trials with ularitide, a synthetically produced urodilatin, showed hemodynamic and clinical benefits in patients with ADHF. Although ularitide has potential to be an alternative to nesiritide or carperitide, much more evidence is needed to evaluate the role of this agent in HF therapy.\n\n\nConclusions and future perspectives\n\nA number of promising compounds for HF therapies are under investigation in addition to the agents we discussed here. However, it is a well-known fact that several drugs have succeeded in preclinical and early-phase clinical trials only to be disappointments in pivotal clinical trials. Therefore, future clinical trials with adequately powered, more appropriate study designs, optimal clinical endpoints, and right patient selection are mandatory to assess the true efficacy of these compounds.\n\n\nAbbreviations\n\nACE, angiotensin-converting enzyme; ADHF, acute decompensated heart failure; ANP, atrial natriuretic peptide; ARB, angiotensin receptor blocker; ARNi, angiotensin receptor-neprilysin inhibitor; BNP, brain natriuretic peptide; bpm, beats per minute; cAMP, cyclic adenosine monophosphate; CI, confidence interval; CKD, chronic kidney disease; FDA, US Food and Drug Administration; HF, heart failure; HFpEF, heart failure with preserved ejection fraction; HFrEF, heart failure with reduced ejection fraction; HR, heart rate; LV, left ventricular; LVEF, left ventricular ejection fraction; MRA, mineralocorticoid receptor antagonist; NP, natriuretic peptide; NT-proBNP, N-terminal of the prohormone brain natriuretic peptide; NYHA, New York Heart Association; PCWP, pulmonary capillary wedge pressure; RAAS, renin-angiotensin-aldosterone system; RELAX-AHF, Relaxin in Acute Heart Failure; RCT, randomized controlled trial; SHIFT, systolic heart failure treatment with If inhibitor ivabradine.",
"appendix": "Competing interests\n\n\n\nTK declares that he has no competing interests.\n\n\nGrant information\n\nWT is funded by a grant from the National Institutes of Health (R01HL103931).\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\nBui AL, Horwich TB, Fonarow GC: Epidemiology and risk profile of heart failure. Nat Rev Cardiol. 2011; 8(1): 30–41. 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N Engl J Med. 2011; 364(1): 11–21. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nYusuf S, Pfeffer MA, Swedberg K, et al.: Effects of candesartan in patients with chronic heart failure and preserved left-ventricular ejection fraction: the CHARM-Preserved Trial. Lancet. 2003; 362(9386): 777–81. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCleland JG, Tendera M, Adamus J, et al.: The perindopril in elderly people with chronic heart failure (PEP-CHF) study. Eur Heart J. 2006; 27(19): 2338–45. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMassie BM, Carson PE, McMurray JJ, et al.: Irbesartan in patients with heart failure and preserved ejection fraction. N Engl J Med. 2008; 359(23): 2456–67. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFlather MD, Shibata MC, Coats AJ, et al.: Randomized trial to determine the effect of nebivolol on mortality and cardiovascular hospital admission in elderly patients with heart failure (SENIORS). Eur Heart J. 2005; 26(3): 215–25. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAhmed A, Rich MW, Fleg JL, et al.: Effects of digoxin on morbidity and mortality in diastolic heart failure: the ancillary digitalis investigation group trial. Circulation. 2006; 114(5): 397–403. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDesai AS, Lewis EF, Li R, et al.: Rationale and design of the treatment of preserved cardiac function heart failure with an aldosterone antagonist trial: a randomized, controlled study of spironolactone in patients with symptomatic heart failure and preserved ejection fraction. Am Heart J. 2011; 162(6): 966–972.e10. 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 | F1000 Recommendation\n\nPfeffer MA, Claggett B, Assmann SF, et al.: Regional variation in patients and outcomes in the Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist (TOPCAT) trial. Circulation. 2015; 131(1): 34–42. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nONTARGET Investigators, Yusuf S, Teo KK, et al.: Telmisartan, ramipril, or both in patients at high risk for vascular events. N Engl J Med. 2008; 358(15): 1547–59. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nParving HH, Brenner BM, McMurray JJ, et al.: Cardiorenal end points in a trial of aliskiren for type 2 diabetes. N Engl J Med. 2012; 367(23): 2204–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFried LF, Emanuele N, Zhang JH, et al.: Combined angiotensin inhibition for the treatment of diabetic nephropathy. N Engl J Med. 2013; 369(20): 1892–903. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWeir MR, Bakris GL, Bushinsky DA, et al.: Patiromer in patients with kidney disease and hyperkalemia receiving RAAS inhibitors. N Engl J Med. 2015; 372(3): 211–21. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPitt B, Anker SD, Bushinsky DA, et al.: Evaluation of the efficacy and safety of RLY5016, a polymeric potassium binder, in a double-blind, placebo-controlled study in patients with chronic heart failure (the PEARL-HF) trial. Eur Heart J. 2011; 32(7): 820–8. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nStavros F, Yang A, Leon A, et al.: Characterization of structure and function of ZS-9, a K+ selective ion trap. PLoS One. 2014; 9(12): e114686. PubMed Abstract | Publisher Full Text\n\nAsh SR, Singh B, Lavin PT, et al.: A phase 2 study on the treatment of hyperkalemia in patients with chronic kidney disease suggests that the selective potassium trap, ZS-9, is safe and efficient. Kidney Int. 2015; 88(2): 404–11. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPackham DK, Rasmussen HS, Lavin PT, et al.: Sodium zirconium cyclosilicate in hyperkalemia. N Engl J Med. 2015; 372(3): 222–31. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKosiborod M, Rasmussen HS, Lavin P, et al.: Effect of sodium zirconium cyclosilicate on potassium lowering for 28 days among outpatients with hyperkalemia: the HARMONIZE randomized clinical trial. JAMA. 2014; 312(21): 2223–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAnker SD, Kosiborod M, Zannad F, et al.: Maintenance of serum potassium with sodium zirconium cyclosilicate (ZS-9) in heart failure patients: results from a phase 3 randomized, double-blind, placebo-controlled trial. Eur J Heart Fail. 2015; 17(10): 1050–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMetra M, Torp-Pedersen C, Swedberg K, et al.: Influence of heart rate, blood pressure, and beta-blocker dose on outcome and the differences in outcome between carvedilol and metoprolol tartrate in patients with chronic heart failure: results from the COMET trial. Eur Heart J. 2005; 26(21): 2259–68. PubMed Abstract | Publisher Full Text\n\nButler J, Arbogast PG, BeLue R, et al.: Outpatient adherence to beta-blocker therapy after acute myocardial infarction. J Am Coll Cardiol. 2002; 40(9): 1589–95. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nde Groote P, Isnard R, Assyag P, et al.: Is the gap between guidelines and clinical practice in heart failure treatment being filled? Insights from the IMPACT RECO survey. Eur J Heart Fail. 2007; 9(12): 1205–11. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKomajda M, Follath F, Swedberg K, et al.: The EuroHeart Failure Survey programme--a survey on the quality of care among patients with heart failure in Europe. Part 2: treatment. Eur Heart J. 2003; 24(5): 464–74. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBorer JS, Fox K, Jaillon P, et al.: Antianginal and antiischemic effects of ivabradine, an If inhibitor, in stable angina: a randomized, double-blind, multicentered, placebo-controlled trial. Circulation. 2003; 107(6): 817–23. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nManz M, Reuter M, Lauck G, et al.: A single intravenous dose of ivabradine, a novel If inhibitor, lowers heart rate but does not depress left ventricular function in patients with left ventricular dysfunction. Cardiology. 2003; 100(3): 149–55. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nThollon C, Cambarrat C, Vian J, et al.: Electrophysiological effects of S 16257, a novel sino-atrial node modulator, on rabbit and guinea-pig cardiac preparations: comparison with UL-FS 49. Br J Pharmacol. 1994; 112(1): 37–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBois P, Bescond J, Renaudon B, et al.: Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells. Br J Pharmacol. 1996; 118(4): 1051–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSimon L, Ghaleh B, Puybasset L, et al.: Coronary and hemodynamic effects of S 16257, a new bradycardic agent, in resting and exercising conscious dogs. J Pharmacol Exp Ther. 1995; 275(2): 659–66. PubMed Abstract\n\nBel A, Perrault LP, Faris B, et al.: Inhibition of the pacemaker current: a bradycardic therapy for off-pump coronary operations. Ann Thorac Surg. 1998; 66(1): 148–52. PubMed Abstract | Publisher Full Text\n\nFox K, Ford I, Steg PG, et al.: Ivabradine for patients with stable coronary artery disease and left-ventricular systolic dysfunction (BEAUTIFUL): a randomised, double-blind, placebo-controlled trial. Lancet. 2008; 372(9641): 807–16. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSwedberg K, Komajda M, Böhm M, et al.: Ivabradine and outcomes in chronic heart failure (SHIFT): a randomised placebo-controlled study. Lancet. 2010; 376(9744): 875–85. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBöhm M, Swedberg K, Komajda M, et al.: Heart rate as a risk factor in chronic heart failure (SHIFT): the association between heart rate and outcomes in a randomised placebo-controlled trial. Lancet. 2010; 376(9744): 886–94. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBorer JS, Böhm M, Ford I, et al.: Effect of ivabradine on recurrent hospitalization for worsening heart failure in patients with chronic systolic heart failure: the SHIFT Study. Eur Heart J. 2012; 33(22): 2813–20. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nEkman I, Chassany O, Komajda M, et al.: Heart rate reduction with ivabradine and health related quality of life in patients with chronic heart failure: results from the SHIFT study. Eur Heart J. 2011; 32(19): 2395–404. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDu XJ, Bathgate RA, Samuel CS, et al.: Cardiovascular effects of relaxin: from basic science to clinical therapy. Nat Rev Cardiol. 2010; 7(1): 48–58. PubMed Abstract | Publisher Full Text\n\nSamuel CS, Du XJ, Bathgate RA, et al.: 'Relaxin' the stiffened heart and arteries: the therapeutic potential for relaxin in the treatment of cardiovascular disease. Pharmacol Ther. 2006; 112(2): 529–52. PubMed Abstract | Publisher Full Text\n\nNovak J, Parry LJ, Matthews JE, et al.: Evidence for local relaxin ligand-receptor expression and function in arteries. FASEB J. 2006; 20(13): 2352–62. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nConrad KP, Novak J: Emerging role of relaxin in renal and cardiovascular function. Am J Physiol Regul Integr Comp Physiol. 2004; 287(2): R250–61. PubMed Abstract | Publisher Full Text\n\nBani D, Failli P, Bello MG, et al.: Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension. 1998; 31(6): 1240–7. PubMed Abstract | Publisher Full Text\n\nJeyabalan A, Novak J, Danielson LA, et al.: Essential role for vascular gelatinase activity in relaxin-induced renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small arteries. Circ Res. 2003; 93(12): 1249–57. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJeyabalan A, Shroff SG, Novak J, et al.: The vascular actions of relaxin. Adv Exp Med Biol. 2007; 612: 65–87. PubMed Abstract | Publisher Full Text\n\nTeerlink JR, Metra M, Felker GM, et al.: Relaxin for the treatment of patients with acute heart failure (Pre-RELAX-AHF): a multicentre, randomised, placebo-controlled, parallel-group, dose-finding phase IIb study. Lancet. 2009; 373(9673): 1429–39. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTeerlink JR, Cotter G, Davison BA, et al.: Serelaxin, recombinant human relaxin-2, for treatment of acute heart failure (RELAX-AHF): a randomised, placebo-controlled trial. Lancet. 2013; 381(9860): 29–39. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMetra M, Cotter G, Davison BA, et al.: Effect of serelaxin on cardiac, renal, and hepatic biomarkers in the Relaxin in Acute Heart Failure (RELAX-AHF) development program: correlation with outcomes. J Am Coll Cardiol. 2013; 61(2): 196–206. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWoodard GE, Rosado JA: Natriuretic peptides in vascular physiology and pathology. Int Rev Cell Mol Biol. 2008; 268: 59–93. PubMed Abstract | Publisher Full Text\n\nPotter LR, Yoder AR, Flora DR, et al.: Natriuretic peptides: their structures, receptors, physiologic functions and therapeutic applications. Handb Exp Pharmacol. 2009: (191): 341–66. PubMed Abstract | Publisher Full Text\n\nSchulz-Knappe P, Forssmann K, Herbst F, et al.: Isolation and structural analysis of \"urodilatin\", a new peptide of the cardiodilatin-(ANP)-family, extracted from human urine. Klin Wochenschr. 1988; 66(17): 752–9. PubMed Abstract | Publisher Full Text\n\nMitrovic V, Seferovic PM, Simeunovic D, et al.: Haemodynamic and clinical effects of ularitide in decompensated heart failure. Eur Heart J. 2006; 27(33): 2823–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDontas ID, Xanthos T, Dontas I, et al.: Impact of nesiritide on renal function and mortality in patients suffering from heart failure. Cardiovasc Drugs Ther. 2009; 23(3): 221–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nForssmann W, Meyer M, Forssmann K: The renal urodilatin system: clinical implications. Cardiovasc Res. 2001; 51(3): 450–62. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11657",
"date": "18 Dec 2015",
"name": "Hector Ventura",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11658",
"date": "18 Dec 2015",
"name": "Marrick Kukin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1475
|
https://f1000research.com/articles/4-1474/v1
|
18 Dec 15
|
{
"type": "Review",
"title": "Recent Advances in Managing Acute Pancreatitis",
"authors": [
"Nigeen Janisch",
"Timothy Gardner",
"Nigeen Janisch"
],
"abstract": "This article will review the recent advances in managing acute pancreatitis. Supportive care has long been the standard of treatment for this disease despite extensive, but ultimately unsuccessful, efforts to develop disease-specific pharmacologic therapies. The primary interventions center on aggressive fluid resuscitation, initiation of early enteral nutrition, targeted antibiotic therapy, and the management of complications. In this article, we will detail treatment of acute pancreatitis with a focus on intravenous fluid resuscitation, enteral feeding, and the current evidence behind the use of antibiotics and other pharmacologic therapies.",
"keywords": [
"Acute Pancreatitis",
"pancreatitis",
"Enteral Feeding",
"Fluid Resuscitation"
],
"content": "Introduction\n\nAcute pancreatitis can be severe with extensive morbidity, mortality, and hospitalization costs1. As the most common inpatient gastrointestinal diagnosis in the United States (over 270,000 inpatient stays in 2009), acute pancreatitis was responsible for USD $2.6 billion in health-care costs in 20092. With an increasing incidence in the last decade and an overall mortality ranging from 5% to 20% depending on severity, extensive efforts have been under way to improve important clinical outcomes in the disease3–5. However, despite these efforts, no targeted pharmacologic therapy specific to acute pancreatitis has been found6,7. In this article, we will discuss advances in supportive care that have contributed to improved outcomes in this disease. In addition, we will highlight the failures of previous studies of targeted pharmacologic therapy. Finally, we will outline opportunities for future research that we feel show promise in the management of acute pancreatitis.\n\n\nFluid resuscitation\n\nThe most effective intervention for acute pancreatitis to date is early aggressive fluid resuscitation. By providing adequate perfusion of the pancreatic microcirculation, fluid resuscitation maintains intravascular volume in the setting of the massive capillary leak associated with the inflammatory response of acute pancreatitis. In turn, preventing ischemia of the microcirculation inhibits the development of local and systemic complications such as pancreatic necrosis, systemic inflammatory response syndrome (SIRS), and multi-system organ failure8,9.\n\nThe pancreatic microcirculation can be defined as the area of vasculature, including the celiac and superior mesenteric arteries, which supplies oxygen-rich blood to the pancreatic acinar cells. Inflammatory mediators released in acute pancreatitis are thought to exert a microangiopathic effect leading to hypercoagulability with microthrombi, subsequent endothelial damage from free radical release, and finally increased capillary permeability promoting hypovolemia10,11. Disruption of the microcirculation therefore is theorized as an important factor responsible for the transition from mild, interstitial edema to severe, necrotizing pancreatitis.\n\nThe most important area of research in terms of developing targeted interventions for acute pancreatitis, in our opinion, involves fully elaborating the inflammatory cascade specific to the disease. Determining the driving stimulus behind pancreatic injury and subsequent inflammatory activation will be the critical step in designing targeted therapy.\n\nData from both retrospective and prospective clinical trials demonstrate that early fluid resuscitation is more effective than delayed fluid resuscitation. One recent study specifically addressed this issue by defining early fluid resuscitation as receiving greater than one third of the total 72-hour fluid volume within the first 24 hours of hospitalization and late as receiving less than one third12. Although the investigation yielded no information on total infused fluid volume, they concluded that patients receiving early fluid resuscitation experienced less mortality than those receiving it late. Additional studies, including a retrospective analysis of 436 patients with acute pancreatitis which found an association between early fluid resuscitation and decreased SIRS, organ failure at 72 hours, length of hospital stay, and a lower rate of intensive care unit admission, support these conclusions13.\n\nEarly fluid resuscitation is agreed upon as an intervention of paramount importance; however, to date, there are no standard guidelines on optimal fluid type or volume, rate, or duration of treatment14. Human studies regarding the rate of hydration consistently show decreased morbidity and mortality with aggressive hydration in the first 24 hours, although total volume of hydration at the 48-hour mark seems to have no effect on patient outcomes. The American College of Gastroenterology guidelines currently recommend 250 to 500 mL per hour of isotonic crystalloid solution in the first 12 to 24 hours, with frequent re-evaluation every 6 hours, ultimately with the therapeutic goal of decreasing the blood urea nitrogen (BUN) level15. Most experts will agree with a starting infusion of 250 to 300 mL/hour or enough to produce a urine output of at least 0.5 mL/kg, in addition to the 1- to 2-L fluid bolus given in the emergency department16. The goal within the first 24 hours is a total infusion of 2.5 to 4 L, with adjustments made on the basis of the patient’s age, weight, physical exam, and comorbid conditions17.\n\nDuration of aggressive resuscitation is difficult to determine and this should be individualized. It is recommended, however, to aim for a decrease in hematocrit or BUN, or both, in the first 24 hours of hospitalization. An increased risk of pancreatic necrosis has been linked with an elevated hematocrit at admission or failure to decrease after 24 hours as well as an increase in creatinine within 48 hours in independent studies18–20. With regard to BUN, a 2011 meta-analysis of 1,043 acute pancreatitis cases showed an increased risk of mortality and death with a BUN of at least 20 mg/dL (odds ratios of 4.6 and 4.3, respectively) at admission or a rise within the first 24 hours21.\n\nThe type of fluid to use for resuscitation has been incompletely studied. In the only randomized study specifically evaluating different colloid resuscitation fluids, Lactated Ringer’s solution had a greater effect on decreasing SIRS and C-reactive protein levels than normal saline22.\n\nIn summary, acute pancreatitis leads to alterations in the pancreatic microcirculation brought about by an intense inflammatory cascade that has yet to be completely delineated. Aggressive fluid resuscitation is used to blunt the capillary leak syndrome associated with this cascade, although the optimal rate, type, and duration of fluid resuscitation have yet to be studied. Further studies are needed to evaluate for any complications related to over-aggressive fluid resuscitation.\n\n\nAntibiotics\n\nGiven the morbidity and mortality associated with infected pancreatic necrosis, it stands to reason that giving antibiotics may serve as a solution to this problem. Pancreatic necrosis complicated by translocated enteric bacteria continues to be the most common cause of mortality in patients with acute pancreatitis that survive the early phase, accounting for up to 70% of all deaths4,6. Though still a controversial topic, prophylactic antibiotic therapy is currently not recommended to prevent pancreatic necrosis associated with acute pancreatitis9.\n\nIn previous years, prophylactic antibiotics were recommended and common in practice, supported by early research showing that broad-spectrum antibiotics improved outcomes and reduced mortality23. However, a 2001 study evaluated three separate randomized controlled trials comparing antibiotic prophylaxis to no prophylaxis in the setting of acute necrotizing pancreatitis. The study found reductions of 21.2% in sepsis and 12.3% in mortality in patients receiving prophylactic antibiotics; however, there was no difference in the incidence of pancreatic infection24.\n\nStudies since this report have continued to show conflicting results. A 2008 meta-analysis, including the same three previously mentioned randomized controlled trials, saw no difference in the rates of pancreatic infection or mortality between the group receiving antibiotics versus placebo25. A subsequent Cochrane review confirmed no difference in mortality but found a significant difference while using imipenem alone26. Most recently, in 2011, an evaluation of 14 randomized controlled trials totaling 841 patients compared those receiving antibiotics versus placebo. In the categories of mortality, incidence of infected pancreatic necrosis, non-pancreatic infection, and surgical intervention, no significant differences were reported27. There may even be an association with antibiotic use and an increased risk of intra-abdominal fungal infections28.\n\nAlthough prophylactic antibiotics are not recommended to prevent infected pancreatic necrosis, there has been some discussion of probiotic prophylaxis with a theorized benefit through selective gut decontamination. This intervention involves giving oral antibiotics to eradicate enteric Gram-negative rods and thus reduce bacterial translocation from the gastrointestinal tract into the pancreas. A 2009 meta-analysis regarding probiotic prophylaxis resulted in no reduction in the risk of pancreatic infection or associated mortality29. One large study from the Dutch Acute Pancreatitis Study group even found that in patients with predicted severe acute pancreatitis, there was increased mortality from bowel ischemia in the group given probiotics30. Further studies need to be performed to assess the efficacy and safety of this possible intervention.\n\nUltimately, prophylactic antibiotics are not recommended for use in acute pancreatitis and should not be administered in the first 24 hours of the episode unless there is clinical suspicion for concurrent infection. Patients may present initially with sepsis, SIRS, multi-organ failure, or a combination of these and thus may have clinical symptoms such as fever that may mimic infection. Treatment with antibiotics is appropriate if after evaluation of the patient via blood cultures and fine needle aspiration of pancreatic necrosis, infection is revealed. However, if there is no obvious source of infection, antibiotics should be stopped15.\n\n\nEnteral feeding\n\nThe standard of care in the past has been to maintain patients on NPO (nil per os, or nothing by mouth) status until pain resolution while encouraging pancreatic rest. Currently, it is widely accepted that early enteral feeding is critical to improving outcomes31. Bowel rest is associated with intestinal mucosal atrophy and increased infectious complications due to bacterial translocation. To maintain gut barrier function, enteral feeding is preferred over parenteral feeding in the management of acute pancreatitis32,33.\n\nIn mild acute pancreatitis, oral intake with a low-fat soft solid diet is often tolerated within 1 week of admission and no interventions are required. However, if the patient is not eating after 1 week, enteral feeding is recommended after cessation of nausea and vomiting, no longer requiring parenteral analgesics, reduction in abdominal pain, and return of bowel sounds15,34,35.\n\nIn severe or predicted severe acute pancreatitis, enteral feeding is recommended to start within the first 72 hours of hospitalization with oral or tube feeding. A recent study in the New England Journal of Medicine demonstrated that tube or oral feedings are equivalent in terms of preventing complications36. Enteral feeding is thought to preserve the enteric gut barrier to prevent bacterial translocation along with avoiding the complications associated with parenteral nutrition. A 2012 meta-analysis of 381 patients with severe acute pancreatitis confirmed the benefit of enteral versus parenteral feeds. With the groups randomly assigned to receive each variation of nutrition, those with enteral feeds benefitted in mortality, infection, and organ failure and had a lower surgical rate37. Nasojejunal feeding has long been preferred, although there is evidence that nasogastric feeds have a similar effect38. Although evidence shows a preference toward enteral feeding, should the patient not tolerate it or not meet nutritional goals, parenteral nutrition should be started while maintaining a slow rate of enteral feeds15.\n\n\nPharmacologic therapies\n\nMany research initiatives have aimed at finding a targeted pharmacologic therapy for acute pancreatitis. Pharmacologic agents that initially presented the most merit were pancreatic anti-secretory agents, including somatostatin, octreotide, atropine, glucagon, and cimetidine. However, experience with these agents has been universally disappointing. For example, in 1994, a randomized controlled trial of 302 patients with acute pancreatitis treated with octreotide, a longer-acting analog of somatostatin, showed no differences in mortality or complications when compared with controls39. A meta-analysis of five randomized controlled trials in 2002 showed cimetidine to be no more effective than placebo in decreasing complications or pain40.\n\nAnti-proteases, owing to their inhibition of pancreatic proteases, which could stimulate pancreatic autodigestion, were also investigated. Studies on such drugs, like gabexate mesilate, nafamostat, and aprotinin, have not consistently demonstrated therapeutic benefit and are not universally employed41–44. Platelet-activating factor antagonists such as lexipafant, antioxidants, corticosteroids, nitroglycerin, anti-interleukin-10 (anti-IL-10) antibodies, and anti-tumor necrosis factor-alpha (anti-TNF-α) antibodies have been shown to be of no value in the treatment of acute pancreatitis.\n\nThus, despite initial promise for many agents, there unfortunately continues to be no adequate targeted pharmacologic option with any proven benefit in randomized clinical trials15. The only exception has been in the treatment of post-endoscopic retrograde cholangiopancreatography (post-ERCP) pancreatitis. In a recent multi-center, double-blind, randomized placebo controlled trial of 602 patients, there was a significant reduction in post-ERCP pancreatitis when high-risk patients received rectal indomethacin45. Clinical trials gleaned similar results with rectal diclofenac46. Therefore, in high-risk patients only, 100 mg of rectal indomethacin is reasonable as prophylaxis15.\n\n\nConclusions\n\nAcute pancreatitis is a devastating disease affecting millions of people worldwide. Despite improvements in supportive care, there is currently no targeted pharmacologic therapy that is used specifically to treat this disease. Medications such as anti-secretory agents and anti-proteases have been studied and failed to improve clinical outcomes. On the horizon, the key to improving outcomes in acute pancreatitis will be to develop therapies that specifically target the immune storm caused by pancreatic autodigestion. Specific immunologic therapies that target specific responses in the disease will be the key to its control.\n\n\nAbbreviations\n\nBUN, blood urea nitrogen; ERCP, endoscopic retrograde cholangiopancreatography; NPO, nil per os; SIRS, systemic inflammatory response syndrome.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nNeoptolemos JP, Raraty M, Finch M, et al.: Acute pancreatitis: the substantial human and financial costs. Gut. 1998; 42(6): 886–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeery AF, Dellon ES, Lund J, et al.: Burden of gastrointestinal disease in the United States: 2012 update. Gastroenterology. 2012; 143(5): 1179–87.e1–3. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMann DV, Hershman MJ, Hittinger R, et al.: Multicentre audit of death from acute pancreatitis. Br J Surg. 1994; 81(6): 890–3. PubMed Abstract | Publisher Full Text\n\nRusso MW, Wei JT, Thiny MT, et al.: Digestive and liver diseases statistics, 2004. Gastroenterology. 2004; 126(5): 1448–53. 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PubMed Abstract | Publisher Full Text\n\nBanks PA, Freeman ML; Practice Parameters Committee of the American College of Gastroenterology: Practice guidelines in acute pancreatitis. Am J Gastroenterol. 2006; 101(10): 2379–400. PubMed Abstract | Publisher Full Text\n\nTakeda K, Mikami Y, Fukuyama S, et al.: Pancreatic ischemia associated with vasospasm in the early phase of human acute necrotizing pancreatitis. Pancreas. 2005; 30(1): 40–9. PubMed Abstract\n\nBang UC, Semb S, Nojgaard C, et al.: Pharmacological approach to acute pancreatitis. World J Gastroenterol. 2008; 14(19): 2968–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGardner TB, Vege SS, Chari ST, et al.: Faster rate of initial fluid resuscitation in severe acute pancreatitis diminishes in-hospital mortality. Pancreatology. 2009; 9(6): 770–6. PubMed Abstract | Publisher Full Text\n\nWarndorf MG, Kurtzman JT, Bartel MJ, et al.: Early fluid resuscitation reduces morbidity among patients with acute pancreatitis. Clin Gastroenterol Hepatol. 2011; 9(8): 705–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNasr JY, Papachristou GI: Early fluid resuscitation in acute pancreatitis: a lot more than just fluids. Clin Gastroenterol Hepatol. 2011; 9(8): 633–4. PubMed Abstract | Publisher Full Text\n\nTenner S, Baillie J, DeWitt J, et al.: American College of Gastroenterology guideline: management of acute pancreatitis. Am J Gastroenterol. 2013; 108(9): 1400–15; 1416. PubMed Abstract | Publisher Full Text\n\nTalukdar R, Swaroop Vege S: Early management of severe acute pancreatitis. Curr Gastroenterol Rep. 2011; 13(2): 123–30. PubMed Abstract | Publisher Full Text\n\nWorking Group IAP/APA Acute Pancreatitis Guidelines: IAP/APA evidence-based guidelines for the management of acute pancreatitis. Pancreatology. 2013; 13(4 Suppl 2): e1–15. PubMed Abstract | Publisher Full Text\n\nBaillargeon JD, Orav J, Ramagopal V, et al.: Hemoconcentration as an early risk factor for necrotizing pancreatitis. Am J Gastroenterol. 1998; 93(11): 2130–4. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBrown A, Orav J, Banks PA: Hemoconcentration is an early marker for organ failure and necrotizing pancreatitis. Pancreas. 2000; 20(4): 367–72. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMuddana V, Whitcomb DC, Khalid A, et al.: Elevated serum creatinine as a marker of pancreatic necrosis in acute pancreatitis. Am J Gastroenterol. 2009; 104(1): 164–70. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWu BU, Bakker OJ, Papachristou GI, et al.: Blood urea nitrogen in the early assessment of acute pancreatitis: an international validation study. Arch Intern Med. 2011; 171(7): 669–76. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWu BU, Hwang JQ, Gardner TH, et al.: Lactated Ringer's solution reduces systemic inflammation compared with saline in patients with acute pancreatitis. Clin Gastroenterol Hepatol. 2011; 9(8): 710–717.e1. PubMed Abstract | Publisher Full Text\n\nHeinrich S, Schäfer M, Rousson V, et al.: Evidence-based treatment of acute pancreatitis: a look at established paradigms. Ann Surg. 2006; 243(2): 154–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharma VK, Howden CW: Prophylactic antibiotic administration reduces sepsis and mortality in acute necrotizing pancreatitis: a meta-analysis. Pancreas. 2001; 22(1): 28–31. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBai Y, Gao J, Zou DW, et al.: Prophylactic antibiotics cannot reduce infected pancreatic necrosis and mortality in acute necrotizing pancreatitis: evidence from a meta-analysis of randomized controlled trials. Am J Gastroenterol. 2008; 103(1): 104–10. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVillatoro E, Mulla M, Larvin M: Antibiotic therapy for prophylaxis against infection of pancreatic necrosis in acute pancreatitis. Cochrane Database Syst Rev. 2010; (5): CD002941. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWittau M, Mayer B, Scheele J, et al.: Systematic review and meta-analysis of antibiotic prophylaxis in severe acute pancreatitis. Scand J Gastroenterol. 2011; 46(3): 261–70. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nTrikudanathan G, Navaneethan U, Vege SS: Intra-abdominal fungal infections complicating acute pancreatitis: a review. Am J Gastroenterol. 2011; 106(7): 1188–92. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSun S, Yang K, He X, et al.: Probiotics in patients with severe acute pancreatitis: a meta-analysis. Langenbecks Arch Surg. 2009; 394(1): 171–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBesselink MG, van Santvoort HC, Buskens E, et al.: Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial. Lancet. 2008; 371(9613): 651–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSteinberg W, Tenner S: Acute pancreatitis. N Engl J Med. 1994; 330(17): 1198–210. PubMed Abstract | Publisher Full Text\n\nPetrov MS, Kukosh MV, Emelyanov NV: A randomized controlled trial of enteral versus parenteral feeding in patients with predicted severe acute pancreatitis shows a significant reduction in mortality and in infected pancreatic complications with total enteral nutrition. Dig Surg. 2006; 23(5–6): 336–44; discussion 344-5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGupta R, Patel K, Calder PC, et al.: A randomised clinical trial to assess the effect of total enteral and total parenteral nutritional support on metabolic, inflammatory and oxidative markers in patients with predicted severe acute pancreatitis (APACHE II > or =6). Pancreatology. 2003; 3(5): 406–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEckerwall GE, Tingstedt BB, Bergenzaun PE, et al.: Immediate oral feeding in patients with mild acute pancreatitis is safe and may accelerate recovery--a randomized clinical study. Clin Nutr. 2007; 26(6): 758–63. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJacobson BC, Vander Vliet MB, Hughes MD, et al.: A prospective, randomized trial of clear liquids versus low-fat solid diet as the initial meal in mild acute pancreatitis. Clin Gastroenterol Hepatol. 2007; 5(8): 946–51; quiz 886. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBakker OJ, van Brunschot S, van Santvoort HC, et al.: Early versus on-demand nasoenteric tube feeding in acute pancreatitis. N Engl J Med. 2014; 371(21): 1983–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nYi F, Ge L, Zhao J, et al.: Meta-analysis: total parenteral nutrition versus total enteral nutrition in predicted severe acute pancreatitis. Intern Med. 2012; 51(6): 523–30. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEatock FC, Chong P, Menezes N, et al.: A randomized study of early nasogastric versus nasojejunal feeding in severe acute pancreatitis. Am J Gastroenterol. 2005; 100(2): 432–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMorimoto T, Noguchi Y, Sakai T, et al.: Acute pancreatitis and the role of histamine-2 receptor antagonists: a meta-analysis of randomized controlled trials of cimetidine. Eur J Gastroenterol Hepatol. 2002; 14(6): 679–86. PubMed Abstract | Publisher Full Text\n\nUhl W, Büchler MW, Malfertheiner P, et al.: A randomised, double blind, multicentre trial of octreotide in moderate to severe acute pancreatitis. Gut. 1999; 45(1): 97–104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndriulli A, Leandro G, Clemente R, et al.: Meta-analysis of somatostatin, octreotide and gabexate mesilate in the therapy of acute pancreatitis. Aliment Pharmacol Ther. 1998; 12(3): 237–45. PubMed Abstract | Publisher Full Text\n\nHeinrich S, Schäfer M, Rousson V, et al.: Evidence-based treatment of acute pancreatitis: a look at established paradigms. Ann Surg. 2006; 243(2): 154–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPiaścik M, Rydzewska G, Milewski J, et al.: The results of severe acute pancreatitis treatment with continuous regional arterial infusion of protease inhibitor and antibiotic: a randomized controlled study. Pancreas. 2010; 39(6): 863–7. PubMed Abstract | Publisher Full Text\n\nHoribe M, Egi M, Sasaki M, et al.: Continuous Regional Arterial Infusion of Protease Inhibitors for Treatment of Severe Acute Pancreatitis: Systematic Review and Meta-Analysis. Pancreas. 2015; 44(7): 1017–23. PubMed Abstract | Publisher Full Text\n\nElmunzer BJ, Scheiman JM, Lehman GA, et al.: A randomized trial of rectal indomethacin to prevent post-ERCP pancreatitis. N Engl J Med. 2012; 366(15): 1414–22. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nMurray B, Carter R, Imrie C, et al.: Diclofenac reduces the incidence of acute pancreatitis after endoscopic retrograde cholangiopancreatography. Gastroenterology. 2003; 124(7): 1786–91. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11652",
"date": "18 Dec 2015",
"name": "Max Petrov",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11656",
"date": "18 Dec 2015",
"name": "Grazyna Rydzewska",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1474
|
https://f1000research.com/articles/4-1473/v1
|
18 Dec 15
|
{
"type": "Review",
"title": "Getting to the core of cadherin complex function in Caenorhabditis elegans",
"authors": [
"Jeff Hardin"
],
"abstract": "The classic cadherin-catenin complex (CCC) mediates cell-cell adhesion in metazoans. Although substantial insights have been gained by studying the CCC in vertebrate tissue culture, analyzing requirements for and regulation of the CCC in vertebrates remains challenging. Caenorhabditis elegans is a powerful system for connecting the molecular details of CCC function with functional requirements in a living organism. Recent data, using an “angstroms to embryos” approach, have elucidated functions for key residues, conserved across all metazoans, that mediate cadherin/β-catenin binding. Other recent work reveals a novel, potentially ancestral, role for the C. elegans p120ctn homologue in regulating polarization of blastomeres in the early embryo via Cdc42 and the partitioning-defective (PAR)/atypical protein kinase C (aPKC) complex. Finally, recent work suggests that the CCC is trafficked to the cell surface via the clathrin adaptor protein complex 1 (AP-1) in surprising ways. These studies continue to underscore the value of C. elegans as a model system for identifying conserved molecular mechanisms involving the CCC.",
"keywords": [
"Caenorhabditis elegans",
"C. elegans",
"classic cadherin-catenin complex",
"cadherin",
"β-catenin"
],
"content": "Caenorhabditis elegans: a simple system for studying cadherins\n\nThe classic cadherin-catenin complex (CCC) is a conserved multiprotein complex found in all metazoans and connects transmembrane cadherins to the actin cytoskeleton1. This strong yet dynamic connection is made possible by linker proteins known as catenins. β-catenin binds the cytoplasmic tail of classic cadherins and binds α-catenin, which in turn binds F-actin. In epithelial tissues in situ, the CCC localizes to adherens junctions, which confer adhesive connections between cells and which allow cells to exert forces across epithelia2. In addition, cell-cell contacts must withstand the stresses generated by actomyosin-mediated contractility3. The importance of fundamental work in vertebrate tissue culture for understanding the CCC is well documented and has provided many insights into CCC function. In living organisms, however, the CCC must function in three dimensions and must be tightly regulated. Perhaps the most dramatic example of the need for such regulation is in developing embryos, where cells must make and break cell-cell contacts in a tightly coordinated fashion to build new tissues. Analyzing CCC function in these more complicated tissue environments is challenging; it requires an ability to manipulate key aspects of CCC function while analyzing the detailed cellular effects of such manipulations in vivo.\n\nThe nematode C. elegans is a powerful tool for in vivo analysis of CCC function4. C. elegans epithelia possess a bona fide adherens junctional complex (4,5; Figure 1A,B), which possesses a single classic cadherin, HMR-1; the short isoform, HMR-1A, forms a complex with HMP-1/α-catenin and HMP-2/β-catenin6 and is bound by the p120ctn homologue, JAC-1, at its juxtamembrane region7. Two key events, driven by morphogenetic movements in the epidermis, critically depend on the CCC. Zygotic loss of function of the cadherin, HMR-1, leads to the Hammerhead phenotype, in which epidermal cells fail to enclose the embryo. Zygotic loss of HMP-1/α-catenin or HMP-2/β-catenin does not lead to enclosure failure, because maternally provided mRNA allows homozygous embryos to complete enclosure. Instead, as the embryo tries to elongate via a coordinated change in shape of its cells from a cuboidal shape to being elongated roughly fourfold along the anterior-posterior axis (reviewed in 8), F-actin-based circumferential filament bundles (CFBs) detach from junctions, leading to the Humpback phenotype. CFB detachment is accompanied by loss or tearing (or both) of the junctional proximal actin network recruited by HMP-16.\n\n(A) Schematic of the C. elegans apical junction. MAGI-1 (membrane-associated guanylate kinase with inverted organization protein 1) localizes to a domain between the CCC and the DLG-1/AJM-1 complex. (B) The core components of the CCC. HMR-1A is the epithelial cadherin, HMP-2 the junctional β-catenin, HMP-1 the C. elegans α-catenin, and JAC-1 the p120ctn homolog. (C) Structures of HMP-2 (Armadillo repeats shown in gold and green) bound to the phosphorylated HMR-1 cytoplasmic domain (magenta). The structure of phosphorylated E-cadherin cytoplasmic domain bound to β-catenin is also shown superimposed on the structure (gray; PDB ID: 1I7W). Phosphoserine 1212 in HMR-1 interacts with residues in HMP-2, including R271, which is mutated to C in the canonical hmp-2 allele, zu364. (D) A model for cadherin-induced cell polarization in blastomeres in the C. elegans embryo, based on 41. PAC-1 is recruited via CCC components to cell contact sites, where its GTPase-activating protein (GAP) domain negatively regulates CDC-42 at cell contacts to inhibit recruitment of PAR-6. The dashed line indicates a role that may not be direct. An inferred function that acts independently of the CCC via the pleckstrin-homology (PH) domain of PAC-1 is not shown for clarity. A is adapted from 13 with permission. B is adapted from 63 with permission. C is adapted with permission from a figure courtesy of Hee-Jung Choi by using data from 25.\n\nRemarkably, other earlier embryonic events, such as cleavage and gastrulation, while modulated by CCC function, can proceed without them. The relatively mild resulting phenotypes have actually provided advantages for identifying molecular pathways that act alongside the CCC during cleavage and gastrulation9,10. During later morphogenesis, the use of weak hmp-1 and hmp-2 mutants has similarly made genome-wide RNA interference (RNAi) screens possible to identify molecular components that act with or alongside the CCC during morphogenesis11–14. This brief review discusses recent work that sheds additional light on the core components of the CCC in C. elegans, how the CCC is deployed in the early embryo, and how it is localized during epithelial tissue remodeling.\n\n\nA conserved phosphorylation switch controls the cadherin/β-catenin interaction\n\nC. elegans is a useful model system for pursuing a structure-function approach to study the CCC. By rescuing strong loss-of-function mutants with green fluorescent protein (GFP)-tagged transgenes, it has been possible to assess several key aspects of CCC function in a living organism. For example, the N-terminal domain of HMP-1/α-catenin, which binds HMP-2/β-catenin, has been shown to be crucial for recruitment of HMP-1 to junctions, and the C terminus, which binds F-actin, is necessary for all key functions of HMP-115, vetting results from prior work in tissue culture (reviewed in 16) and consistent with similar recent work in Drosophila17.\n\nBecause of the mechanical stress experienced by the C. elegans epidermis during elongation, its morphogenesis requires robust cell-cell adhesion. However, adhesion in embryos must also be dynamically regulated as cell-cell contacts are extensively remodeled during these processes. One possible mechanism for regulation of CCC components is phosphorylation. Studies in cell culture have revealed that a serine-rich region of the cadherin tail is phosphorylated18, and in vitro phosphorylation of the cadherin tail strengthens its affinity for β-catenin by about 800 fold19–21. Mutation of the phosphorylated serines in the cadherin tail reduces cell-cell adhesion when these constructs are introduced into NIH 3T3 cells20. McEwen et al. recently narrowed these phosphorylation sites to three residues that are required for high-affinity β-catenin binding and cell adhesion in cultured cells22. In contrast, phosphorylation of β-catenin by Src at Tyr654 reduces affinity for cadherin23, and CKII phosphorylation of β-catenin regulates its interaction with α-catenin24.\n\nRecent work by Choi et al.25 rigorously tests the importance of phosphorylation of the cadherin cytoplasmic tail and β-catenin in C. elegans. As with vertebrate E-cadherin, phosphorylation of the HMR-1/cadherin tail dramatically increases its affinity for HMP-2/β-catenin. Solving the structure of an HMR-1/HMP-2 co-crystal reveals that a key conserved serine, S1212, which appears in the structure only when it is phosphorylated, is positioned precisely to allow interaction with several key residues in HMP-2 (Figure 1C). In some conformations, three additional downstream residues (T1215, S1218, and S1221) are phosphorylated. HMR-1 S1212 and S1218 are equivalent to two of the three residues which have been recently identified as bearing the majority of vertebrate E-cadherin phosphorylation and which have been proposed to be sequentially phosphorylated in tissue culture22. Significantly, S1212 and downstream residues are phosphorylated in vivo as well, based on results of co-immunoprecipitation/mass spectrometry analysis performed by Callaci et al.26. Independent assessment of binding affinities via reconstitution on liposomes confirms the work of Choi et al. showing that S1212 is crucial for HMR-1/HMP-2 association26.\n\nThe ideal test of necessity, of course, is in vivo requirement. Choi et al. found that a full-length hmr-1::gfp transgene driven by the endogenous promoter rescues hmr-1 deletion mutants to viability but that a non-phosphorylatable HMR-1::GFP S1212A construct cannot, despite localizing to junctions25. Although it is difficult to show that sequential phosphorylation actually occurs in vivo, Choi et al. assessed the possibility that pS1212 acts as a priming site for downstream phosphorylations by creating an hmr-1(T1215A, S1218A)::gfp construct in which S1212 remains phosphorylatable, but the C-terminal phosphorylation cascade is blocked. This construct is able to partially rescue hmr-1(zu389) embryonic lethality but cannot rescue larval lethality, indicating that, although S1212 is a key phosphoswitch, other phosphosites also have some importance.\n\nIn addition to examining the cadherin cytoplasmic tail, Choi et al. examined key residues in HMP-2/β-catenin. A gratifying result of this analysis was that sequencing of a canonical strong hmp-2 allele (zu364) revealed that one of the coordinating residues in HMP-2 visible in the HMR-1/HMP-2 co-crystal, Arg271, is mutated to a cysteine (Figure 1C). Since previous in vitro work had shown that Src phosphorylation of vertebrate β-catenin at Tyr654 reduces its affinity for E-cadherin23, Choi et al.25 mutated the equivalent residue in HMP-2, Tyr599, to create phosphomimetic and phospho-null mutations. They found that both constructs were able to rescue hmp-2(zu364) to viability and almost as efficiently as wild-type constructs, although the distribution of the phosphomimetic (predicted to have weaker binding to cadherin) becomes punctate and appears to cluster near sites where CFBs insert orthogonally into the junctional-proximal actin network. This suggests that the CCC in such embryos may be more susceptible to mechanical tension during morphogenesis, since CFBs are thought to transmit tension throughout the epidermis8.\n\nIn addition to the sites identified in the work of Choi et al., Callaci et al. found phosphosites in HMP-2 and HMP-1 in embryonic extracts. In vitro, HMP-2 protein harboring phosphomimetic mutations at the sites identified from extracts reduced its affinity for HMR-1. In vivo, constructs carrying identical phosphomimetic mutations rescue, but somewhat more weakly than wild-type transgenes. In contrast, perturbations of phosphosites identified in HMP-1 from embryonic extracts did not affect the ability of HMP-1 to bind actin or its comformation26. These results are similar to those in vertebrate tissue culture and Drosophila, which found only weak effects of numerous phosphosites in α-catenin27.\n\nTaken together, these results demonstrate the fruitfulness of an “angstroms to embryos” approach in C. elegans (i.e., correlation of changes in single residues identified as important from detailed molecular structural studies with aggregate effects on embryogenesis). There are several limitations to this approach, however. First, it leaves an important “meso-level” analysis of CCC function, such as the role of subcellular mechanics, largely unexplored (e.g., 28). Second, basic structure-function studies cannot address dynamic phosphorylation and dephosphorylation events. It remains to be seen whether such dynamic events can fine-tune CCC function or whether the functionally important sites identified in these studies are constitutively phosphorylated.\n\n\nThe PAR/aPKC complex as an upstream regulator of the CCC\n\nThere is abundant evidence that cadherin localization in metazoan epithelia is heavily influenced by molecular pathways that establish and maintain apicobasal polarity. In particular, polarity crucially depends on the function of the PAR/aPKC complex29–31. PAR proteins PAR-3 and PAR-6 were originally discovered in C. elegans through their maternal role in partitioning components in the one-cell zygote32. Subsequent work in worms, Drosophila, and tissue culture added molecular players and further clarified the role of this important complex in the establishment of cell polarity. A conserved cassette includes activated Cdc42, which binds Par6, which in turn recruits aPKC. Par3 (Bazooka in Drosophila) and Cdc42/Par6/aPKC engage in a complicated interplay at the apical ends of cells in many contexts (reviewed in 29,31,33–35).\n\nRecent work in C. elegans has added insight into PAR/aPKC function, especially as it relates to CCC function. Previous work had shown that during C. elegans intestinal differentiation, CCC and polarity complex proteins colocalize to foci that accumulate at the apical surface36,37, eventually coalescing to form bona fide junctions. Examining the role of the PAR/aPKC complex during this process was made possible by using maternally provided, modified PAR-3 and PAR-6 proteins that are present during polarization of the one-cell zygote but that are rapidly degraded thereafter37. Curiously, in the absence of PAR-3, intestinal HMR-1 is initially mislocalized, but HMP-1 is still present in foci36 that now localize with the more basal junctional component, DLG-1/Discs large38. Similar localization defects are seen in the pharynx in par-3 mutants36. Although par-3 function is initially dispensable in the epidermis, apical localization of HMR-1 and DLG-1 is progressively lost and the epidermis eventually tears36. In both the intestine and the epidermis, PAR-6 is required for junctional maturation but not for targeting of the CCC to initial spot junctions37. This result is largely consonant with similar previous work in Drosophila showing that the relationship between Par3 and Par6 is complex. There, Par3/Bazooka is initially upstream of Par6, but later Par3 and Par6/aPKC become spatially separated (reviewed in 35).\n\nRecent work on the C. elegans pharynx extends previous work by showing that PAR-6 is necessary for polarization of arcade cells39. HMR-1/cadherin, the ERM protein, ERM-1, and the DLG-1/Discs large binding partner, AJM-1, all fail to localize in par-6(M/Z) embryos. In contrast to the crucial requirement for par-6 function, embryos knocked down for hmr-1/cadherin and pat-3/β-integrin do not show major defects in localization of apical proteins, suggesting that neither the CCC nor adhesion to the basal lamina is essential for apical polarization in arcade cells. In the future, identifying other cell adhesion pathways that act in the absence of these two crucial adhesion systems should yield insights into how epithelia regulate polarity downstream of the PAR/aPKC system in metazoa.\n\n\nThe CCC as a reciprocal regulator of the PAR/aPKC complex in blastomeres\n\nThe relationship between the CCC and the PAR/aPKC proteins, based on the work described thus far, is admittedly somewhat complex in detail but comports well with work in many other systems, in which the PAR/aPKC complex acts upstream of the CCC in epithelial polarization (reviewed in 29,31,33). It is surprising, then, that the PAR/aPKC complex is not essential for blastomere adhesion in the early embryo38. This lack of absolute requirement, however, has proven to be an advantage for teasing apart the complex interplay between the CCC and the PAR/aPKC complex during embryogenesis. To gain insight into how blastomere polarity is established, Anderson et al.40 screened for mutations that prevent the normal localization pattern of PAR-6::GFP, which is confined to the outer (non-contact) surfaces of blastomeres in early C. elegans embryos. They identified PAC-1 (ARHGAP21 in humans), a Rho-family GTPase-activating protein (GAP). PAR-6, PAR-3, and aPKC/PKC-3 all show a symmetric cortical localization in pac-1 blastomeres. As in embryos with M/Z loss of PAR proteins, pac-1 mutant embryos show delayed ingression of cells during gastrulation but no defects in epithelial polarity in the later embryo, suggesting that PAC-1 is dispensable for other types of PAR asymmetries. GFP::PAC-1 localizes to inner (contact) surfaces but not the outer surfaces of blastomeres (i.e., in a pattern complementary to PAR-6). The complementary distributions of PAR-6 and PAC-1 suggest that PAC-1 controls PAR-6 localization by inhibiting CDC-42 at contact surfaces between blastomeres. This supposition was confirmed by making mutant PAR-6 protein that cannot bind CDC-42. The mutant protein mislocalizes, indicating that activated CDC-42 leads to recruitment of PAR-6, whereas PAC-1 at cell-cell contacts prevents PAR-6 accumulation there through downregulation of CDC-4240.\n\nRecent work from the Nance group extends this PAC-1/ARHGAP21 story41. Given that PAC-1 localizes to the same sites in the early embryo where the CCC is found, Klompstra et al.41 examined the distribution of full-length PAC-1 in hmr-1/cadherin loss-of-function embryos and found that it is reduced, although its localization is not abolished. N-terminal fragments of PAC-1 completely fail to localize in hmr-1 mutants, indicating that this region of the protein is regulated by the CCC. Deleting the pleckstrin-homology (PH) domain of PAC-1 abolished this residual PAC-1 in hmr-1 mutant embryos, indicating that this domain is required for the putative redundant localization mechanism. Taken together, these results suggest that both the CCC and other unidentified mechanisms localize PAC-1 to contact sites between blastomeres.\n\nKlompstra et al. then examined the catenins. Loss of jac-1/p120ctn function led to partial loss of GFP–PAC-1N from cell contacts. Removing HMP-1 in jac-1 embryos caused a complete loss of GFP–PAC-1N from contacts, as in hmr-1 embryos; these results suggest that JAC-1 and HMP-1 may work together in localizing PAC-1. Yeast two-hybrid screening eventually identified a possible mechanism linking PAC-1 and JAC-1 involving PICC-1 (PAC-1-interacting coiled-coil protein 1), the homologue of a poorly characterized human coiled-coil protein known as CCDC85B/DIPA, which has been shown to bind p120ctn42. GFP::PICC-1 localizes in an HMR-1- and JAC-1-dependent manner but persists in hmp-1(RNAi) embryos. Taken together, these data suggest a model in which PAC-1 is localized to cell surfaces in contact via both JAC-1/PICC-1 and HMP-1 (Figure 1D). To test this CCC-dependent model for Cdc42/PAR polarization, Klompstra et al. forced expression of the HMR-1 cytoplasmic tail ubiquitously at the cell surface by using a PH-domain chimera. The result was loss of polarization and greatly reduced PAR-6 at the cortex. These studies suggest a model whereby CCC-dependent contact between blastomeres leads to localized downregulation of Cdc42 via PAC-1/ARHGAP21 (Figure 1D).\n\nThe findings of Klompstra et al. may also help to explain puzzling results from the previous literature. As stated above, phenotypes associated with loss of function of the single p120ctn family member in both flies and worms are remarkably mild, given the clearly essential roles that p120ctn plays in vertebrates43–46. One potential explanation for this discrepancy is that the δ-catenin/p120ctn gene family in vertebrates has apparently arisen because of gene duplication from a single ancestral gene that encoded a protein more similar to δ-catenin. Subsequently, a gene encoding p120ctn arose in vertebrates, which has evolved essential roles in numerous processes (reviewed in 47). The work of Klompstra et al. may have uncovered an ancestral role for p120ctn/δ-catenin in spatial control of GAPs for Rho family GTPases. These findings comport well with previous studies of p120ctn, which have implicated it in recruitment of p190RhoGAP to junctions48–51. However, in worms, the role of PAC-1 is modulatory, since loss of pac-1 function is not lethal on its own (40 and Jeff Hardin and Allison Lynch, unpublished data).\n\n\nThe AP-1 complex regulates localization of the CCC during tissue remodeling\n\nIn addition to regulation of the core components of the cadherin/catenin complex by phosphorylation and through interactions with the partitioning-defective (PAR)/atypical protein kinase C (aPKC) polarity system, a well-known regulatory node affecting the CCC is intracellular trafficking. Here again, much of this work has been done in cultured cells (reviewed in 52–54); C. elegans has promise as a system for examining what aspects of trafficking of CCC components to and from the plasma membrane are essential in a metazoan. Some previous results have been surprising in the context of endocytic recycling of cadherins. For example, p120ctn has been shown to regulate endocytic trafficking of VE-cadherin by masking a conserved motif in its cytoplasmic tail55. If such regulation occurs in the case of HMR-1/cadherin in C. elegans, it must only be modulatory, given the mild phenotypes observed in the absence of JAC-1/p120ctn7,41. Similar results have been obtained in Drosophila, in which very mild phenotypes have been reported under laboratory conditions following p120ctn loss of function56,57. The AP-2 clathrin adaptor complex is likewise surprisingly dispensable in C. elegans58,59, although loss of AP-2 components has been reported to lead to synergistic lethality in a weak hmp-1/α-catenin mutant background in a genome-wide RNAi screen13.\n\nWork from the Michaux group60 has shown that, in contrast to the AP-2 complex, which is involved in endocytic recycling, the clathrin adaptor protein complex 1 (AP-1), well known to be involved in the export of proteins to the plasma membrane, is required for normal apicobasal polarity in the embryonic intestine of C. elegans. Knockdown of AP-1 subunits leads to homogeneous distribution of apical and basally localized proteins, including, significantly, PAR/aPKC complex proteins and CDC-42. Interestingly, knockdown embryos have ectopic lumenal vesicles, including components of both the CCC and the more basal complex that contains the C. elegans homologue of Discs large, DLG-1, and its binding partner, AJM-160.\n\nRecent work by the Michaux group61 extends this work on AP-1 to the C. elegans epidermis. To identify genes required for polarized localization of HMR-1 in the epidermis, Gillard et al.61 used RNAi of candidate genes to identify genes whose knockdown led to lateral mislocalization of HMR-1. These included chc-1/clathrin heavy chain, the σ and γ subunits of AP-1 complex (aps-1 and apg-1), the functionally redundant AP-1 μ subunits, and the AP-1-interacting protein HEATR5B/p200/Laa1p (in C. elegans known as soap-1, for sorting of apical proteins). In contrast, depletion of dynamin, RAB-5, RAB-11, or exocyst complex components led to changes in overall levels of HMR-1 but not its apicobasal localization. Thus, although these proteins play an essential role in HMR-1 secretion or recycling (or both) at the apical membrane, they—unlike AP-1 components—are not involved in apicobasal polarity. Gillard et al. went on to examine interactions among AP-1, clathrin subunits, and SOAP-1. Loss of AP-1 led to a strong decrease in recruitment of clathrin subunits to membranes. Similarly, loss of SOAP-1 led to an overall reduction in AP-1 components, suggesting that SOAP-1 is involved in targeting of AP-1 to sites of protein export to the membrane.\n\nAdditional observations, however, suggest that there are more global disruptions in apicobasal polarization in epidermal cells following loss of AP-1 components that include, but are not restricted to, the CCC. RNAi against the σ, γ, or both μ subunits of AP-1 induces a shift in the position of the DLG-1/AJM-1 complex to a more lateral position. In addition, in normal embryos, a small amount of HMR-1/cadherin is visible in the lateral membrane, but this lateral cadherin disappears during subsequent morphogenesis as the embryo elongates. In contrast, in aps-1(RNAi) embryos, HMR-1 accumulates laterally. Given the crucial role of the CCC during morphogenesis, this leads to disruption of F-actin during elongation. How the AP-1 is distinctively involved in maintenance of apicobasal polarity remains unclear.\n\nTaken together, the findings of Gillard et al. also raise interesting questions regarding the selectivity of effects of perturbing the AP-1 complex and other apicobasal cues in various epithelia in C. elegans. The specific mislocalization of HMR-1 in the epidermis, for example, contrasts markedly with the effects of depletion of AP-1 component in the intestine, in which no such mislocalization is observed. Previous work on the PAR complex similarly showed differential requirements in different tissues. While PAR-3 is critical for CCC establishment in the intestine, it is surprisingly dispensable in the epidermis, although localization of HMR-1 and DLG-1 is eventually lost, along with the mechanical integrity of the epidermis (36; reviewed in 62). What might account for such differences? The answer is unclear, but a key difference between the intestine and epidermis is the presence of a cuticle in the latter. Identifying molecular differences in CCC trafficking and apicobasal polarization in these tissues will be interesting in the future.\n\n\nConclusions\n\nC. elegans is a useful system for studying both the core functions and regulation of the CCC. On the one hand, the rapidity with which the function of single amino acids within each of the core CCC proteins can be assessed has allowed incisive experiments to be performed that link single amino acid changes in CCC proteins to essential, highly specific morphogenetic processes. On the other hand, the surprising lack of stringent requirements for CCC components or their modifiers in some developmental events has provided an opportunity to uncover conserved pathways shared by all metazoans. There is every reason to expect C. elegans to continue to be a useful model system for analyzing the CCC in the future.\n\n\nAbbreviations\n\nAP-1, clathrin adaptor protein complex 1; aPKC, atypical protein kinase C; CCC, cadherin-catenin complex; CFB, circumferential filament bundle; GAP, GTPase-activating protein; GFP, green fluorescent protein; PAR, partitioning-defective; PH, pleckstrin-homology; RNAi, RNA interference; SOAP-1, sorting of apical proteins-1.",
"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 R01GM58038 and R21HD072769. I apologize to many fine colleagues, whose work I could not cover at the level of detail it deserves, especially as it relates to work outside of C. elegans.\n\n\nReferences\n\nTakeichi M: Dynamic contacts: rearranging adherens junctions to drive epithelial remodelling. Nat Rev Mol Cell Biol. 2014; 15(6): 397–410. PubMed Abstract | Publisher Full Text\n\nHarris TJ, Tepass U: Adherens junctions: from molecules to morphogenesis. Nat Rev Mol Cell Biol. 2010; 11(7): 502–14. PubMed Abstract | Publisher Full Text\n\nLecuit T, Yap AS: E-cadherin junctions as active mechanical integrators in tissue dynamics. Nat Cell Biol. 2015; 17(5): 533–9. 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Prog Mol Biol Transl Sci. 2013; 116: 409–32. PubMed Abstract | Publisher Full Text\n\nAnastasiadis PZ, Reynolds AB: The p120 catenin family: complex roles in adhesion, signaling and cancer. J Cell Sci. 2000; 113(Pt 8): 1319–34. PubMed Abstract\n\nReynolds AB: p120-catenin: Past and present. Biochim Biophys Acta. 2007; 1773(1): 2–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarnahan RH, Rokas A, Gaucher EA, et al.: The molecular evolution of the p120-catenin subfamily and its functional associations. PLoS One. 2010; 5(12): e15747. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnastasiadis PZ, Moon SY, Thoreson MA, et al.: Inhibition of RhoA by p120 catenin. Nat Cell Biol. 2000; 2(9): 637–44. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWildenberg GA, Dohn MR, Carnahan RH, et al.: p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho. Cell. 2006; 127(5): 1027–39. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nZebda N, Tian Y, Tian X, et al.: Interaction of p190RhoGAP with C-terminal domain of p120-catenin modulates endothelial cytoskeleton and permeability. J Biol Chem. 2013; 288(25): 18290–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNoren NK, Liu BP, Burridge K, et al.: p120 catenin regulates the actin cytoskeleton via Rho family GTPases. J Cell Biol. 2000; 150(3): 567–80. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDelva E, Kowalczyk AP: Regulation of cadherin trafficking. Traffic. 2009; 10(3): 259–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWirtz-Peitz F, Zallen JA: Junctional trafficking and epithelial morphogenesis. Curr Opin Genet Dev. 2009; 19(4): 350–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Beco S, Amblard F, Coscoy S: New insights into the regulation of E-cadherin distribution by endocytosis. Int Rev Cell Mol Biol. 2012; 295: 63–108. PubMed Abstract | Publisher Full Text\n\nNanes BA, Chiasson-MacKenzie C, Lowery AM, et al.: p120-catenin binding masks an endocytic signal conserved in classical cadherins. J Cell Biol. 2012; 199(2): 365–80. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMagie CR, Pinto-Santini D, Parkhurst SM: Rho1 interacts with p120ctn and alpha-catenin, and regulates cadherin-based adherens junction components in Drosophila. Development. 2002; 129(16): 3771–82. PubMed Abstract | Faculty Opinions Recommendation\n\nMyster SH, Cavallo R, Anderson CT, et al.: Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component. J Cell Biol. 2003; 160(3): 433–49. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMayers JR, Wang L, Pramanik J, et al.: Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane. Proc Natl Acad Sci U S A. 2013; 110(29): 11857–62. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGu M, Liu Q, Watanabe S, et al.: AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis. eLife. 2013; 2: e00190. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nShafaq-Zadah M, Brocard L, Solari F, et al.: AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine. Development. 2012; 139(11): 2061–70. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGillard G, Shafaq-Zadah M, Nicolle O, et al.: Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells. Development. 2015; 142(9): 1684–94. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLoveless T, Hardin J: Cadherin complexity: recent insights into cadherin superfamily function in C. elegans. Curr Opin Cell Biol. 2012; 24(5): 695–701. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPettitt J: The cadherin superfamily. WormBook. 2005; 1–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11653",
"date": "18 Dec 2015",
"name": "Albert Reynolds",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11654",
"date": "18 Dec 2015",
"name": "David Sherwood",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11655",
"date": "18 Dec 2015",
"name": "Carien Niessen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1473
|
https://f1000research.com/articles/4-1472/v1
|
18 Dec 15
|
{
"type": "Review",
"title": "FGF23 as a calciotropic hormone",
"authors": [
"María E. Rodríguez-Ortiz",
"Mariano Rodríguez",
"María E. Rodríguez-Ortiz"
],
"abstract": "Maintaining mineral metabolism requires several organs and hormones. Fibroblast growth factor 23 (FGF23) is a phosphatonin produced by bone cells that reduces renal production of calcitriol – 1,25(OH)2D3 – and induces phosphaturia. The consequences of a reduction in 1,25(OH)2D3 involve changes in calcium homeostasis. There are several factors that regulate FGF23: phosphorus, vitamin D, and parathyroid hormone (PTH). More recently, several studies have demonstrated that calcium also modulates FGF23 production. In a situation of calcium deficiency, the presence of 1,25(OH)2D3 is necessary to optimize intestinal absorption of calcium, and FGF23 is decreased to avoid a reduction in 1,25(OH)2D3 levels.",
"keywords": [
"FGF23",
"calcium",
"FGF receptor"
],
"content": "Introduction\n\nThe regulation of calcium (Ca) and phosphorus (P) is controlled mainly by the parathyroid hormone (PTH), vitamin D, and, to a lesser extent, calcitonin. Fibroblast growth factor 23 (FGF23) was discovered in the early 2000s, initially identified as the cause of several congenital and acquired diseases1–4. Shimada and collaborators demonstrated the involvement of FGF23 in mineral homeostasis as a regulator of P and vitamin D metabolism2,5.\n\nFGF23 is a 32 KDa protein produced primarily in bone by osteocytes and osteoblasts. FGF23 acts mainly in the kidney inducing phosphaturia by decreasing the expression of renal cotransporters NaPiIIa and NaPiIIc5,6. In addition, FGF23 reduces calcitriol – 1,25(OH)2D3 – by both decreasing 1α-hydroxylase and increasing 24-hydroxylase activities; these enzymes catalyze the synthesis and catabolism of 1,25(OH)2D3, the active metabolite of vitamin D, respectively6. Parathyroid glands have been shown to be another target for FGF23, as it suppresses both PTH synthesis and secretion7.\n\nIn order to exert its biological actions, FGF23 targets a FGF receptor (FGFR). FGF23 belongs to the family of endocrine FGFs, molecules with a very low affinity for their receptors. Thus, the FGFR requires a co-receptor that eases the ligand-receptor binding: αKlotho is the co-receptor for FGF238.\n\nIn chronic kidney disease (CKD), the accumulation of P stimulates FGF23 production from the early stages of CKD. High levels of FGF23 are thought to contribute to the reduction in renal production of 1,25(OH)2D3, a key factor in the development of uremic hyperparathyroidism9.\n\nThe regulation of FGF23 production has been investigated by several groups. It is well known that vitamin D upregulates FGF23 in vivo and in vitro10. PTH also stimulates FGF23 production11,12. An increase in dietary P stimulates FGF23, although acute increases in serum P fail to increase FGF239,13. PHEX and DMP1, factors involved in the regulation of bone mineralization, also modulate FGF23 expression14. Interestingly, intravenous iron induces transient elevations in the levels of FGF2315, and recent findings suggest that both leptin and estrogens also influence FGF23 expression16,17. The main regulators of FGF23 levels are summarized in Figure 1.\n\nRecent studies reveal that the FGF23-Klotho axis is involved in renal ion transport through different mechanisms. FGF23 regulates sodium transport by modulating the sodium-chloride channel (NCC) in distal renal tubules18, and Klotho influences the secretion of potassium by regulating the abundance of the renal outer medullary potassium channel 1 (ROMK1)19.\n\nOver the last few years, experimental and association studies have assessed the role of Ca as a modulator of FGF23 expression. Conversely, FGF23 also modulates renal Ca handling. Both aspects of the association between Ca and FGF23 will be discussed here.\n\n\nModulation of FGF23 expression by calcium\n\nSeveral lines of evidence point out that Ca is a direct regulator of FGF23. In vitamin D receptor (VDR)-null mice, with almost undetectable levels of FGF23, administration of high dietary Ca induced a marked elevation of FGF23 at both mRNA and protein levels20. We obtained similar results using a different experimental approach21. In rats fed with a diet deficient in Ca and vitamin D, the serum levels of FGF23 were extremely low despite very high PTH, which should stimulate FGF23 production. Analyzing the association between FGF23 and Ca (Figure 2), we found a positive and significant correlation (r2=0.73; P<0.001); interestingly, those rats with serum Ca below 1 mM had reduced FGF23, suggesting a threshold level of Ca above which an increase in FGF23 is allowed. In parathyroidectomized (PTx) rats that presented hypocalcemia, serum FGF23 concentration was low. In these PTx animals, an elevation of serum Ca after an acute (6-hour) infusion of Ca or after the chronic administration of a high-Ca diet produced an increase in serum FGF23. In these experiments, the increase in FGF23 was not explained by an increase in serum concentration of 1,25(OH)2D3 or P. Gravesen et al.22 analyzed the effects of acute changes in serum Ca on FGF23 levels over a shorter period of time. After 60 minutes of hypocalcemic and hypercalcemic clamps, no changes were detected in FGF23, and the acute infusion of Ca in PTx rats did not modify serum FGF23 either. Therefore, it seems that the regulation of FGF23 by Ca is not produced in such a short time.\n\nAdapted from reference 21 (Rodríguez-Ortiz et al., 2012).\n\nIn recent work by David et al.23, the levels of Ca and 1,25(OH)2D3 were positively associated with FGF23 in wild-type, 1α-hydroxylase-/- and GCM2-/- mice (P=0.048 and P=0.011, respectively) in a multivariate analysis of pooled data. This model is characterized by the absence of 1,25(OH)2D3 and PTH, and the administration of a rescue diet with a high content of Ca stimulated FGF23 and FGF23 mRNA expression. Importantly, the authors also observed that high Ca stimulated the activity of the FGF23 promoter in osteoblastic cells as well as the levels of FGF23 secreted. The effect of Ca on promoter activity was blocked by the addition of a Ca channel blocker to the culture medium. Conversely, treatment with Ca ionophores increased promoter activity. Although previous work by this group failed to find an effect of Ca on FGF23 promoter activity10, the reason for such apparent disparity of results might reside in the characteristics of the cell lines used and/or the different Ca concentrations used in the experiments.\n\nStudies by Dr Brown’s laboratory examined the role of the calcium-sensing receptor (CaSR) in the regulation of FGF23 by Ca24, as it constitutes the main mechanism of Ca sensing in the tissues involved in mineral homeostasis. They found that the stimulatory effect of Ca on FGF23 was present in the double knockout mice PTH-CaSR. Therefore, they concluded that the CaSR did not mediate the stimulation of FGF23 by Ca in bone cells. In addition, they found that concentrations of 5 mg/dl of P were required to enable the stimulation of FGF23 by Ca. Conversely, regulation of FGF23 by P is abolished when Ca is lower than 8 mg/dl; in fact, they obtained a better correlation between FGF23 and Ca × P (r=0.70) than between FGF23 and Ca or P individually (r=0.65 and 0.58, respectively).\n\nIn healthy humans, Vervloet and collaborators analyzed the effect of a Ca and P-rich diet on serum FGF23. Although the effects of the dietary Ca and P were not analyzed separately, the authors found an increase in FGF23 after 36 hours of consumption of the Ca and P-rich diet25. More recently, a cross-sectional study performed in a cohort of middle-aged subjects analyzed the influence of demographic, clinical, and dietary factors on FGF23. In this work, intake of Ca and protein were analyzed individually, with dietary Ca intake significantly associated with higher levels of FGF23 (P=0.01)26. These results are in concordance with previous findings in animals, with higher FGF23 associated with Ca intake.\n\nThe effects of acute infusions of sodium citrate and Ca have also been tested in parallel in healthy humans and in uremic patients. No changes in FGF23 were observed after an acute change (120 minutes) in serum Ca concentration in subjects with either normal or reduced renal function27. According to these results, the regulation of FGF23 by Ca does not seem to be as rapid as that of PTH.\n\nAlso in the context of CKD, it is worth mentioning that association studies have found significant relationships between FGF23 and Ca. For instance, Imanishi and collaborators reported a positive correlation between FGF23 and Ca (r=0.355, P<0.0001)28. In a prospective study performed in transplant patients, Ca was independently associated with FGF23 levels (P=0.01)29.\n\nTaken together, these data support the idea of Ca as a modulator of FGF23 levels. However, the effect of Ca on FGF23 does not seem to be acute and may not be mediated by CaSR. The meaning of this regulation gains especial relevance in case of hypocalcemia associated with vitamin D deficiency. In such context, the over-suppression of vitamin D by FGF23 would further reduce the calcemia. Therefore, it seems logical that the level of serum Ca conditions that of FGF23. This would therefore constitute a defense mechanism against hypocalcemia.\n\n\nInvolvement of FGF23 in calcium homeostasis\n\nDespite the fact that FGF23 is considered essentially as a phosphatonin or hormone regulator of P metabolism, there is growing evidence about the involvement of FGF23 in the maintenance of Ca homeostasis.\n\nThree of the four types of FGFR (FGFR1, 3, and 4) can be found in the proximal tubule of the kidney6,30. The deletion of any of them does not completely block the phosphaturic effect of FGF23, which suggests that this action is not mediated by a single receptor. Gattineni et al. identified FGFR1 and 4 as key elements for the phosphaturic response to FGF2331. While the action of FGF23 increasing phosphaturia takes places upon the interaction with its receptors in proximal tubules of the kidney, FGF23 increases Ca reabsorption by augmenting the expression of the transient receptor potential vanilloid type 5 (TRPV5) in distal tubules. TRPV5 is a glycoprotein essential for the handling of Ca at the kidney level.\n\nIt has been reported that Klotho itself controls TRPV5 in an FGF23-independent manner, therefore regulating renal Ca transport32. In addition, Klotho also promotes the trafficking of TRPV5 from inside the epithelial cell33. FGF23 has also been shown to increase Ca reabsorption by regulating the abundance of TRPV5, in an action that is mediated by the signaling pathways ERK1/2, SGK1, and WNK434. It might be speculated that the FGF23-independent effects of Klotho appear to be related to the activation and trafficking of the TRPV5, whereas the actions of Klotho acting as a co-receptor of FGFR are involved in the maintenance of P and vitamin D levels. The phenotypical similarities between FGF23-/- and Klotho-/- mutant mice would support this notion.\n\nKao et al. have reported the dysregulatory effect that FGF23 exerts at the cardiovascular level35. FGF23 promotes the phosphorylation of proteins involved in Ca handling in HL-1 atrial cells, such as Ca/calmodulin-dependent protein kinase II (CaMKII) and phospholamban at threonine 17 (PLB). This effect may underlie the relationship between FGF23 and atrial fibrillation described by Seiler and collaborators36.\n\nFGF23 and Klotho contribute to conserving the level of Ca in the organism. This fact might have pathophysiological consequences in the context of CKD. Uremia is characterized by the presence of extraordinarily high levels of FGF23, which may help prevent the Ca loss.\n\n\nConclusion\n\nSystems regulating Ca and P homeostasis are closely related. This is illustrated by FGF23, which regulates Ca and P metabolism and is modulated by both elements. The effect of Ca as a regulator of FGF23 has been widely demonstrated. In studies carried out in experimental animals, Ca deficiency is associated with low FGF23, whereas Ca administration increases its levels. In healthy humans, higher dietary Ca is associated with higher FGF23, although this effect is not observed when Ca is administered acutely. In uremia, some studies point out an association between FGF23 and Ca. On the other hand, very recent work has shown how FGF23 regulates Ca homeostasis, increasing renal reabsorption in a mechanism involving TRPV5. The association between FGF23 and Ca might be relevant in CKD, when there is an imbalance in FGF23 production and risk of unfavorable effects associated with high Ca.\n\nForthcoming research should be focused on studying in depth the nature of the relationship between FGF23 and Ca, particularly in the context of CKD and its derangements in mineral metabolism. In addition, it is of outstanding importance to unravel the molecular mechanisms and signaling pathways underlying this regulatory feedback loop.\n\n\nAbbreviations\n\nCa, calcium; P, phosphorus; PTH, parathyroid hormone; FGF23, fibroblast growth factor 23; 1,25(OH)2D3, calcitriol; FGFR, fibroblast growth factor receptor; CKD, chronic kidney disease; PTx, parathyroidectomized; CaSR, calcium-sensing receptor; TRPV5, transient receptor potential vanilloid type 5.",
"appendix": "Competing interests\n\n\n\nM.E. Rodríguez has nothing to declare.\n\n\nGrant information\n\nM. Rodríguez has received research grants from Amgen and Fresenius, and lecture fees from Abbvie, Amgen, Fresenius, and Shire.\n\n\nReferences\n\nADHR Consortium: Autosomal dominant hypophosphataemic rickets is associated with mutations in FGF23. Nat Genet. 2000; 26(3): 345–8. PubMed Abstract | Publisher Full Text\n\nShimada T, Mizutani S, Muto T, et al.: Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci U S A. 2001; 98(11): 6500–5. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJonsson KB, Zahradnik R, Larsson T, et al.: Fibroblast growth factor 23 in oncogenic osteomalacia and X-linked hypophosphatemia. N Engl J Med. 2003; 348(17): 1656–63. PubMed Abstract | Publisher Full Text\n\nLarsson T, Yu X, Davis SI, et al.: A novel recessive mutation in fibroblast growth factor-23 causes familial tumoral calcinosis. J Clin Endocrinol Metab. 2005; 90(4): 2424–7. PubMed Abstract | Publisher Full Text\n\nShimada T, Hasegawa H, Yamazaki Y, et al.: FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004; 19(3): 429–35. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGattineni J, Bates C, Twombley K, et al.: FGF23 decreases renal NaPi-2a and NaPi-2c expression and induces hypophosphatemia in vivo predominantly via FGF receptor 1. Am J Physiol Renal Physiol. 2009; 297(2): F282–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBen-Dov IZ, Galitzer H, Lavi-Moshayoff V, et al.: The parathyroid is a target organ for FGF23 in rats. J Clin Invest. 2007; 117(12): 4003–8. PubMed Abstract | Free Full Text | Faculty Opinions Recommendation\n\nUrakawa I, Yamazaki Y, Shimada T, et al.: Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006; 444(7120): 770–4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLarsson T, Nisbeth U, Ljunggren O, et al.: Circulating concentration of FGF-23 increases as renal function declines in patients with chronic kidney disease, but does not change in response to variation in phosphate intake in healthy volunteers. Kidney Int. 2003; 64(6): 2272–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLiu S, Tang W, Zhou J, et al.: Fibroblast growth factor 23 is a counter-regulatory phosphaturic hormone for vitamin D. J Am Soc Nephrol. 2006; 17(5): 1305–15. PubMed Abstract | Publisher Full Text\n\nLavi-Moshayoff V, Wasserman G, Meir T, et al.: PTH increases FGF23 gene expression and mediates the high-FGF23 levels of experimental kidney failure: a bone parathyroid feedback loop. Am J Physiol Renal Physiol. 2010; 299(4): F882–9. PubMed Abstract | Publisher Full Text\n\nLópez I, Rodríguez-Ortiz ME, Almadén Y, et al.: Direct and indirect effects of parathyroid hormone on circulating levels of fibroblast growth factor 23 in vivo. Kidney Int. 2011; 80(5): 475–82. PubMed Abstract | Publisher Full Text\n\nAntoniucci DM, Yamashita T, Portale AA: Dietary phosphorus regulates serum fibroblast growth factor-23 concentrations in healthy men. J Clin Endocrinol Metab. 2006; 91(8): 3144–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMartin A, Liu S, David V, et al.: Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling. FASEB J. 2011; 25(8): 2551–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWolf M, Koch TA, Bregman DB: Effects of iron deficiency anemia and its treatment on fibroblast growth factor 23 and phosphate homeostasis in women. J Bone Miner Res. 2013; 28(8): 1793–803. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTsuji K, Maeda T, Kawane T, et al.: Leptin stimulates fibroblast growth factor 23 expression in bone and suppresses renal 1alpha,25-dihydroxyvitamin D3 synthesis in leptin-deficient mice. J Bone Miner Res. 2010; 25(8): 1711–23. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nCarrillo-López N, Román-García P, Rodríguez-Rebollar A, et al.: Indirect regulation of PTH by estrogens may require FGF23. J Am Soc Nephrol. 2009; 20(9): 2009–17. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAndrukhova O, Slavic S, Smorodchenko A, et al.: FGF23 regulates renal sodium handling and blood pressure. EMBO Mol Med. 2014; 6(6): 744–59. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCha SK, Hu MC, Kurosu H, et al.: Regulation of renal outer medullary potassium channel and renal K+ excretion by Klotho. Mol Pharmacol. 2009; 76(1): 38–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShimada T, Yamazaki Y, Takahashi M, et al.: Vitamin D receptor-independent FGF23 actions in regulating phosphate and vitamin D metabolism. Am J Physiol Renal Physiol. 2005; 289(5): F1088–95. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRodriguez-Ortiz ME, Lopez I, Muñoz-Castañeda JR, et al.: Calcium deficiency reduces circulating levels of FGF23. J Am Soc Nephrol. 2012; 23(7): 1190–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGravesen E, Mace ML, Hofman-Bang J, et al.: Circulating FGF23 levels in response to acute changes in plasma Ca2+. Calcif Tissue Int. 2014; 95(1): 46–53. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDavid V, Dai B, Martin A, et al.: Calcium regulates FGF-23 expression in bone. Endocrinology. 2013; 154(12): 4469–82. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nQuinn SJ, Thomsen AR, Pang JL, et al.: Interactions between calcium and phosphorus in the regulation of the production of fibroblast growth factor 23 in vivo. Am J Physiol Endocrinol Metab. 2013; 304(3): E310–20. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nVervloet MG, van Ittersum FJ, Büttler RM, et al.: Effects of dietary phosphate and calcium intake on fibroblast growth factor-23. Clin J Am Soc Nephrol. 2011; 6(2): 383–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\ndi Giuseppe R, Kühn T, Hirche F, et al.: Potential Predictors of Plasma Fibroblast Growth Factor 23 Concentrations: Cross-Sectional Analysis in the EPIC-Germany Study. PLoS One. 2015; 10(7): e0133580. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWesseling-Perry K, Wang H, Elashoff R, et al.: Lack of FGF23 response to acute changes in serum calcium and PTH in humans. J Clin Endocrinol Metab. 2014; 99(10): E1951–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nImanishi Y, Inaba M, Nakatsuka K, et al.: FGF-23 in patients with end-stage renal disease on hemodialysis. Kidney Int. 2004; 65(5): 1943–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nEvenepoel P, Naesens M, Claes K, et al.: Tertiary 'hyperphosphatoninism' accentuates hypophosphatemia and suppresses calcitriol levels in renal transplant recipients. Am J Transplant. 2007; 7(5): 1193–200. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAndrukhova O, Zeitz U, Goetz R, et al.: FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway. Bone. 2012; 51(3): 621–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGattineni J, Alphonse P, Zhang Q, et al.: Regulation of renal phosphate transport by FGF23 is mediated by FGFR1 and FGFR4. Am J Physiol Renal Physiol. 2014; 306(3): F351–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChang Q, Hoefs S, van der Kemp AW, et al.: The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science. 2005; 310(5747): 490–3. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWolf MT, An SW, Nie M, et al.: Klotho up-regulates renal calcium channel transient receptor potential vanilloid 5 (TRPV5) by intra- and extracellular N-glycosylation-dependent mechanisms. J Biol Chem. 2014; 289(52): 35849–57. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAndrukhova O, Smorodchenko A, Egerbacher M, et al.: FGF23 promotes renal calcium reabsorption through the TRPV5 channel. EMBO J. 2014; 33(3): 229–46. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKao YH, Chen YC, Lin YK, et al.: FGF-23 dysregulates calcium homeostasis and electrophysiological properties in HL-1 atrial cells. Eur J Clin Invest. 2014; 44(8): 795–801. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSeiler S, Cremers B, Rebling NM, et al.: The phosphatonin fibroblast growth factor 23 links calcium-phosphate metabolism with left-ventricular dysfunction and atrial fibrillation. Eur Heart J. 2011; 32(21): 2688–96. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11650",
"date": "18 Dec 2015",
"name": "Reinold G Erben",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11651",
"date": "18 Dec 2015",
"name": "Jordi Bover",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 not only provides a top-of-art review on the issue but it is also of outstanding quality, in correspondence with the reputation of the authors.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1472
|
https://f1000research.com/articles/4-40/v1
|
10 Feb 15
|
{
"type": "Research Article",
"title": "Developing a wintering waterfowl community baseline for environmental monitoring of Narragansett Bay, Rhode Island",
"authors": [
"Betty J. Kreakie",
"Kristopher Winiarski",
"Richard McKinney",
"Kristopher Winiarski",
"Richard McKinney"
],
"abstract": "In 2004, the Atlantic Ecology Division of the US Environmental Protection Agency’s Office of Research and Development began an annual winter waterfowl survey of Rhode Island’s Narragansett Bay.\n\nHerein, we explore the survey data gathered from 2004 to 2011 in order to establish a benchmark understanding of the Narragansett Bay’s waterfowl communities and to establish a statistical framework for future environmental monitoring. Abundance and diversity of Narragansett Bay’s wintering waterfowl were relatively stable during the initial years of this survey, except in 2010 when there was a large spike in abundance and a reciprocal fall in diversity. There was no significant change in ranked abundance of most waterfowl species, with only Bufflehead (Bucephala albeola) and Hooded Merganser (Lophodytes cucllatus) showing a slight yet significant upward trend during the course of our survey period. Nonmetric multidimensional scaling was used to examine the community structure of wintering waterfowl in Narragansett Bay. The results of the Nonmetric multidimensional scaling indicate that there is a spatial structure to the waterfowl communities of Narragansett Bay and this structure has remained relatively stable since the survey began. Our Nonmetric multidimensional scaling analysis helps to solidify what is known anecdotally about Narragansett Bay’s waterfowl ecology, and provides a formalized benchmark for long-term monitoring of Narragansett Bay’s waterfowl communities. Birds, including waterfowl, are preferred bioindicators and we propose using our multivariate approach to monitor the future health of Narragansett Bay.",
"keywords": [
"Baseline Data",
"Community",
"Environmental Monitoring",
"Narragansett Bay",
"Nonmetric Multidimensional Scaling",
"Waterfowl"
],
"content": "Introduction\n\nAs modern environmental pressures continue to adversely impact natural habitats, global waterfowl populations are declining at accelerated rates. According to the Millennium Ecosystem Assessment, approximately 12% of all bird species are presently threatened with extinction, and 41% of all waterfowl populations are declining in abundance (Assessment, 2003; Assessment, 2005). Narragansett Bay’s waterfowl communities are not immune to the global drivers of waterfowl population decline and modifications of waterfowl communities; Narragansett Bay is exposed to habitat conversion, shoreline hardening, increased sedimentation and pollution, and increased threats from climate change (Nixon & Fulweiler, 2012). Even though we know that these changing conditions are having a global impact on waterfowl populations, we are uncertain about the specific ramifications on Narragansett Bay’s wintering waterfowl communities. Twenty-three North American waterfowl species have been observed wintering in Narragansett Bay, including 11 of the 15 known species of sea ducks, a guild of waterfowl that breed in boreal Canada and winter as far south as Chesapeake Bay (McKinney, 2004). Understanding or predicting deviations from normal is not possible without baseline monitoring data on waterfowl communities (Temple & Wiens, 1989).\n\nIn 2004, the Atlantic Ecology Division (AED) of the US Environmental Protection Agency’s (EPA’s) Office of Research and Development, in collaboration with state wildlife agencies and local environmental groups, began an annual winter waterfowl survey of Rhode Island’s Narragansett Bay (hence forth referred to as the Bay). Every year in January, local wildlife biologists and environmental scientists conduct a comprehensive survey of the Bay’s waterfowl. The waterfowl survey was implemented in an attempt to fill critical gaps in our ecological knowledge about the Bay’s waterfowl communities. While there have been numerous waterfowl studies conducted in this area (Caron & Paton, 2007; Loring et al., 2013; McKinney & McWilliams, 2005), we are unaware of any long-term multispecies studies. Consequently, we are still relatively ill-informed about long-term trends of the Bay’s waterfowl populations and communities. The survey data can also provide us a means to monitor the Bay’s overall environmental health by using waterfowl as a bioindicator. Due to waterfowl’s comparatively high trophic status, waterfowl communities provide insight about local food webs’ relative health and stability.\n\nTo determine the underlying waterfowl community structure, we used a multivariate ordination technique, Nonmetric multidimensional scaling (NMDS) (Austin, 1976; Clarke, 1993). Additionally, we propose employing the NMDS as a statistical framework for environmental monitoring of the Bay (Gabrey & Afton, 2004; Urban, 2006). Like other ordination methods, the NMDS approach reduces data complexity, which is critical when analyzing data that are complex and highly variable. Yet unlike other ordination methods, NMDS requires few, if any, a priori assumptions about the distribution of the data. This multivariate approach allows us to detect any relative shifts in community composition between sites and years, and also to explore relationships with potential environmental drivers of change (Clarke & Ainsworth, 1993; Karydis, 1992).\n\nBirds, especially waterfowl, are often the logical candidates for monitoring environmental health (Amat & Green, 2010; Kushlan, 1993). Since waterfowl are high trophic level foragers, the environmental stressors of all lower trophic levels accumulate in waterfowl (Matsinos & Wolff, 2003; O’Connell et al., 2000). Waterfowl have, therefore, been used successfully to monitor a wide array of environmental stressors. For example, these species have been used to monitor a range of heavy metals, including cadmium, mercury, and lead (Di Giulio & Scanlon, 1984; Mochizuki et al., 2002; Monteiro & Furness, 1995; Ribeiro et al., 2005), and the impacts of habitat conversion, e.g., forested land to agriculture or road (Austin, 1976; Koper & Schmiegelow, 2006; Maisonneuve et al., 2006). Birds respond to habitat conversion at multiple temporal and spatial scales (DeLuca et al., 2004; Findlay et al., 2000). Waterfowl respond to these stressors at the local up to the regional scale and their responses can be apparent nearly immediately and continue after substantial time lags. These species have a high detection probability and are easy to identify by even novice birders (Pagano & Arnold, 2009), which further strengthen the argument of using waterfowl as bioindicators of environmental health.\n\nIt is nearly impossible to overstate the economic and environmental significance of Narragansett Bay to New England. Narragansett Bay contributes meaningfully to the economy through recreation, tourism, fishing, and shipping (Pastore, 2011; Tyrrell et al., 1994). Its economic contributions are equally matched by its environmental contributions. The Bay serves as critical habitat to numerous species and provides innumerable ecosystem services. This study analyzes the first eight years of our survey data in order to develop a baseline understanding of waterfowl community spatial and temporal structure in the Bay, which can be used to track future changes in the overall health of the Bay.\n\n\nMethods\n\nThe Narragansett Bay Winter Waterfowl Survey (NBWWS) is conducted throughout Narragansett Bay, Rhode Island (Figure 1). Narragansett Bay is a well-mixed embayment containing a complex of estuaries. The Bay is 234 km2 in area and has a mean depth of 8.7m (Calabretta & Oviatt, 2008; Nixon et al., 2009). In the northern portion of the Bay, the Seekonk and Providence Rivers are the main freshwater sources. This area is also more urban, and exposed to periodic hypoxia, especially in the summer months (Codiga et al., 2009). The southern portion of the Bay is deeper and has more intense oceanic influences. Additionally, the land surrounding the southern portion tends to be less densely developed and populated.\n\nInset of Rhode Island (USA). Sections are color-coded and site boundaries delineated. The points represent the approximate observation locations for the surveys.\n\nInitiated in 2004, the NBWWS is completed annually by eight teams composed of 2–4 observers who survey waterfowl at 67 site locations throughout Narragansett Bay (McKinney, 2004). Survey locations were determined by dividing the bay first into sections, and then further into sites within the sections (Figure 1). The number of sites in each section ranged from eight sites in sections 4 and 5 to 15 sites in section 6. The division of sections and sites was based on the geography of the Bay; this layout ensures that all areas of the Bay will be visible from the survey locations (Figure 1).\n\nCoordinated sampling occurs at all sites during a single day in early to mid-January, beginning at approximately 0730 in the morning and ending by 1645 in the afternoon. This is a shore-based survey and observers use direct counts to record all waterfowl present at a location at the time of the observation. We define waterfowl species as ducks, geese, and swans. Because of the difficulty of distinguishing between Lesser and Greater Scaup to the species level, these species are simply categorized into a single Scaup (Aythya spp.) taxonomic group.\n\nCounting is completed from a stationary point from which the entire area (i.e., cove or embayment) is scanned with binoculars or a spotting scope. Every bird seen on the water surface or on the adjoining shoreline up to 50 m from the water is counted; when possible sex and age were also noted. Large flocks of greater than 100 birds are estimated by counting in groups of ten or one hundred. Observers take as much time as necessary to accurately count and record all waterfowl observed. Most locations require between 10–20 minutes to survey.\n\nWe initially explored basic population trends for each waterfowl species. This included average abundance and standard deviations between years. We calculated ranked abundance for non-rare species and fitted regression trends and tested significance for each species. Ranked abundance allowed us to examine the relative dominance or rarity of a species given the current community. Furthermore, we assessed total waterfowl abundance and Shannon diversity index by year.\n\nWe used nonmetric multidimensional scaling to identify the community level structure of wintering waterfowl in Narragansett Bay (Austin et al., 2001; Clarke & Ainsworth, 1993; Kruskal, 1964). Nonmetric multidimensional scaling is a nonparametric ordination technique used to reduce the dimensionality of a complex data set while maintaining the relative relationship between species composition of sites (Dixon, 2009). This specific ordination method does not require a prior constraining of axes or assumptions of normality. NMDS fits all ordination axes simultaneously not by sequentially finding orthogonal linear axes. Therefore, calculating variance explained by axis or linear goodness of fit measures are not applicable to this specific ordination method.\n\nNonmetric multidimensional scaling was conducted using survey data collected from 2004 to 2011. A Wisconsin double transform, standardized by species percent abundance and by maximum for each species, was conducted on the species data, and Bray-Curtis distance was used to calculate community distance for in-depth discussion of methods see (Faith et al., 1987; Minchin, 1987). We iteratively fit NMDS solutions of increasing dimensionality to determine the solution with adequate levels of decreased stress. Stress is a measure of goodness of fit; it is a measure of the agreement between the distance in ordination space and observed waterfowl community distance (Kruskal, 1964). Our goal was to minimize stress while avoiding superfluous ordination axes. To find the global stress minimization of an NMDS, random configurations of start locations were interjected into the fitting algorithm (Kruskal & Wish, 1978). All analysis was conducted in R version 2.13.1 (R Development Core Team, 2013) and NMDS was conducted with the Vegan package (Oksanen et al., 2007).\n\nTo explore relationships between environmental conditions and the waterfowl community structure, we tested for correlations between the NMDS axes and ancillary variables (Table 1). Ancillary variable selection was hypothesis driven. Initially, we examined the area effect and spatial structure of the waterfowl community composition. Also, we explored the impacts of near shore habitat conversion. Furthermore, we hypothesized that winter intensity and extreme wind events might be driving inter-annual variation. All ancillary variables (Table 1) were initially tested for significance to the final four-dimensional NMDS structure and then cross correlation between variables. Among correlated variables (greater than 0.8), only the most significant variables to the NMDS structure were included in the final analysis.\n\nLocation was measured as the latitude and longitude of each site’s centroid. Delineated site boundaries were used to calculate area and site perimeter length (Figure 1). Degraded wetland area was calculated using RI Department of Environmental Management (RI DEM) Statewide Planning Program’s impacted wetland digital vector data, which were downloaded from the RI Geographic Information System (RIGIS) (http://www.edc.uri.edu/rigis). Total wetland area was calculated from the US Fish and Wildlife Service (US FWS) National Wetland Inventory (http://www.fws.gov/wetlands). National Oceanic and Atmospheric Administration’s (NOAA) bathymetry data were also downloaded from RIGIS.\n\nThe North Atlantic Oscillation (NAO) is a large-scale climate index that measures the atmospheric pressure at sea level between the Icelandic low and Azores high, which captures information about the relative intensity of the winter (Hurrell, 1995). A strongly positive NAO index is related to above normal temperatures in the study region, whereas a negative NAO index is associated with colder, more severe winters (Visbeck et al., 2001). The NAO index data were obtained from the National Center for Atmospheric Research (NCAR) (climatedataguide.ucar.edu). We used the NAO index to investigate whether winter waterfowl habitat selection was impacted by the relative severity of the winter weather. Wind speed variables were calculated from data downloaded from the NOAA National Climatic Data Center (http://www.ncdc.noaa.gov).\n\n\nResults\n\nA total of 23 waterfowl taxa were recorded and included in the analysis presented within this study (Table 2). The total waterfowl population for the Bay averaged approximately 20,000 individuals annually. Total count was lowest in 2006 (15,090 individuals) and highest in 2010 (26,503 individuals) (Figure 2). The 2010 peak in abundance was due to a spike in the number of Scaup spp. present in the Bay. This jump in Scaup spp. abundance corresponded to a reciprocal dip in the 2010 Shannon diversity index (Figure 2). Ranked abundances of individual species showed no significant trends of increase or decrease over the course of this study, except for slight increases in Bufflehead (Bucephala albeola) and Hooded Merganser (Lophodytes cucullatus) (Figure 3).\n\nMean is the average abundance for each species throughout the entire study area. Proportion represents the fraction that each species contributes to the total for duration of the study. *Species making up less than 1% of the community, and considered rare. Trend analysis (Figure 3) were not be conducted on these rare species.\n\nOur final NMDS fit had four dimensions and a stress value of 0.1449 (Figure 4A), and is well within the acceptable stress limits of NMDS (Clarke, 1993; Kruskal & Wish, 1978). Sites in the NMDS plot that are closer in ordination space are more similar in species composition. As the distance between points increases, the species composition becomes more dissimilar. Our final presentation of the NMDS rotated the data so that greatest distance between site scores are plotted on NMDS axis 1. There is some spatial clustering of survey sections across the first two NMDS axes. Sections 1 and 6 are concentrated on the left side of the NMDS axis 1, while sections 2 and 3 are predominantly on the right side. Sections 4 and 5 are located approximately in the middle, as they are in actual Bay position.\n\nThe circles illustrate the location of a single site for each surveyed year. The sites are color-coded by section. Figure 4A: Species locations are illustrated with four-letter abbreviation. Figure 4B: Bi-plot of NMDS axis one and two with vectors of significant environmental variables overlaid. The arrow’s direction illustrates the environmental gradient and the length is proportional to the correlation strength between the variable and the NMDS (See Table 3).\n\nSpecies location in the ordination space can approximate how species sort into habitat types along the spatial gradient in the Bay (Figure 4a). The upper left of the ordination space reflects predominantly open ocean, deep-water sites (sections 1 and 6). In line with the habitat location in ordination space, indicator species of deeper water oceanic habitat (e.g. Harlequin Duck (Histrionicus histrionicus) and Common Eider (Somateria mollissima)) are also positioned in the upper left hand corner of the ordination. Northern sites in the Bay are more shallow and marsh-like (sections 2 and 3), with species indicative of this habitat type (e.g. Atlantic Brant (Branta bernicla) and Canada Goose (Branta canadensis)).\n\nAfter the removal of correlated variables, ten ancillary variables were fit to the NMDS results (Table 3). Only latitude, mean bathymetry, area, and the average wind speed three days prior to the survey were significantly correlated to the NMDS (Figure 4B). Wind speed the day of the survey and day before were slightly less significant. Although not significant, the NAO index for November, December, and winter average showed strong relationships with NMDS axis 2.\n\nSignificance codes: “***” < 0.001, “**” <0.01, “*” <0.05\n\nMost sections’ locations in the ordination space did not change drastically between years; they remained in the same relative location of the ordination (Figure 5). The relative stability of sections in ordination space among years indicates that communities had consistent species composition between years. Section 2 shifted the most among years, especially between 2007 and 2008 (Figure 5). The relatively large shifts of section 2 may be a characteristic of this particular section; it is thought that waterfowl communities in this part of the Bay vary greatly according to the intensity of winter and amount of ice. However, large shifts among years or over the course of several years may be an indicator of environmental change.\n\nIllustrates where the section exists in the overall ordination space and how that position changes through the study period.\n\n\nDiscussion\n\nOur community level analysis allowed us to establish the expected spatial and temporal structure of the Bay’s winter waterfowl communities which can be used to monitor future environmental changes. Spatially, the NMDS analysis formalized several aspects of the Bay’s waterfowl community that were previously understood only anecdotally, and provided a clear depiction of the community spatial structure across the Bay. NMDS as a monitoring tool in conjunction with the baseline conditions identified in this study could be particularly useful in identifying any future changes in waterfowl community structure in this region and change in the Bay’s overall health.\n\nIn the upper portions of the Bay (sections 2 and 3), waterfowl habitat is characterized by shallow, salt-marsh dominated coves and sheltered coves and shorelines with ample freshwater inputs. These sites mapped predominantly on the right-side of the NMDS (Figure 4). Dabbling duck species such as Mallard (Anas platyrhynchos) and American Black Duck (Anas rubripes) use salt-marsh sites at high tide for cover, protection from predators, and feeding, and as sites for roosting at low tide (Bellrose, 1980). Several smaller diving ducks, including benthic-feeding Bufflehead and piscivorous Hooded Merganser use sheltered coves and shorelines for feeding during the day (McKinney, 2004). Canada Goose and Mute Swan (Cygnus olor) also utilize these sites as they presumably provide ready access to submerged aquatic vegetation on which these species feed (Mowbray, 2002). An urban center (city of Providence) and several expansive suburban cities are located in close proximity to the upper Bay sites, and it is in this region of the Bay where urban development would be expected to most influence waterfowl distribution.\n\nThe middle portion of the Bay is characterized by an increase in deeper, open water habitats, which continue towards the Bay mouth where they are supplemented by rocky shoreline habitats. These sites concentrated on the left-side of the NMDS (Figure 4, color-coded red and yellow). Open-water species such as Red-breasted Merganser (Mergus serrator), Common Goldeneye (Bucephal clangula), Scoter species (Melanitta spp.), and Common Eider use this region of the Bay. These species have larger body sizes that enable them to dive in deeper water to take advantage of prey not found in shallow water areas (e.g., Blue Mussel, Mytilus edulis), and of benthic prey such as crabs that migrate to deeper water during winter (Ehrlich et al., 1988). Harlequin Duck uses rocky shoreline habitats found near the mouth of the Bay that reflect their northern rocky stream breeding sites and where they can feed on benthic invertebrates such as amphipods (Robertson, 1999). The lower Bay sites are currently less impacted by adjacent urbanization effects as shorelines tend to be more sparsely populated. However, knowledge of baseline conditions could lend insight into interpreting data from future monitoring and aid in identifying any impacts of increased urbanization, or changes in waterfowl community structure resulting from displacement of upper Bay species.\n\nIn addition to species’ life history strategies, environmental factors can potentially influence waterfowl distribution in estuaries. In our study, wind speed, averaged over the three day period before sampling, was the only significant dynamic variable included in the final NMDS. This too reinforces what was communicated anecdotally about the movement of waterfowl through the Bay. Large groups of birds will shift their location in the Bay during prolonged high wind events, such as nor’easters. Typically before and during large storms, birds will relocate to the leeward side of islands or into wind protected coves. Due to global climate change, nor’easter frequency and intensity have increased on a global scale (Yohe & Hope, 2013). It is assumed that Narragansett Bay will be impacted by this increase in predicted probability of severe winter events. Our study suggests waterfowl respond to short-term changes in wind speed, and it will be of interest to monitor how waterfowl respond to any increase in both intensity and frequency of extreme wind events.\n\nWe observed a small, yet significant, increase in the ranked abundance of Hooded Merganser, and an even more apparent upward trend of the Bufflehead population. The Bufflehead trend may be the effect of increased level of protection for this species. In the early 20th century, Bufflehead populations were in decline due to over harvest and in response received increased protection through such means as reduced bag limits (Gauthier, 1993). In addition, Bufflehead nesting boxes were installed to compensate for loss of breading habitat (Erskine, 1960; Owen & Black, 1990). Population numbers have been growing steadily since, and perhaps this rebound could explain the increasing trend we saw in Narragansett Bay. Since we used ranked abundance, we hypothesize that the elevated conservation status of Bufflehead and subsequent population growth is providing the Bufflehead a competitive advantage in the Bay.\n\nAs we move forward with our monitoring, the NMDS approach can provide a useful means to compare future survey data with baseline conditions established during the first ten years of the survey. The NMDS provides a statistical framework to analyze monitoring data at the site and section level, but in context of the entire Bay (Faith et al., 1987). Due to random noise, we expect the position of the site or the average position of the section (as in Figure 5) to undergo relatively small changes in ordination space from year to year. In contrast, locations that jump from one year to the next, or that display a trajectory through NMDS space through time may indicate environmental forcing that is more than random.\n\nAlthough the NMDS shows promise as an effective statistical framework, there are obvious limitations to our approach. First and foremost, annual surveys provide discrete snapshots of the Bay’s waterfowl community through time. We survey one day a year, and only in the winter when these species are present. We do not have a quantitative estimate of the amount of variability data arising from either short-term (i.e., daily) or longer-term (weekly or monthly) movements of individuals among sites, or into and out of our study area. Because of this, our ability to detect change in community composition may require data collection over an extended time period. Yet despite these shortcomings, the ease of data collection and robustness of the NMDS method make it a viable long-term monitoring option.\n\nIn this study we proposed an approach to analyzing long-term waterfowl monitoring data in order to establish baseline conditions against which future trends in community composition and habitat utilization can be compared. This approach provides a quantitative yet visual means to represent baseline community structure and observed patterns of waterfowl distribution, and provides an easily interpreted series of templates against which future observations or patterns of change can be evaluated. Our approach was developed for waterfowl Narragansett Bay, but can be applied to other estuaries and potentially other species, although environmental factors in the model may need to be modified to reflect those relevant to the species investigated. Overall, our approach will help facilitate the use of waterfowl populations, as well as other relevant species, to monitor the environmental health of a large bay.\n\n\nData availability\n\nF1000Research: Dataset 1. Environmental data, 10.5256/f1000research.6080.d42953 (Kreakie et al., 2014a)\n\nF1000Research: Dataset 2. Winter waterfowl survey, 10.5256/f1000research.6080.d42954 (Kreakie et al., 2014b)",
"appendix": "Author contributions\n\n\n\nRM designed, implemented, and supervised all waterfowl surveys. Additionally, RM curates all survey data. BK conducted coding and analysis, and prepared the manuscript for publication. KW has participated in waterfowl surveys. All authors were involved in developing conclusions and revising the manuscript. All authors 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\nAcknowledgements\n\nWe would like to thank the myriad of biologists and environmental scientists who participated in the Narragansett Bay Winter Waterfowl Survey during the time of the study especially Peter Paton, Scott McWilliams, Jay Osenkowski, Kenny Raposa, Tim Gleason, Jay Winiarski, Kathy Vigness-Raposa, Bob Kenney, Walter Berry, Jonathan Serbst, Denise Champlin, Barbara Sherman, Wayne Munns, Ed Dettman, Carol Trocki, Heather Hopkins, and Cathy Wigand for special assistance in collecting waterfowl survey data. Additionally, we would like to thank Dwaine Kreakie, Anne Kuhn, Nathan Smucker, and Bryan Milstead for their helpful comments during the development of this manuscript. This paper has not been subjected to Agency review. Therefore, it does not necessary reflect the views of the Agency. This is contribution number ORD-006396 of the Atlantic Ecology Division, Office of Research and Development, National Health and Environmental Effects Research Laboratory.\n\n\nReferences\n\nAmat JA, Green AJ: Waterbirds as Bioindicators of Environmental Conditions. Conservation Monitoring in Freshwater Habitats. 2010; 45–52. Publisher Full Text\n\nAssessment ME: Millennium ecosystem assessment. Ecosystems. 2003.\n\nAssessment ME: Ecosystems and human well-being: Synthesis. Island Press Washington, DC, 2005. Reference Source\n\nAustin JE, Buhl TK, Guntenspergen GR, et al.: Duck populations as indicators of landscape condition in the prairie pothole region. Environ Monit Assess. 2001; 69(1): 29–47. PubMed Abstract | Publisher Full Text\n\nAustin M: On non-linear species response models in ordination. Vegetatio. 1976; 33(1): 33–41. 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}
|
[
{
"id": "7881",
"date": "30 Mar 2015",
"name": "L. Ignacio Vilchis",
"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\nGeneral CommentsThis study provides an excellent contribution to our knowledge of baseline species diversity and abundance for the waterfowl community in Narragansett Bay, Rhode Island. The authors analyze longitudinal survey data from the Narragansett Bay Winter Waterfowl Survey (NBWWS) in order to detect shifts in the species composition of the water waterfowl community and to explore if environmental drivers might be driving any change. On a regional scale the study covers almost the entire bay, and considering the general paucity of such monitoring programs, this study is important in that it documents baseline knowledge of the species diversity and abundance of the areas waterfowl and seabird species. Specific CommentsIntroduction:The main purpose of the introduction was to argue the need for baseline monitoring data for waterfowl communities. The authors do achieve this objective in first and fourth paragraphs, leading to the purpose of study in the last sentence of the fifth paragraph of the introduction. However, I did not find all the information in the introduction leading directly to the statement of purpose of the study, with this statement no matching what was stated in the abstract. The second and third paragraphs for example, include information that would be better suited for the methods. Also, while the authors do a great job in explaining why waterfowl are logical candidates for monitoring environmental health, I found the excessive references of the citations in the fourth paragraph a bit distracting. I am not certain of the editorial guidelines of F1000Research (I looked these up but failed to find the citation/reference guidelines), but I think a generally accepted rule of thumb is to not list more than three references per citation (although I could very well be wrong here). Nevertheless, when excessive references and citations are used it distracts the reader from the written message and disrupts the flow of the paragraph. I understand that you have to cite certain statements, but I would advice the authors to go through their citations and only cite essential sources. Doing so along with moving the information from the second and third paragraphs to the methods would help streamline the introduction. Methods:A general comment on the survey technique: As written, the survey methodology explains that complete counts are made in each of the areas corresponding to the site locations. It is not clear however, how observers would delineate the off shore boundaries of the survey areas, particularly those facing each other or off shore boundaries in general. How were these distances discerned? I am curious to how the observers gauged the distance from shore so that birds in the survey area from the opposite shoreline were not recounted. Did they use range finders? Adding some text explaining this would benefit the methods. I downloaded the data that is made available with this study via the F1000Research website and found that in the data set describing the winter waterfowl survey (10.5256/f1000research.6080.d42954) the column corresponding to buffleheads is missing. Was a different dataset used for this study? If not, why are buffleheads not included in the dataset but in the result of the study? First paragraph: In the fourth sentence the authors refer to “This area”. I would suggest clearing up what 'area' is being referred to. Also, in the fifth sentence the authors state that the southern portion of the bay is deeper. However, they did not mention deeper ‘than what’. Typically the reader expects to see the full comparison. I know these are nit picky grammar and style comments, but clearing these two minor points would make the paragraph read much better. Fourth paragraph: In the first sentence the authors start by saying “Counting is completed” when the rest of the verbs in the paper are in the past tense. I think changing this to ‘counting was completed’ would be better. In the second sentence, the authors explain: “Every bird seen on the water surface or on the adjoining shoreline up to 50 m from the water is counted; when possible sex and age were also noted”. Is this vertically or horizontally from the water? I am assuming that they mean 50 meters from the waters edge or shoreline – or along the coast. Please state this more clearly. Seventh paragraph: Please explain why the data was transformed and why the Wisconsin double transformation was chosen. Because non-metric multidimensional scaling uses rank orders, it is not clear why the transformation was used. Stating this more clearly in the text would benefit the paper. Eighth paragraph. In the last sentence, I would suggest using ‘>’ instead of ‘greater than’. Results:It is not obvious to the reader why the data presented in figures 1 & 2 show survey data up to 2012, while the ordination only used data up to 2011. Stating this reason more clearly in the methods would benefit the paper. The legend of Table 2 is not clear. Changing the wording could help clear up the message of the legend. For example, the last two sentences state: “*Species making up less than 1% of the community, and considered rare. Trend analysis (Figure 3) were not be conducted on these rare species.” The tense is wrong here, please change analysis to analyses. Also I would suggest saying this a bit clearer using fewer words, with perhaps something like this? “Species with overall species compositions < 1% were considered rare and excluded from trend analysis”. On this note, if I understand correctly, the legend states that trend analyses were not conducted on rare species and then refers to Figure 3. Figure 3 however shows trends in rank abundance of all 23 species listed in Table 2. This is confusing. Clearing this up in the legend or text would benefit the paper. Regarding the non-metric multidimensional scaling (NMDS), why were four dimensions chosen over two or three? Did solutions with fewer dimensions have too large stress value? Was this assessed with a scree plot? Stating these details would benefit the paper and help the reader understand why four dimensions were the best choice for the NMDS analysis. Furthermore, in Figure 4, where the results of the NMDS are first shown, it is not clear why are king eiders and long-tailed ducks not shown in the plot. I am assuming from table two that this is because these two species have proportions of the total waterfowl community abundance that are close to zero. Using this logic however, barrow’s goldeneyes should also be excluded from the plot—but they are not. As the NMDS analysis is the central component of the study, the authors should either include all 23 species or clearly state why only 20 were used. As is, the results are somewhat confusing and distract the reader from the overall message of the study. The authors use Figure 5 to detect shifts in the waterfowl community structure of Narragansett Bay during the 8-yr period of the study (2004 and 2011). It is confusing why the NMDS plots in Figure 5 are different to those in Figure 4. I might be missing something, but should not the ordination plots in Figure 5 have the same points and axes as in Figure 4? Are these plots showing the other two dimensions of the ordination? The details of Figure 5 should be made more elaborate explanation in the text. I am assuming the plots in Figure 5 show the average ordination positions for each section-year combination, with these being color coded for each year. Instead of this exercise, I would suggest authors use analysis of similarity (ANOSIM) to tests hypotheses of community change in each of the sections or the entire bay throughout the 8-yr study period. In this way the authors could obtain an objective measure indicating if there has been community change (or no change) in the waterfowl of Narragansett Bay during the 8-yr study period. Using ANOSIM here would be analogous to using ANOVAs to assess before and after changes at certain breaks in the 8-yr study period. ANOSIM however, uses the same similarity matrices imputed into NMDS and is philosophically allied with NMDS ordination. The functions need to perform this analysis are included in the R vegan package, which the authors have shown to have an excellent handle on using. Including ANOSIM in the study to objectively assess temporal changes in the beta diversity of the Narragansett Bay waterfowl community would therefore fit in nicely in the study as as it complements NMDS. In the last paragraph of the results the authors state: “it is thought that waterfowl communities in this part of the Bay vary greatly according to the intensity of winter and amount of ice”. This statement should have a citation referring to the reference thinking or hypothesizing this mechanism. Discussion:First paragraph. In the first sentence, I would suggest replacing “expected” for “baseline”. In the third sentence the authors refer to NMDS as a monitoring tool. NMDS is an ordination analysis used to interpret ecological data, and not a monitoring tool. I think this statement could be reworded better saying for example: “Using NMDS to analyzing monitoring data is a valuable approach to assess…” Sixth paragraph: Again, NMDS is not an approach—it is an analytical tool. Using this ordination technique is the approach the authors took to assess the community structure of the Narragansett Bay waterfowl community. Addressing and fixing this accordingly, will benefit the flow of the text. The second objective of the of the study was to explore if environmental drivers that might be driving changes in the species composition of the waterfowl species composition. I think this objective needs to be addressed a bit more in the discussion. It is only touched upon lightly. Furthermore, what do the authors think is the reason why they found strong correlations of the average wind speed for the three days prior to survey, area, mean bathymetry of site and latitude with the ordination, and yet the wintering waterfowl species composition of the bay seemed to remain stable through time.",
"responses": [
{
"c_id": "1669",
"date": "18 Dec 2015",
"name": "Betty Kreakie",
"role": "Author Response",
"response": "We greatly appreciate your feedback on our manuscript. Your thorough and thoughtful review has allowed us to significantly improve the manuscript. Below are point-by-point responses to your comments. Your concerns will be reflected in version 3 of the manuscript. Thank you again for all your help. Please let us know if you have any additional concerns. 1. The main purpose of the introduction was to argue the need for baseline monitoring data for waterfowl communities. The authors do achieve this objective in first and fourth paragraphs, leading to the purpose of study in the last sentence of the fifth paragraph of the introduction. However, I did not find all the information in the introduction leading directly to the statement of purpose of the study, with this statement no matching what was stated in the abstract. The second and third paragraphs for example, include information that would be better suited for the methods. Response: This concern was also expressed by the first reviewer of the manuscript. For version 2, we added considerable text to the introduction about how this method could have broader application. In the first version, you are correct in suggesting this text should be moved to the Methods section. However, the second version expands the statement of purpose. Hopefully these edits will address your concern. 2. Also, while the authors do a great job in explaining why waterfowl are logical candidates for monitoring environmental health, I found the excessive references of the citations in the fourth paragraph a bit distracting. I am not certain of the editorial guidelines of F1000Research (I looked these up but failed to find the citation/reference guidelines), but I think a generally accepted rule of thumb is to not list more than three references per citation (although I could very well be wrong here). Nevertheless, when excessive references and citations are used it distracts the reader from the written message and disrupts the flow of the paragraph. I understand that you have to cite certain statements, but I would advice the authors to go through their citations and only cite essential sources. Doing so along with moving the information from the second and third paragraphs to the methods would help streamline the introduction.Response: We completely agree that the amount of references were distracting. We have selectively reduced the number of citations in this part. Specifically, we removed several of the older citations. Most of the material from the older works was more comprehensively covered in the more recent work. 3. As written, the survey methodology explains that complete counts are made in each of the areas corresponding to the site locations. It is not clear however, how observers would delineate the off shore boundaries of the survey areas, particularly those facing each other or off shore boundaries in general. How were these distances discerned? I am curious to how the observers gauged the distance from shore so that birds in the survey area from the opposite shoreline were not recounted. Did they use range finders? Adding some text explaining this would benefit the methods.Response: To clarify this issue, we added text the first paragraph of the Survey Method subsection of the Methods.4. I downloaded the data that is made available with this study via the F1000Research website and found that in the data set describing the winter waterfowl survey (10.5256/f1000research.6080.d42954) the column corresponding to buffleheads is missing. Was a different dataset used for this study? If not, why are buffleheads not included in the dataset but in the result of the study?Response: The wrong data set did get uploaded to the F1000Research page. Thank you for pointing out this mistake. The correct data are now available. 5. First paragraph: In the fourth sentence the authors refer to “This area”. I would suggest clearing up what 'area' is being referred to.Response: We have changed to this test to “The land surrounding the northern portion of Narragansett Bay.”6. Also, in the fifth sentence the authors state that the southern portion of the bay isdeeper. However, they did not mention deeper ‘than what’. Typically the reader expects to see the full comparison. I know these are nit picky grammar and style comments, but clearing these two minor points would make the paragraph read much better.Response: We have added ”than the portion near Providence, Rhode Island.” Also, we completely agree that this paragraph now reads much better. 7. In the first sentence the authors start by saying “Counting is completed” when the rest of the verbs in the paper are in the past tense. I think changing this to ‘counting was completed’ would be better.Response: This error has been corrected.8. In the second sentence, the authors explain: “Every bird seen on the water surface or on the adjoining shoreline up to 50 m from the water is counted; when possible sex and age were also noted”. Is this vertically or horizontally from the water? I am assuming that they mean 50 meters from the waters edge or shoreline – or along the coast. Please state this more clearly. Response: We have incorporated this recommendation into the text. 9. Please explain why the data was transformed and why the Wisconsin double transformation was chosen. Because non-metric multidimensional scaling uses rank orders, it is not clear why the transformation was used. Stating this more clearly in the text would benefit the paper.Response: We have added text to clarify why this transformation was performed. 10. In the last sentence, I would suggest using ‘>’ instead of ‘greater than’. Response: This change was added to the text.11. It is not obvious to the reader why the data presented in figures 1 & 2 show survey data up to 2012, while the ordination only used data up to 2011. Stating this reason more clearly in the methods would benefit the paper.Response: At the time of the analysis, all the environmental variables were not available through 2012. The waterfowl survey for 2012 was available and had gone through our QA/QC process. We decided to include another year into the population, since we were specifically looking for significant trends. We have added clarification to the text about this issue. 12. The legend of Table 2 is not clear. Changing the wording could help clear up the message of the legend. For example, the last two sentences state: “*Species making up less than 1% of the community, and considered rare. Trend analysis (Figure 3) were not be conducted on these rare species.” The tense is wrong here, please change analysis to analyses. Also I would suggest saying this a bit clearer using fewer words, with perhaps something like this? “Species with overall species compositions < 1% were considered rare and excluded from trend analysis”. On this note, if I understand correctly, the legend states that trend analyses were not conducted on rare species and then refers to Figure 3. Figure 3 however shows trends in rank abundance of all 23 species listed in Table 2. This is confusing. Clearing this up in the legend or text would benefit the paper.Response: We apologize for the confusion. These was a change in approach that obviously did not get corrected in all parts of the final manuscript. Previous reviewers request that all species be presented in Figure 3 and therefor that figure was edited accordingly. The manuscript has been corrected to reflect the presented data. 13. Regarding the non-metric multidimensional scaling (NMDS), why were four dimensions chosen over two or three? Did solutions with fewer dimensions have too large stress value? Was this assessed with a scree plot? Stating these details would benefit the paper and help the reader understand why four dimensions were the best choice for the NMDS analysis. Response: We explained our process for selecting the number of dimensions in the Methods (third paragraph of the Data Analysis subsection). In addition, we added text to the Results (second paragraph). 14. Furthermore, in Figure 4, where the results of the NMDS are first shown, it is not clear why are king eiders and long-tailed ducks not shown in the plot. I am assuming from table two that this is because these two species have proportions of the total waterfowl community abundance that are close to zero. Using this logic however, barrow’s goldeneyes should also be excluded from the plot—but they are not. As the NMDS analysis is the central component of the study, the authors should either include all 23 species or clearly state why only 20 were used. As is, the results are somewhat confusing and distract the reader from the overall message of the study.Response: All 23 species were indeed used to construct the plot. Both long-tailed duck and king eider plot very low on the y-axis. Since this is a very complicated plot, we decided to zoom in slightly to have a better view of the main cluster. This also allowed us to make our labels more legible. However, we failed to explain this in text. We have added text to the figure capture to explain this decision and to report values for long-tailed duck and king eider. 15. The authors use Figure 5 to detect shifts in the waterfowl community structure of Narragansett Bay during the 8-yr period of the study (2004 and 2011). It is confusing why the NMDS plots in Figure 5 are different to those in Figure 4. I might be missing something, but should not the ordination plots in Figure 5 have the same points and axes as in Figure 4? Are these plots showing the other two dimensions of the ordination? The details of Figure 5 should be made more elaborate explanation in the text.Response: The subplots in figure 5 are the same plots as in figure 4. For figure 5 we greyed out the site locations, and added the average section location with a square symbol. However, as you pointed out, this is not intuitively clear. We added text in the figure caption to clarify this issue. 16. I am assuming the plots in Figure 5 show the average ordination positions for each section-year combination, with these being color coded for each year. Instead of this exercise, I would suggest authors use analysis of similarity (ANOSIM) to tests hypotheses of community change in each of the sections or the entire bay throughout the 8-yr study period. In this way the authors could obtain an objective measure indicating if there has been community change (or no change) in the waterfowl of Narragansett Bay during the 8-yr study period. Using ANOSIM here would be analogous to using ANOVAs to assess before and after changes at certain breaks in the 8-yr study period. ANOSIM however, uses the same similarity matrices imputed into NMDS and is philosophically allied with NMDS ordination. The functions need to perform this analysis are included in the R vegan package, which the authors have shown to have an excellent handle on using. Including ANOSIM in the study to objectively assess temporal changes in the beta diversity of the Narragansett Bay waterfowl community would therefore fit in nicely in the study as it complements NMDS.Response: We agree that ANOSIM would tell us if the groups are different and different by year. In figure 5 we are trying to show how the sections change through the years in context to the of the entire Bay waterfowl community. Hopefully, the additional text will clarify our intent to the audience. Additional, we added considerable text to the discussion in version 2 that adds to the interpretation of this figure. 17. In the last paragraph of the results the authors state: “it is thought that waterfowl communities in this part of the Bay vary greatly according to the intensity of winter and amount of ice”. This statement should have a citation referring to the reference thinking or hypothesizing this mechanism. Response: This material was moved to the Discussion in version 2. We have added a citationto this state. 18. In the first sentence, I would suggest replacing “expected” for “baseline”.Response: This concern was corrected in version 2.19. In the third sentence the authors refer to NMDS as a monitoring tool. NMDS is an ordination analysis used to interpret ecological data, and not a monitoring tool. I think this statement could be reworded better saying for example: “Using NMDS to analyzing monitoring data is a valuable approach to assess…”Response: In version 2, we added text to clarify how we feel that this method could have broader appeal. However, we do agree that the NMDS is an ordination analysis. We have edited the text to address this concern. 20. Again, NMDS is not an approach—it is an analytical tool. Using this ordination technique is the approach the authors took to assess the community structure of the Narragansett Bay waterfowl community. Addressing and fixing this accordingly, will benefit the flow of the text.Response: See above for explanation about how this issue was corrected. 21. The second objective of the study was to explore if environmental drivers that might be driving changes in the species composition of the waterfowl species composition. I think this objective needs to be addressed a bit more in the discussion. It is only touched upon lightly.Response: The first reviewer of the manuscript agreed with your concern. Therefore we add material to the Discussion in the second version to further explore our interpretation on the environmental drivers.22. Furthermore, what do the authors think is the reason why they found strong correlations of the average wind speed for the three days prior to survey, area, mean bathymetry of site and latitude with the ordination, and yet the wintering waterfowlspecies composition of the bay seemed to remain stable through time.Response: As mentioned above, we did add text to Discussion about these issues."
}
]
},
{
"id": "8808",
"date": "09 Jul 2015",
"name": "Nigel Yoccoz",
"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\nGeneral commentsEnvironmental monitoring means different things to different people. It always involves a long-term commitment – that is, a given system is followed up over time, years to decades – but how much this is driven by hypotheses about what drive the changes of the system, and/or about the consequences of these changes, vary. One could use one of the most famous examples of environmental monitoring – the monitoring of CO2 concentrations initiated by Charles David Keeling in 1958 at Hawaii’s Mauna Loa observatory. Keeling was expecting a rise as a consequence of the combustion of fossil fuels, and there were different hypotheses regarding the rate of increase depending on in particular exchange between atmosphere and ocean (see e.g., Revelle and Suess 1957). Keeling concluded for example in 1960 “At the South Pole the observed rate of increase is nearly that to be expected from the combustion of fossil fuel (1.4 p.p.m.), if no removal from the atmosphere takes place” (p. 203; Keeling used measurements from both hemispheres, not just Mauna Loa). This was therefore a strongly hypothesis-driven monitoring – but that did not exclude surprises, in particular the large seasonal variations due to photosynthetic activity. But such surprises often came because there are rather precise expectations about possible changes (see Lindenmayer et al. 2010 for a related discussion). Many environmental monitoring programs, however, do not have such underlying hypotheses, and are often characterized as being more of the surveillance type (see Nichols and Williams 2006). The main goal is to identify changes – other than due to the stochastic variability of the system. This paper falls in this category – the main purpose is to describe waterfowl communities in a given area, the Narragansett Bay, and identify possible changes (as e.g., an upward trend in two species, Bufflehead and Hooded Merganser). Variables that could drive these changes, and with a relevance for local or regional management, are not included in the analyses – environmental data used refer either to site variables which are unlikely to change (latitude/longitude, bathymetry, area) or that rather reflect short-term temporal variation (wind speed on the day of the survey or just prior to it, North Atlantic Oscillation – a measure of regional atmospheric patterns – in the month of the survey or the months prior to it). That in fact identifying the causes of observed changes in waterfowl communities will be hard without having more explicit hypotheses is made clear in some parts of the discussion. How can we know for example if an observed increase in a wintering duck population is due to 1) improved reproductive success on the breeding grounds, 2) lower hunting pressure in the fall, and 3) change in wintering areas (these hypotheses are not exclusive, so the problem is to assess their relative importance, as for CO2 concentrations: burning fuels, exchange atmosphere-ocean and photosynthetic activity all play a role). Monitoring environmental systems should, in my opinion, aim at characterizing the state of the system but also the drivers of changes (Yoccoz et al. 2001; Yoccoz 2012; see Lindenmayer et al 2015 for a thorough discussion). Doing this without formulating hypotheses (and if possible quantitative models) is difficult. Even if the paper focuses on developing a baseline for environmental monitoring, it also proposes “a statistical framework for future environmental monitoring”. I do not see how the proposed framework will allow for such a quantitative evaluation of hypotheses.Another concern is the reference to a baseline – using the 2004-2011 data. It is most likely that waterfowl communities during that period have already been heavily impacted by important environmental drivers, either positively (think about the increase of some goose populations) or negatively. Taking those years as a baseline might lead to what Pauly (1995) in the context of fisheries call the “syndrome of shifting baselines” (see Clavero 2014 for an example with non-native species). What we see as a baseline is already very different from historical conditions – irrespective of the reference time used to define “historical”. Monitoring requires we determine the state of the system, but calling it a baseline may be rather misleading without at least a comment.A final general comment relates to waterfowl as “logical candidates for monitoring environmental health” (p. 2) because they respond to “a wide array of environmental stressors [...] at the local up to the regional scale”. Clearly, what we monitor should reflect the changes we expect, but they should also allow for disentangling causes of change – for example if managers want to act on these causes. Knowing if for example causes are local or regional will determine where policy decisions should be made. Monitoring system variables, such as waterfowl communities, that are influenced by a large number of drivers at very different scales may make them too “integrative” for management (or scientific) purposes. Of course, it may be that there will be a main driver of change (think of DDT and birds of prey) but that seems to be unlikely in this case. I personally think we should aim at monitoring directly state variables and rely less on indicators, but I understand also the practical (and financial) difficulties. But if one relies upon indicators, or surrogates, at least simple conceptual models linking indicators and state variables would help. It would also make transparent assumptions regarding the stability of the links.To conclude, the authors have done a great job organizing and synthesizing a waterfowl survey over nearly 10 years. The nest step – “future environmental monitoring” – should go further and provides us with a conceptual model of expected changes in the waterfowl community as stressors change at different spatial scales, and if one has the necessary information to assess the relative impact of these stressors.Specific comments:NMDS is one among many multivariate methods that can be used for analyzing spatio-temporal community data (see Legendre and Gauthier 2014 for a recent review). These methods do not make assumptions about the distribution of the data (p.2), but they all make assumptions about what is a “good” measure of e.g. community similarity (for example one could consider the number of species the two communities share, and how many species that are unique to each community, and combine these numbers in different similarity indices). If one wants to make inference (for the number of axes to be interpreted, or relationships with environmental variables), most analyses rely upon permutation tests which do not rely upon distribution of the data but may be sensitive to dependence among samples. Particularly to identify relationships with environmental variables, alternative approaches to NMDS such as Canonical Correspondence Analysis, would have been interesting to use (ter Braak 1987, Legendre et al. 2011).p. 4, it is written that the choice of ancillary (environmental) variables was “hypothesis driven”. It would have helped to be more explicit about which component of the variation could be explained by these variables, and in what way it was related to the stressors described e.g. p.2. Clearly the first 10 variables (latitude to wetland area) are unlikely to change during the 8 years of the survey, so they would explain the spatial variation only. Wind speed the day of the survey might explain the day to day variation (but given there is no replicate survey within a year, it will be confounded with the yearly variation), whereas NAO.fall might reflect weather conditions during migration and early winter.Table 2, p. 5: for counts with skewed distributions, the standard deviation (SD) might be a poor descriptor of the variability (the mean might also be a poor descriptor of location). It might be useful to give the range and median in addition to mean and SD.Table 3: what is meant by “results”? Correlations? How are P-values calculated?p. 7: Bucephalap. 8: species’ life history strategies: use traits rather, you do not really investigate strategies here.",
"responses": []
},
{
"id": "7882",
"date": "27 Jul 2015",
"name": "José Manuel Arcos",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper describing a waterfowl winter community and its evolution through time. Authors use non-metric dimensional scaling to assess changes in community structure, taking into account environmental features (habitat, climatic, metheorological). The work seems adequately conducted and is based on a sound dataset that extends along 8 years. I find therefore the work worthy to be indexed. However, I have some concerns about how the work is presented. Overall, it has a too local focus and should be put in a wider context, with clear aims. I hope the following comments, by sections, will help authors to improve the current version. Abstract This section is particularly too local it should give a wider view of the work and its potential contribution beyond the study area. The authors propose a methodological approach that could be extended to other areas, to solve similar questions (i.e. how to assess changes in wintering waterfowl communities over time, in a scenario of climate change and increasing coastal development). The last paragraph of the discussion is written under this philosophy, and part of it could be used here. Of course it’s also important to depict the results, but they need to be better contextualized.Another issue to address is the excess of repetitions (the same problem is applicable to other sections of the manuscript). For instance, Narragansett Bay is quoted eight times in the abstract alone, and the analytical tool \"nonmetric multidimensional scaling\" is written in full three times (worth introducing here the acronym NMDS). Introduction Again I find the text too local. Only the first two sentences of paragraph 1, plus paragraph 4 attempt to put the study in a wider context. I suggest to join (and slightly extend) these wider issues at the beginning, as well as introduce afterwards the proposed methodological approach, and leave the local considerations for the end of the introduction (except for a final paragraph stating the aims of the paper, trying again to give a wider view and to describe clear objectives). The text in the current last paragraph related to the economic and environmental significance of Narrangansett Bay could be better suited (and slightly extended) in the discussion. On the other hand, some of the detailed information of the Bay could be moved to the Methods (study site/study method). As a last minor comment, it’d be appropriate to provide a reference for the statement that Waterfowl respond to these stressors at the local up to the regional scale and their responses can be apparent nearly immediately and continue after substantial time lags (4th paragraph). Methods Study site Remove the first sentence from this section (it describes the waterfowl survey, not the study site). Also consider, if necessary, to move some of the information provided in the introduction here, avoiding repetitions. Survey method Overall, try to be consistent with the verb forms. Most of this section is written in present tense, but some sentences use the past simple tense. In the 2nd paragraph of this section, quote grebes along with ducks, geese and swans (Horned Grebe is also considered). Also in this paragraph, I suggest rewriting the last sentence, to start saying that in most cases birds were identified to species level. For instance: “All birds were identified to the species level, except Lesser and Greater Scaups, that were simple categorized into a single Scaup (Aythya spp.) taxonomic group due to the difficulty of distinguishing them.” Data analysis Just as a comment, I find that there are too many pre-selected ancillary variables, particularly NAO-related variables. I understand that this has a minor effect, however, as most of them were removed from the final model. On this regard, it’d be worth explaining why a threshold of r = 0.8 was adopted.\n\nIn the third paragraph of this section, I’d suggest to change “for in-depth discussion of methods see (Faith et al., 1987; Minchin, 1987)” to “(for in-depth discussion of methods see Faith et al., 1987; Minchin, 1987)”. Also change “All analysis was conducted in R” to “All analyses were conducted in R” Results Table 2. Check wording. For instance, at the end of the table caption, change “were not be conducted” to “were not conducted”. I’d suggest changing the last column of the table to %, as this would be more visual for the reader. The last two sentences of the results’ section seem more appropriate for the discussion. This issue, on the other hand (stability of most sections/changes in section 2 over time) deserve some further attention in the discussion. Discussion Again I’d slightly change the structure, and try to avoid excessive description. I’d condensate the description of the waterfowl community site by site, trying to avoid references to conservation issues (e.g. implications of highly urbanized areas) here (see below). I feel a bit uneasy with the sentence “In addition to species’ life history strategies, environmental factors can potentially influence waterfowl distribution in estuaries”. I understand that some environmental factors (such as habitat features) are closely related to life history. So maybe change “environmental” to “climatic”/”meteorological”? I’d add a paragraph regarding conservation-related issues, including coastal-development issues (i.e. the higher pressure in the inner Bay), as well as other relevant local considerations, plus references to climate change. Regarding the increase of Hooded Merganser and Bufflehead, why only the latter is discussed? I miss an explanation for the increase of Hooded Merganser too. On the other hand, I miss some more precision regarding the dates of eventsw regarding Bufflehead (e.g. when –roughly- protection measures started to be in place?). And last regarding this issue, I don’t understand why increased protection to buffleheads provides them a competitive advantage in the Bay. They increase in relative numbers, and therefore their rank position changes, but is this an actual advantage in the wintering area? I appreciate the considerations on the limitations of the study, as described in the penultimate paragraph.",
"responses": [
{
"c_id": "1594",
"date": "24 Sep 2015",
"name": "Betty Kreakie",
"role": "Author Response",
"response": "Thank you so much for taking the time to give us such a thoughtful review. It is clear that your recommendations have greatly improved the text. Below we can included point-by-point responses to your review and have posted a new version based on your comments to F1000Research. Hopefully, this version of the manuscript will be met with your approval. Please let us know if you have any additional concerns or questions. This section is particularly too local it should give a wider view of the work and its potential contribution beyond the study area. The authors propose a methodological approach that could be extended to other areas, to solve similar questions (i.e. how to assess changes in wintering waterfowl communities over time, in a scenario of climate change and increasing coastal development). The last paragraph of the discussion is written under this philosophy, and part of it could be used here. Of course it’s also important to depict the results, but they need to be better contextualized.Response: We completely agree with this statement. n fact, early drafts of the manuscript were more focused on developing this method for wide use in environmental monitoring. However, previous reviewers and co-authors felt that we were overreaching and felt it was more interesting to just focus on this particular study area. To compromise, we have added back some text about the broad application of this method. Another issue to address is the excess of repetitions (the same problem is applicable to other sections of the manuscript). For instance, Narragansett Bay is quoted eight times in the abstract alone, and the analytical tool \"nonmetric multidimensional scaling\" is written in full three times (worth introducing here the acronym NMDS).Response: Thank you for pointing this out. We have rework the abstract (and the remaining text) to remove several of the redundancies and introduced the NMDS acronym to the abstract. Again I find the text too local. Only the first two sentences of paragraph 1, plus paragraph 4 attempt to put the study in a wider context. I suggest to join (and slightly extend) these wider issues at the beginning, as well as introduce afterwards the proposed methodological approach, and leave the local considerations for the end of the introduction (except for a final paragraph stating the aims of the paper, trying again to give a wider view and to describe clear objectives). The text in the current last paragraph related to the economic and environmental significance of Narragansett Bay could be better suited (and slightly extended) in the discussion. On the other hand, some of the detailed information of the Bay could be moved to the Methods (study site/study method).Response: We agree that the previous version of manuscript appears to be very local. As noted in the responses to comment #1, we have added and edited to manuscript to broaden the scope and present the application of the methods. Hopefully the reviewer will feel that we accomplished the goal of broaden the manuscripts scope. Yet coauthors and previous reviewer still feel that this research is in essence simple an analysis of bird communities in Narragansett Bay. We feel that their concern of overreaching is valid. Hopefully with these edits, we have found a balance. As a last minor comment, it’d be appropriate to provide a reference for the statement that Waterfowl respond to these stressors at the local up to the regional scale and their responses can be apparent nearly immediately and continue after substantial time lags (4th paragraph).Response: This error has been corrected. Remove the first sentence from this section (it describes the waterfowl survey, not the study site). Also consider, if necessary, to move some of the information provided in the introduction here, avoiding repetitions.Response: This edited has been included into the recent version. Due to convention, we have decided to leave some of material about the study area in this section. Overall, try to be consistent with the verb forms. Most of this section is written in present tense, but some sentences use the past simple tense.Response: We have reread this section and made edits as needed. However, this section does require the both the past and present tense. Since this survey is ongoing and yet the survey design was completed at the beginning of the study, we used verb tense to indicate which decision were made historically and which still are being used. In the 2nd paragraph of this section, quote grebes along with ducks, geese and swans (Horned Grebe is also considered). Also in this paragraph, I suggest rewriting the last sentence, to start saying that in most cases birds were identified to species level. For instance: “All birds were identified to the species level, except Lesser and Greater Scaups, that were simple categorized into a single Scaup (Aythya spp.) taxonomic group due to the difficulty of distinguishing them.”Response: Both of these suggestions have been incorporated. Just as a comment, I find that there are too many pre-selected ancillary variables, particularly NAO-related variables. I understand that this has a minor effect, however, as most of them were removed from the final model. On this regard, it’d be worth explaining why a threshold of r = 0.8 was adopted.Response: We agree that there were a lot of pre-selected ancillary variables. We knew that we wanted examine the correlations between climatic and other variables and our NMDS structure, but there was not enough literature on the subject to reduce those variables a priori. Since these variables were computational easy to derived, we decided to error on the side of caution. We also change the wording to thresholding to reflect more accurately how we selected variables. In the third paragraph of this section, I’d suggest to change “for in-depth discussion of methods see (Faith et al., 1987; Minchin, 1987)” to “(for in-depth discussion of methods see Faith et al., 1987; Minchin, 1987)”. Also change “All analysis was conducted in R” to “All analyses were conducted in R”Response: We have corrected both of these errors. Table 2. Check wording. For instance, at the end of the table caption, change “were not be conducted” to “were not conducted”. I’d suggest changing the last column of the table to %, as this would be more visual for the reader.Response: This error has been corrected and proportions have been converted to percentages. The last two sentences of the results’ section seem more appropriate for the discussion. This issue, on the other hand (stability of most sections/changes in section 2 over time) deserve some further attention in the discussion.Response: This comment had been completely incorporated into the text. We have deleted the last two sentences from results. This text has been added the discussion along with additional explanation. Again I’d slightly change the structure, and try to avoid excessive description. I’d condensate the description of the waterfowl community site by site, trying to avoid references to conservation issues (e.g. implications of highly urbanized areas) here (see below).Response: This concern has been addressed through the edits to other comments. I feel a bit uneasy with the sentence “In addition to species’ life history strategies, environmental factors can potentially influence waterfowl distribution in estuaries”. I understand that some environmental factors (such as habitat features) are closely related to life history. So maybe change “environmental” to “climatic”/”meteorological”?Response: We have incorporated this suggestion into the text. I’d add a paragraph regarding conservation-related issues, including coastal-development issues (i.e. the higher pressure in the inner Bay), as well as other relevant local considerations, plus references to climate change.Response: This concern has been addressed through the additional paragraph in the discussion. Regarding the increase of Hooded Merganser and Bufflehead, why only the latter is discussed? I miss an explanation for the increase of Hooded Merganser too. On the other hand, I miss some more precision regarding the dates of eventsw regarding Bufflehead (e.g. when –roughly- protection measures started to be in place?). And last regarding this issue, I don’t understand why increased protection to buffleheads provides them a competitive advantage in the Bay. They increase in relative numbers, and therefore their rank position changes, but is this an actual advantage in the wintering area?Response: This paragraph is only intended to speculative about possible reason behind these shifts. We did not explicitly test. It was an interest discovery and we did not want to simply ignore it. We have add some text to this paragraph to it clear that we simply presenting some thoughts about what might be behind this pattern. Clearly more research is needed on this."
},
{
"c_id": "1665",
"date": "23 Oct 2015",
"name": "Betty Kreakie",
"role": "Author Response",
"response": "I now realize that I should have waited to post the response after the new version was published. Version 2, which addresses your concerns, is now published. Sorry for any confusion that this may have caused."
}
]
}
] | 1
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https://f1000research.com/articles/4-40
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https://f1000research.com/articles/4-1469/v1
|
17 Dec 15
|
{
"type": "Review",
"title": "Recent Advances in Preventing Adverse Reactions to Transfusion",
"authors": [
"Thomas S Rogers",
"Mark K Fung",
"Sarah K Harm",
"Mark K Fung",
"Sarah K Harm"
],
"abstract": "The spectrum of adverse reactions to blood product transfusion ranges from a benign clinical course to serious morbidity and mortality. There have been many advances in technologies and transfusion strategies to decrease the risk of adverse reactions. Our aim is to address a few of the advancements in increasing the safety of the blood supply, specifically pathogen reduction technologies, bacterial contamination risk reduction, and transfusion associated acute lung injury risk mitigation strategies.",
"keywords": [
"Blood Transfusion",
"Transfusion Related Acute Lung Injury",
"TRALI",
"Transfusion Associated Circulatory Overload",
"TACO",
"Human leukocyte antigens",
"Human leukocyte antigen antibodies"
],
"content": "Introduction\n\nThe transfusion of blood products is never without risk. Complications such as transfusion-transmitted infectious diseases (TTIDs), antibody formation to red and white blood cells, preformed cytokines, and sudden increases in intravascular volume, to name a few, may result in severe health consequences to the transfusion recipient. A large focus of the transfusion medicine community has been to decrease the risk of TTIDs such as human immunodeficiency virus (HIV), hepatitis B, and hepatitis C (see Table 1) through extensive donor infectious disease testing, removal of monetary compensation to blood donors, and enhancement of the donor health history questionnaire1–4. Despite the successes in reducing the risk of TTIDs, there remain the risks of sepsis due to bacterial contamination, transmission of unknown pathogens, and numerous non-infectious complications, some of which have emerged as leading causes of fatalities due to blood product transfusion.\n\nCurrent testing of blood donors miss these viruses if the donors are in the “window period” when they are infected but do not yet test positive1–4.\n\nIn 2014, transfusion-related acute lung injury (TRALI) was the leading cause of death due to blood product transfusion in the United States, followed by transfusion-associated circulatory overload (TACO)5. Since 2010, 41% of transfusion-related fatalities were due to TRALI, 22% due to TACO, and microbial infection accounted for 8% (see Table 2). Unfortunately, there are no laboratory tests to prevent TACO in transfusion recipients. Thus, transfusion services are left with tools such as physician and patient education to recognize clinical signs and symptoms of TACO and help clinicians identify which patients might be most sensitive to sudden increases in intravascular volume. On the other hand, there are newer testing-based strategies to help prevent both TRALI and microbial infection, which once universally incorporated should further reduce the risk of these complications of transfusion.\n\nOther: FY2010 and FY2011: Graft vs. host disease\n\nFY2014: Hypotensive reaction\n\nAdditional non-infectious complications of transfusion, while not the leading causes of death, pose serious risks to recipients of blood products. Immunomodulation, nosocomial infection, and other consequences of biologic response modifiers (i.e. byproducts of the red blood cell and platelet storage lesion) may lead to transfusion-related morbidity and mortality6–8. Mitigation of these risks may include leukoreduction, byproduct removal by saline washing blood products, and/or using a restrictive transfusion strategy9–11. A recent meta-analysis showed that a restrictive transfusion strategy in patients with critical illness or bleeding, using a hemoglobin transfusion trigger of <7 g/dL, resulted in a significant reduction in cardiac events, rebleeding, bacterial infections, and total mortality when compared to a less restrictive (more liberal) strategy12. However, it is well known that certain patient populations (e.g. acute coronary syndrome) may require higher hemoglobin transfusion triggers13. In addition, recent randomized controlled clinical trials in critical care and cardiac surgery patients showed no difference in mortality when receiving fresh versus older red blood cell units14,15. Therefore, judicious use of blood products and avoidance of unnecessary transfusion in combination with leukoreduction (and saline washing when clinically indicated) provides the best defense against many of the non-infectious complications of transfusion.\n\nDespite the best efforts of the transfusion medicine community, infectious and non-infectious risks of transfusion remain a problem for transfusion recipients. Ongoing studies continue to discover the consequences of blood product storage, the impact biologic response modifiers have on patient outcomes, the optimal triggers for transfusion, and the detection of pathogens in the blood supply. As we cannot address each and every improvement the transfusion medicine community has made to make the blood supply as safe as possible, we will briefly describe some of the newer strategies adopted by blood centers and hospital transfusion services to help prevent adverse reactions to blood product transfusions. Specifically, we will discuss general pathogen reduction (PR) technologies, improvements that increase the sensitivity of screening for bacterial contamination in platelet products, and the newest TRALI risk mitigation strategies for plasma and platelet products.\n\n\nPathogen reduction technologies\n\nThe safety of blood product transfusion has increased greatly due to an extensive donor health history questionnaire and sophisticated donor infectious disease testing, yet the risk of pathogen-related complications in blood product recipients remains. PR technologies include using solvent and detergent, a psoralen compound, or riboflavin, the latter two combined with ultraviolet light, to render pathogens non-infectious (see Table 3)16. The goal of PR is zero risk from existing and emerging pathogens in blood products. New technologies reduce but unfortunately do not eliminate the risk of viruses and microbial infection, mentioned in both Table 1 and Table 2. For example, certain Klebsiella pneumonia and Bacillus cereus microbes are not 100% killed by PR17,18. PR technologies have been slow to market in the United States as opposed to Europe, where PR has been in use for over 10 years (see Table 3)16,19,20. While PR technology for platelets and plasma is in its infancy in the United States, the European experience has been positive, with only two transfusion-transmitted infections out of 1681 transfusion-related incidents reported in 201421. In addition, a report on over 50,000 PR plasma transfusions showed no significant difference in adverse events (mostly allergic in nature) compared to non-PR plasma22. PR for red blood cells is still in United States phase 2 and European phase 3 studies. Potential benefits of PR red blood cells include reducing the risk of current transfusion-transmitted infections to essentially zero, albeit with risks including alloimmunization and increased cost; however, the true rate of transfusion-transmitted infections has yet to be determined4.\n\nCE = Conformité Européenne\n\nFDA = Food and Drug Administration\n\nThere is evidence for the PR systems available for platelets and plasma that most pathogens are inactivated, except for some non-enveloped viruses and certain bacterial strains18,19. Thus, there remains a need to screen donors for pathogens that may not be inactivated by the PR method. Additional critiques of PR technologies include a sizable cost increase compared to untreated products, negative effects on platelet function and count, acute respiratory distress syndrome (ARDS), and the potentially associated increased risk of bleeding in transfusion recipients17,23. The cost increase of PR technologies has been shown to be offset by the ability to extend the storage time of platelet products and the decreased rate of transfusion-related sepsis24. While in vitro measures of adhesion and aggregation in PR platelets is comparable to untreated platelets, in vivo measures of post-transfusion corrected count increments and recovery are lower with PR platelets than untreated platelets25–27. Despite in vitro and in vivo study results, there remains equipoise over the clinical bleeding risk in patients who receive PR platelets compared to untreated platelets. For recipients of PR platelets, a Cochrane Review found no significant difference in clinically significant or severe bleeding, mortality, transfusion reactions, or adverse events (including sepsis and transfusion-transmitted infection) compared to recipients of untreated platelets27. However, recipients of PR platelets generally require more platelet transfusions, thus more donor exposure, due to poor post-transfusion increments.\n\nThe numerous PR technologies available reflect the complexity of finding a balance between effective PR and preserving acceptable quality and functionality of the blood components. In addition to the decreased risk of bacterial contamination and TTIDs, PR technologies provide additional benefits of prevention of transfusion-associated graft-versus-host disease (GVHD), prevention of cytomegalovirus (CMV) disease transmission, and possible reduction of alloimmunization due to inactivation of white blood cells that remain in the blood products. While PR technologies may be a very good defense against emerging TTIDs, there remain concerns over cost, toxicity, alloimmunization, and cellular function (i.e. bleeding risk). As with most new technologies, additional studies are needed to ensure PR blood products are as effective as untreated products in preventing bleeding and adverse events in transfusion recipients. Current PR technologies are a step in the right direction; however, there remains a need to develop safer and better technologies that kill all pathogens.\n\n\nBacterial contamination risk reduction\n\nTransfusion-related microbial infections range in severity from a mild, transient temperature increase to acute lung injury, fulminant septic shock, and death. While there was only one fatality due to sepsis reported to the Food and Drug Administration (FDA) in fiscal year 2014, clinical sepsis is reported after 1 in every 100,000 platelet transfusions5,16. Blood collection centers have implemented a combination of techniques to reduce the risk of microbial contamination in the final blood product, including improved disinfection methods for the venipuncture site, introduction of the diversion pouch during the blood collection procedure, and automated bacterial culture of platelet products, as well as platelet additive solutions and PR systems. Newer developments in bacterial detection (i.e. nucleic acid testing and bio-chip technology) may be on the horizon but unfortunately have not been widely adopted or approved for use throughout the world. At the receiving end, hospital transfusion services have the ability to perform either culture or rapid, non-culture-based bacterial screening tests for platelet products that are at high risk for bacterial contamination at the time of issue. While the interventions described attempt to reduce the risk of fatal bacterial infection in Table 2, they do not provide protection from viral, protozoan, or other pathogens.\n\nThe leading sources of blood product contamination are skin bacteria from the venipuncture site during the blood collection procedure. Platelet products are especially susceptible to bacterial growth due to room temperature storage for up to 5 days. After disinfection with povidone iodine or isopropyl alcohol plus iodine tincture, only half of all donors will have remaining bacterial colonies when the venipuncture site is cultured1. Unfortunately, bacteria in the deeper skin layers cannot be removed by simple skin disinfection, thus a diversion pouch is required in any collection system intended for preparation of a platelet product28. The diversion pouch is integrally attached to the blood product collection set and collects the first few milliliters of whole blood, including any potential skin plug within the access needle, thus diverting the contaminating skin bacteria away from the final blood product container. The combination of skin disinfection with iodine-containing solutions and use of the diversion pouch effectively decreases the risk of bacterial contamination in platelet products29.\n\nUnfortunately, there remains a residual risk of bacterial contamination in platelet products despite the improved arm preparation techniques and use of the diversion pouch. Since 2004, the AABB Standards require testing of 100% of platelet products for bacterial contamination28. For apheresis platelets, aliquots from the platelet product are generally drawn 18–24 hours after collection and used to inoculate aerobic cultures using one of the two bacterial detection systems cleared by the FDA for quality control testing of platelets. However, culture data and clinical reports show that even bacterial culture, the gold standard for bacterial detection, can fail to detect all contaminated platelet units30,31. For whole-blood-derived platelet products, an alternative to culture is the use of an FDA-cleared rapid bacterial detection test at the time of product issue. Unfortunately, these tests carry a high false-positive rate and result in the unnecessary discard of platelet products32. However, the rapid bacterial detection tests have been successfully used to allow for the extension of apheresis platelet shelf life in times of medical necessity and help ensure that platelet products, especially day 4 and 5, are free of clinically significant levels of bacterial contamination33. Thus, the Blood Product Advisory Committee (BPAC) has made recommendations to take precautions in order to decrease the risk of bacterial contamination of blood products.\n\nIn 2012, BPAC released a report recommending that blood centers and transfusion services either modify or perform additional testing on platelet products to help reduce the risk of recipient fatality due to bacterial contamination34. Then, in 2014, the FDA released industry guidance in line with the BPAC report to enhance the safety of platelet transfusions35. Recommendations for apheresis and pre-storage pooled platelets include testing for bacterial contamination using an FDA-cleared device no sooner than 24 hours post-collection and inoculating at least an aerobic culture medium, sampling the maximum volume permitted by the manufacturer of the culture device and considering increased sample volumes in proportion to collection volume (i.e. double and triple platelet collections) to maximize primary culture sensitivity, and adhering to the minimal incubation period described by the bacterial detection device manufacturer. Recommendations for whole-blood-derived platelets that are pooled at the time of issue and not previously tested for bacterial contamination include either culture as described for apheresis platelets or the use of a rapid bacterial detection device no sooner than 72 hours after collection. Thus, both the timing and the volume of the sample drawn for bacterial culture is important to help decrease the risk of sepsis due to bacterial contamination in platelet products.\n\nWhile blood centers can adopt strategies to improve detection of bacterial contamination in platelet products, hospital transfusion services can take additional precautions to prevent septic transfusion reactions. As septic transfusion reactions and fatalities are more common on days 4 and 5 of storage, after initially low inoculums of bacteria are allowed to grow to clinically significant levels, the BPAC report and FDA guidance document also recommended the use of either a rapid bacterial screening test or re-culture of the platelet product on days 4 and 5 of storage34,35. Transfusion services have also successfully adopted a strategy of screening for bacterial contamination on day 4 or 5 of storage to extend the expiry of platelet products to 7 days when there is urgent clinical need with no increase in the rate of septic transfusion reactions33. While no bacterial detection system is perfect, the combination of culture and rapid screening tests may provide the best risk reduction strategy for platelet transfusion-related septic reactions and safely extend the expiry date of the transfusion service’s most limited resource.\n\n\nTRALI risk mitigation strategies\n\nTRALI is a severe transfusion reaction characterized by the acute onset of new, non-cardiogenic pulmonary edema that occurs within 6 hours of transfusion. Most cases of TRALI result from cognate antibodies between recipient human leukocyte antigens (HLA) on white blood cells and HLA antibodies in donor plasma. HLA antibodies are formed after exposure to foreign white blood cells. Thus, recipients of blood products replete with white blood cells, females who have been pregnant and exposed to fetal white blood cells, and recipients of solid organ or bone marrow transplants are most likely to develop HLA antibodies.\n\nThe prevalence of HLA antibodies in female donors is related to the number of prior pregnancies36. Consequently, when comparing men and nulliparous women to previously pregnant donors, transfusion of blood components, especially apheresis plasma, carries a higher risk of inducing TRALI37. While previous transfusion increases the overall risk of developing HLA antibodies, a study of donors who had received a transfusion greater than 12 months prior to enrollment revealed that the incidence of HLA antibodies was not significantly increased compared to non-transfused donors38. Thus, a remote transfusion history in donors does not significantly contribute to an increased risk of HLA antibodies and these donors should not be excluded as part of TRALI risk mitigation strategies. Given the available evidence, in 2006, blood centers in the United States began restricting a large portion of plasma collections to males and nulliparous females. Subsequently, two separate analyses showed that a male-predominant plasma strategy has been associated with a reduction in TRALI cases39,40.\n\nThen, in 2014, due to the observed residual risk of TRALI, the AABB Standard 5.4.1.2 stated that “plasma and whole blood for allogeneic transfusion shall be from males, females who have not been pregnant, or females who have been tested since their most recent pregnancy and results interpreted as negative for HLA antibodies”28. Any donor who is found to be HLA antibody positive is not eligible for future donations of apheresis plasma or whole-blood-derived plasma for transfusion, while a negative result permits the release of all subsequent plasma components unless or until the donor becomes pregnant.\n\nTherefore, blood collection facilities have two options to meet the AABB Standard: either perform HLA antibody screening on all female donors instead of taking a pregnancy history on all donors whose donation produces transfusable plasma components, or target HLA antibody testing for any female who has had any number of pregnancies carried to term or delivered. The estimated impact in loss of parous female donors must be weighed against targeted HLA testing to arrive at the best TRALI risk reduction strategy.\n\nThere has been some evidence suggesting human neutrophil antigen (HNA) antibodies may play a role in the development of TRALI, but due to their low prevalence in the donor population and the fact that they require specialized testing not conducive to large-scale screening, there is no compelling data to adopt screening for HNA antibodies41. In addition, the fact that HLA antibody co-occurred in the majority of HNA antibody-positive donors suggests HNA-positive blood products may already be reduced as a consequence of HLA antibody screening.\n\nDespite the current decreased incidence of TRALI with modified transfusion practices, it is still the leading cause of transfusion-related fatalities in the United States5. However, it should be recognized that the FDA fatality reports through fiscal year 2014 include both TRALI and possible TRALI cases, the latter of which are most likely ARDS cases and not related to transfusion42. Thus, plasma mitigation strategies will not decrease the incidence of possible TRALI. It is therefore paramount that clinicians recognize, diagnose, and report TRALI and TRALI-related mortality to the blood bank so that incidence, pathophysiology, and strategies to prevent this leading cause of transfusion-associated mortality can be further studied.\n\n\nConclusion\n\nThe blood supply is the safest it has been in decades, yet blood centers and transfusion services continue to pursue massive efforts to prevent the infectious and non-infectious complications associated with blood product transfusion. One major challenge is to identify and stay one step ahead of emerging pathogens that threaten the safety of transfusable blood components. While blood centers continue to harness PR technologies and improve upon current pathogen detection techniques to enhance the safety of blood products, a challenge will be to keep the cost-benefit ratio of new technologies in check. Meanwhile, transfusion services will continue to struggle with the many non-infectious complications of transfusion. For example, despite the risk mitigation strategies implemented to date for plasma and platelet products, TRALI remains the leading cause of transfusion-related mortality, with red blood cell units now emerging as the implicated blood product. A challenge for the transfusion medicine community will be to further decrease the risk of TRALI while maintaining a healthy balance between the eligible donor pool and blood product inventory. While challenges remain for both blood centers and hospital transfusion services, the recent successes and strides made towards increasing the safety of the blood supply are noteworthy.",
"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\nFung MK: Technical Manual. 18th ed: AABB; 2014.\n\nRoth WK, Busch MP, Schuller A, et al.: International survey on NAT testing of blood donations: expanding implementation and yield from 1999 to 2009. Vox Sang. 2012; 102(1): 82–90. PubMed Abstract | Publisher Full Text\n\nZou S, Stramer SL, Dodd RY: Donor testing and risk: current prevalence, incidence, and residual risk of transfusion-transmissible agents in US allogeneic donations. Transfus Med Rev. 2012; 26(2): 119–28. PubMed Abstract | Publisher Full Text\n\nKleinman S, Stassinopoulos A: Risks associated with red blood cell transfusions: potential benefits from application of pathogen inactivation. Transfusion. 2015. PubMed Abstract | Publisher Full Text\n\nFatalities Reported to FDA Following Blood Collection and Transfusion: Annual Summary for Fiscal Year 2014. 2014. Reference Source\n\nGoubran HA, Burnouf T, Stakiw J, et al.: Platelet microparticle: a sensitive physiological \"fine tuning\" balancing factor in health and disease. Transfus Apher Sci. 2015; 52(1): 12–8. PubMed Abstract | Publisher Full Text\n\nVaron D, Shai E: Role of platelet-derived microparticles in angiogenesis and tumor progression. Discov Med. 2009; 8(43): 237–41. PubMed Abstract\n\nRubin O, Delobel J, Prudent M, et al.: Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation. Transfusion. 2013; 53(8): 1744–54. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRohde JM, Dimcheff DE, Blumberg N, et al.: Health care-associated infection after red blood cell transfusion: a systematic review and meta-analysis. JAMA. 2014; 311(13): 1317–26. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLannan KL, Sahler J, Spinelli SL, et al.: Transfusion immunomodulation--the case for leukoreduced and (perhaps) washed transfusions. Blood Cells Mol Dis. 2013; 50(1): 61–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCholette JM, Henrichs KF, Alfieris GM, et al.: Washing red blood cells and platelets transfused in cardiac surgery reduces postoperative inflammation and number of transfusions: results of a prospective, randomized, controlled clinical trial. Pediatr Crit Care Med. 2012; 13(3): 290–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSalpeter SR, Buckley JS, Chatterjee S: Impact of more restrictive blood transfusion strategies on clinical outcomes: a meta-analysis and systematic review. Am J Med. 2014; 127(2): 124–131.e3. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCarson JL, Brooks MM, Abbott JD, et al.: Liberal versus restrictive transfusion thresholds for patients with symptomatic coronary artery disease. Am Heart J. 2013; 165(6): 964-971.e1. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLacroix J, Hébert PC, Fergusson DA, et al.: Age of transfused blood in critically ill adults. N Engl J Med. 2015; 372(15): 1410–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSteiner ME, Ness PM, Assmann SF, et al.: Effects of red-cell storage duration on patients undergoing cardiac surgery. N Engl J Med. 2015; 372(15): 1419–29. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSnyder EL, Stramer SL, Benjamin RJ: The safety of the blood supply--time to raise the bar. N Engl J Med. 2015; 372(20): 1882–5. PubMed Abstract | Publisher Full Text\n\nJacobs MR, Lazarus HM, Maitta RW: The Safety of the Blood Supply--Time to Raise the Bar. N Engl J Med. 2015; 373(9): 882. PubMed Abstract | Publisher Full Text\n\nSchmidt M, Hourfar MK, Sireis W, et al.: Evaluation of the effectiveness of a pathogen inactivation technology against clinically relevant transfusion-transmitted bacterial strains. Transfusion. 2015; 55(9): 2104–12. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nProwse CV: Component pathogen inactivation: a critical review. Vox Sang. 2013; 104(3): 183–99. PubMed Abstract | Publisher Full Text\n\nAuBuchon JP, Prowse CV, AABB: Pathogen inactivation: the penultimate paradigm shift. Bethesda, Md.: AABB Press; 2010; 294. Reference Source\n\nBolton-Maggs P (Ed), Poles D, Thomas D, et al.: On behalf of the Serious Hazards of Transfusion (SHOT) Steering Group: The 2014 Annual SHOT Report. 2015. Reference Source\n\nBost V, Chavarin P, Boussoulade F, et al.: Independent evaluation of tolerance of therapeutic plasma inactivated by amotosalen-HCl-UVA (Intercept™) over a 5-year period of extensive delivery. Vox Sang. 2015; 109(4): 414–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLozano M, Cid J: Analysis of reasons for not implementing pathogen inactivation for platelet concentrates. Transfus Clin Biol. 2013; 20(2): 158–64. PubMed Abstract | Publisher Full Text\n\nGirona-Llobera E, Jimenez-Marco T, Galmes-Trueba A, et al.: Reducing the financial impact of pathogen inactivation technology for platelet components: our experience. Transfusion. 2014; 54(1): 158–68. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSnyder E, Raife T, Lin L, et al.: Recovery and life span of 111indium-radiolabeled platelets treated with pathogen inactivation with amotosalen HCl (S-59) and ultraviolet A light. Transfusion. 2004; 44(12): 1732–40. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSlichter SJ, Raife TJ, Davis K, et al.: Platelets photochemically treated with amotosalen HCl and ultraviolet A light correct prolonged bleeding times in patients with thrombocytopenia. Transfusion. 2006; 46(5): 731–40. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nButler C, Doree C, Estcourt LJ, et al.: Pathogen-reduced platelets for the prevention of bleeding. Cochrane Database Syst Rev. 2013; 3: CD009072. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nStandards for blood banks and transfusion services. 29th ed. Bethesda, MD: AABB; 2014. Reference Source\n\nde Korte D, Curvers J, de Kort WL, et al.: Effects of skin disinfection method, deviation bag, and bacterial screening on clinical safety of platelet transfusions in the Netherlands. Transfusion. 2006; 46(3): 476–85. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDumont LJ, Kleinman S, Murphy JR, et al.: Screening of single-donor apheresis platelets for bacterial contamination: the PASSPORT study results. Transfusion. 2010; 50(3): 589–99. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJacobs MR, Smith D, Heaton WA, et al.: Detection of bacterial contamination in prestorage culture-negative apheresis platelets on day of issue with the Pan Genera Detection test. Transfusion. 2011; 51(12): 2573–82. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHarm SK, Delaney M, Charapata M, et al.: Routine use of a rapid test to detect bacteria at the time of issue for nonleukoreduced, whole blood-derived platelets. Transfusion. 2013; 53(4): 843–50. PubMed Abstract | Publisher Full Text\n\nDunbar NM, Kreuter JD, Marx-Wood CR, et al.: Routine bacterial screening of apheresis platelets on Day 4 using a rapid test: a 4-year single-center experience. Transfusion. 2013; 53(10): 2307–13. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\n(BPAC) BPAC: Considerations for Options to Further Reduce the Risk of Bacterial Contamination in Platelets. 2012. Reference Source\n\nU.S. Department of Health and Human Services FDA: Bacterial Detection Testing by Blood Collection Establishments and Transfusion Services to Enhance the Safety and Availability of Platelets for Transfusion. 2014. Reference Source\n\nTriulzi DJ, Kleinman S, Kakaiya RM, et al.: The effect of previous pregnancy and transfusion on HLA alloimmunization in blood donors: implications for a transfusion-related acute lung injury risk reduction strategy. Transfusion. 2009; 49(9): 1825–35. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKleinman SH, Triulzi DJ, Murphy EL, et al.: The Leukocyte Antibody Prevalence Study-II (LAPS-II): a retrospective cohort study of transfusion-related acute lung injury in recipients of high-plasma-volume human leukocyte antigen antibody-positive or -negative components. Transfusion. 2011; 51(10): 2078–91. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKakaiya RM, Triulzi DJ, Wright DJ, et al.: Prevalence of HLA antibodies in remotely transfused or alloexposed volunteer blood donors. Transfusion. 2010; 50(6): 1328–34. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSchmickl CN, Mastrobuoni S, Filippidis FT, et al.: Male-predominant plasma transfusion strategy for preventing transfusion-related acute lung injury: a systematic review. Crit Care Med. 2015; 43(1): 205–25. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMüller MC, van Stein D, Binnekade JM, et al.: Low-risk transfusion-related acute lung injury donor strategies and the impact on the onset of transfusion-related acute lung injury: a meta-analysis. Transfusion. 2015; 55(1): 164–75. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGottschall JL, Triulzi DJ, Curtis B, et al.: The frequency and specificity of human neutrophil antigen antibodies in a blood donor population. Transfusion. 2011; 51(4): 820–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nToy P, Bacchetti P, Grimes B, et al.: Recipient clinical risk factors predominate in possible transfusion-related acute lung injury. Transfusion. 2015; 55(5): 947–52. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "11481",
"date": "17 Dec 2015",
"name": "Erhard Seifried",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11480",
"date": "17 Dec 2015",
"name": "Pearl Toy",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11482",
"date": "17 Dec 2015",
"name": "Jerard Seghatchian",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11483",
"date": "17 Dec 2015",
"name": "Neil Blumberg",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 relatively recently described complications of nosocomial infection, thrombosis and multi-organ failure that are caused, in part, by allogeneic red cell transfusion affect 1-2% of transfused patients, at minimum (1-4). These are severe and life threatening acute morbidities that are rarely mentioned in current textbooks and reviews, but well characterized in randomized trials of restrictive transfusion practices (2,3), autologous transfusion (5) and leukoreduced (1) transfusions. Thus mitigating the side effects of transfusion includes strategies such as restrictive red cell transfusion where validated in randomized trials, universal leukoreduction of transfusions, and implementation of blood management programs including techniques for minimizing the need for allogeneic transfusion. Such techniques can include various types of autologous transfusion (normovolemic hemodilution, cell salvage) and minimization of iatrogenic or surgical blood loss, as well as anemia management and pharmacologic approaches that mitigate anemia and thrombocytopenia. There is also preliminary data that removal of supernatant from transfused red cells and platelets may benefit patients undergoing cardiac surgery (6) and those treated for acute leukemia (7). Animal models support these possible benefits of washed transfusions (8). In addition, there is a growing body of evidence that ABO non-identical transfusions interfere with normal hemostasis, increase bleeding and mortality in hospitalized patients (9-13). Use of ABO identical transfusions is associated with reduced transfusion reactions, lung injury, alloimmunization and death in epidemiologic observational studies. While these data are still preliminary, they derive from multiple institutions and clinical settings, and have credible mechanistic explanations. ABO identical transfusions may be one key strategy to improve clinical outcomes in the future. (14)",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1469
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https://f1000research.com/articles/4-1468/v1
|
17 Dec 15
|
{
"type": "Research Article",
"title": "Systematics of Chasmosaurus - new information from the Peabody Museum skull, and the use of phylogenetic analysis for dinosaur alpha taxonomy",
"authors": [
"Nicholas Longrich"
],
"abstract": "The horned dinosaur Chasmosaurus from the late Campanian Dinosaur Park Formation of Alberta, is known from numerous skulls and skeletons, but over a century after its description, the taxonomy of the genus is controversial. Two species, Chasmosaurus belli and C. russelli, are currently recognized, with a third species, C. irvinensis, recently placed in a new genus, Vagaceratops. Here, the Yale Chasmosaurus skull is described, and implications for Chasmosaurus systematics are explored. The Yale skull is intermediate between typical C. belli and C. irvinensis. C. belli-like features include large, triangular lateral epiparietals, large parietal fenestrae, and an emarginate parietal. Yet the skull also exhibits derived features of C. irvinensis, including a posteriorly inclined narial strut, brow horns replaced by rugose bosses, reduced parietal emargination, five pairs of epiparietals, and epiparietals that fuse at their bases and hook forward over the frill. Specimen-level phylogenetic analysis provides a hypothesis of relationships upon which to base the taxonomy of Chasmosaurus. C. belli is paraphyletic with respect to C. irvinensis, and the Peabody skull is closer to C. irvinensis than to other C. belli. The holotype of C. russelli clusters with C. belli, making C. russelli a junior synonym of C. belli. Accordingly, Chasmosaurus can be divided into three species: C. belli, C. irvinensis, and C. priscus sp. nov, including specimens previously referred to C. russelli. The systematics of Chasmosaurus show how specimen level phylogeny can provide an evolutionary framework upon which to establish taxonomies. However, the resulting phylogenies may lead to paraphyletic species and genera.",
"keywords": [
"Chasmosaurinae",
"Dinosaur Park Formation",
"Campanian"
],
"content": "Institutional abbreviations\n\nAMNH, American Museum of Natural History, New York, New York; CMN, Canadian Museum of Nature, Ottawa, Ontario; NHMUK, Natural History Museum, London; ROM, Royal Ontario Museum, Toronto, Ontario; TMP, Royal Tyrrell Museum of Palaeontology, Drumheller, Alberta; UALVP, University of Alberta Laboratory for Vertebrate Paleontology; YPM, Yale Peabody Museum, New Haven, Connecticut.\n\n\nIntroduction\n\nThe Chasmosaurinae is a diverse group of horned dinosaurs that flourished during the Late Cretaceous of western North America1,2. Chasmosaurines were especially diverse in the late Campanian. Different species are found up and down the coast of the Western Interior Seaway1,3–6, and multiple species frequently occur in a single habitat. This diversity and a rich fossil record make chasmosaurines an ideal system for studying dinosaur diversity and biogeography.\n\nAmong the best-known genera is the Chasmosaurus from the Dinosaur Park Formation (DPF) of Alberta. Chasmosaurus is known from numerous skulls and skeletons7–12 but despite abundant material there is little agreement about how many species, or even genera are present4–8,10,13.\n\nUntil recently, Chasmosaurus was split into three species: Chasmosaurus belli, Chasmosaurus russelli, and Chasmosaurus irvinensis7. The three succeed each other in stratigraphic section, with primitive species occurring at the base of the DPF and more derived forms appearing higher up7, suggesting a lineage evolving through time. Recently, however, it has been proposed that C. irvinensis represents a distinct lineage, and it was placed in a new genus, Vagaceratops4.\n\nHere, a skull from the Yale Peabody Museum of Natural History, YPM 2016, is described. Until now, this specimen has only received cursory description11 but its unique morphology may help sort out the systematics of Chasmosaurus. YPM 2016 has previously been referred to C. belli5,7,8,10,11, but closer examination shows apomorphies that are absent in other specimens of this species. These features include five pairs of epiparietals, widely divergent lateral rami of the parietal caudal bar, and a caudally inclined premaxillary narial strut. These characters occur, however, in the highly derived C. irvinensis7.\n\nThis paper uses YPM 2016 as a starting point to examine Chasmosaurus’ systematics. Here, a revised taxonomy is proposed. Rather than starting with a taxonomy and then identifying diagnostic characters, this paper uses a bottom-up approach to taxonomy. Specimen-level phylogenetic analysis was conducted first, then phylogeny is used as a framework to delimit and diagnose species. Specimen-level phylogenies have previously been used to examine the systematics of other groups, including apatosaurine sauropods14, thescelosaurid ornithopods15, and the leptoceratopsid Montanoceratops16. Most recently, Scannella et al.17 have used specimen-level phylogenetic analysis to examine the systematics of Triceratops, and concluded that Triceratops prorsus represented the direct descendant of Triceratops horridus. This study represents the first application of this approach to Chasmosaurus. First, however, a history of the genus is presented.\n\nThe genus Chasmosaurus has a complex taxonomic history. Lambe12 originally described Monoclonius belli on the basis of a partial parietal (CMN 491). This species would then be referred to Hatcher et al.18 to the now defunct Ceratops, as Ceratops belli. Later, with more complete fossils, Lambe showed that the animal was distinct from previously known ceratopsids and created a new genus, Protorosaurus19. After Protorosaurus proved to be preoccupied, Lambe proposed Chasmosaurus20 as a replacement. And so, within the space of a dozen years, the species bore four different genus names. Despite being fragmentary, the holotype is diagnostic, as the morphology of the posterior bar of the parietal is unique to this species5.\n\nLambe named a second species in the 1902 paper, Monoclonius canadensis, which was later renamed Eoceratops canadensis21. This species was later placed in Chasmosaurus, as Chasmosaurus canadensis22,23. As discussed below, it appears to be distinct from Chasmosaurus, but the type is nondiagnostic.\n\nNew specimens led to new species. In 1933, Lull described a skull (ROM 839) as Chasmosaurus brevirostris11. The specimen has an unusually short rostrum, but this feature is typical of juvenile chasmosaurs24 and other features of C. brevirostris, including unfused epoccipitals and open cranial sutures24–26 indicate immaturity. As differences between C. brevirostris and C. belli likely reflect ontogeny, C. brevirostris is considered a junior synonym of C. belli8,10,22.\n\nThat year, another skull, AMNH 5401, was described as Chasmosaurus kaiseni27. AMNH 5401 has traditionally been referred to C. belli8,10, but the resemblance of the frill to that of C. belli is the result of plaster reconstruction. As discussed below, the skull appears to be distinct from Chasmosaurus and has been referred to a new genus, Mojoceratops5.\n\nSternberg9 named a fourth species, C. russelli. The type (CMN 8800) consists of a crushed skull. Sternberg interpreted the frill as having a V-shaped caudal margin; based on this and other characters, he regarded CMN 8800 as a distinct species. Sternberg referred three more specimens to C. russelli, with additional specimens referred by Godfrey and Holmes8, including CMN 2280, a largely complete skeleton. C. russelli has been regarded as a valid species by all recent studies1,4,5,7,8,10,13.\n\nIn 1989, a fifth species, Chasmosaurus mariscalensis, was named from bonebed material from the upper Campanian Aguja Formation of Texas22. Phylogenetic analyses fail to group this species and Chasmosaurus to the exclusion of other chasmosaurs3–6,28–30 however, and so it was referred to a separate genus, as Agujaceratops mariscalensis31.\n\nIn 2001, a sixth species, C. irvinensis, was described7. C. irvinensis is characterized by absence of brow horns, a straight posterior margin of the frill, and five pairs of epiparietals7 that fuse at their bases and curl forward. Holmes et al. interpreted C. irvinensis as closely related to C. belli7. The animal also occurs above C. belli in section7, raising the possibility that C. irvinensis descends from C. belli.\n\nPlacement of this species within Chasmosaurus has been contested, however. A species-level analysis by Sampson et al.4 recovered C. irvinensis as sister to Kosmoceratops richardsoni, an arrangement recovered in subsequent analyses using variants of the same character-taxon matrix13,29. Because this analysis placed C. irvinensis outside of the other species of Chasmosaurus, the animal was put in a new genus, as Vagaceratops irvinensis4. The phylogeny recovered appears well-supported in light of the similarity between the frills of C. irvinensis and Kosmoceratops. In each, the frill’s posterior margin bears ten epoccipitals, with the four middle pairs coalesced and hooked forward. Yet the frill as described by Holmes et al.7 differs from that of Kosmoceratops4 in its construction. In C. irvinensis, the back of the frill is formed by the parietal, which bears five epiparietals (P1–P5). In Kosmoceratops, the back of the frill is composed of parietal and squamosals, and the ornament consists of three epiparietals (P1–P3), an episquamosal (S1), and an epiparietal crossing the parietal-squamosal border (EPS)4. These differences suggest that resemblance between the two is convergent. However, the matrix used by Sampson et al.4 codes the posterior margin of the frill in C. irvinensis as being composed of the parietals and squamosals, as in Kosmoceratops. More recently, Longrich6 recovered irvinensis within Chasmosaurus.\n\nIn addition, Chasmosaurus’ systematics are further complicated by referral of a number of long-horned chasmosaurines to the genus. These include CMN 1254, holotype of Ceratops canadensis, AMNH 5401, holotype of C. kaiseni, and TMP 1989.83.25.1, a partial skull referred to C. russelli. Although traditionally referred to Chasmosaurus8,10,22,23,27 phylogenetic analyses show that these specimens represent a distinct lineage, named Mojoceratops5. The reader is referred to the literature5,28 for a discussion of this issue.\n\nFinally, one specimen referred to Chasmosaurus, CMN 8801, does not appears to belong to that species. Instead, phylogenetic analysis recovers it as sister to K. richardsoni6.\n\n\nGeological setting\n\nYPM 2016 and all other specimens of Chasmosaurus are from the DPF of southern Alberta32. The formation is late Campanian in age, and is dated to 77.0 Ma - 75.47 Ma33. DPF strata are 70–80 m thick, and consist of river sands and fine-grained overbank deposits. Thick river sandstones are well-developed basally, while towards the top, mudstones and coals document swamps and estuaries formed during the transgression of the Bearpaw Sea34 which overlies the DPF.\n\nAt the time, the area was located further north than it is today35, at 58°N, roughly the same latitude as southeast Alaska. The climate would have been seasonal, but warm Cretaceous temperatures supported a diverse biota. The formation contains a rich herpetofauna of fish36, amphibians37, lizards38, turtles35, alligators and choristoderes39. Non-avian dinosaurs were especially diverse, with over 45 known species6, making this the most diverse known dinosaur assemblage. Ornithischians included hadrosaurs, ceratopsians, ankylosaurs, thescelosaurs, and pachycephalosaurs5,6,40,41 and theropods included tyrannosaurids, ornithomimids, caenagnathids, troodontids, and dromaeosaurs42–45 and eight species of ornithurine bird46. A diverse mammalian fauna occurs as well47,48.\n\nWhile the formation contains a high diversity of dinosaurs, not all species coexisted7,40,49. Instead, many species have restricted stratigraphic distributions. Thus, the DPF assemblage is not a single fauna, but a succession of faunas spanning 1.5 million years.\n\nThe lowermost fauna is the Centrosaurus-Corythosaurus fauna; succeeded by a Styracosaurus-Lambeosaurus fauna, and finally a Lambeosaurus magnicristatus-Chasmosaurus/Vagaceratops irvinensis faunal zone. Chasmosaurus itself is known to show turnover through the formation. Holmes et al.7 suggested that three species occur in the formation- C. russelli near the base, C. belli higher up, and C. irvinensis at the top.\n\nReports of stratigraphic data for YPM 2016 conflict. Lull11 reports that YPM 2016 was “collected by C. M. Sternberg in 1919, on the south side of the west branch of Little Sandhill Creek, 60 feet below the overlying Pierre shales” i.e. the marine shales of the Bearpaw Formation, which overly the top of the DPF. Assuming that the formation is 70–80 m thick here, this puts the skull high up in section, in the muddy interval just below the Lethbridge Coal Zone (LCZ)34, and around 50–60 m above the base of the DPF. These data place YPM 2016 above C. belli, just below C. irvinensis in section7, and near the transition between the Styracosaurus-Lambeosaurus faunal zone and the L. magnicristatus-C. irvinensis faunal zone.\n\nLater, in 1935 and 1936, Sternberg staked quarries and published maps of localities in 1935. YPM 2016 was reported as coming from Quarry 110. In contrast to the original reports, Quarry 110 lies low in section. Godfrey and Holmes8 place the quarry at 31.4 m above the base of the DPF contact, putting it several meters below the highest C. belli (CMN 2245, ROM 839, and ROM 843), and about 20 m below the level estimated in Sternberg’s notes. Currie and Russell32 report that the specimen comes from 684.5 meters above sea level (MASL), versus 682.4-702.7 MASL for other C. belli. This would put the specimen near the bottom of the Styracosaurus-Lambeosaurus faunal zone or at the top of the Centrosaurus-Corythosaurus faunal zone.\n\nThe reason for the discrepancy is unclear, but there are several possible explanations.\n\nFirst, Sternberg may simply have committed an error with his original notes— perhaps misidentifying the top of the DPF, estimating the distance incorrectly, or simply recording the numbers incorrectly. Slumping of the overlying Bearpaw, for example, could create the impression that the contact with the Bearpaw was lower than it actually was.\n\nSecond, stratigraphy in the DPF is complicated by a number of factors. The position of the DPF-Oldman contact varies regionally and locally. Within a distance of 250 m, the height of the contact can vary by up to 5 m and it may vary by as much as 10 m over 1.5 km (Currie and Russell, 2005). The thickness of the formation also varies locally by up to 10 m, depending on whether the strata are composed of sandstones, which resist compaction, or mudstones, which readily compact34. These factors mean that distance above the DPF-Oldman formational contact does not correspond precisely with geological age. However, such factors seem unlikely to explain the 20–30 m discrepancy between Sternberg’s notes and more recent measurements. Another possibility is that the overlying marine strata are slumped down- making the contact appear lower than it is.\n\nThird, Sternberg’s notes could be correct, but the specimen may be assigned to the wrong quarry. Quarry maps were not published until 1935 and staking efforts did not occur until 1935 and 1936, 16–17 years after the original excavation50. Given that (i) a number of quarries are known to be misidentified in the original map50, (ii) many known quarries cannot be assigned to any known specimen, and (iii) the original notes are inconsistent with the stratigraphic data for Quarry 110, a very real possibility exists that Sternberg’s original notes are correct and that the specimen does not come from Quarry 110. Sternberg may have simply misremembered where the quarry was. Further work is needed to resolve this issue. As discussed below there are different implications depending on whether YPM 2016 sits low or high in the formation.\n\n\nSystematic paleontology\n\nYPM 2016 is a virtually complete skull (Figure 1, Figure 2), but some details are obscured by plaster and many bones are fused, obscuring sutures. The skull is also crushed dorsoventrally, complicating comparisons. YPM 2016 is now on display, and so the palate could not be studied, but was figured in ventral view by Lull11. Throughout, comparisons with other chasmosaurines are based on a revision of chasmosaur systematics (Longrich 20146, this paper). A list of specimens examined and literature studied for this analysis is given in the Supplementary material.\n\nChasmosaurus YPM 2016 in A, right lateral view; B, dorsal view; C, anterior view. Abbreviations: j, jugal; mb, median bar of parietal; nhc, nasal horn core; ns, narial strut; pb, posterior bar of parietal; pf, parietal fenestra; phc, postorbital horn core; pm, premaxilla; pmf, premaxillary fossa; spf, narial septum posterior flange; s1–s8, episquamosals s1–s8; p1–p5, epiparietals p1–p5.\n\nPremaxillae and rostral. The premaxillae and rostral form a beak that is low, broad, and triangular in lateral view (Figure 3). The tip of the rostral projects anteriorly, similar to C. irvinensis CMN 413577. By comparison, the dorsal margin of the rostral is less triangular and more rounded in C. belli (e.g. AMNH 2402) and in CMN 2280, the snout is much deeper and the rostral has a distinctly rounded anterodorsal margin. These differences may be taxonomically significant. Holmes et al.7 diagnosed C. irvinensis in part on the basis of the low, broad rostrum, although the referred specimen (TMP 87.45.1) has a taller, narrower snout7.\n\nRostrum of Chasmosaurus YPM 2016 in A, left lateral view, and B, right lateral view. Abbreviations: cf, caudal flange; naf, narial fossa; np, narial process; nst, narial strut; pfo, premaxillary fossa; pmx, premaxilla; ros, rostral.\n\nAnteriorly, the rostral is fused to the premaxillae, a feature of mature chasmosaurines26. The premaxilla bears a broad narial fossa pierced by an ovoid premaxillary fossa, as in other chasmosaurs1. It is unclear if an interpremaxillary fenestra is present; this area is covered with plaster. Below the premaxillary fossa is a shallow anteroventral fossa, as in other chasmosaurines.\n\nA narial strut separates the premaxillary fossa from the naris as in other chasmosaurs1 (Figure 3). In contrast to C. belli (e.g., AMNH 5402, ROM 839, CMN 8800) and CMN 2280, where the strut is anteriorly inclined, the strut is posteriorly inclined in YPM 2016. Posterior inclination of the narial strut is a derived feature shared with C. irvinensis7 and convergently in Arrhinoceratops51 and Anchiceratops13. While the rostrum is crushed, the orientation of the narial strut does not appear to result from distortion of the skull. Reorienting the strut from an anteriorly inclined orientation to a posteriorly inclined orientation would require rotating the premaxilla up and/or shearing the premaxilla, however the premaxillae do not appear to be either rotated upward or sheared.\n\nThe posterior margin of the narial strut bore a thin flange. As in other primitive chasmosaurs and centrosaurines, the flange is dorsoventrally extensive, running up the narial strut and onto the ventral margin of the nasal process. The posteroventral margin of the narial strut bears a narial process, a chasmosaurine apomorphy1. The flange is damaged, but apparently had the same subrectangular shape as other Chasmosaurus8.\n\nThe nasal processes of the premaxillae are long and slender, and insert into a narrow median slot formed by the premaxillary processes of the nasals. The nasal processes are horizontal anteriorly, but their posterior ends are upturned where they approach the nasal horn. The tips of the nasal processes are fused to the nasals posteriorly, but anteriorly the premaxilla-nasal suture is open.\n\nThe maxillary processes are long and slender, as in other chasmosaurines. The ventral margin of the maxillary process is medially inset, producing an anterior extension of the buccal fossa onto the premaxilla. Damage to the skull makes it difficult to ascertain details of the premaxilla-nasal contact.\n\nNasals. The nasals fuse to each other, the premaxillae, and the frontals. This extensive fusion is seen in other chasmosaurines, e.g. Triceratops26 but is generally absent in centrosaurs. The nasals arch transversely, forming a broad, rounded muzzle (Figure 2, Figure 4) as in other ceratopsids. Their dorsal surface is rugose and gnarled, but their lateral surfaces are smooth.\n\nSkull roof of Chasmosaurus YPM 2016 in A, dorsolateral view and B, anterodorsal view. Abbreviations: ff, frontal fontanelle; nhc, nasal horncore; phc, postorbital horncore. C, anterodorsal view of the skull, showing the frontal fontanelle.\n\nAnteriorly, each nasal bears a long, finger-like premaxillary process. These extend lateral to the nasal processes of the premaxillae to clasp the premaxillae between them, as in other ceratopsians. The maxillary process of the nasal forms the posterodorsal margin of the external naris, and has a long, fingerlike anteroventral extension as in other chasmosaurines.\n\nDorsally, the nasals bear a conical horn. In other chasmosaurs, the nasal horn develops from an epinasal ossification1,25. This was presumably the case here, but during ontogeny, the epinasal-nasal suture fuses, and so a discrete epinasal is not seen in adults. The nasal horn is shorter and blunter than in most specimens of C. belli (e.g., ROM 843, AMNH 5402) but taller and narrower than in C. irvinensis (CMN 41357).\n\nMaxillae. Maxillae are typical of chasmosaurines in being subtriangular in lateral view (Figure 2). As in other ceratopsians, the toothrow is medially inset and the maxilla bears a broad buccal fossa. Dorsally, the maxilla bounds a small, narrow antorbital fossa. As in other Ceratopsidae, the antorbital fossa extends ventrally into the maxilla to form a dorsally opening pocket, concealing the antorbital fenestra.\n\nThe maxillae are long and low. Protoceratops and Centrosaurinae have proportionately shorter, taller maxillae, but chasmosaurs tend to have a low maxilla. Chasmosaurus takes this feature to an extreme7, resulting in an elongate rostrum, with the rostrum depth being 50% or less the skull’s preorbital length. This feature is exaggerated by crushing in YPM 2016, but even so the rostrum would have been elongate as in other Chasmosaurus.\n\nCircumorbital bones. Circumorbital bones (Figure 4), including the prefrontals, frontals, and postorbitals, are fused and the sutures obliterated. The orbit is subcircular, as in most specimens of Chasmosaurus7, although some individuals do have an orbit that is taller than wide10. In almost all other ceratopsids, the orbit is taller than wide. The orbit’s anterior margin bears a rugose preorbital boss as in other ceratopsids.\n\nThe postorbitals lack a horn core; instead each bears a massive, gnarled supraorbital boss. The postorbital bosses seen in YPM 2016 also occur in the holotype of C. irvinensis7 and one specimen of C. belli (ROM 843). The holotype of C. russelli (CMN 8800) has a boss over the left orbit, but a short horncore with a resorbed tip over the right.\n\nThe top of each supraorbital boss bears a series of pits, up to 20 mm in diameter. Such pits also occur in C. irvinensis7 and in short-horned centrosaurines, e.g. Centrosaurus. They apparently result from resorption of the postorbital horncore as the animals grew52.\n\nSkull roof. The frontals resemble those of other ceratopsids (Figure 4). They are platelike bones that slope down from the orbits toward the midline, making the skull roof dorsally concave. The frontals fuse to the postorbitals and nasals with no trace of a suture. Posteriorly, the frontals border a large frontal fontanelle. The frontal fontanelle is long, narrow, and expanded anteriorly to give it the keyhole shape that characterizes basal chasmosaurines such as Chasmosaurus8 and Mojoceratops5. Unusually, the frontal fontanelle is closed posteriorly by a small midline contact of the frontals (Figure 4B); no other specimen of Chasmosaurus exhibits this feature.\n\nPosteriorly, the frontoparietal fontanelle communicates with the supratemporal fossae via a pair of narrow grooves, the dorsotemporal channels53. Posterior to this, the supratemporal fenestrae open upwards and the supratemporal fossae extend backwards onto the upper surface of the frill as a pair of broad, concave channels between the parietal and squamosal. These channels are partially roofed over by medial extension of the squamosals, which curve in over the supratemporal fossae as in other ceratopsids. The channels do not reach the parietal fenestrae; instead, the space between the parietal fenestrae and the fossae is covered by bone with a highly rugose, vascularized texture; presumably this part of the frill was covered by skin.\n\nCheek region. The jugal is typical of Ceratopsidae in being a triradiate element (Figure 2A, Figure 4A). It contacts the maxilla anteriorly, the quadratojugal posteroventrally, and the squamosal posterodorsally, and forms the ventral border of the orbit. The jugal is partly covered by paint and plaster, but apparently bore a prominent posterior flange that extends beneath the lateral temporal fenestra, a primitive feature seen in other Chasmosaurus. The epijugal hornlet is reduced to a blunt protuberance. This reduced epijugal is a derived character seen in most but not all other specimens of Chasmosaurus7,8, and convergently in Triceratops18,54.\n\nThe quadratojugal is overlapped by the jugal laterally, and overlaps the quadrate laterally. The jugal and quadratojugal, together with the squamosal, define a small lateral temporal fenestra. It is narrow and subtriangular, with its long axis inclined posteriorly, as in C. irvinensis7. The quadrate is similar to that of other chasmosaurs in being anteroposteriorly flattened and mediolaterally expanded to form a broad, platelike bone.\n\nSquamosal. The squamosals (Figure 2, Figure 5A) resemble other chasmosaurines in being subtriangular, with and a long caudal blade and a broad ventrolateral wing. The base of the squamosal projects posteriorly from the back of the skull in lateral view, as in other specimens of Chasmosaurus8 and other basal chasmosaurs such as Mojoceratops5. By contrast, the base of the squamosal projects slightly downward in Pentaceratops where it contacts the back of the skull, and strongly downward in Triceratops, Torosaurus, and centrosaurines.\n\nChasmosaurus YPM 2016, A, frill in dorsal view; B, closeup of right ramus of posterior bar of parietal. Abbreviations: mb, median bar of parietal; pb, posterior bar of parietal; pf, parietal fenestra; s1–s6, episquamosals s1–s6; p1–p5, epiparietals p1–p5.\n\nThe caudal blade has a concave dorsal face, with the medial edge curving upwards and the ventrolateral edge projecting down and out. This creates a trough-like concavity along the length of the blade, a derived feature of chasmosaurines. The caudal blade is thin but as in other chasmosaurs it has a thick lateral edge, and a thickened, rounded medial bar where it overlaps the squamosal. The squamosals are distinctly asymmetrical: the tip of the right squamosal is strongly tapered and thickened distally, and has a relatively flat dorsal surface. The end of the right squamosal is broader and thinner, with a more concave dorsal surface. This fluctuating asymmetry- i.e. random deviation from perfect bilateral symmetry- is commonly seen in other chasmosaurs such as Mojoceratops5 and also in pachycephalosaurs55 and is typical of structures under strong sexual selection55.\n\nThe left squamosal bears eight epoccipitals. There are seven or eight on the right side, but it is unclear whether the anterolateral corner bears two epoccipitals or one. Epoccipitals are large and crescentic anteriorly, decrease in size posteriorly, then become larger and more triangular towards the tip of the squamosal. The first few epoccipitals are completely fused to the squamosal with no trace of a suture. More posterior epoccipitals are fused to the frill but a suture is visible.\n\nParietal. The parietal posterior wing is long, broad, and triangular (Figure 5). As in other Chasmosaurus8, and other basal chasmosaurines, parietal fenestrae are expanded so that the frill is reduced to a frame of struts around them. Fenestrae are defined by a median, posterior, and lateral bars. The anterior end of the median bar is a broad, platelike element that is arched transversely, as in C. irvinensis. C. belli is similar, but the prominent dorsal ridge seen in C. belli (e.g. CMN 491) is absent in YPM 2016.\n\nAt its midlength, the median bar is wider than tall, measuring 52.5 mm wide by 47 mm tall. The bar becomes slightly narrower and thinner posteriorly. The dorsal surface of the bar is broadly rounded and rugose, and its lateral surface bears smooth, broad sulci. As it joins the caudal bar of the parietal, the median bar becomes wider again but continues to taper in lateral view, measuring 90 mm wide by 32 mm tall.\n\nThe lateral bar is incomplete, tapering posteriorly and then disappearing such that the squamosals participate in the parietal fenestrae. A similar condition occurs in CMN 2280. In other specimens referred to C. belli, the lateral bar may be either slender but well developed (CMN 2245; ROM 843) or reduced to a thin lamina (AMNH 5402), but is complete. The anterior part of the lateral bar bears a thin lamina of bone that projects inward to constrict the parietal fenestra, similar to other specimens of Chasmosaurus8.\n\nThe parietal caudal bar resembles that of other Chasmosaurus in being straplike in dorsal view. The lateral rami of the caudal bar meet along the midline at an angle of 165°. This angle is intermediate between that of C. belli, where the angle ranges from 150° to 160° (CMN 2245 and ROM 843, respectively), and C. irvinensis, where the lateral rami diverge at an angle of 180° (CMN 41357). As in both C. irvinensis and C. belli, the lateral rami are straight for most of their length, then near their lateral ends they bend anteriorly to underlie the posteromedial end of the squamosal, giving the lateral rami a distinctive ‘L’ shape. By contrast, the lateral rami are straight medially and strongly arched laterally in CMN 2280, giving them a ‘J’ or fishhook shape.\n\nThe caudal bar has the distinctive, comma-shaped cross section that characterizes Chasmosaurus. The anterior margin of the caudal bar is thin, measuring about 5–8 mm in thickness, but it becomes thicker posteriorly. It measures 10–12 mm thick a few centimeters behind the fenestra, then becomes much thicker along its caudal margin, measuring up to 25 mm thick near the posterolateral corner and 36 mm thick near the midline.\n\nThe posterior edge of the caudal bar curls upwards, such that the dorsal surface of the parietal bar is strongly concave, forming a trough across the midline. A similar condition is present in C. irvinensis7. In other specimens of C. belli, the posterior margin of the bar is curved upwards and there is a dorsal trough, but it is more poorly developed; for example in AMNH 5402, the trough extends across the midline but is relatively shallow, and in ROM 843 the trough is developed laterally but does not extend across the midline. In CMN 2280, the caudal margin of the bar curves upwards, but the dorsal surface is only weakly concave, rather than trough-like, and this concavity does not extend across the midline.\n\nThe parietal epoccipitals consist of a large, subtriangular lateral epoccipital, and a series of low epoccipitals medial to this. That the epoccipitals are low and tightly coalesced to the frill obscures their number and arrangement. Because the epoccipitals are upturned, they are best seen in posterior view, where four low, rounded epoccipitals (p1–p4) are bordered laterally by a triangular epoccipital (p5). The arrangement appears to have been similar on the left; p1 and p2 are developed in the same fashion as on the right, but p3–p5 are damaged. Thus, YPM 2016 bears five pairs of epoccipitals. This feature is unusual among chasmosaurs, occurring only in C. irvinensis7 and Torosaurus latus56. By comparison, C. belli bears four pairs of epoccipitals (AMNH 5402, ROM 843), and CMN 2280 has three pairs.\n\nP1–P4 coalesce at their bases to form a continuous, rugose ridge, a derived condition seen in C. irvinensis (CMN 41357)7. The epiparietals are also coalesced at their bases in K. richardsoni4.\n\nIn mature C. belli, P1 and P2 fuse (AMNH 5402, CMN 491, ROM 843) but P3 is separate (AMNH 5402, ROM 843, condition unknown for CMN 491). The holotype of C. russelli (CMN 8800) appears to exhibit the same condition as in C. belli; however P1 and P2 are unfused in CMN 2280.\n\nP1 is slightly pointed but P2–P4 are low and rounded. By contrast, P1–P4 are long and pointed in C. irvinensis. Both P1 and P2 are low and rounded in C. belli.\n\nThe forward curvature of the epiparietals is intermediate between the condition seen in C. irvinensis and that of C. belli. P1 hooks anteriorly over the frill; P2 and P3 are oriented anterodorsally, P4 projects dorsally, and P5 projects posteriorly. The curvature of the epoccipitals is less extreme than in C. irvinensis, where P1–P3 project anteriorly, and P4 projects anterodorsally. However, the curvature is more extreme than in C. belli and CMN 2280, where P1 and P2 project dorsally, but do not curve forward over the frill.\n\nP5 is large and triangular. A large, triangular lateral epiparietal is seen in other specimens referred to C. belli (e.g. AMNH 5402, ROM 843, CMN 2245) although it appears that the position at which the character is expressed may vary (i.e. a ‘frame shift’): in AMNH 5402 the large epiparietal corresponds to P4; in ROM 843, P3 is enlarged.\n\nOcciput. The occiput exhibits little variation in ceratopsids, and it is typical of ceratopsids in YPM 2016. The exoccipitals form the borders of an ovoid foramen magnum. Above the foramen magnum, the supraoccipital bears a pair of deep depressions, which are separated by a bony lamina. Lateral to the foramen magnum, the exoccipitals extend outwards to form a pair of broad paroccipital processes, contacting the parietals dorsally and the squamosals laterally. The paroccipital processes are expanded distally to give them a triangular shape, and twist to face posteroventrally. Below the foramen magnum, the exoccipitals and basioccipital fuse to form a ball-shaped occipital condyle that is offset from the braincase by a distinct neck. The basal tubera are robust and mediolaterally expanded.\n\n\nPhylogenetic analysis\n\nTo assess the relationships of Chasmosaurus, YPM 2016 was placed in a phylogenetic analysis along with specimens referred to Chasmosaurus and Mojoceratops, to create a specimen-level phylogeny. The matrix (Appendix 2) is modified from Longrich6 which in turn draws on several previous studies, primarily Dodson et al.1 and its derivatives3,5,28,57, but also Holmes7 and Sampson et al.4.\n\nThe matrix includes 203 characters; characters were treated as ordered where a transitional sequence could be identified (e.g., postorbital horns absent, short, moderately elongate, elongate). Character descriptions are given in the Supplementary material (Appendix 1). The character-taxon matrix (Appendix 2) was coded using Mesquite 2.7558. Following Longrich6, Ojoceratops, Nedoceratops, and Tatankaceratops are referred to Triceratops horridus and T. prorsus and excluded from analysis; in particular, characters previously used to diagnose Nedoceratops, including the orientation of the postorbital horns and the position of the squamosal59 are present only on one side of the skull (compare Farke 2011, Figure 1A,B vs. Figure 1C,D), indicating that they are artifacts resulting from postmortem distortion.\n\nAnalyses were conducted using PAUP* 4.0 b 1060 for phylogenetic analysis and TNT 1.1a61 to calculate support values. A specimen-level analysis was conducted including individual specimens referred to Chasmosaurus and Mojoceratops. Next, species were delimited based on the results of the specimen-level analysis and composite codings for each Chasmosaurus species were used for a species level-analysis.\n\nThen, to test whether the results are contingent on the matrix used, the analysis of Sampson et al.4 was rerun with YPM 2016 included. For this study, the codings of the frill and epiparietals for C. irvinensis were revised. Following Holmes et al.7 and this study, the posterior margin of the frill in C. irvinensis is formed of the parietal, with five pairs of epiparietals. Characters were recoded for the matrix as follows: 60:2->1 (squamosal subequal to parietal->squamosal slightly shorter than parietal), 61:1->0 (squamosal forms part of posterior margin of frill, present->absent), 89:2->1 (P2 elongate->P2 D-shaped), 91:1->0 (EPS present->absent), 92:1->? (EPS is absent and therefore its shape cannot be scored), 93:0-1 (3 epiparietals->5 epiparietals). Characters 60, 61, 91, and 92 are recoded to reflect the fact that the posterior margin of the frill appears to be composed of the parietal (as reconstructed by Godfrey et al.) whereas Sampson et al. code the frill as being composed of the parietal and squamosal. The reader is referred to the discussion below for the rationale for these codings.\n\nThis published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the International Commissions on Zoological Nomenclature. The ZooBank LSID (Life Science Identifier) is:\n\nurn:lsid:zoobank.org:pub:DD4D2E55-7974-4C16-BE01-BD433ABD0000 (the information associated with this LSID can be viewed through any standard web browser by adding the LSID to the prefix http://zoobank.org/)\n\nPhylum: Chordata\n\nSubclass: Dinosauria\n\nFamily: Ceratopsidae\n\nSubfamily: Chasmosaurinae\n\nGenus: Chasmosaurus\n\nDerivation of name: chasma, Greek, wide opening, sauros, Greek, lizard.\n\nDiagnosis: Chasmosaurine ceratopsid characterized by the following derived characters. Supraorbital horncores short, length 200% basal diameter or less. Posterior bar of parietal with a thickened caudal margin and a thin lamina extending anteriorly. Posterior margin of parietal upturned and dorsal surface of posterior bar concave, epoccipitals P1 and P2 projecting dorsally or anterodorsally.\n\nSpecies: priscus.\n\nDerivation of name: priscus, Latin, ancient or primitive\n\nDiagnosis: Rostrum rounded anteriorly in lateral view. Orbital horncores short, length 150%–200% basal diameter. Parietal bar with a well-developed caudal emargination, with lateral rami of caudal bar diverging at an angle of 120º. Lateral rami of posterior bar straight medially, but arched laterally, giving them a fishhook shape. Dorsal surface of lateral rami weakly concave, producing a shallow trough that does not extend across the midline. Three pairs of short, broad epiparietals, which do not coalesce at their bases. C. priscus is also distinguished by from C. belli and C. irvinensis by the absence of the derived features characterizing those species, described below.\n\nHolotype: CMN 2280\n\nType locality: Dinosaur Provincial Park, Alberta\n\nSpecimen-level analysis. A specimen-level analysis was run using a heuristic search algorithm until 69201 trees were obtained. The resulting tree statistics were tree length (TL) = 389; consistency index (CI) = .5979; retention index (RI) = .8298.\n\nIn the strict consensus (Figure 6) Chasmosaurus-like skulls fall into two clusters. One includes chasmosaurines with long, anterolaterally projecting horns, an expanded, triangular frontal fontanelle, a deeply notched parietal, a parietal caudal bar with a concave anterior surface and tuberosities on the anterodorsal margin, and posteriorly projecting epiparietals. This clade corresponds to specimens previously referred to Mojoceratops5. Variation exists within this cluster5, but there is no resolution within this grouping, because so many specimens are fragmentary.\n\nA, strict consensus tree showing all taxa; B, strict consensus showing Chasmosaurus specimens and proposed taxonomy; C, adams consensus showing the relationships of Chasmosaurus specimens. Treelength = Consensus of 50184 trees. Treelength = 389; CI = .5979; RI = .8298.\n\nThe second cluster contains chasmosaurines with short postorbital horns and a parietal caudal bar with a thickened, upturned posterior margin. This group corresponds to Chasmosaurus as defined by Longrich5. Within Chasmosaurus, phylogenetic structure exists in the strict (Figure 6B) and Adams (Figure 6C) consensus trees. Three main groupings can be identified. The first group, including the most primitive specimens, TMP 1981.19.175, CMN 2280 and UALVP 40, lie basal to all other Chasmosaurus. They have moderately short brow horns and in CMN 2280, strong caudal emargination of the parietal. The second group includes specimens previously referred to C. belli, including the holotype, CMN 491, and AMNH 5402, ROM 843, CMN 2245, and NHMUK R4948. This group also includes the holotypes of C. russelli, CMN 8800 and C. brevirostris, ROM 839. They are united by very short brow horns, weak caudal emargination of the parietal, an L-shaped lateral ramus of the caudal bar, and a low and coalesced P1 and P2.\n\nThe third group includes C. irvinensis, including CMN 41357, TMP 87.45.1, and CMN 41357. This clade is characterized by a posteriorly inclined narial strut, loss of brow horns, an expanded caudal end of the squamosal, a straight posterior bar of the parietal, and five parietal epoccipitals. The parietal ornament of these animals is specialized, with P1-P4 being fused, elongate, and turned forward over the frill.\n\nThe Peabody Chasmosaurus, YPM 2016, does not fit easily into any group. It lies just outside of the grouping formed by C. irvinensis, in both strict and Adams consensus trees, reflecting the intermediate nature of its morphology. It shares derived features with C. irvinensis, and plesiomorphic features with C. belli. Derived features include a posteriorly inclined narial strut and loss of the brow horns, and P1–P4 are fused. The parietal emargination is reduced compared to C. belli, and is therefore derived here, but not to the degree seen in C. irvinensis. Similarly, P1 and P2 are turned forward, a derived feature, but not P3 and P4, a primitive feature. P1 is slightly elongated, a derived feature, but P2–4 are not, which is primitive. P5 is smaller than in typical C. belli, but larger than in C. irvinensis. The parietal fenestrae are large and the median bar is subrectangular, both plesiomorphies shared with C. belli.\n\nWhen all specimens are included, bootstrap and Bremer support indices are poor (Figure 7A). This seems to result from inclusion of a high number of incomplete specimens, rather than a lack of signal in the data: when the more incomplete specimens are excluded and the analysis is rerun, the resulting tree has high support values for most nodes, but especially for the inclusion of C. irvinensis within Chasmosaurus.\n\nA, support values for specimen-level analysis; B, support values for analysis focusing on complete specimens. Low support values in the first analysis seem to result primarily from missing data, rather than conflicting data or lack of support for particular nodes.\n\nResults of species-level analysis. The species-level analysis, with YPM 2016 included, produced 9 most parsimonious trees (Figure 8) (Treelength = 377, Consistency Index = .6435, Retention Index = .8184, Rescaled Consistency Index = .5266). Results do not differ from the specimen-analysis, but conducting the analysis at species rather than specimen level speeds up analysis and makes it possible to use a branch-and-bound search. Results broadly agree with a number of previous analyses1,3–5,13,28,29,57 in putting Chasmosaurus basal within the Chasmosaurinae.\n\nStrict consensus of 9 most parsimonious trees (TL = 377, CI = .6435, RI = .8184, RC = .5266).\n\nReanalysis of Sampson et al. (2010) matrix. Reanalysis of the Sampson et al. matrix (Figure 9) produced a total of 6 trees (Treelength = 270, Consistency Index = .6426, Retention Index = .7804, Rescaled Consistency Index = .5087). In this analysis, irvinensis clusters with Chasmosaurus (Figure 9B). This result is contingent on the inclusion of YPM 2016. When YPM 2016 is deleted (Figure 9A), C. irvinensis again clusters with Kosmoceratops, consistent with the original tree4.\n\nA, results of analysis using Sampson et al. (2010) with C. irvinensis recoded, with polyphyletic Chasmosaurus. Strict consensus of 6 most parsimonious trees; TL = 267, CI = .6500, RI = .7791, RC = .5136. B, results of analysis with YPM 2106 included, strict consensus of 6 most parsimonious trees; TL = 270, CI = .6426, RI = .7804, RC = .5087. Inclusion of the transitional morphology represented by YPM 2016 breaks up the long branch separating C. belli and C. irvinensis, resulting in a monophyletic Chasmosaurus.\n\n\nDiscussion\n\nThe phylogenetic results presented here provide a framework for interpreting the taxonomy of Chasmosaurus. Critical issues addressed here include the assignment of C. irvinensis to the genus Vagaceratops, the diagnosis of C. belli, validity of C. russelli, and the identity of CMN 2280.\n\nPhylogenetic analysis places the species irvinensis not as the sister taxon of C. belli but nested within C. belli. Numerous synapomorphies unite specimens referred to C. belli and C. irvinensis, including reduced brow horns, a frill that is weakly inclined in lateral view, a parietal posterior bar that is thickened posteriorly and upturned such that the bar is concave dorsally, and an upturned P1 and P2. It shares with C. belli further reduction of the orbital horns, the L-shaped lateral bars of the parietal, reduced midline emargination of the frill, and coalesced P1 and P2. Finally, C. irvinensis shares with YPM 2018 the further reduction of the posterior emargination of the frill, the five pairs of parietal epoccipitals, coalescence of P1–P4 at the bases, and strong forward curvature of P1 and P2.\n\nThe relationships found here do not result from the characters selected for this study. As discussed above, when C. irvinensis is recoded in the Sampson et al.4 matrix following the original description and YPM 2016 is included, irvinensis is again recovered with Chasmosaurus.\n\nPart of the argument for uniting C. irvinensis with Kosmoceratops concerns frill morphology. As discussed above, in the description of C. irvinensis, the back of the frill was interpreted as being composed of the parietals, with five epiparietals7, as in the Peabody Chasmosaurus. However, Sampson et al.4 interpreted the frill as being composed of the parietals and squamosals, with three epiparietals, as in Kosmoceratops. The suture between the parietal and squamosal is difficult to trace distally in C. irvinensis, but it appears to curve laterally towards the end of the frill (Figure 10), such that the parietal forms the back of the frill, as suggested by Holmes et al.7. The interpretation advanced by Sampson et al.4 would instead require that the ends of the squamosals make a 90° bend and hook in medially behind the parietal fenestrae. No other ceratopsid exhibits such a configuration; more importantly, there is no evidence for the parietosquamosal suture here.\n\nAbbreviations: par, parietal; sq, squamosal; p1–p5, epiparietals 1 – 5.\n\nThe results of the phylogenetic analysis mean that referring irvinensis to Chasmosaurus is a reasonable taxonomic decision given that the animal nests within specimens previously referred to Chasmosaurus. That being said, the animal is also very distinct from C. belli, to the point that the relationship between the two has been controversial. Given this, treating the animal as a distinct genus, Vagaceratops is also reasonable. One emphasizes the continuity of the lineage, the other approach emphasizes the marked differences.\n\nNeither is more correct, in a strictly scientific sense, than the other. Given the current tree topology, the difference between C. irvinensis and V. irvinensis is semantic, not scientific. There is no objective way of deciding which name to use, because there is no objective, scientific criterion for deciding how distinctive a species must be to merit its own genus. The choice is up to the taxonomist; it is an issue of taxonomic bookkeeping, and not a scientific debate. Here, Chasmosaurus is used because it emphasizes the continuity of the lineage, in part because of tradition, and in part because it is simply more euphonious. Admittedly, these are not scientific reasons, but the argument is not a scientific argument. The debate will need to be settled through the consensus of the palaentological community. For this paper, I have chosen to use Chasmosaurus. Other paleontologists can either follow this approach or not, as they see fit.\n\nIncluding Vagaceratops within a clade of Chasmosaurus does have an interesting consequence: Chasmosaurus becomes paraphyletic. Although some authors argue that genera should be monophyletic62, others do not recognize monophyly as necessary or desirable; Lehman30 presented a phylogeny in which Chasmosaurus and Torosaurus were both paraphyletic genera. In fact, the International Code of Zoological Nomenclature does not require monophyletic taxa and, as discussed below, paraphyletic taxa are an inevitable consequence of a ranked taxonomy.\n\nSpecimens previously referred to C. belli are recovered as a paraphyletic grouping. No derived features were found to unite C. belli that are not also found in C. irvinensis. Instead, C. belli is diagnosed only by the absence of apomorphies diagnosing C. irvinensis. Furthermore, if YPM 2016 is included in C. belli, then C. belli ranges in form from primitive animals that retain short orbital horns and strongly emarginate parietals with four epoccipitals, to derived animals with orbital bosses, and weakly emarginated parietals with five epoccipitals. Excluding YPM 2016 from C. belli and referring it to C. irvinensis would result in a more limited range of variation in the species, but would not result in a monophyletic C. belli. The only way to create a monophyletic C. belli would be to create new species for all forms that lie either above the holotype or below the holotype on the tree.\n\nAs with the decision to refer C. irvinensis to Chasmosaurus, the decision to leave YPM 2016 in C. belli is largely arbitrary. Here, YPM 2016 is referred to C. belli because it lacks many of the apomorphies of C. irvinensis. Yet one could just as easily refer YPM 2016 to C. irvinensis because it exhibits a number of the apomorphies of this species that are absent in C. belli.\n\nChasmosaurus russelli (Figure 11) emerges as a problematic taxon in the current analysis. Sternberg9 diagnoses C. russelli as follows:\n\nA, left lateral view; B, close up of frill; C, close up of face; D, close up of rostrum, E, close up of posterior margin of frill. Abbreviations: aaf, accessory antorbital fenestra; aof, antorbital fenestra; ff, frontal fontanelle; mbs, medial bar of squamosal; nf, narial fossa; nhc, nasal horn core; np, narial process; pbp, posterior bar of parietal; pf, parietal fenestra, pmf, premaxillary fossa; phc, postorbital horn core; stf, supratemporal fossa; sul, sulcus; vf, ventral fossa; p1–p4, epiparietals 1–4; s1–s9, episquamosals 1–9.\n\n“1) Skull large, 2) relatively high and short in front of orbits; 3) rostral straight inferiorly, not hooked downward at tip, 4) nasal horncore massive, broad between orbits; 5) no brow horncores; 6) well-developed epijugal 7) parietals deeply indented posteriorly, 8) squamosal border not strongly scalloped, 9) epoccipitals small, 10) mandible massive.” (numbers are inserted for the reader).\n\nCharacter 1 applies to all ceratopsids. Character 2 is possibly due to crushing. Character 3 cannot be assessed in the type. Character 4 does not differ markedly from the condition in C. belli. Character 5 is found on the left side of the skull only, and is seen in some C. belli and C. irvinensis. Character 6 is developed to a comparable degree in some specimens of C. belli (e.g., ROM 843). Character 7, parietal emargination, is debatable. The skull is crushed almost flat, with the squamosals displaced to lie posterior to the parietals, rather than lateral to them, and the right horn lies ahead of the left, showing that the entire skull has been not only crushed but sheared, with the left side of the skull moved posteriorly relative to the right. It is therefore impossible to know if the emargination is real, or simply a result of crushing. Character 8 is also seen in C. irvinensis. Character 9, reduction of the epiparietals, is seen in the median epiparietals of C. belli, although the reduction of the lateral epiparietals seems to be unique to this specimen. Character 10 is not preserved in the holotype.\n\nThere are real differences between CMN 8800 and characteristic C. belli, including the weakly scalloped squamosal and small lateral epiparietals, but they do not appear to lie outside the range expected for intraspecific variation, and so the differences identified by Sternberg appear insufficient to separate the type from C. belli.\n\nC. russelli has continued to be treated as a distinct species, however. The reason is that CMN 2280 was referred to C. russelli, and the species has then been diagnosed based on this highly complete and well-described specimen8. Godfrey and Holmes diagnose C. russelli as follows: “1) Posterior margin of parietal frill broadly arched on either side of median emargination. 2) Each side bears three low, triangular roughly equal-sized epoccipitals. 3) The lateral ramus of the parietal is reduced and does not completely encircle the fenestra in all but one specimen, permitting the squamosal to form a part of its lateral border” (numbers added here for clarity). Again, this diagnosis is based on CMN 2280, not on the holotype of C. russelli. CMN 8800, the holotype, clearly lacks characters 1 and 2 (Figure 11). Character 3 cannot be assessed in the holotype because the skull is embedded in the matrix; at any rate it is present in C. belli (e.g. YPM 2016) and therefore does not differentiate C. russelli.\n\nLongrich5 provided the following diagnosis: “Chasmosaurus exhibiting the following combination of characters: 1) lateral rami of parietal posterior bar weakly arched in dorsal view, 2) well-developed caudal emargination of the frill, with an angle between the lateral rami of 120°, 3) three broad, moderately elongate parietal epoccipitals.” Again, the diagnosis is based on CMN 2280, not the holotype, CMN 8800, and characters 1 and 3 (= Godfrey and Holmes’ characters 1 and 2) are absent in CMN 8800, and as discussed above, the emarginated parietal of CMN 8800 may be a preservational artifact. While these problems were recognized during the writing of the 2010 paper, it was felt that dealing with Chasmosaurus russelli was beyond the scope of that particular project.\n\nLast, Maidment and Barrett10 diagnose C. russelli as follows: “taxon displaying the combination of characters unique to the genus Chasmosaurus along with the following features: 1) parietal posterior bar bearing a median emargination and is broadly arched either side so that it forms an ‘M’ shape with the parietal median bar; 2) each side of the posterior parietal bar bears three roughly equally sized epiparietals (after Godfrey & Holmes, 1995). Both characters are autapomorphic for C. russelli within Chasmosaurus.” This diagnosis follows Godfrey and Holmes8, and again is based on CMN 2280, not the type of C. russelli.\n\nSternberg’s diagnosis of C. russelli does not provide a basis for recognizing a distinct species, and the holotype cannot readily be separated from C. belli. Meanwhile, the diagnoses of Godfrey and Holmes8, Longrich5 and Maidment and Barrett10 do potentially diagnose a distinct species, but the diagnosis is based on CMN 2280, and the holotype of C. russelli, CMN 8800, does not fit this diagnosis, as it has reduced medial epiparietals and lateral episquamosals. CMN 8800 instead appears to represent a crushed C. belli, and the reduction of the epoccipitals may be related to the age of the specimen, as seen in other chasmosaurs24. This conclusion is supported by phylogenetic analysis (Figure 6). However, the diagnoses provided by Godfrey and Holmes8, Longrich5 and Maidment and Barrett (2011) do diagnose a distinct taxon, in that they separate CMN 2280 from the remaining Chasmosaurus.\n\nA number of plesiomorphies distinguish CMN 2280 from C. belli and C. irvinensis (excellent figures and a description of CMN 2280 were presented by Godfrey and Holmes; so readers are referred to that paper for a thorough description). First, while the brow horns are reduced in CMN 2280, they are markedly longer than in C. belli (e.g. AMNH 5402). Although horn length changes with age24,52, this feature is difficult to explain in terms of ontogeny. Because CMN 2280 is near maturity: the frill is rugose and some of the epoccipitals are fused, with only the rostral and some epiparietals remaining unfused. By comparison, AMNH 5402 is comparable in terms of maturity, but has shorter horns.\n\nThe posterior margin of the frill also has a distinct M-shape, similar to that of Mojoceratops, although not developed to the same degree. The posterior bar of the parietal is straight medially, but strongly curved laterally, such that each ramus of the bar is shaped like a ‘J’ or a fishhook. By contrast, the posterior bar is straight along most of its length in C. belli, then sharply bends forward near the parietal-squamosal junction, giving it a shape like an L or a 7. The arching of the posterior bar also means that the parietal projects well posterior to the squamosals, approaching the condition in Mojoceratops. By contrast, the parietal does not project as far in C. belli. The emargination of the parietal is also well developed; the lateral rami of the posterior bar diverge at an angle of 120°, versus 150° or more in C. belli.\n\nIn addition, the parietal is more primitive in the weak development of the distinctive upturned edge of the frill. The posterior margin of the parietal bar is slightly upturned in CMN 2280 and its dorsal surface is weakly concave. By comparison, the posterior margin is strongly upturned in C. belli, resulting in a trough along the dorsal surface of the parietal posterior bar.\n\nFinally, the parietal ornament is more primitive. There are only three parietal epoccipitals, versus four or more in C. belli. P1 and P2 are also prominent and separate, versus low and coalesced in C. belli.\n\nThus, there appears to be a primitive form of Chasmosaurus present in the DPF, which is represented by CMN 2280. TMP 1981.19.175 and UALVP 40 may also represent this species, as they retain relatively long brow horns. TMP 1981.19.175 may be more primitive than CMN 2280, because the parietal fenestrae appear to extend further anteriorly, as in Mojoceratops, whereas there is a broad sheet of bone behind the supratemporal fenestra in CMN 2280, as in C. belli and C. irvinensis.\n\nThe differences seen here appear to be sufficient to warrant a distinct species, but the referral of CMN 8800, the holotype of C. russelli, to C. belli leaves this species without a name. Accordingly, the name C. priscus is proposed as a replacement. Following the results of the analyses presented above, the following is proposed as a revised taxonomy of the genus.\n\n\nRevised taxonomy of Chasmosaurus\n\nTaxonomy\n\nDinosauria Owen 1842\n\nOrnithischia Seeley 1888\n\nCeratopsia Marsh 1890\n\nEuceratopsia new taxon\n\nCeratopsidae Marsh 1888\n\nChasmosaurinae Lambe 1915\n\nChasmosaurus Lambe 1914\n\nEuceratopsia is a node-based clade defined as the most recent common ancestor of Zuniceratops, Turanoceratops, and Triceratops, and all of its descendants.\n\nChasmosaurus Lambe 1914\n\nChasmosaurine ceratopsid characterized by the following derived characters. Supraorbital horncores short, length 200% basal diameter or less. Posterior bar of parietal with a thickened caudal margin and a thin lamina extending anteriorly. Posterior margin of parietal upturned and dorsal surface of posterior bar concave, epoccipitals P1 and P2 projecting dorsally or anterodorsally.\n\nType species. Chasmosaurus belli Lambe 1902.\n\nDistribution. Dinosaur Park Formation, Alberta, Canada.\n\nChasmosaurus priscus sp. nov.\n\nDiagnosis. Rostrum rounded anteriorly in lateral view. Orbital horncores short, length 150%–200% basal diameter. Parietal bar with a well-developed caudal emargination, with lateral rami of caudal bar diverging at an angle of 120°. Lateral rami of posterior bar straight medially, but arched laterally, giving them a fishhook shape. Dorsal surface of lateral rami weakly concave, producing a shallow trough that does not extend across the midline. Three pairs of short, broad epiparietals, which do not coalesce at their bases. C. priscus is also distinguished by from C. belli and C. irvinensis by the absence of the derived features characterizing those species, described below.\n\nHolotype. CMN 2280.\n\nReferred specimens. TMP 1981.19.175 (?), UALVP 40 (?)\n\nDistribution. Lower Dinosaur Park Formation, Alberta, Canada.\n\nChasmosaurus belli Lambe 1902\n\nSynonyms\n\nMonoclonius belli Lambe 1902\n\nCeratops belli Hatcher 1907\n\nProtorosaurus belli Lambe 1914\n\nChasmosaurus brevirostris Lull 1933\n\nChasmosaurus russelli Sternberg 1940\n\nDiagnosis. Chasmosaurus distinguished from C. priscus by the following derived characters. Rostrum with subtriangular beak in lateral view. Orbital horncores very short or absent, with length subequal to or less than basal diameter. Parietal short, with limited projection posterior to squamosals. Caudal emargination of parietal reduced, lateral rami of caudal bar diverging at an angle of 140°–165°. Lateral rami of posterior bar straight medially and strongly bent at the corner of the frill, giving them the shape of the numeral 7. Lateral rami of caudal bar with a strongly concave dorsal surface, which may or may not extend across the midline. At least four parietal epoccipitals. Epiparietals P1 and P2 short and their bases coalesced. Large, subtriangular lateral epiparietals. C. belli is typically distinguished from C. irvinensis by the absence of the derived features characterizing that species, which are described below. However, some of these features are in fact found in YPM 2016.\n\nHolotype. CMN 0491\n\nReferred specimens. AMNH 5402, CMN 2245, NHMUK R4948, ROM 839 (holotype of “Chasmosaurus brevirostris”), ROM 843, CMN 8800 (holotype of “Chasmosaurus russelli”), YPM 2016.\n\nDistribution. Lower Dinosaur Park Formation, Alberta, Canada.\n\nChasmosaurus irvinensis Holmes et al. 2001\n\nSynonyms\n\nVagaceratops irvinensis Sampson et al. 2010\n\nDiagnosis. Narial strut of premaxilla inclined posteriorly. Orbital horncores reduced to low, rugose bosses. Squamosal broad distally. Parietal with limited projection beyond squamosals. Parietal fenestrae reduced, broader than long. Caudal emargination of parietal absent, posterior bar of parietal straight. Caudal margin of parietal strongly upturned, defining a deep, trough-like concavity developed on the dorsal surface of the lateral rami of the parietal bar and extending across the midline across the junction of the caudal bar and median bar. Five pairs of parietal epoccipitals. Parietal epoccipitals strongly curved forward, with apices of median parietal epoccipitals projecting anteriorly. Medial parietal epoccipitals elongate.\n\nHolotype. CMN 41357.\n\nReferred specimens. TMP 87.45.1, TMP 98.102.8\n\nDistribution. Upper Dinosaur Park Formation (Lethbridge Coal Zone), Alberta, Canada.\n\nSpeciation of Chasmosaurus could potentially involve anagenesis, cladogenesis, or some combination of the two. The two different mechanisms make distinct predictions about both tree structure and stratigraphic distribution. Overall, the structure of the tree found in the current analysis (Figure 6) is consistent with the hypothesis that specimens referred to Chasmosaurus represent a single, evolving population, i.e. anagenesis. Rather than forming discrete clusters as expected for reproductively isolated populations, the specimens form a pectinate array, with primitive C. priscus specimens branching off near the base, followed by more derived C. belli specimens, then YPM 2016, until the highly derived C. irvinensis appears.\n\nStratigraphic distribution of Chasmosaurus specimens is largely consistent with the idea of anagenesis (Figure 13), with more derived specimens tending to occur higher in section. Specimens referred to C. priscus lie low in section in the DPF. C. belli lies above, and finally, C. irvinensis lies at the top7,32.\n\nA, Skulls of the three Chasmosaurus species and YPM 2016 compared. Abbreviations: p1–p5, epiparietals 10–5. B, hypothesized transitional series and transformations.\n\nA, Phylogeny of Chasmosaurus specimens calibrated against elevation (meters above sea level, MASL), above, and meters above the Oldman, below. In general, the more advanced specimens tend to lie higher in stratigraphic section, but the stratigraphy of Dinosaur Park is complicated by the fact that the formational contact dips from East to West, and from South to North.\n\nWhere YPM 2016 fits into this picture is unclear. In terms of phylogenetic position, YPM 2016 lies between classic C. belli and C. irvinensis morphotypes. If C. irvinensis is the product of anagenesis, then YPM 2016 should also lie between the two in terms of stratigraphy. The original data reported by Sternberg place the specimen relatively high in section, which is consistent with the hypothesis of anagenesis.\n\nHowever, measurements for Quarry 110 place the site low in stratigraphic section. Assuming this is correct, it implies cladogenesis, not anagenesis. If YPM 2016 lies low in section, then one lineage must split off and ultimately produce C. irvinensis, while one or more morphologically conservative lineages of C. belli persist, before ultimately being replaced by the C. irvinensis lineage at the top of the formation. Thus, Chasmosaurus may not have been a single, evolving population but a collection of many populations evolving more or less in isolation, with one lineage- the one leading to C. irvinensis- ultimately winning out.\n\nAs discussed above, Sternberg’s stratigraphic and provenance data are in conflict; YPM 2016 is reported as coming from high in section but Quarry 110 lies low in section. In light of conflicting data, we cannot be certain that the specimen comes from Quarry 110- although additional study of the matrix, palynology, or the quarry might help settle the issue. DPF stratigraphy is complicated, such that elevation and stratigraphic position are not perfect proxies for age. In general, specimens lying higher up in elevation lie higher up in section, but the base of the DPF dips from east to west and south to north32, such that specimens lying at similar elevations might not lie at the same stratigraphic position. Measuring distance from the formational contact is also problematic, for two reasons. First, the contact is not even; even adjacent boundary sections may differ in elevation by almost 10 m (Figure 15), which raises questions about whether the onset of deposition was simultaneous across the park. Second, the thickness of the formation varies34 locally. Because sandstone resists compaction better than mudstones, sequences with large basal sandstones will tend to be thicker than those with more mudstones34. In effect, the compression of the mudstones will compress time.\n\nThis might help explain the apparent discrepancy between the original stratigraphic data, putting the specimen 20 m below the Bearpaw, and more recent data which put it 31.4 m above the base of the formation. Additional stratigraphic work will be required to resolve this issue, but is beyond the scope of this paper.\n\nThe case of Chasmosaurus provides a case study in the evolution of dinosaurs. Altogether, the Chasmosaurus lineage spans a period of roughly 1.8 million years34. Given this, it would be unsurprising if the lineage adapted to selective pressures exerted by the environment, flora, predators, and other members of the species. One of the more striking changes is seen in the rostrum. In C. priscus (TMP 1981.19.175; CMN 2280) the snout is broadly rounded in lateral view. In C. belli (e.g., AMNH 5402, YPM 2016) and especially C. irvinensis (CMN 41357) the rostrum is more tapered in lateral view, with a more triangular rostral. YPM 2016 and C. irvinensis also differ in having a posteriorly inclined narial strut, which would presumably alter the ability of the beak to resist bending and shearing stresses. Such changes imply changes in dietary preferences and/or feeding strategies. Given that the flora underwent rapid changes during the Campanian63, it would be unsurprising to find that the jaws responded to shifts in floral composition, much as the beak of Galapagos finches adapts to changes in available food64,65. Similar patterns are seen in Triceratops, where changes in beak morphology distinguish the derived T. prorsus from the primitive T. horridus66, with the short-beaked T. prorsus appearing higher in section.17. Insofar as the beaks of extinct dinosaurs (like the beaks of living dinosaurs such as Galapagos finches) evolved rapidly in response changes in diet, dinosaur beaks may be useful for species-level diagnosis44,66, as much or more than the cranial ornament.\n\nThe horns in Chasmosaurus also evolved rapidly. The ancestor of Chasmosaurus would have had moderately long postorbital horns, as in Mojoceratops. In C. priscus, the horns are shortened; in primitive C. belli they are reduced to blunt protuberances, and in advanced C. belli and C. irvinensis the horns are replaced by low bosses. Horns in modern animals- including deer, bovids, and rhinos - are used for combat, sometimes against predators, but primarily against conspecifics67 and the same presumably held for horned dinosaurs as well68. Long-horned chasmosaurines presumably engaged in combat by locking their horns together and engaging in wresting and shoving matches68. Reduction of the brow horns suggests that Chasmosaurus did not engage in such bouts; however the robust nasal horn, extended by a horny sheath, would have remained an effective weapon for goring the flanks of opponents or defending against predators.\n\nFinally, and most conspicuously, the shape of the frill and parietal ornament underwent marked changes. From C. priscus to C. irvinensis, the frill evolved from a heart-shaped structure to a subrectangular shield. The epiparietals increased in number; the medial epoccipitals were fused, reduced, then were elongated again. The frill of ceratopsians is likely to have evolved in response to sexual selection69 as a display structure. Sexually selected structures tend to exhibit high variability between species69 which indicates rapid evolution; the rapid evolution of the frill structure can therefore be explained by- and also be considered evidence of- sexual selection.\n\nTaken together, the information suggests that Chasmosaurus represents a lineage evolving through time. Few other examples are known for dinosaurs, although recent work on Triceratops has presented compelling evidence for evolution in this genus17. Although such examples are rare, they are hardly unexpected. Dinosaurs appear to have evolved rapidly, given the profusion of species seen in a short period of time; it stands to reason that dense sampling of a taxon through a single formation could reveal evidence of evolution within that taxon.\n\n\nImplications for dinosaur systematics\n\nAlong with previous, specimen-level analyses14–17,66, the results presented here show the advantages of applying phylogenetic analysis to individual specimens. Many studies have previously taken on the taxonomy of Chasmosaurus5,7,8,10,22,70, but consensus has been elusive. The traditional approach has been to focus on supposed key characters such as frill shape8,10,22,70; characters inconsistent with the proposed taxonomy, such as horn length, are then interpreted as intraspecific variation. The problem with this approach should be obvious: how can we know that parietal emargination captures the true signal, rather than horn length, or some other character entirely? With this approach, homology of the characters in question is assumed, instead of tested.\n\nPhylogenetic analysis instead examines all characters and then produces the most parsimonious relationship given the data. Taxonomic groupings and diagnostic characters are therefore inferred from the phylogeny, rather than defined beforehand. This approach is not foolproof: the results will depend on which characters are included, how characters are defined, and depend on accurate coding and identifying informative characters. The advantage of the approach is that it is explicit and repeatable; evidence and assumptions used to classify specimens will be evident, along with any errors.\n\nA potential issue is that ontogenetic changes in morphology could confound the phylogenetic analysis. Ceratopsians experience marked morphological changes as they mature24–26,52, and clustering analysis using ontogenetic characters will group specimens on the basis of maturity, rather than common ancestry26. It follows that either juveniles should be excluded, or ontogenetically variable characters must only be coded for adults. This problem is not unique to a phylogenetic approach; classic approaches to taxonomy are likewise misled when ontogeny is not taken into account (e.g., misidentification of the juvenile Chasmosaurus “brevirostris” as a distinct species).\n\nThe use of a specimen-level phylogeny reveals a conflict between evolution and taxonomy. Species are identified by the existence of discontinuities between populations71 either in terms of reproductive isolation or morphological differences71. Yet if evolution proceeds incrementally via the successive accumulation of slight variation71,72, we should expect variation to be continuous, rather than discrete, and the boundaries between species should be blurry.\n\nIt follows that when discontinuities do exist such that an extant population is separated from other populations, this must be an artifact of extinction, while discontinuities in the fossil record must be an artifact of sampling72. But where the fossil record is well sampled over a long period of time, we should expect intermediates between recognized species. The endpoints of a lineage, such as C. belli and C. irvinensis, may look distinct, but intermediates such as YPM 2016 will prove difficult to classify, and as additional intermediates are discovered, species boundaries will only become more blurred.\n\nWhere the lines are drawn is somewhat arbitrary. One could reasonably place the boundary between C. irvinensis and C. belli such that the Peabody specimen fell into either. Including YPM 2016 in C. irvinensis emphasizes that it forms a monophyletic assemblage with irvinensis specimens to the exclusion of other Chasmosaurus, and would emphasize their apomorphies. Including YPM 2016 in C. belli emphasizes the plesiomorphies shared with that species. All species concepts are ultimately statements about evolutionary lineages73, but to be more specific, all species concepts involve identifying lineages and then ranking them. Biologists or paleontologists must first identify lineages, and then decide whether these lineages differ enough from other segments of the tree to merit rank as a species. Phylogenies are a useful tool for the taxonomist because they allow us to assign organisms to lineages. However, this does nothing to resolve the issue of ranking. The question of how different one segment of a lineage must be from another to merit species or genus rank is still up to the taxonomist, and it remains as much an art as a science, subjective rather than objective, and always will.\n\nThe divisions are not, however, wholly arbitrary. As taxonomists, we want our names to apply to real entities. In the Linnaean worldview, these ‘real’ entities reflected the underlying logic of a Creator, who (like his human creations) thought about the world in terms of discrete categories. In a post-Darwinian worldview, groupings such as genera and species reflect common ancestry72. While the boundaries chosen between species may be human constructs, they must reflect real patterns of descent; species and genera must represent groups united by common ancestry74.\n\nAnother issue raised by specimen-level analysis is that recognized species and genera may fail to emerge as monophyletic. Two of the three species of Chasmosaurus form paraphyletic assemblages in the current analysis. Instead, the three species lie inside one another like Russian dolls, with C. irvinensis nesting inside of C. belli, which in turn lies inside of C. priscus.\n\nIf one advocates a taxonomy based on monophyletic taxa74,75, one can attempt to either split or lump lineages to create monophyletic species. In the case of Chasmosaurus, one approach would be to name the side branches that diverge on the way to C. irvinensis; the Peabody skull would then be given its own species. However, splitting the Peabody specimen off fails to render the remaining C. belli monophyletic (Figure 6C); instead all of the lineages branching off below C. irvinensis must become species to create a monophyletic taxonomy. Furthermore, each time a new specimen was recognized as either intermediate or an outgroup, another species would be necessary, and so on.\n\nAnother approach is to lump the specimens and treat the entire clade as one species, C. belli, evolving through time. This approach might better characterize the diversity of the DPF, in that there may have been only one species of present at any given time7. However, it obscures the marked differences in the frill, horns, and jaws within Chasmosaurus, and if more primitive or more derived members of Chasmosaurus were identified, these would have to be included to maintain monophyly, no matter how distinct they appeared. If an even more primitive specimen was discovered- part of the ancestral population that gave rise not only to Chasmosaurus but also to the lineage including Pentaceratops and Triceratops- then it would be necessary to subsume all the genera into one to maintain monophyly.\n\nSplitting or lumping to enforce monophyly may be effective up to a point, particularly when the record is poorly sampled. But both are half-measures and fail to deal with the fundamental problem: enforcing the criterion of monophyly in a classificatory scheme74,75 is inherently incompatible with ranked taxa such as genera and species76,77. The heart of the issue is the existence of ancestors. Species and genera must descend from other species and other genera72. The ancestral taxon, by definition, does not include its descendants, and so is paraphyletic. Ancestors may be difficult to identify with confidence but this does not mean that they did not exist. They must exist if our understanding of evolution is correct, and the completeness of the fossil record is such that ancestor-descendant pairs should be relatively common78.\n\nAmong chasmosaurines, potential ancestors include C. priscus as an ancestor for C. belli, and C. belli as an ancestor to C. irvinensis. T. horridus lacks autapomorphies26,66 and T. prorsus is nested within T. horridus17, and therefore may be ancestral to T. prorsus. Titanoceratops ouranos lacks autapomorphies3 and therefore could potentially be ancestral to the remainder of the Triceratopsini. Among modern dinosaurs, the common Canada Goose (Branta canadensis) is an extant ancestral taxon: mitochondrial DNA indicates that various populations of the species are paraphyletic with respect to a Hawaiian clade containing the Nene (B. sandvicensis) and two extinct flightless species of Branta79. The existence of such ancestors means that without abandoning ranked taxa – including genera and species- it is logically impossible to create a complete taxonomy based on the criterion of monophyly76,77.\n\nFinally, the taxonomy presented also demonstrates that the common practice of using autapomorphies to diagnose taxa is impractical. The nature of an ancestral species is that it will be distinguished from its descendants by plesiomorphies. C. belli for example, can be distinguished from C. priscus by apomorphies, but can only be distinguished from C. irvinensis by plesiomorphic characters. Similarly, the features used to distinguish T. horridus from T. prorsus26,66 are all plesiomorphies. Ancestral taxa will be diagnosed by a unique combination of apomorphies and plesiomorphies, rather than a list of autapomorphies.\n\nAs shown by the case of Chasmosaurus, specimen-level phylogenetic analysis is a powerful tool for creating phylogenies, which can in turn be used for delimiting and diagnosing species. The best approach to taxonomy is to start with phylogeny; the tree should come first, and names and diagnoses after. The inevitable consequence of this approach is that it will create paraphyletic taxa. A degree of arbitrariness is unavoidable; there is no scientific way to determine how distinct a taxon must be to merit its own genus, and the boundaries between species will only be distinct insofar as our record of evolution is poor.",
"appendix": "Author contributions\n\n\n\nNRL conceived, designed, and executed the research, and wrote the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests.\n\n\nGrant information\n\nNRL was funded by a Donnelley Postdoctoral Fellowship and the Yale Institute for Biospheric Studies.\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\nThanks to the curators and staff of the Yale Peabody Museum, American Museum of Natural History, Canadian Museum of Nature, and Royal Ontario Museum for specimen access.\n\n\nReferences\n\nDodson P, Forster CA, Sampson SD: Ceratopsidae. In: Weishampel DB, Dodson P, Osmolska H, editors. The Dinosauria. Second ed. Berkeley: University of California Press; 2004; 494–513.\n\nDodson P: The Horned Dinosaurs. 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Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Bloomington: Indiana University Press. 2005; 221–34. Reference Source\n\nRyan MJ, Evans DC: Ornithischian Dinosaurs. In: Currie PJ, Koppelhus EB, editors. Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Bloomington: Indiana University Press. 2005; 312–48. Reference Source\n\nArbour VM, Currie PJ: Euoplocephalus tutus and the diversity of ankylosaurid dinosaurs in the Late Cretaceous of Alberta, Canada, and Montana, USA. PLoS One. 2013; 8(5): e62421. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLongrich NR, Currie PJ: A microraptorine (Dinosauria-Dromaeosauridae) from the Late Cretaceous of North America. Proc Natl Acad Sci U S A. 2009; 106(13): 5002–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCurrie PJ: Theropods, including birds. In: Currie PJ, Koppelhus EB, editors. Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Bloomington: Indiana University Press, 2005; 367–97. Reference Source\n\nLongrich NR, Barnes K, Clark S, et al.: Caenagnathidae from the upper Campanian Aguja Formation of west Texas, and a revision of the Caenagnathinae. Peabody Museum Bulletin. 2013; 54(1): 23–49. Publisher Full Text\n\nLongrich N: A new, large ornithomimid from the cretaceous Dinosaur Park Formation of Alberta, Canada: implications for the study of dissociated dinosaur remains. Palaeontology. 2008; 51(4): 983–97. Publisher Full Text\n\nLongrich N: An ornithurine-dominated avifauna from the Belly River Group (Campanian, Upper Cretaceous) of Alberta, Canada. Cretaceous Res. 2009; 30(1): 161–77. Publisher Full Text\n\nSankey JT, Brinkman DB, Fox RC, et al.: Patterns of Distribution of Mammals in the Dinosaur Park Formation and the Paleobiological Significance. In: Currie PJ, Koppelhus EB, editors. Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Bloomington: Indiana University Press; 2005; 437–49. Reference Source\n\nFox RC: Late Cretaceous mammals. In: Currie PJ, Koppelhus EB, editors. Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Bloomington: Indiana University Press; 2005; 417–35. Reference Source\n\nMallon JC, Evans DC, Ryan MJ, et al.: Megaherbivorous dinosaur turnover in the Dinosaur Park Formation (upper Campanian) of Alberta, Canada. Palaeogeogr Palaeoclimatol Palaeoecol. 2012; 350–352: 124–38. Publisher Full Text\n\nTanke DH: Identifying Lost Quarries. In: Currie PJ, Koppelhus EB, editors. Dinosaur Provincial Park: A Spectacular Ancient Ecosystem Revealed. Bloomington: Indiana University Press, 2005; 34–53. Reference Source\n\nMallon JC, Holmes R, Anderson JS, et al.: New information on the rare horned dinosaur Arrhinoceratops brachyops (Ornithischia: Ceratopsidae) from the Upper Cretaceous of Alberta, Canada. Can J Earth Sci. 2014; 51(6): 618–34. Publisher Full Text\n\nSampson SD, Ryan MJ, Tanke DH: Craniofacial ontogeny in centrosaurine dinosaurs (Ornithischia: Ceratopsidae): taxonomic and behavioral implications. Zool J Linn Soc. 1997; 121(3): 293–337. Publisher Full Text\n\nFarke AA: Evolution, homology, and function of the supracranial sinuses in ceratopsian dinosaurs. J Vertebr Paleontol. 2010; 30(5): 1486–500. Publisher Full Text\n\nForster CA: New information on the skull of Triceratops. J Vertebr Paleontol. 1996; 16(2): 246–58. Publisher Full Text\n\nLongrich NR, Sankey JT, Tanke DH: Texacephale langstoni, a new genus of pachycephalosaurid (Dinosauria: Ornithischia) from the upper Campanian Aguja Formation, southern Texas, USA. Cretaceous Res. 2010; 31(2): 274–84. Publisher Full Text\n\nFarke AA: Cranial osteology and phylogenetic relationships of the chamosaurine ceratopsid, Torosaurus latus. In: Carpenter K, editor. Horns and Beaks: Ceratopsian and Ornithopod Dinosaurs. Bloomington: Indiana University Press, 2006; 235–57.\n\nWu XC, Brinkman DB, Eberth DA, et al.: A new ceratopsid dinosaur from the uppermost Horseshoe Canyon Formation (upper Maastrichtian), Alberta, Canada. Can J Earth Sci. 2007; 44(9): 1243–65. Reference Source\n\nMaddison WP, Maddison DR: Mesquite: a modular system for evolutionary analysis. Version 2.75. 2011. Reference Source\n\nFarke AA: Anatomy and taxonomic status of the chasmosaurine ceratopsid Nedoceratops hatcheri from the Upper Cretaceous Lance Formation of Wyoming, U.S.A. PLoS One. 2011; 6(1): e16196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwofford DL: Paup*. Phylogenetic Analysis Using Parsimony (*and other methods). 4.0b10 ed. Sunderland, Massachusetts: Sinauer Associates; 2002. Reference Source\n\nGoloboff PA, Farris JS, Nixon KC: TNT, a free program for phylogenetic analysis. Cladistics. 2008; 24(5): 774–86. Publisher Full Text\n\nSampson SD: Two new horned dinosaurs from the Upper Cretaceous Two Medicine Formation of Montana; with a phylogenetic analysis of the Centrosaurinae (Ornithischia: Ceratopsidae). J Vertebr Paleontol. 1995; 15(4): 743–60. Publisher Full Text\n\nBraman DR, Sweet AR: Biostratigraphically useful Late Cretaceous–Paleocene Terrestrial palynomorphs from the Canadian Western Interior Sedimentary Basin. Palynology. 2012; 36(Supp 1): 8–35. Publisher Full Text\n\nGrant PR, Grant BR: Unpredictable evolution in a 30-year study of Darwin's finches. Science. 2002; 296(5568): 707–11. PubMed Abstract | Publisher Full Text\n\nGrant PR: Ecology and Evolution of Darwin's Finches. Princeton: Princeton University Press; 1986. Reference Source\n\nForster CA: Species resolution in Triceratops: cladistic and morphometric approaches. J Vertebrate Paleontol. 1996; 16(2): 259–70. Publisher Full Text\n\nGeist V: The Evolution of horn-like organs. Behaviour. 1966; 27(3/4): 175–214. Reference Source\n\nFarlow JO, Dodson P: The behavioral significance of frill and horn morphology in ceratopsian dinosaurs. Evolution. 1975; 29(2): 353–61. Publisher Full Text\n\nKnell RJ, Naish D, Tomkins JL, et al.: Sexual selection in prehistoric animals: detection and implications. Trends Ecol Evol. 2012; 28(1): 38–47. PubMed Abstract | Publisher Full Text\n\nKonishi T: Redescription of UALVP 40, an unusual specimen of Chasmosaurus Lambe, 1914 (Ceratopsidae: Chasmosaurinae) bearing long postorbital horns, and its implications for ontogeny and alpha taxonomy of the genus1. Can J Earth Sci. 2015; 52(8): 608–19. Publisher Full Text\n\nSimpson GG: Tempo and Mode in Evolution. New York: Columbia University Press; 1944; 237.\n\nDarwin CR: The Origin of Species. London: John Murray; 1859.\n\nde Queiroz K: The general lineage concept of species, species criteria, and the process of speciation: A conceptual unification and terminological recommendations. In: Howard DJ, Berlocher SH, editors. Endless Forms Species and Speciation. Oxford, England: Oxford University Press. 1998; 57–75. Reference Source\n\nde Queiroz K, Gauthier J: Toward a phylogenetic system of biological nomenclature. Trends Ecol Evol. 1994; 9(1): 27–31. PubMed Abstract | Publisher Full Text\n\nHennig W: Phylogenetic Systematics. Urbana: University of Illinois Press; 1966.\n\nSosef MSM: Hierarchical models, reticulate evolution and the inevitability of paraphyletic supraspecific taxa. Taxon. 1997; 46(1): 75–85. Publisher Full Text\n\nBrummit RK: How to chop up a tree. Taxon. 2002; 51: 31–41. Reference Source\n\nFoote M: On the probability of ancestors in the fossil record. Paleobiology. 1996; 22(2): 141–51. Reference Source\n\nPaxinos EE, James HF, Olson SL, et al.: mtDNA from fossils reveals a radiation of Hawaiian geese recently derived from the Canada goose (Brantacanadensis). Proc Natl Acad Sci U S A. 2002; 99(3): 1399–404. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11622",
"date": "19 Jan 2016",
"name": "Jordan Mallon",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper tackles a difficult subject: the taxonomy of the horned dinosaur Chasmosaurus. Researchers have been debating the matter for years, such that Chasmosaurus is arguably among the most problematic ceratopsid genera to date. In this sense, I applaud Longrich for being brave enough to take on such a difficult problem. However, I have many issues with his paper that I think should preclude its acceptance. These are as follows:1. On my first read through the paper, I found numerous typos and sentence fragments throughout. Longrich also waffles in his use of terminology (e.g., posterior vs. caudal; epiparietals vs. epoccipitals; chasmosaurs vs. chasmosaurines; centrosaurs vs. centrosaurines; P1 vs. p1 epiparietals, etc., etc., etc.). Also, the Systematic Paleontology section appears twice. This made for difficult and confusing reading, and the paper could have used a thorough going-over before submission.2.The figures are okay, but not great (e.g., Figure 3B appears out of focus; Figure 4C is washed out; sometimes the line overlays don't connect the labels to their respective anatomical details; some figure abbreviations are not provided). I felt that additional interpretive line drawings would have been helpful because the skull sutures can be difficult to see on the small figures. For example, it would be nice to see a tracing of the parietal-squamosal sutures of YPM 2016, details of the maxilla/nasal/premaxilla/jugal intersection, bones surrounding the infratemporal fenestra, etc. This specimen is crucial to Longrich's argument, and so should be illustrated properly. A figure of the occiput would be hepful for the sake of completeness, too, even if the area isn't particularly diagnostic. For such an important specimen, the table of measurements is pretty bare.3. Much of the terminology is out of date and/or confusing. For example, we now use 'epiparietals'/'episquamosals'/'epiossifiations', not the old and inaccurate 'epoccipitals'. Frontoparietal fossa, not frontal fossa. Where Longrich relies on his own terminology, it quickly becomes confusing (e.g., narial process vs. nasal process; talk of 'lateral rami' of the posterior parietal bar vs. lateral parietal bars). The figure labels help, but they're not always reliable (e.g., where is the 'anteroventral fossa' of the premaxilla that he mentions?).4. Longrich doesn't interact with the latest literature -- particularly two recent papers of special relevance:Konishi, T. (2015). Redescription of UALVP 40, an unusual specimen of Chasmosaurus Lambe, 1914 (Ceratopsidae: Chasmosaurinae) bearing long postorbital horns, and its implications for ontogeny and alpha taxonomy of the genus. Canadian Journal of Earth Sciences, 52(8), 608-619.Campbell, J. A., Ryan, M. J., Holmes, R. B., & Schröder-Adams, C. J. (2016). A Re-Evaluation of the Chasmosaurine Ceratopsid Genus Chasmosaurus (Dinosauria: Ornithischia) from the Upper Cretaceous (Campanian) Dinosaur Park Formation of Western Canada. PloS one, 11(1), e0145805.These papers are critical to Longrich's recent work on chasmosaurines and, in the case of the latter paper, already does much of what Longrich has set out to do by identifying YPM 2016 as an evolutionary intermediate between C. belli and C. irvinensis (unfortunately, making much of Longrich's work less original/impactful). It is crucial that Longrich interacts with these articles and addresses their arguments regarding Chasmosaurus taxonomy and systematics. Admittedly, the last paper was still in press by the time this one was submitted.5. The anatomical descriptions are rife with interpretation (e.g., mention of 'primitive' and 'derived' features, talk of evolutionary convergence, etc.) that should be relegated to the Discussion.6. Longrich's cladistic methodology is unclear. For example, he says that he ran a heuristic algorithm until 69201 MPTs were obtained. But did he run the analysis to completion? Did he stop the analysis there? If so, why? As one of the commenters of the online version of the manuscript mentioned, the number of MPTs mentioned in Figure 6 is at odds with the number he gives in the text. Further, what was the outgroup? Were any Chasmosaurus specimens excluded from the analysis (e.g., C. canadensis type), and why? These outstanding questions, and the egregious absence of a character matrix provided as supplementary data, make replicating Longrich's results very difficult.7. In an offhand comment, Longrich mentions that \"characters previously used to diagnose Nedoceratops, including the orientation of the postorbital horns and the position of the squamosal are present only on one side of the skull... indicating that they are artifacts resulting from postmortem distortion.\" He does this to support his exclusion of this taxon from his cladistic analysis. However, it isn't enough to simply say this is the case. He needs to show it. How do we know that the diagnostic characters (whatever they are -- he doesn't say) weren't taken from the undistorted side of the skull?8. As mentioned above, Chasmosaurus taxonomy is a bit of a mess, but I'm afraid that Longrich's solution creates more confusion than clarity, and this is my biggest grievance with his latest contribution. His erection of Mojoceratops perifania (in 2010) to receive specimens traditionally assigned to Chasmosaurus canadensis/kaiseni was a mistake. At the time, Longrich reasoned that the types of C. canadensis/kaiseni were undiagnostic, but the C. kaiseni type falls out with Mojoceratops in this latest cladistic study (I'm not sure why the C. canadensis type was excluded), thereby contradicting his initial assertion that the specimen is undiagnostic. Therefore, Mojoceratops should rightly be called Chasmosaurus kaiseni (if not C. canadensis), but Longrich doesn't recognize this problem in his paper, and continues to use Mojoceratops without warrant.Further, I would argue that erecting yet another new species of Chasmosaurus (his C. priscus) is presently unwise. By Longrich's own admission, his tree topology for Chasmosaurus is only weakly supported (very low bootstrap and Bremer support values), so it's difficult to say with any confidence that CMN 2280 (his new type for C. priscus) is as 'primitive' as he thinks. I imagine it would only take one or two more steps to unite CMN 2280 with his C. belli.Erecting C. priscus as a new species is also problematic because it is at odds with the philosophy adopted in his paper. Longrich (I think rightly) wants to pay tribute to evolutionary heritage by sinking Vagaceratops into Chasmosaurus, but he is being inconsistent by then wanting to erect a new paraphyletic species (which isn't even figured in the paper, except for a small thumbnail in Figure 12, which I find irritating).Coining the name 'Euceratopsia' in this paper, which isn't even referred to in the main text, is also both extravagant and unnecessary.(None of this is to say anything about the preferability of naming paraphyletic taxa, which is a highly contentious issue of its own, and one that merits careful consideration.)At the end of the day, I don't think the matter of Chasmosaurus taxonomy is going to be settled without careful consideration of both ontogeny and biostratigraphy, both of which garner short shrift from Longrich in this paper, and are more thoroughly treated in Campbell, 2016 (see above). One of the outstanding issues is that many of the original Chasmosaurus quarries are still unidentified and/or not located in section. This is the necessary groundwork that is going to need to be done before we can get a handle on correlating morphology (with all its individual variation) with stratigraphy. The fact that the C. russelli holotype (CMN 8800) comes from high in section near the Lethbridge Coal Zone (alongside C. irvinensis; see Campbell, 2016), and not low in section as Longrich indicates, suggests that Longrich's new taxonomy is at odds with the fossil record and that his proposed anagenetic scenario may be overly simplistic.In light of the totality of these concerns, I think this study needs major reconsideration and should not be indexed in its present form.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1468
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https://f1000research.com/articles/4-337/v1
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23 Jul 15
|
{
"type": "Case Report",
"title": "Case Report: A case report of unstable Hangman fracture in a eighty year old male",
"authors": [
"Sunil Munakomi",
"Binod Bhattarai",
"Binod Bhattarai"
],
"abstract": "Herein we discuss a rare variant of hangman’s fracture in an eighty year old male presenting without any neurological deficits. We performed X-ray and magnetic resonance imaging (MRI) of the cervical spine to confirm the diagnosis. The patient was placed on a cervical traction which showed good reduction. We performed posterior occipitocervical fusion with bone graft fusion followed by early mobilization. A postoperative scan showed good reduction and purchase of the screws. This case highlights the importance of choosing the correct therapeutic attitude for the management of the geriatric population especially in those who do not have any significant co-morbid conditions.",
"keywords": [
"hangman’s fracture",
"unstable",
"management"
],
"content": "Introduction\n\nRigid immobilization alone is sufficient for most cases of hangman’s fracture (traumatic spondylolisthesis of C2) classified as Effendi type I and some of type II. Effendi type III fractures are very rare and invariably have neurological deficits because of impingement due to the facet dislocation on the spinal cord posteriorly1. Fracture instability is the presence of complete disruption of the annular and/or posterior ligament with forward and/or rotatory vertebral body slip of axis2. Surgical stabilization and rigid immobilization together is recommended in such cases, such as Levine-Edwards type IIa and III fractures. Here we discuss the management of an unstable type III hangman’s fracture in an aged patient without any neurological deficits. Most doctors choose traction and prolonged immobilization in a halo vest due to associated medical comorbidities and the anesthetic risks involved in this group3,4. However there is a high risk of nonunion, instability, persistent pain and a need for a prolonged period of halo immobilization5. Since our patient had a good Karnofsky performance score6, we opted for only posterior fusion so as to minimize the anesthetic risk involved with both anterior and posterior approaches. However, we chose a long segment occipitocervical screw and graft fusion so as to aid the healing process in the aged bone.\n\n\nCase report\n\nAn 80 year old man from the Tarai region of Nepal was brought to emergency with the chief complaint of falling from a swing after being pushed by his grandson 2 days prior. He complained of pain at the nape of his neck. Neurological examination did not reveal any features of radiculomyelopathy. The patient was placed in a cervical collar and an urgent X-ray of the cervical spine revealed presence of spondylolysthesis of the axis with significant translation and angulation (Figure 1). Magnetic resonance imaging (MRI) of the cervical spine revealed a type III hangman’s fracture with presence of pinching effect on the cord without any significant signal changes (Figure 2).\n\nThe patient was an ex-army serviceman and was in good health with good Karnofsky performance score6. There was no significant past medical or surgical illnesses. He had a habit of smoking marijuana previously. After explaining the disease condition, treatment options and the risks involved the patient was placed on cervical traction with a 6 kg load and was observed for features of realignment. Stringent care was taken to observe for features of over distraction. Because the fracture was an unstable type III variant, the decision of surgical fixation was taken. However, routine screening echocardiography revealed a cardiac ejection fraction of only 33%. Therefore we decided to go for occipitocervical fusion so as to minimize the anesthetic risk imposed to the patient from both anterior and posterior approaches. Intra-operatively there was fracture of the pars and the lamina of C2. Since there was no atlantoaxial dislocation, we opted for occipital and C1 and C3 lateral mass fixation. There is evidence of good results with short fixation of C1 and C3 only, but keeping in mind the risk of osteoporosis in this case, we wanted further anchorage from occipital fusion as well. Since there was good posterior realignment of the spinal lines after traction (Figure 3) and intra-operatively, we choose the posterior approach only to minimize the added risk of the anterior approach. Lateral mass screws were placed in C1 and C3 (Figure 4 and Figure 5). Bone graft harvested from iliac bone was placed in the C1 and C2 inter-space to further enhance the fusion process. The patient was started on dexamethasone (8 mg intravenously and then rapidly tapered off in the following 2 days). The patient was safely extubated. Neurological examination was normal. The patient was in complete bed rest for a week and then mobilized with support. A CT spine check after one week revealed good screw purchase (Figure 6) and good reduction of fracture segment (Figure 7). The patient was restricted to light weight bearing and was advised to keep the cervical collar for at least 6 weeks. The patient was started on calcium supplementation (tablet calcium 500 mg orally every 12 hours. The patient followed up in the outpatient department after 1.5 months walking on his own without any deficits.\n\n\nDiscussion\n\n“Hangman's fracture”, first coined by Schneider et al. in 19657 results from hyperextension of the upper cervical spine. There is fracture of the lateral mass and the pedicle of the axis with simultaneous disruption of the anterior longitudinal ligament allowing C2-C3 listhesis. Traumatic hangman’s fracture, in contrast to the judicial hangman's fracture, is caused from extension and compression of the upper cervical spine with rare cord injury8.\n\nThe most widely used classification for hangman's fractures was firstly described by Effendi et al.9 and later modified by Levine et al.10,11. Anterior approaches include anterior cervical disectomy and graft fusion12; posterior approaches include lateral mass, pedicle or transarticular screw placement13.\n\nAnterior discectomy and screw plate fixation is an effective, but not very popular technique due to difficulty in exposing the C2-C3 region14 and the elimination of C2-C3 rotation15. Direct screw fixation of C2 pars adds to the risk of injury to the vertebral artery15 and also there is the need for complete manual reduction of the fracture intra-operatively15,16.\n\nFusion of lateral masses of C1 and C3 for hangman's fractures minimizes risk of vertebral artery injury and displacement of fractured segments into the canal. The efficacy of this approach has been validated in a biomechanical study by Chittiboina et al.17\n\nThis study hereby highlights the importance of the treatment algorithm chosen for the management of unstable hangman’s fracture in geriatric patients. Patients with good Karnofsky performance score would benefit from long segment posterior fusion, rather than both anterior and posterior approaches which might increase the intra-operative risk. Managing such patients with a prolonged period of immobilization in a halo imposes a higher risk of nonunion.\n\n\nConclusion\n\nAge alone should not determine a doctor’s approach to the treatment of geriatric patients. By taking only age into account when deciding on treatment, we risk compromising effective management in elderly patients. Karnofsky performance scale6 is one reliable marker that helps in making such treatment decisions.\n\n\nConsent\n\nBoth written and verbal informed consent for publication of images and clinical data related to this case was sought and obtained from the son of the patient.",
"appendix": "Author contributions\n\n\n\nSM reviewed the literature, designed the study and formatted the paper. BB revised and edited the final format.\n\n\nCompeting interests\n\n\n\nNo competing interests.\n\n\nGrant information\n\nNo funding was involved in supporting this work.\n\n\nReferences\n\nChoi WG, Vishteh AG, Baskin JJ, et al.: Completely dislocated hangman’s fracture with a locked C2-3 facet. Case report. J Neurosurg. 1997; 87(5): 757–760. PubMed Abstract | Publisher Full Text\n\nMoon MS, Moon JL, Moon YW, et al.: Traumatic spondylolisthesis of the axis: 42 cases. Bull Hosp Jt Dis. 2001–2002; 60(2): 61–66. PubMed Abstract\n\nHadley MN, Browner C, Sonntag VK, et al.: Axis fractures: a comprehensive review of management and treatment in 107 cases. Neurosurgery. 1985; 17(2): 281–290. PubMed Abstract | Publisher Full Text\n\nSchneider RC, Livingston KE, Cave AJ, et al.: “Hangman’s fracture” of the cervical spine. J Neurosurg. 1965; 22: 141–154. PubMed Abstract | Publisher Full Text\n\nVaccaro AR, Madigan L, Bauerle WB, et al.: Early halo immobilization of displaced traumatic spondylolisthesis of the axis. Spine (Phila Pa 1976). 2002; 27(20): 2229–2233. PubMed Abstract | Publisher Full Text\n\nPéus D, Newcomb N, Hofer S: Appraisal of the Karnofsky Performance Status and proposal of a simple algorithmic system for its evaluation. BMC Med Inform Decis Mak. 2013; 13: 72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoullosa JL, Colli BO, Carlotti CG Jr, et al.: Surgical management of axis’ traumatic spondylolisthesis (Hangman's fracture). Arq Neuropsiquiatr. 2004; 62(3B): 821–826. PubMed Abstract | Publisher Full Text\n\nWilliams TG: Hangman’s fracture. J Bone Joint Surg Br. 1975; 57(1): 82–88. PubMed Abstract\n\nEffendi B, Roy D, Cornish B, et al.: Fractures of the ring of the axis. A classification based on the analysis of 131 cases. J Bone Joint Surg Br. 1981; 63-B(3): 319–327. PubMed Abstract\n\nLevine AM, Edwards CC: The management of traumatic spondylolisthesis of the axis. J Bone Joint Surg Am. 1985; 67(2): 217–226. PubMed Abstract\n\nLevine AM, Rhyne AL: Traumatic spondylolisthesis of the axis. Semin Spine Surg. 1991; 3: 47–60.\n\nDuggal N, Chamberlain RH, Perez-Garza LE, et al.: Hangman’s fracture: a biomechanical comparison of stabilization techniques. Spine (Phila Pa 1976). 2007; 32(2): 182–187. PubMed Abstract | Publisher Full Text\n\nPapagelopoulos PJ, Currier BL, Hokari Y, et al.: Biomechanical comparison of C1-C2 posterior arthrodesis techniques. Spine (Phila Pa 1976). 2007; 32(13): E363–370. PubMed Abstract | Publisher Full Text\n\nTraynelis VC, Fontes RB: Anterior fixation of the axis. Neurosurgery. 2010; 67(3 Suppl Operative): ons229–36; discussion ons236. PubMed Abstract | Publisher Full Text\n\nBristol R, Henn JS, Dickman CA: Pars screw fixation of a Hangman’s fracture: Technical case report. Neurosurgery. 2005; 56(1 Suppl): E204; discussion E204. PubMed Abstract\n\nEIMiligui Y, Koptan W, Emran I: Transpedicular screw fixation for type II Hangman’s fracture: A motion preserving procedure. Eur Spine J. 2010; 19(8): 1299–305. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChittiboina P, Wylen E, Ogden A, et al.: Traumatic spondylolisthesis of the axis: A biomechanical comparison of clinically relevant anterior and posterior fusion techniques. J Neurosurg Spine. 2009; 11(4): 379–87. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "9613",
"date": "11 Aug 2015",
"name": "Rajiv R. Ratan",
"expertise": [],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report describes a case of a Hangman's fracture without associated neurological symptoms. There are several problems with this report that limit its usefulness and message, First, a Hangman's fracture, which is defined as fractures of both pedicles or pars interarticularis of the C2 vertebra (axis), is not defined in the abstract. There is a classification protocol that has been developed by Levine and Edwards that is based on etiology and guides treatment, this is also not mentioned. Second, neurological impairment is only seen in 25% of cases, so the absence of neurological symptoms is the rule not the exception as implied by this abstract. I am not convinced that this case report adds significantly to our understanding of how to diagnose and treat Hangman's fracture.",
"responses": [
{
"c_id": "1517",
"date": "11 Aug 2015",
"name": "Sunil Munakomi",
"role": "Author Response",
"response": "Thank you for the report. We totally agree with your comments, but the purpose of our paper was to highlight the clinical importance of choosing the correct surgical management, barring the age factor, which do significantly play a major role in decision making in developing countries like ours. So, we wanted to highlight the importance of neurological status and the Karnofsky score in making such decisions in geriatric patients."
}
]
},
{
"id": "11119",
"date": "06 Nov 2015",
"name": "Virendra Deo Sinha",
"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\nWe have read with interest the case report \"A case report of unstable Hangman fracture in a eighty year old male\" by Munakomi et al .The authors need to be congratulated for highlighting their view in managing unstable Hangman fracture in the geriatric age group.The patient in your report was put on traction with six kilogram loading. It is an accepted fact that patients with type 3 Hangman fracture should not be subjected to traction due to risk of “iatrogenic hanging’’.Secondly, the radiology images published do not point towards severe osteoporosis. A DEXA scan could have been more informative as the decision to do occipito-cervical fusion in Hangman fracture exposes the patient to severe motion restriction in an already spondylotic spine.Finally, I presume C2 -C3 anterior cervical discectomy and fixation with casper plates would have been preferable than to subjecting the patient with an ejection fraction of 33% to surgery in prone position.",
"responses": []
},
{
"id": "9611",
"date": "03 Dec 2015",
"name": "George Wittenberg",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this is a well-written and interesting case report. As a single case report, it cannot be used to guide treatment. The outcome could have been different even though the authors suggest a very reasonable approach to the situation. I would agree with Dr. Ratan's comments, although I may be less of an expert on the surgical management of hangman's fracture. I think there would be a small benefit to medical knowledge to indexing this paper, as the management of this condition would likely be guided by both exam, radiological findings, and patient medical condition.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-337
|
https://f1000research.com/articles/4-1466/v1
|
17 Dec 15
|
{
"type": "Method Article",
"title": "The manufacture of filtered cannabis cigarettes: uniform particle distribution and combustion properties for consistent cannabinoid delivery",
"authors": [
"Jason A. Cranford",
"Donald C. Cooper",
"Jason A. Cranford"
],
"abstract": "Here we describe a manufacturing process for the production of commercial filtered Cranfords cannabis cigarettes (CN). Unlike production of filtered tobacco cigarettes, standardization in the manufacture of cannabinoid containing cigarettes is lacking. The numerous cannabis strains with variable cannabinoid content, differences in cultivation methods and variability in assembly associated with hand-rolled cannabis cigarettes makes consistent cannabinoid inhalation dosing challenging. To address the growing need for standardization in the manufacture of cannabis cigarettes we developed a process for the production of filtered CN using machine-rolled tobacco cigarette equipment. The processed CN packing density, particle size distribution and curing procedures were designed to produce filtered CN that were identical in appearance and qualitatively similar in combustion properties to market-leading filtered commercial tobacco cigarettes. Quality control procedures were implemented to assure consistency in the manufacturing process and minimize variability associated with cigarette production such as inconsistencies in packing density, particle size, and combustion rate of mainstream smoke. Passive inverted smoldering assessment indicated that CN cigarettes burned at a faster rate compared to commercial filtered tobacco cigarettes of similar density. Overall, it is expected that machine-rolled standardized cannabis cigarettes with control over filler particle sizes, packing density and smoldering rates will contribute to making inhalation dosing of cannabis cigarettes more effective for therapeutic use.",
"keywords": [
"Cranfords",
"cannabis cigarette",
"tobacco",
"cannabis",
"filler particle size",
"smoldering rate",
"cannabinoid delivery"
],
"content": "Introduction\n\nCannabis-based preparations have been used for the treatment of a variety of medical conditions. Although there are dozens of molecular variants of cannabinoids in a typical cannabis plant, the main psychoactive component of cannabis is Δ9 tetrahydrocannabinol (THC) converted from the decarboxylation of the biosynthetic precursor tetrahydrocannabinolic acid (THCA) during combustion. In 1997, the NIH reviewed scientific data concerning potential therapeutic uses for marijuana and hemp and found there may be beneficial medicinal effects and recommended that researchers develop alternative dosage forms (NIDA, 1997). Since then, at least four clinical trials have examined smoked cannabis compared with placebo for therapeutic uses (Abrams et al., 2007; Ellis et al., 2009; Ware et al., 2010; Wilsey et al., 2008). All four trials found a positive treatment effect with no serious adverse effects. Despite the wealth of data showing the medicinal effectiveness of cannabis and THC, the FDA has not approved the marijuana or hemp plant as medicine.\n\nThe most common route of THC administration is inhalation of smoke. The concentration of THC that may be inhaled from smoking a cigarette is determined by the THC content itself or the non psychoactive biosynthetic precursor THCA, which is present in the processed dried cannabis buds and small leaves. Each 700 mg CN cigarette contains less than 0.4% (~3 mg) THC and ~11% (77 mg) THCA by weight. More than 95% of the potentially active THC resulting from smoking is derived from THCA, which is converted to active THC by heat-induced decarboxylation at temperatures above 115°C during combustion (Dussy et al., 2005). The direct relationship between temperature-dependent conversion of THCA to THC indicates that consistency in combustion across cigarettes is important for reliable THC dosing.\n\nOnce inhaled, the THC laden smoke is absorbed in the lungs within seconds and rapidly exerts its pharmacological activity via actions on the cannabinoid receptors located throughout the brain and body. Although several routes of administration exist, inhalation via smoking is the most common, least expensive and most effective route (Ohlsson et al., 1980). Compared to oral administration, inhalation administers more rapid peak plasma levels and pharmacological effects. By comparison, oral administration produces variable peak plasma levels in humans between 60–90 minutes and pharmacological actions between 120–180 minutes after intake, while inhalation produces peak plasma levels in 3 minutes and pharmacological effects in 10 minutes (Ohlsson et al., 1980). The rapid onset and dissipation of effects enables users to better self-titrate dosing. Unfortunately, it is difficult to determine the effective dosage of smoked cannabis, since the concentration of THC from smoking cannabis can be variable and subject to several factors related to the preparation including, growing conditions, the relative concentration of cannabinoids, the packing density of the material, the puff volume and frequency, the depth of inhalation into the lungs, additives that influence combustion and absorption of the cannabinoids in the lungs and temperature of the smoke.\n\nThe goal of this report is to describe a simple standardized procedure for the commercial production of machine-rolled filtered Cranfords cannabis cigarettes (CN) in a manner similar to commercial tobacco filtered cigarettes. To benefit from the therapeutic actions of inhaled THC it is important to produce a cigarette with consistent cannabinoid content and delivery. Improved THC dose consistency resulting from machine-rolling a filtered CN as an alternative to the variable unfiltered hand-rolled cannabis cigarettes is important because many of the therapeutic effects of inhaled THC arise within a narrow therapeutic low-dose window while at larger doses the psychoactive intoxicating actions can impair cognitive function, mood and judgment. The inclusion of a filter in the CN has advantages based on the utility of filters in tobacco cigarettes. For example, it provides a firm mouthpiece and permits the smoker to avoid direct contact with the cannabis. It can prevent particulates from entering the lungs and it reduces the temperature of the smoke entering the mouth. It is known that filters can selectively remove certain constituents of tobacco smoke, including phenols and alkylphenols (Baggett & Morie, 1973; Hoffmann & Wynder, 1963).\n\nWe describe a standardized method for the commercial production and manufacture of machine-rolled filtered CN with combustion properties similar to commercial tobacco cigarettes. Recent medicinal use legalization of cannabis cultivation, production and usage in Colorado and at least 23 other States has resulted in a growing industry that lacks coherence in cultivation and the type of regulatory oversight that exists in the pharmaceutical, food safety or tobacco industries. Establishing low-cost quality control tests is important, not only from a therapeutic THC dosing perspective, but also from a consumer protection perspective because contaminants like pesticides, herbicides, additives and toxins from fungi have been reported in processed cannabis (Llewellyn & O’Rear, 1977). It has been reported that few cannabis cultivation and processing operations follow industry best practices, largely because a historically illicit market led producers to compromise on plant health and contaminant safety in order to maximize yield instead of a safe, reliable product (Cohen & Ziskind, 2013). More reliable standardized cultivation and processing of cannabinoid delivery systems is important for both recreational and medicinal consumption. Tighter control and automation of the manufacturing process leading to a reliable commercial cigarette cannabinoid delivery device is a useful step towards this objective.\n\n\nMethods\n\nThe production of CN filtered cigarettes from Cannabis sativa L. are described as follows:\n\n1. Cannabis strains (Ghost train haze & Larry OG) of high THC (10–11% by weight) cannabis are organically grown without pesticides or added chemicals using nutrients/ingredients registered with OMRI (http://www.omri.org). OMRI offers independent review of brand name input products intended for organic farming and processing. OMRI's standards are based on the U.S. National Organic Program (NOP) and on the Canada Organic Regime (COR) standards. The plants are grown for ~120 days before hanging inverted under humidity and temperature controlled conditions (60% humidity, at 24°C for 11–13 days).\n\n2. After removing stems and seeds the plant buds, trichomes and small leaves (<3 cm) are chopped into small particles. Process chopping is accomplished using a rotating food processor blade for 5–7 minutes such that the final chopped material is between 0.04–10 mm in diameter before sifting with a 10 mm/10 mm opening/aperture stainless steel filter mesh screen.\n\n3. The plant material is homogenized in a batch cement-style mixer containing ~11.3 kg of material rotating ~20 turns/minute for 60 minutes.\n\n4. The plant material is humidified in a climate-controlled environment set at 60% humidity and 24°C under fan-mediated circulating air in 1 m × 0.6 m 0.6 m containers filled 75% full and rotated by inverting the storage container into an empty container every 12 hours for a period 2–3 days. The rotation is used to prevent the growth of fungi.\n\n5. The RYO cigarette rolling machine (RYO Machines LLC, Girard, OH) is prepared by lightly hand spraying cannabis contact points with food grade oil-based lubricant (e.g. vegetable, soy, canola, hemp, corn, etc) before loading the cigarette tube blanks. Unlike tobacco processing machines lubricant is required to prevent adherence of cannabis resin to the machine parts.\n\n6. To prevent plant material adhering to the pistons of the machine, the pistons that pack the plant material into the paper blanks are constructed of Teflon.\n\n7. The cigarette rolling machines are loaded with blank paper tubes (8 mm diameter, 70 mm length and a 12 mm filter) containing a ~1 cm cellulose acetate or cotton based filter. Filter tubes are loaded and the plant material is then filled into the tubes using a custom-made (Cranfords LLC) filling spout at a pressure of ~80 PSI designed to fill the tubes completely with processed plant material.\n\n8. The filling spouts are inserted into blank paper cigarette tubes to fill them with processed plant material.\n\n9. Filled cigarettes are collected in a tray approximately 0.3 m long × 0.1 m wide × 0.1 m deep and stored at ~30% humidity at 24°C for 12 hrs before packaging.\n\nParticle measurement in CN was determined by digital caliper measurements of individual randomly sampled cigarettes. Individual cigarettes containing 700 mg of material were emptied into a conical tube and shaken vigorously to separate the dried plant matter. A subsample was then emptied to a plate and 50 particles/cigarette were randomly selected and measured from a cross-sectional area of 0.04 to 90 mm2.\n\nInverted passive smoldering was determined by hanging single CN, Camel Blue (CB) (RJR Winston-Salem, NC), or Newport (NP) (RJR Winston-Salem, NC) cigarettes (82 mm length × 8 mm diameter) by the filter region in an open Ball® 16 oz canning jar immediately after igniting the cigarette for a period of 10–11 seconds. The smoldering coal for each cigarette was allowed to burn to a demarcated point where 5 cm of the cigarette was converted to ash with ambient temperature between 22–26°C and relative humidity of 20% and the smoldering rate was measured using a digital stopwatch.\n\n\nResults & discussion\n\nCigarette smoke from tobacco or cannabis is formed by the condensation of chemicals formed by the combustion of dried plant material, pyrolysis and pyrosynthesis, and aerosolized particles in the cooler region immediately behind the burning coal (Browne, 1990). The tobacco coal temperature reaches between 800–900°C, and the temperature of the smoke during a puff drops rapidly as it passes through the cigarette rod (Touey & Mumpower, 1957). Burning finer-cut tobacco creates an aerosol with smaller particles, which are easier to inhale. So changing the filler cut particle size can influence the aerosol and chemistry (Centers for Disease Control and Prevention, 2010). In general, a courser cut width of shredded tobacco increases the number of puffs per cigarette compared to finer cut widths. This is due to the fact that cigarettes containing more coarsely cut tobacco burn less efficiently than those with finer cut shreds (Geiss & Kotzias, 2007). CN packing particle size distribution was determined by measuring a random sample of the length and width of the processed cannabis particles in CN cigarettes (n = 6) from different batches (n = 3, n = 2/batch). The sorted CN cigarette plant material filler particle area distribution for the 50 sampled particles/cigarette is shown in Figure 1. Using this analysis we determined that 50% of the CN cigarette filler particles had an area between 0.04 mm2 and 3 mm2 and 50% had an area between 3 mm2 and 90 mm2. Approximately 90% of the CN particles measured were between 0.5 mm2 and 25 mm2 in area. The normalized particle distribution represents visually selected samples of the particle size range for each cigarette and was not intended to represent the weighted size distribution of the particle areas within each cigarette. Randomized non biased imaging or sorting methods of cigarette particles to establish the relative weighted distribution of particle areas are planned for future experiments.\n\nCN cigarettes were sampled (n = 6) for particle area distribution of the processed dried plant material and normalized to the largest particle observed. The minimum particles sampled were 0.04 mm2 and the largest sampled were 90 mm2. Coefficient values for the double exponential curve fit (solid black line) were y0 =1.0004, A1 = -0.89904, tau1 = 2.9679, A2 = -0.17192, tau2 =19,9972. Chi2 p = 0.03 using IGOR pro software (Wavemetrics, Inc). The sorted particle measurements were averaged and plotted with standard error bars for each of the 50 particles measured/cigarette.\n\nThe inverted smoldering rates for CB, NP and CN cigarettes are shown in Figure 2. CB burned the slowest followed by NP and CN. The faster smoldering rate observed in the CN cigarettes may be due to the packing density and differences in the shape of the processed leaves and buds in the CN cigarette compared to the more uniform linear filler-cut tobacco packing. It is also possible that the CN cigarettes burn at a higher temperature resulting in a faster rate of smoldering, however preliminary measurements do not support this interpretation. Lastly, tobacco cigarettes contain additives that may retard smoldering rate. Additives, like humectants (e.g. polyethylene glycol) are added to alter combustion and smoke quality whereas CN cannabis cigarettes contain no additives. This is the first report of a method for processing cannabis buds and short leaves as cannabis cigarette filler for a machine-rolled CN cigarette. The process is an important determinant of plant filler particle size and smoldering rate, which, in turn can regulate the THCA to THC conversion during combustion and ultimately the inhaled THC dose.\n\nCamel Blue cigarettes (CB) burned at a rate of 673 sec/5 cm, Newport (NP) cigarettes burned significantly faster at a rate of 588 sec/5 cm (t-test; n=10, ***p<0.001), Cranfords (CN) cigarettes burned the fastest at a rate of 527 sec/5 cm compared to CB (t-test; n=10, ***p<0.001) and NP (t-test; n=10, ## p<0.01). Bars represent the average and standard error bars. Two-tailed, t-tests were performed using Excel for Mac version 15.12 (Microsoft).\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for Figure 1, 10.5256/f1000research.7472.d109091 (Cranford & Cooper, 2015a).\n\nF1000Research: Dataset 2. Raw data for Figure 2, 10.5256/f1000research.7472.d109092 (Cranford & Cooper, 2015b).",
"appendix": "Author contributions\n\n\n\nJAC designed the protocol for manufacture and production of the cannabis and CN cigarettes and wrote the manuscript. DCC designed, executed and performed the experiments and statistical analysis and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nJAC is the owner and founder of Cranfords LLC, the manufacturer of Cranfords cannabis cigarettes. DCC has no declared competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbrams DI, Jay CA, Shade SB, et al.: Cannabis in painful HIV-associated sensory neuropathy: a randomized placebo-controlled trial. Neurology. 2007; 68(7): 515–21. PubMed Abstract | Publisher Full Text\n\nBaggett MS, Morie GP: Quantitative determination of phenol and alkylphenols in cigarette smoke and their removal by various filters. Tobacco Science. 1973; 17: 30–2. Reference Source\n\nBrowne CL: The Design of Cigarettes. 3rd ed. Charlotte (NC): Hoechst Celanese Corporation, 1990.\n\nCenters for Disease Control and Prevention (US); National Center for Chronic Disease Prevention and Health Promotion (US); Office on Smoking and Health (US): How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General. Atlanta (GA): Centers for Disease Control and Prevention (US). 2010. PubMed Abstract\n\nCohen M, Ziskind J: Preventing Artificial Adulterants and Natural Contaminants in Cannabis Production: Best Practices. BOTEC Analysis Corp. I-502 Project #430-1b, 2013. Reference Source\n\nCranford JA, Cooper DC: Dataset 1 in: The manufacture of filtered cannabis cigarettes: Uniform particle distribution and combustion properties for consistent cannabinoid delivery. F1000Research. 2015a. Data Source\n\nCranford JA, Cooper DC: Dataset 2 in: The manufacture of filtered cannabis cigarettes: Uniform particle distribution and combustion properties for consistent cannabinoid delivery. F1000Research. 2015b. Data Source\n\nDussy FE, Hamberg C, Luginbühl M, et al.: Isolation of Delta9-THCA-A from hemp and analytical aspects concerning the determination of Delta9-THC in cannabis products. Forensic Sci Int. 2005; 149(1): 3–10. PubMed Abstract | Publisher Full Text\n\nEllis RJ, Toperoff W, Vaida F, et al.: Smoked medicinal cannabis for neuropathic pain in HIV: a randomized, crossover clinical trial. Neuropsychopharmacology. 2009; 34(3): 672–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGeiss O, Kotzias D: Tobacco, Cigarettes and Cigarette Smoke: An Overview Institute for Health and Consumer Protection. 2007. Reference Source\n\nHoffmann D, Wynder EL: Filtration of phenols from cigarette smoke. J Natl Cancer Inst. 1963; 30: 67–84. PubMed Abstract\n\nLlewellyn GC, O’Rear CE: Examination of fungal growth and aflatoxin production on marihuana. Mycopathologia. 1977; 62(2): 109–112. PubMed Abstract | Publisher Full Text\n\nNational Institute of Drug Abuse, Report to the Director, United States of America: Workshop on the Medical Utility of Marijuana; Bethesda, MD: National Institutes of Health. 1997. Reference Source\n\nOhlsson A, Lindgren JE, Wahlen A, et al.: Plasma delta-9 tetrahydrocannabinol concentrations and clinical effects after oral and intravenous administration and smoking. Clin Pharmacol Ther. 1980; 28(3): 409–416. PubMed Abstract | Publisher Full Text\n\nTaylor DN, Wachsmuth IK, Shangkuan YH, et al.: Salmonellosis associated with marijuana: a multistate outbreak traced by plasmid fingerprinting. N Engl J Med. 1982; 306(21): 1249–1253. PubMed Abstract | Publisher Full Text\n\nTouey GP, Mumpower RC: Measurement of the Cigarette Zone Temperature of Cigarettes. Tobacco Science. 1957; 1: 33–37.\n\nWare MA, Wang T, Shapiro S, et al.: Smoked cannabis for chronic neuropathic pain: a randomized controlled trial. CMAJ. 2010; 182(14): E694–701. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilsey B, Marcotte T, Tsodikov A, et al.: A randomized, placebo-controlled, crossover trial of cannabis cigarettes in neuropathic pain. J Pain. 2008; 9(6): 506–21. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11614",
"date": "19 Jan 2016",
"name": "Kurt R. Illig",
"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\nCranford and Cooper describe a method for manufacturing cannabis cigarettes with the aim of providing a way to better control the reliability of cigarettes as a cannabinoid-delivery device. The research is conducted expertly, the methods are understandable, and the manuscript is easily readable. As a methods paper, there is sufficient detail for the reader to follow the processes described and how these were developed. I have only two concerns about this work. First, Cranford and Cooper measure particle sizes for plant material from six cigarettes (two cigarettes from each of three batches) and find that particle sizes are relatively uniform (Figure 1). However, there is a problem with their sampling procedure, which the authors themselves point out, namely that the \"normalized particle distribution represents visually selected samples of the particle size range for each cigarette...\". By visually selecting particle sizes, rather than random sampling, the authors bias their findings. The result is that the data shown in Figure 1 shows only how consistently these samples are selected, not the actual distribution of particle sizes within the cigarettes, nor how consistently particles of various sizes appear in each cigarette. To address this, the authors should sample particle sizes in Cranford cigarettes using random, unbiased sampling methods. A further enhancement to these results could be made by using image analysis software to determine particle sizes, which would also allow the authors to sample many more than 50 particles/cigarette (which seems like a very low proportion of the total particles available, considering the sizes of the particles relative to the cigarette).My second concern is that the effects reported are based on a fairly small number of cigarettes tested. For example, the particle size results are based on only six cigarettes (only two from each of three batches). The concern here is that the small number of cigarettes introduces too little (or, perhaps, too much) variability to know whether the results will be generalizable to large runs during manufacturing, or what the variability will be among different batches.",
"responses": []
},
{
"id": "11613",
"date": "20 Jan 2016",
"name": "James Bibb",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 describes a defined study which is informative and useful, and addresses an important need. It is clearly written and easy to understand.As a suggestion, the authors might consider showing the data for the quantitation which states \"each 700 mg CN cigarette contains less than 0.4% (~3 mg) THC and ~11% (77 mg) THCA by weight\".This is just a suggestion.Otherwise, the paper should be indexed as it now appears, in my opinion.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1466
|
https://f1000research.com/articles/4-1465/v1
|
17 Dec 15
|
{
"type": "Review",
"title": "Regulation of IL-10 and IL-12 production and function in macrophages and dendritic cells",
"authors": [
"Xiaojing Ma",
"Wenjun Yan",
"Hua Zheng",
"Qinglin Du",
"Lixing Zhang",
"Yi Ban",
"Na Li",
"Fang Wei",
"Wenjun Yan",
"Hua Zheng",
"Qinglin Du",
"Lixing Zhang",
"Yi Ban",
"Na Li",
"Fang Wei"
],
"abstract": "Interleukin-10 and Interleukin-12 are produced primarily by pathogen-activated antigen-presenting cells, particularly macrophages and dendritic cells. IL-10 and IL-12 play very important immunoregulatory roles in host defense and immune homeostasis. Being anti- and pro-inflammatory in nature, respectively, their functions are antagonistically opposing. A comprehensive and in-depth understanding of their immunological properties and signaling mechanisms will help develop better clinical intervention strategies in therapy for a wide range of human disorders. Here, we provide an update on some emerging concepts, controversies, unanswered questions, and opinions regarding the immune signaling of IL-10 and IL-12.",
"keywords": [
"IL-12",
"IL-10",
"immune signaling"
],
"content": "Interleukin-12 signaling\n\nInterleukin-12 (IL-12) is the first member of a family of heterodimeric cytokines identified1. It is a pro-inflammatory molecule produced primarily by professional antigen-presenting cells (APCs), including monocytes/macrophages and dendritic cells (DCs)2. IL-12 is composed of p35 (encoded by Il12a) and p40 (encoded by Il12b) chains, and it principally activates natural killer (NK) cells and induces the differentiation of naïve CD4+ T lymphocytes to become interferon-gamma (IFN-γ)-producing T helper 1 (Th1) effectors in cell-mediated immune responses to intracellular pathogens2. IFN-γ, in turn, acts on APCs to augment IL-12 secretion in a positive feedback loop3,4. The p40 chain can also form a dimer with p19 to give rise to IL-235, which is required for Th17 differentiation, function, and maintenance6. Similarly, the p35 chain can combine with Epstein-Barr-induced 3 (EBI3) to form IL-357, the latest addition to the IL-12 family, in induced regulatory T-cell population (referred to as iTr358) and in tolerogenic human DCs9. IL-12 and IL-23 have overlapping as well as distinct immunostimulatory activities6. IL-12 signals through the IL-12 receptor (IL-12R) comprised of the IL-12Rβ1 and IL-12Rβ2 subunits that are expressed on T cells, NK cells, and DCs10,11. IL-12 stimulates non-receptor Janus kinase 2 (JAK2) and tyrosine kinase 2 (TYK2) activities, leading to the phosphorylation of signal transducers and activators of transcription (STATs) (in particular, STAT4 homodimers)12,13. IL-35 is an immunosuppressive cytokine that signals through IL-12β2 and gp130, resulting in the heterodimeric formation and activation of STAT1 and STAT4, which in turn bind to the unique promoter regions of Ebi3 and Il12a14.\n\n\nRegulation of interleukin-12 production\n\nBoth Il12a and Il12b genes need to be expressed coordinately in the same cells to produce biologically active IL-1215. Paradoxically, the mRNA of Il12a is widely expressed in many cell types, albeit at low levels in some cells, most of which do not even produce IL-12. The Il12b mRNA is restricted to cells that can produce biologically active heterodimer16. Synthesis of the p35 chain was proposed to be a rate-limiting step for IL-12 production for its low abundance of transcripts in cells under steady-state conditions17. Over the past 20 years, a large number of molecular analyses have identified numerous transcription factors that bind to the promoter regions of Il12a and Il12b. The promoters of Il12a have been shown to bind transcription factors such as nuclear factor kappa B (NFκB) c-Rel (in DCs)18, c-Maf (as an inhibitor)19, and IFN regulatory factor 1 (IRF-1)20 in activated macrophages. Goriely et al. showed that lipopolysaccharide (LPS)- and IFN-γ-induced human Il12a gene activation was immediately preceded by a selective and rapid remodeling of a single positioned nucleosome within the -396/-241 region of the promoter containing critical Sp1-binding sites21. The same group also reported that, in human DCs activated through Toll-like receptor 3 (TLR3) and TLR4 but not TLR2, IRF-3 was recruited to an IFN-stimulated response element (ISRE) between -251 and -242 in the Il12a gene promoter. Accordingly, DCs from IRF-3-deficient mice were impaired in TLR4-induced Il12a mRNA expression and IL-12p70 synthesis22.\n\nInterestingly, a novel nuclear protein called GC-binding protein (GC-BP) was found in macrophages that engulf apoptotic cells via phagocytosis. GC-BP is activated via tyrosine phosphorylation induced by interactions between the phagocyte and the apoptotic cell expressing externalized phosphatidylserine. GC-BP has a direct and selective inhibitory activity on the transcription of the Il12a gene and IL-12 production23. It is speculated that this is part of the mechanisms that help suppress autoimmune responses to self-antigens during the clearance of apoptotic cells. This notion is consistent with the converse observation of the induction of IL-10 production during phagocytosis of apoptotic cells24.\n\nCompared with Il12a, the Il12b promoter has been more extensively studied, and numerous transcriptional factors have been identified as regulators for Il12b transcription. When murine macrophages are stimulated with LPS, nucleosome 1 is selectively remodeled so that the transcription factor CCAAT enhancer-binding protein β (C/EBPβ)/LAP could gain access to this region25. However, remodeling of nucleosome 1 alone is not sufficient for Il12b transcription and more factors are required for its induced expression. These factors include NFκB26,27, PU.128, IRF-129, nuclear factor in activated T cells (NFAT)30, and IFN consensus sequence-binding protein (ICSBP, also called IRF-8)31 in human or murine macrophages or both. Activation protein 1 (AP-1) has been reported to be an activator of Il12b transcription in LPS-stimulated macrophages32, whereas in tumor-derived prostaglandin E2 (PGE2)-treated macrophages, it appears to play the opposite role: inhibiting Il12b transcription and promoting tumor progression in vivo33. The controversy has not been resolved to date.\n\nGoodridge et al. observed that whilst LPS-induced p38 mitogen-activated protein kinase (MAPK) activation is required for the induction of both p40 and p35 subunits, extracellular signal-regulated kinase (ERK) signaling mediates negative feedback regulation of p40, but not p35, production34. Such ERK activation is downstream of calcium influx and targets LPS-induced Il12b transcription by suppressing the synthesis of the transcription factor IRF-1. In contrast, the negative regulation of the p35 subunit of IL-12 occurs via a calcium-dependent, but ERK-independent, mechanism, which was thought to involve NFκB signaling.\n\nCpG oligodeoxynucleotides (ODN) activates the TLR9/MyD88/TRAF6 (TNF receptor-associated factor 6) cascade leading to the activation of I kappa B kinase (IKK) -NFκB and JNK, which are critical for the production of pro-inflammatory cytokines. Ma et al. reported that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is involved in this activation process35. DNA-PKcs-deficient DCs exhibited a defect in the IL-6 and IL-12p40 expression in response to CpG-ODN in a dose- and time-dependent manner. Loss of DNA-PKcs impaired phosphorylation of IKK, IκBα, NFκB, and JNK in response to CpG-ODN35. TLR2-mediated production of IL-12p40 in monocytes and macrophages triggered by the synthetic ligand Pam3csk4 has been shown to activate the phosphorylation of JNK-1/2. Blocking JNK with a chemical inhibitor resulted in inhibition of Pam3csk4-induced p40 production36. However, the further downstream signaling is not clear.\n\nAt the transcriptional level, the differential regulation of Il12a and Il12b genes is well illustrated in macrophages derived from C/EBPβ-deficient mice. In sharp contrast to the enhanced induction of Il12b mRNA, C/EBPβ−/− primary macrophages derived from both the bone marrow and the peritoneal cavity displayed a totally defective expression of Il12a mRNA. This may explain the defective production of bioactive IL-12 and the impaired Th1 responses of C/EBPβ-deficient mice to Candida albicans, a pathogen that requires Th1-mediated control37. The enhanced p40 production in C/EBPβ-deficient macrophages is in direct contradiction to an earlier molecular study25. It cautions against directly extrapolating in vitro data for its in vivo relevance.\n\nAn important pathway in robust IL-12 induction is the requirement for “priming” of LPS-activated macrophages and DCs by IFN-γ for the expression of maximal amounts of Il12a and Il12b mRNAs and for IL-12 production4,20,38. The IFN-γ priming is a positive feedback mechanism for more robust IL-12 production in certain immune responses, as the primer IFN-γ is derived principally from NK cells and activated Th1 lymphocytes, cells that are initially activated by APC-derived IL-12 upon pathogen infection. Overall, inadequate investigations have been performed to elucidate this important feedback amplification mechanism in a comprehensive manner.\n\nNegishi et al. reported that MyD88-associated IRF-1 migrates into the nucleus more efficiently than non-MyD88-associated IRF-1. The critical role of MyD88-dependent “IRF-1 licensing” is underscored by the observation that the induction of a specific gene subset downstream of the TLR-MyD88 pathway, such as IFN-β, inducible nitric oxide (NO) synthase, and IL-12p35, is impaired in Irf1-deficient cells39. The study places IRF-1 as an additional member participating in MyD88 signaling and provides a mechanistic explanation for the enhancement of the TLR-dependent IL-12p35 induction program by IFN-γ.\n\nThe TLR-NFκB-dependent pathway inducing IL-12 and the IFN-dependent pathway inducing type I IFN (α and β) and IFN-regulated genes have also been shown to cooperate for the robust production of IL-12 in DCs. Gautier et al. reported that R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly (I:C) (TLR3 ligand) or LPS (TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-β mRNA accumulation by mouse bone marrow-derived DCs (BMDCs). Strikingly, IL-12p70, but not IL-12p40, secretion was strongly reduced in BMDCs from STAT1−/− and IFNAR−/− mice. STAT1 tyrosine phosphorylation, IL-12p35, and IFN-γ mRNA accumulations were strongly inhibited in IFNAR−/− BMDCs activated with the TLR ligand combinations. Similar observations were made by using neutralizing anti-IFNAR2 antibodies in human TLR8-expressing peripheral blood monocyte-derived DCs40. This study suggests that TLR engagement on DC induces endogenous IFNs that cooperate with the NFκB-inducing machinery for optimal IL-12p70 secretion.\n\nSignaling events from distinct classes of pathogen recognition receptors (PRRs) affect each other in modulating innate and adaptive immunity through modulating IL-12 production. Activation of cytosolic RIG-I-like receptors (RLRs) results in the selective suppression of TLR-induced transcription of the Il12b gene through the binding of RLR-activated transcription factor IRF-3 to the Il12b promoter, where it competitively edges out IRF-5, a transcriptional activator of Il12b that binds to the same sequence motif, the ISRE. IRF-5 binding in this region is usually accompanied with chromatin remodeling of both regulatory regions and the formation of a productive transcriptional complex containing other transcription factors41. Consequently, the activation of RLRs in mice attenuated TLR-induced Th1 and Th17 responses against viral infection of mice42. Similarly, Kim et al. identified a crosstalk between TLR4- and nucleotide-binding oligomerization domain 2 (NOD2)-mediated activities in the regulation of intestinal mucosal defense and tissue homeostasis via NOD2 signaling selectively interfering with TLR-induced Il12a gene expression and IL-12 production via the transcriptional regulator C/EBPα43.\n\nEmerging evidence has demonstrated that mammalian target of rapamycin (mTOR) is an important regulator of immunity by modulating the differentiation, activation, and function of lymphocytes and APCs44. In exploring the long-held “puzzle” of low levels of IL-12 induced through TLR4 signaling in macrophages and DCs, which implied the existence of stringent regulatory mechanisms, He et al. identified the critical regulatory roles of three protein kinases, mTOR, phosphoinositide-3 kinase (PI3K), and ERK, in TLR-induced Th1 responses by reciprocally controlling IL-12 and IL-10 production in innate immune cells of murine origin45. Moreover, it was revealed that c-fos was a key molecule that mediated the kinase-regulated IL-12 and IL-10 expression in TLR4 signaling by regulating c-fos expression and NFκB binding to the promoters of IL-12 and IL-10 in a differential manner45. These findings confirmed the role of c-fos in this capacity reported in an earlier study by Mitsuhashi et al.33 and were corroborated by a similar study in human DCs with an additional delineation of the opposing activities of the two components of the mTOR complex, mTORC1 and mTORC2, in this signaling pathway46. Thus, by controlling the balance between IL-12 and IL-10, mTOR can specifically regulate the TLR-induced T-cell response in vivo. Indeed, blockade of mTOR by rapamycin efficiently boosted TLR-induced antigen-specific T- and B-cell responses to hepatitis B virus and hepatitis C virus vaccines45. This study links a ubiquitously present and fundamentally important pathway of cellular survival, proliferation, and function to the production of a highly restricted specialist molecule in the immune system. Notably absent from the study is the answer to an obvious question: is the induction of IL-10 via mTOR signaling responsible for the inhibition of IL-12 production? Figure 1 summarizes our current understanding of the transcriptional mechanisms regulating the IL-12p40 promoter47.\n\nThe data are drawn primarily from macrophage studies. In dendritic cells, c-Rel is not required for IL12B transcription. F1 denotes a large molecular complex containing multiple transcription factors binding to the human IL12b promoter47. Green-arrowed lines indicate a stimulatory role for IL12b transcription, whereas red-arrowed lines denote the reverse. Continuous short arrows denote multiple steps involved that are not specified in details. Dashed lines indicate undetermined signaling pathway. The promoter coordinates are with respect to the transcription start site, designated +1, of the human IL12b gene. GAP-12 is a putative transcriptional repressor of unidentified nature that is induced by IL-4 or PGE2 treatment of human monocytes28. The asterisks denotes controversial transcriptional factors that are defined as repressors by mouse knockout studies but as activators in some in vitro studies (see text for details). Akt, Ak strain transforming; AP-1; activating protein 1; cAMP, cyclic adenosine monophosphate; C/EBP, CCAAT enhancer-binding protein; CpG, cytosine-phosphate-guanine; ds, double-stranded; Ets2, E26 2; GAP-12, GATA sequence in the IL-12 promoter; IRF, interferon regulatory factor; JNK, c-Jun N-terminal kinase; MyD88, myeloid differentiation primary response gene 88; mTOR, mammalian target of rapamycin; PGE2, prostaglandin E2; PK, protein kinase; Pol, polymerase; PU.1, purine.1; RLR, retinoic acid-inducible gene-I-like receptor; STAT, signal transduction and transcription; TLR, Toll-like receptor.\n\n\nInterleukin-10 signaling\n\nIL-10 was first discovered by complementary DNA clone-based screening for secreted factors by established Th2 cells that regulate cytokine production by activated Th1 cells48,49. IL-10 is a major immunosuppressive cytokine. It is a critical component in the maintenance of the fine balance between swift and potent immune responses against invading pathogens and the control of detrimental pathological injury. Almost all cells of the innate and adaptive arms of the immune system can produce IL-10, including DCs, macrophages, mast cells, NK cells, eosinophils, neutrophils, B cells, CD8+ T cells, CD4+ Th1, Th2, and Th17 cells50–58, and regulatory T (Treg) cells53,57,59. The major role of IL-10 is to limit the extent of the activation of both the innate and the adaptive immune cells to maintain a homeostatic state. This role of IL-10 is vitally important in protecting the host from infection-associated immunopathology, autoimmunity, and allergy, such as sepsis, arthritis, insulitis, inflammatory bowel disease (IBD), and so on. In addition to these activities, IL-10 regulates growth or differentiation (or both) of B cells, NK cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells51.\n\nThe IL-10 receptor is composed of at least two subunits that are members of the IFN receptor (IFNR) family, the ligand-binding subunit (IL-10Rα and IL-10R1)60,61, and the accessory subunit for signaling, IL-10R2 (IL-10Rβ)62,63. IL-10, produced from various cellular sources upon exposure to pathogens and inflammatory insults, binds to its receptor on target cells. Activation of the IL-10 receptor complex induces a tetramer consisting of two IL-10R1 and two IL-10R2 chains, which bind homodimeric IL-10 to the extracellular domains of IL-10R164. Upon the receptor-ligand engagement, phosphorylation of the receptor-associated protein tyrosine kinase JAK1 is recruited to the intracellular domain by the IL-10R1 chain, while non-receptor TYK2 is recruited to the receptor complex by IL-10R262. These kinases serve as a temporary docking site for inactive cytosolic STAT1 or STAT3 or both62, which are recruited by JAK1 and TYK2 to the site upon phosphorylation of the IL-10R1 chain at two tyrosine residues64. The STATs bind to the IL-10R1 chain via the Src homology 2 (SH2) domain and are tyrosine-phosphorylated by the receptor-associated JAKs. Activation of STAT3 leads to its homodimerization, similarly to STAT165,66. Translocation of activated STATs to the nucleus renders high-affinity binding to the promoter regions of IL-10-responsive genes. Successful engagement of the IL-10 receptor complex subsequently activates distinct JAK-STAT pathways and downstream signaling events that converge through various mechanisms to influence nuclear transcriptional events such as those mediated by NFκB67, resulting in the initiation of extensive anti-inflammatory and homeostatic programs.\n\nIt is important to note that the cellular source of IL-10 production is critical to its immunological activities in a cell-specific manner. Mice with a specific deletion in T cells generated by Cre/loxP-mediated targeting showed heightened contact hypersensitivity reactions and succumbed to severe immunopathology upon infection with Toxoplasma gondii. Splenocytes from these mice secreted increased amounts of pro-inflammatory cytokines after activation in vitro compared with wild-type (WT) control splenocytes. However, in contrast to complete IL-10 deficiency, sensitivity to endotoxic shock and skin irritant responses of the skin in the T-specific IL-10-deficent mice were not greater than those of the WT controls68. A critical role of B cell-derived IL-10 has been demonstrated in the mouse model of experimental autoimmune encephalomyelitis (EAE). Mice with a disruption in the Ig μ heavy chain (μMT), which results in a lack of B cells, develop a non-remitting form of EAE. Transfer of WT B cells restored remission, whereas B lymphocytes from IL-10-deficient mice were unable to suppress the disease progression52. Together, these studies highlight the distinctiveness of IL-10 derived from different cellular origins that determines its unique range of activities.\n\n\nRegulation of interleukin-10 production\n\nIL-10 production by macrophages and DCs through pathogen-associated molecular patterns (PAMPs) has been most widely studied. Macrophages produce IL-10 as a consequence of the recognition of PAMPs by its PRRs. Several classes of PRRs are expressed by macrophages, including TLRs, C-type lectin receptors, RIG-1 (retinoic acid-inducible gene 1) receptors, and NOD-like receptors69,70. The PAMPs bind to the TLRs with its TLR-interacting (TIR) domain, initiating signaling into macrophages with the help of intracellular adaptors that lead to the activation of multiple members of the MAPKs and subsequently transcription factors Sp171, C/EBPβ and δ72, c-Maf73, NFκB74, and phosphorylated cyclic AMP element-binding protein (CREB)75. TLRs can also act in synergy with other agonists such as IL-473 and PGE276,77 to augment IL-10 production. TLR3 or TLR4 activation results in the production of IFNβ, which sets up a feedback loop to sustain IL-10 mRNA induction78.\n\nB cells express a number of TLRs. Agonists that act via TLR2, TLR4, or TLR9 have all been shown to promote IL-10 production79–82. TLR9 activation in B cells stimulates activation of Bruton’s tyrosine kinase (Btk), and B cells from Btk knockout mice fail to secrete IL-10 following TLR9 stimulation. However, the molecular mechanism downstream of Btk is not clear. The role of Btk is not restricted to B cells, as Btk-deficient macrophages also secrete less IL-10 than WT cells83.\n\nCD4+ T cells have been identified as an important source of IL-10 in vivo84. Various transcription factors have been reported to induce IL-10 in T cells, including SP1, c-Jun, c-Maf, SMAD4, GATA3, and STATs84. However, the molecular signaling pathways that regulate IL-10 induction have not been fully delineated. The studies in this area have been complicated by the existence of multiple Th cell subsets, many of which can produce IL-10, including Th1, Th2, Th17, and Treg cells, albeit with different capacities. These observations have prompted the hypothesis that the IL-10 locus becomes differentially modified during Th cell polarization, which then invokes subtly different molecular mechanisms that drive IL-10 transcription in a quantitatively variable manner in the various T-cell subtypes85.\n\nIn contrast to the host response to infectious agents, clearance of apoptotic cells of a self-nature by phagocytes results predominantly in anti-inflammatory reactions characterized by the production of immunoregulatory cytokines IL-10, PGE2, and transforming growth factor beta (TGFβ)86, which are critical to ensuring cellular homeostasis and suppression of autoimmunity as an evolutionarily well-preserved mechanism. Chung et al. reported that the production of IL-10 in response to apoptotic cells is dependent on CD36, p38 MAPK, and the transcription factor TALE homeoprotein Pre-B-cell leukemia homeobox 1 (Pbx1)24. The study establishes a novel role of a developmentally critical factor in the regulation of homeostasis in the immune system and opens up a new area for future exploration at the intersection between cellular homeostasis and immune responses to exogenous pathogens as well as to endogenous danger signals.\n\n\nRegulation of interleukin-12 production by interleukin-10\n\nThe potency of IL-12 in host defense makes it a target for stringent regulation. Indeed, the temporal, spatial, and quantitative expression of IL-12 during an immune response in a microenvironment contributes critically to the determination of the type, extent, and ultimate resolution of the reaction. Breaching of the delicate control and balance frequently leads to immunologic disorders and pathogenesis. One of the most important and well-studied negative regulators of TLR-induced IL-12 production is IL-1087. IL-10 suppression of both IL12a and Il12b genes is seen primarily at the transcriptional level, and the inductions of the two genes have different requirements for de novo protein synthesis88. How IL-10 suppresses Il12a transcription is unknown at present. IL-10 targets an enhancer 10 kb upstream of the Il12b transcriptional start site that is bound by nuclear factor, interleukin 3-regulated (NFIL3), a B-ZIP transcription factor. Myeloid cells lacking NFIL3 produce excessive IL-12p40 and increased IL-12p7089. Thus, the STAT3-dependent expression of NFIL3 is a key component of a negative feedback pathway in myeloid cells that suppresses pro-inflammatory responses.\n\nKobayashi et al. observed that acetylated histone H4 transiently associated with the Il12b promoter in WT bone marrow-derived macrophages (BMDMs), whereas association of these factors was prolonged in Il10−/− BMDMs. Experiments using histone deacetylase (HDAC) inhibitors and HDAC3 short hairpin RNA indicate that HDAC3 is involved in histone deacetylation of the Il12b promoter by IL-10. These results suggest that histone deacetylation on the Il12b promoter by HDAC3 mediates the homeostatic effect of IL-10 in macrophages90. More details clearly need to be worked out to understand the important homeostatic regulation of IL-12 production by IL-10. In this context, the IL-4-inducing transcription factor c-Maf is an interesting molecule that can directly and conversely regulate IL-12 and IL-10 gene expression in activated macrophages19,91. Conversely, IRF-5 is a driver of the “M1” polarization of macrophages promoting Th1 and Th17 activities with activated transcription of inflammatory genes, including Il12a, Il12b, and Il23a, and repressed Il10 transcription92.\n\n\nInterleukin-12 in adoptive cell therapy for cancer\n\nIL-12 is able to activate all major cytotoxic killer and helper cell types of the immune apparatus (NK, NKT, CD4+, and CD8+ T cells) that are crucially important for immunosurveillance of and resistance to cancer development and progression93. The extraordinary anti-tumor efficacy of IL-12 has been demonstrated in animal models of cancer of diverse types94–105, and its use in various forms is now involved in a large number of human cancer clinical trials106. Adoptive cell therapy of malignant diseases takes advantage of the cellular immune system to recognize specific tumor-associated antigens and destroy cancer cells. This is remarkably demonstrated by redirecting T cells with a chimeric antigen receptor (CAR) toward CD19, inducing complete remission of leukemia in more than two thirds of patients in early-phase trials107. After initial tumor reduction by CAR T cells, antigen-negative cancer cells not recognized by CAR may give rise to tumor relapse. Fortunately, the “quagmire” may be overcome by CAR-mediated activation of T cells in the tumor, releasing inducible IL-12, which augments T-cell activation and attracts and activates innate immune cells to eliminate antigen-negative cancer cells in the targeted lesion. Chmielewski et al. demonstrated the feasibility of this strategy by redirecting T cells with a carcinoembryonic antigen (CEA)-targeting CAR and engineering with the inducible recombinant IL-12 expression cassette under the control of the NFAT/IL-2 minimal promoter108. In this context, IL-12 release was triggered by CAR signaling upon tumor antigen recognition and no IL-12 was detected in vitro without CAR signaling. The production capacity of such modified CAR T cells was sufficient to reach therapeutic levels without the need of repetitive drug application109. The therapeutic advantage is indicated by the fact that a dose of 105 IL-12 modified tumor-specific CAR T cells was more effective against established tumors than 106 T cells without IL-12 in a pre-clinical model110.\n\nTo date, despite the enhanced anti-tumor efficacy of IL-12-secreting CAR T cells in this model, the mechanisms associated with this enhanced tumor eradication remain unclear. Previous work showed that IL-12 reversed Treg cell-mediated suppression of CD4+ Foxp3− T-cell proliferation111. IL-12 was shown to induce IFN-γ production by Treg cells in vitro and in vivo112,113. However, IFN-γ expression did not decrease the ability of Treg cells to suppress T-cell proliferation114. Rather, IL-12 treatment decreased Treg cell frequency and Foxp3 levels in Treg cells. Furthermore, IL-12 increased IL-2R expression on effector CD4+ and CD8+ T cells, diminished its expression on Treg cells, and decreased IL-2 production by CD4+ and CD8+ T effectors. Together, these IL-12-mediated changes favored the outgrowth of non-Treg cells114. Kerkar et al. demonstrated that engineering tumor-specific CD8+ T cells to secrete IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma115. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b+F4/80hi macrophages, CD11b+MHCIIhiCD11chi DCs, and CD11b+Gr-1hi MDSCs, causing them to enhance the effects of adoptively transferred CD8+ T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. MHC I expression on host cells was essential for IL-12-mediated anti-tumor enhancements115. These studies point to the potential immunological mechanisms of the T cell-secreted IL-12 in tumor models.\n\nBased on prior pre-clinical studies demonstrating that IL-12-secreting CAR T cells are protected from inhibition by endogenous Treg cells (unpublished results), it is conceivable that IL-12-producing CAR T cells may be refractory to Treg cell-mediated inhibition and that previously requisite CAR-mediated T-cell “co-stimulation” (through CD28 or CD40L) may be overcome by CAR T cell-derived IL-12 secretion. In other words, CAR T cell-derived IL-12 may render the effectors independent of the “second signal” requirement “engraved” in classic T-cell activation paradigms. Furthermore, it is possible that IL-12 secretion within the tumor microenvironment can reverse the anergic state of endogenous tumor-infiltrating lymphocytes (TILs) and blunt the immune suppression by myeloid-derived suppressor cells (MDSCs) as well as modulation of the tumor-associated macrophages (TAMs) from a suppressive M2 phenotype to a pro-inflammatory M1 phenotype116–119.\n\n\nFuture perspectives\n\nIL-10 is a pleiotropic cytokine with a strong role in limiting the scope and extent of immune activation. Loss of IL-10 function has deleterious effects. Therefore, IL-10 could be a potential therapeutic agent for many inflammatory or autoimmune disorders. However, systemic IL-10 administration has proven to be of limited value120 and this indicates that IL-10 production would need to be carefully targeted to be efficacious therapeutically. This is evidenced by adoptive transfers of specific types of IL-10-producing immune cells in some autoimmune disease models that result in protection against the development of inflammatory pathologies121–127. Thus, a far more comprehensive and precise understanding of which IL-10-producing cells are important in vivo, and what the critical target cells of this IL-10 are would be instrumental in the future development of the therapeutic potentials of IL-10. The increased use of conditional gene targeting in mice will help in these future studies85.\n\nIn the intestinal mucosa, IL-10 is a well-established regulator of tissue inflammation and homeostasis. Mutations in the NOD2 gene are strongly associated with Crohn’s disease, a form of IBD believed to be driven by uncontrolled Th1 and Th17 responses128. There has been long a debate on the nature of the IBD-associated NOD2 mutations: “loss of function” or “gain of function”? Noguchi et al. showed that a common disease-related NOD2 mutation, 3020insC, displayed a “gain of function” property in that it suppressed IL10 transcription by blocking the phosphorylation of the nuclear ribonucleoprotein hnRNP-A1 (heterogeneous nuclear ribonucleoprotein A1) via the p38 MAPK129. This effect of 3020insC appears to be unique on the human IL-10 gene but not on its murine counterpart. The study challenges the present paradigms about the influence of the 3020insC mutation on Crohn’s disease, cautioning against deriving conclusions about the human disease on the basis of data from NOD2 knockout mice. It may provide a novel way of thinking about efforts to identify therapeutic targets for the treatment of Crohn’s disease and other Th1/Th17-mediated autoimmune diseases associated with the 3020insC mutation.\n\nAlthough a tremendous amount of knowledge has been gained about the signaling and function of IL-12 in immune cells since its discovery in 1989, many important questions remain. It is widely believed that the majority of the immunological activities of IL-12 are mediated through IFN-γ produced by activated NK and Th1 cells that have been exposed to APC-derived IL-12. However, considerable levels of IFN-γ-independent activities of IL-12 have been reported in many infectious disease and cancer models130–136. The cellular and molecular basis of the non-canonical activities of IL-12 await further elucidation. In immunotherapy of cancers, it has been long noted that the repeated administration of recombinant IL-12 could contribute to increased immunosuppressive properties of the tumor by the induction of IL-10137–139. Although the underlying molecular mechanism for the negative feedback is lacking, the finding that IL-12 is capable of potently inducing its own inhibitor reiterates the concept that the immune system is inherently equipped with an intrinsic negative feedback device that limits ongoing T-cell activation. This also indicates that the kinetics of T-cell responses may be regulated by the ratio of IL-12 and IL-10 levels, which may gradually decline during the immune response.\n\nEndotoxin tolerance, the transient, secondary downregulation of a subset of endotoxin-driven responses after exposure to bacterial products, is thought to be an adaptive response providing protection from pathological hyperactivation of the innate immune system during bacterial infection. IL-12 production is subjected to such a control mechanism. Wysocka et al. examined the development of IL-12 suppression during endotoxin tolerance in mice. The basis for decreased IL-12 production in vivo is clearly multifactorial, involving both loss of CD11chigh DCs as well as alterations in the responsiveness of macrophages and remaining splenic DCs. There is no demonstrable mechanistic role for B or T lymphocytes, the soluble mediators IL-10, TNF-α, IFN-α/β, nitric oxide, or the NFκB family members p50, p52, or RelB140. To date, the tolerance mechanism that inhibits IL-12 production by APCs remains elusive. The need for the understanding is underscored by the frequent occurrence of “immunological paralysis” subsequent to septic shock in patients. In the broad context, Foster et al. have provided some major insights into this phenomenon by proposing a model for the gene-specific regulation of class “T” (for tolerant) and “NT” (for non-tolerant) genes mainly through preferential transcription factor recruitment, histone acetylation, H3K4 trimethylation, and chromatin remodeling in tolerant versus non-tolerant macrophages141.\n\nThe acute-phase proteins, C-reactive protein and serum amyloid A (SAA), are biomarkers of infection and inflammation. He et al. reported a novel property of SAA in the differential induction of IL-12 and IL-23 in human peripheral blood monocytes142. SAA-induced IL-12p40 production was accompanied by a sustained expression of IL-23p19, but not IL-12p35, resulting in preferential secretion of IL-23, but not IL-12. The study identified SAA as a novel endogenous ligand that potentially activates the IL-23/IL-17 pathway, representing a novel mechanism for regulation of inflammation and immunity by an acute-phase protein. The differential production of IL-12 versus IL-23 was also observed in myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) stimulated via TLR ligands. Only mDCs but not pDCs secreted IL-23. Although pDCs produced both mRNA and protein of the p40 subunit, the lack of bioactive heterodimeric IL-23 protein release was due to the absence of translation of the p19 mRNA into protein143. These findings support the hypothesis of a coordinated adaptive immune response based on a finely tuned contribution of these cytokines by different DC subsets. How these endogenous and exogenous ligands induce IL-12 and IL-23 differentially at the molecular level bears both great scientific interests and practical implications.\n\nThe immunological activities of IL-12 are further complicated by the existence of IL-12p40 homodimer, IL-12p80, which acts as an IL-12 antagonist by binding to the IL-12R but which does not mediate a biological response144,145. Secretion of IL-12 is associated with excess production of IL-12p8016. For example, in contrast to the dogma about the restrictive nature of IL-12-producing cell types, meaningful amounts of IL-12p40 monomer and IL-12p80 have been observed in human breast cancer cells146, which could potentially thwart the IL-12-induced anti-tumor responses in vivo. Approximately 20% to 40% of the p40 in the serum of normal and endotoxin-treated mice is in the form of IL-12p80147. In IL-12-dependent shock models, exogenous IL-12p80 inhibits IL-12-induced cell-mediated immune response and protects mice from sepsis-associated death148. However, IL-12p80 has also been reported to stimulate, rather than inhibit, the differentiation of CD8+ Tc1 (type I cytotoxic T) cells in vitro, contrary to its suppressive activity on Th1 function149. The divergent functions of the various forms of p40 highlight our lack of understanding of its true range of biological activities.\n\nRecent pre-clinical studies demonstrated that treatment with CD19-specific, CAR T cells that secrete IL-12 is able to safely eradicate established disease without the sophisticated and laborious prior conditioning of subjects150. Moreover, in severe combined immunodeficient (SCID)-Beige mice with human ovarian cancer xenografts, IL-12-secreting CAR T cells exhibited enhanced anti-tumor efficacy as determined by an increased survival rate, prolonged persistence of T cells, a higher level of systemic IFN-γ, and modulated tumor microenvironment151. How the locally released IL-12 contributes to the highly favorable clinical efficacy and immunological modifications to numerous cell types in the tumor environment is an urgent and challenging task for the benefit of further improving this revolutionary therapeutic strategy for cancers of diverse types and progression states.\n\nIn summary, the complexity of the heterodimeric nature of both the cytokines and their receptors in the IL-12 family (also including IL-27) associated with the activation of different combinations of tyrosine kinases and STATs underlies the overlapping as well as distinct immunological consequences of the regulation and signaling in this cytokine group. Greater efforts are called for to better decipher the intricacies. In the meantime, more caution is needed in interpreting data derived from studies of individual cytokine or receptor chains.",
"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 a grant from the Natural Science Foundation of China (31370903) to XM and by a grant from the National Institutes of Health of the US (1R21AI101841-01) to XM.\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\nKobayashi M, Fitz L, Ryan M, et al.: Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med. 1989; 170(3): 827–45. PubMed Abstract | Free Full Text\n\nTrinchieri G: Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood. 1994; 84(12): 4008–27. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nO'Garra A, Barrat FJ, Castro AG, et al.: Strategies for use of IL-10 or its antagonists in human disease. Immunol Rev. 2008; 223(1): 114–31. PubMed Abstract | Publisher Full Text\n\nBaglaenko Y, Manion KP, Chang NH, et al.: Suppression of autoimmunity by CD5+ IL-10-producing B cells in lupus-prone mice. Genes Immun. 2015; 16(5): 311–20. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFrenkel D, Huang Z, Maron R, et al.: Neuroprotection by IL-10-producing MOG CD4+ T cells following ischemic stroke. J Neurol Sci. 2005; 233(1–2): 125–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGangi E, Vasu C, Cheatem D, et al.: IL-10-producing CD4+CD25+ regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis. J Immunol. 2005; 174(11): 7006–13. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLavasani S, Dzhambazov B, Nouri M, et al.: A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10 producing regulatory T cells. PLoS One. 2010; 5(2): e9009. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMatsushita T, Yanaba K, Bouaziz JD, et al.: Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J Clin Invest. 2008; 118(10): 3420–30. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nYang M, Deng J, Liu Y, et al.: IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation. Am J Pathol. 2012; 180(6): 2375–85. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nYang S, Li W, Liu W, et al.: IL-10 gene modified dendritic cells induced antigen-specific tolerance in experimental autoimmune myocarditis. Clin Immunol. 2006; 121(1): 63–73. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBrand S: Crohn's disease: Th1, Th17 or both? The change of a paradigm: new immunological and genetic insights implicate Th17 cells in the pathogenesis of Crohn's disease. Gut. 2009; 58(8): 1152–67. PubMed Abstract | Publisher Full Text\n\nNoguchi E, Homma Y, Kang X, et al.: A Crohn's disease-associated NOD2 mutation suppresses transcription of human IL10 by inhibiting activity of the nuclear ribonucleoprotein hnRNP-A1. Nat Immunol. 2009; 10(5): 471–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWang ZE, Zheng S, Corry DB, et al.: Interferon gamma-independent effects of interleukin 12 administered during acute or established infection due to Leishmania major. Proc Natl Acad Sci U S A. 1994; 91(26): 12932–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTaylor AP, Murray HW: Intracellular antimicrobial activity in the absence of interferon-gamma: effect of interleukin-12 in experimental visceral leishmaniasis in interferon-gamma gene-disrupted mice. J Exp Med. 1997; 185(7): 1231–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZilocchi C, Stoppacciaro A, Chiodoni C, et al.: Interferon gamma-independent rejection of interleukin 12-transduced carcinoma cells requires CD4+ T cells and Granulocyte/Macrophage colony-stimulating factor. J Exp Med. 1998; 188(1): 133–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoilides E, Tsaparidou S, Kadiltsoglou I, et al.: Interleukin-12 enhances antifungal activity of human mononuclear phagocytes against Aspergillus fumigatus: implications for a gamma interferon-independent pathway. Infect Immun. 1999; 67(6): 3047–50. PubMed Abstract | Free Full Text\n\nHafner M, Falk W, Echtenacher B, et al.: Interleukin-12 activates NK cells for IFN-gamma-dependent and NKT cells for IFN-gamma-independent antimetastatic activity. Eur Cytokine Netw. 1999; 10(4): 541–8. PubMed Abstract\n\nAfanasyeva M, Wang Y, Kaya Z, et al.: Interleukin-12 receptor/STAT4 signaling is required for the development of autoimmune myocarditis in mice by an interferon-gamma-independent pathway. Circulation. 2001; 104(25): 3145–51. PubMed Abstract | Publisher Full Text\n\nShi X, Liu J, Xiang Z, et al.: Gene expression analysis in Interleukin-12-induced suppression of mouse mammary carcinoma. Int J Cancer. 2004; 110(4): 570–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeyaard L, Hovenkamp E, Otto SA, et al.: IL-12-induced IL-10 production by human T cells as a negative feedback for IL-12-induced immune responses. J Immunol. 1996; 156(8): 2776–82. PubMed Abstract\n\nPortielje JE, Lamers CH, Kruit WH, et al.: Repeated administrations of interleukin (IL)-12 are associated with persistently elevated plasma levels of IL-10 and declining IFN-gamma, tumor necrosis factor-alpha, IL-6, and IL-8 responses. Clin Cancer Res. 2003; 9(1): 76–83. PubMed Abstract\n\nGerosa F, Paganin C, Peritt D, et al.: Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-gamma and interleukin-10. J Exp Med. 1996; 183(6): 2559–69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWysocka M, Robertson S, Riemann H, et al.: IL-12 suppression during experimental endotoxin tolerance: dendritic cell loss and macrophage hyporesponsiveness. J Immunol. 2001; 166(12): 7504–13. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFoster SL, Hargreaves DC, Medzhitov R: Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature. 2007; 447(7147): 972–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHe R, Shepard LW, Chen J, et al.: Serum amyloid A is an endogenous ligand that differentially induces IL-12 and IL-23. J Immunol. 2006; 177(6): 4072–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWaibler Z, Kalinke U, Will J, et al.: TLR-ligand stimulated interleukin-23 subunit expression and assembly is regulated differentially in murine plasmacytoid and myeloid dendritic cells. Mol Immunol. 2007; 44(7): 1483–9. PubMed Abstract | Publisher Full Text\n\nGillessen S, Carvajal D, Ling P, et al.: Mouse interleukin-12 (IL-12) p40 homodimer: a potent IL-12 antagonist. Eur J Immunol. 1995; 25(1): 200–6. PubMed Abstract | Publisher Full Text\n\nLing P, Gately MK, Gubler U, et al.: Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J Immunol. 1995; 154(1): 116–27. PubMed Abstract\n\nHeckel MC, Wolfson A, Slachta CA, et al.: Human breast tumor cells express IL-10 and IL-12p40 transcripts and proteins, but do not produce IL-12p70. Cell Immunol. 2011; 266(2): 143–53. PubMed Abstract | Publisher Full Text\n\nHeinzel FP, Hujer AM, Ahmed FN, et al.: In vivo production and function of IL-12 p40 homodimers. J Immunol. 1997; 158(9): 4381–8. PubMed Abstract\n\nMattner F, Ozmen L, Podlaski FJ, et al.: Treatment with homodimeric interleukin-12 (IL-12) p40 protects mice from IL-12-dependent shock but not from tumor necrosis factor alpha-dependent shock. Infect Immun. 1997; 65(11): 4734–7. PubMed Abstract | Free Full Text\n\nPiccotti JR, Chan SY, Li K, et al.: Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gamma-producing cells. J Immunol. 1997; 158(2): 643–8. PubMed Abstract\n\nPegram HJ, Lee JC, Hayman EG, et al.: Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood. 2012; 119(18): 4133–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoneru M, Purdon TJ, Spriggs D, et al.: IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo. Oncoimmunology. 2015; 4(3): e994446. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11601",
"date": "17 Dec 2015",
"name": "Raymond P. Donnelly",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11602",
"date": "17 Dec 2015",
"name": "Stanislas Goriely",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11606",
"date": "17 Dec 2015",
"name": "Tadamitsu Kishimoto",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1465
|
https://f1000research.com/articles/4-1464/v1
|
17 Dec 15
|
{
"type": "Review",
"title": "Dihydrofolate reductase as a model for studies of enzyme dynamics and catalysis",
"authors": [
"Amnon Kohen"
],
"abstract": "Dihydrofolate reductase from Escherichia coli (ecDHFR) serves as a model system for investigating the role of protein dynamics in enzyme catalysis. We discuss calculations predicting a network of dynamic motions that is coupled to the chemical step catalyzed by this enzyme. Kinetic studies testing these predictions are presented, and their potential use in better understanding the role of these dynamics in enzyme catalysis is considered. The cumulative results implicate motions across the entire protein in catalysis.",
"keywords": [
"Dihydrofolate reductase",
"Escherichia coli",
"catalysis"
],
"content": "Introduction\n\nEnzymes are large and flexible proteins that catalyze most chemical reactions in life. The dynamics of a protein's folding from a linear polymer to the globular and active form of the enzyme are rather well understood. The role of the motions and vibrations of the folded enzyme throughout its catalytic cycle, on the other hand, is a matter of intensive investigation. Here we will present studies of E. coli dihydrofolate reductase, or DHFR, that focus on the role of dynamics across the protein in the chemical step catalyzed by this enzyme. DHFR from E. coli is a preferred model system for such studies because it is a small monomeric enzyme, has no metals or S-S bonds, and folds reversibly. Additionally, as this DHFR has been used in many diverse studies, the body of available information on this enzyme opens the door to in-depth physical studies such as those presented below.\n\nWe must first address some controversy that has arisen in the field regarding terminology. The terms “dynamics” and “catalysis” are defined differently by different researchers. Most biochemists and several chemists use the term “dynamics” to refer to any vibration or motion of the protein complex with its ligands and solvent. That definition includes statistical and non-statistical dynamics (motions and vibrations). By contrast, several physical chemists define the term “dynamics” more narrowly, using it to refer only to chemical, or non-statistical, dynamics (motions or vibrations that are not in thermal equilibrium with their environment). As for the term “catalysis”, it is formally defined as the ratio of catalyzed and uncatalyzed turnover rate constants under the same conditions. Unfortunately, performing studies of the uncatalyzed reactions is frequently challenging or even unrealistic for both experimentalists and theoreticians. Computer-based calculations that have attempted to compare catalyzed to uncatalyzed reactions have usually begun with the enzymatic reactive complex, substituted water for the protein, and restricted the reactants to the orientation found in the enzyme in order to calculate the uncatalyzed reaction. However, in reality, the statistics of bringing reactants to the reactive orientation in question do not agree with the calculations, and some of these uncatalyzed reactions never actually occur experimentally without the catalyst. Consequently, most studies of “enzyme catalysis” address only the enzyme-catalyzed reaction, not the comparison to the uncatalyzed reaction. Since these researchers (probably a majority) still use the term “catalysis” (rather than “enzyme-catalyzed reaction”), they and their titles are condemned by some who assume they refer to the comparison with the uncatalyzed reaction. In reality, very few experimental studies have compared the catalyzed to the uncatalyzed reactions1, and (as far as I am aware) no experiment today can distinguish between statistical and non-statistical dynamics in an enzyme-catalyzed reaction2.\n\nReturning to ecDHFR, to the best of my knowledge, no relevant uncatalyzed reaction has yet been reported for this enzyme. Since non-statistical dynamics cannot be tested on their own experimentally, no such dynamics are proposed below when addressing protein dynamics that participate in catalysis. Instead we use a thermally equilibrated model that seems to be in accordance with all experimental findings2, and “dynamics” here will mean all vibrations or motions in the protein complex (including solvent and ligands).\n\n\nStudies of dihydrofolate reductase\n\nMapping a network of enzyme-wide motions involved in catalyzing the chemical conversion requires the ability to experimentally probe the chemical step within the enzyme’s complex kinetic cascade. The chemical step catalyzed by DHFR is a C–H→C hydride transfer, shown in Figure 1. The enzyme catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H2folate) to 5,6,7,8-tetrahydrofolate (H4folate), which is the reactive form of folic acid, and is a critical one-carbon carrier in DNA nucleotides’ biosynthesis and other cellular processes. It has been shown that N5 of H2folate is protonated by the enzyme prior to the hydride transfer step3. This fact greatly simplifies the calculations and data interpretation for this enzyme, as these can focus on a single barrier event. This fact, however, makes the assessment of the experimental rate constant of the C–H→C hydride transfer very challenging. While computer-based molecular calculations address only that chemical step, representing a single kinetic barrier, the rate for that single step is quite impossible to assess experimentally, leaving little room for direct examination of the theoretical predictions. To emphasize this last point, Figure 2 presents a minimal kinetic scheme for this enzyme4. As one can see in this scheme, most of the enzyme is never free. The release of the first product (NADP+) is followed by the binding of the substrate NADPH, and only then is the product H4folate released, prior to the binding of the second substrate, H2folate, to form the reactive complex. This scheme indicates that steady-state kinetic parameters (i.e., kcat and kcat/KM) do not always reflect the chemical step, which can be much faster than other kinetic steps.\n\nThe reaction catalyzed by dihydrofolate reductase (DHFR). R = adenine dinucleotide 2’ phosphate and R’ = (p-aminobenzoyl) glutamate. It has been shown that the protonation of the N5 position of DHF occurs prior to hydride transfer, at all relevant media pH (5–11.5)3.\n\nDuring its turnover, Escherichia coli dihydrofolate reductase (ecDHFR) cycles through 5 kinetic intermediates. The rate constants of all steps are from 4. The pH-independent rate (950 s-1 in red), sometimes addressed as the hydride-transfer rate, was obtained from non-linear regression of the pH dependence of observed single-turnover rate constants4. The overall rate-limiting step on the catalytic turnover number, kcat, is 12.5 s-1 (in blue).\n\nTo handle this type of problem it is usual to employ pre-steady-state kinetics, in which the substrate under investigation (e.g., NADPH) is pre-bound to the enzyme, and the reaction is initiated by a very high concentration of the second substrate (in this case H2folate). The conversion of NADPH to NADP+ on the enzyme is followed spectroscopically, so neither substrate binding nor product release affects the rate constant. However, the second substrate, H2folate, is not protonated in solution (at physiological pH). Thus, after its very fast binding to the enzyme-NADPH complex, major changes in the active site are required (involving at least residues D27 and Y1003) in order to protonate this substrate prior to the hydride transfer, and bring it into the reactive conformation in the ternary complex. This problem with using pre-steady-state rate constants is also apparent from the pH-dependence of these observed rate constants4,5. The only way to assess a pH-independent pre-steady-state rate constant is by measuring the pH dependence across a broad pH range, and extrapolating to infinitely low pH4. Unfortunately, this has been a common practice only in Benkovic’s lab4,6.\n\nFigure 2 also makes plain another, more serious problem with the pre-steady-state approach for ecDHFR: the measured pre-steady-state rate constant, even when extrapolated to a low pH, is at the millisecond timescale (e.g., 950 s-1 in Figure 2). This rate constant is much slower than the C–H→C hydride transfer per se, which takes place at the picosecond to femtosecond timescale.\n\nA more direct experimental method of investigating only the chemical step is to study kinetic isotope effects (KIEs), comparing the rates or rate constants of two substrates in which the cleaved C-H bond carries different isotopes (e.g., H/D, H/T, or D/T). In such a study, the complex kinetic expression that constitutes the absolute rate is greatly simplified, as many steps that are not isotopically sensitive (i.e., steps that do not include the C-H cleavage in this case) fall out of the KIE equation (ratio of rates). This point is emphasized by Figure 3, which indicates how difficult it is to determine rate constants on the H-transfer step from either steady-state kinetics (e.g., kcat or kcat/KM) or pre-steady-state kinetics. The scheme shows that steps other than the C–H→C hydride transfer under study are frequently rate-limiting for the kinetic parameter measured. Kinetic isotope effects (KIEs) are also not complexity-free, and the observed KIEs are often smaller than their intrinsic value on the chemical conversion itself7,8. This said, KIEs are a useful way to examine the chemical step when a method is used to assess their intrinsic value9–11.\n\nBoth steady-state parameters (e.g., kcat/KM (V/K) and kcat) and pre-steady-state rates (e.g., single-turnover rate) involve several microscopic rate constants, which may not represent the rate of the chemical step. In Escherichia coli dihydrofolate reductase (ecDHFR), pH 7, kcat mostly represents the release of the product H4folate, kcat/KM mostly represents the binding of the substrate H2folate4, and the single-turnover rate mostly represents the conversion of the initial ternary complex (Enz.NADPH.H2folate) to its reactive form with protonated-H2folate3. The intrinsic kinetic isotope effects (KIEs) (whether resulting from differences in zero-point-energy or from quantum tunneling or both) better reflect the chemical step per se, but assessing these from their observed values is quite challenging (see text).\n\nAs illustrated in Figure 3, most of the KIE results from differences between the zero point energies (ZPE) of the ground state and the transition state, and can be also affected by nuclear-quantum-mechanical tunneling (a phenomenon in which the atom is transferred under the classical energy barrier via its wave-like properties). In the case of H-tunneling, the transition state becomes the tunnelingready state (TRS), or the chemically reactive state, which is the quantum mechanically delocalized transition state2. Importantly, the intrinsic KIE is an experimentally measurable ratio of rates that can be directly compared to molecular calculations of the chemical step per se.\n\nTo better assess the intrinsic KIEs for DHFR, we used all three isotopes of hydrogen (i.e., the Northrop-method)12,13. In contrast to most other methods, this method makes assumptions that might slightly affect the size of the KIE, but are not likely to alter its temperature dependence. This is useful because the temperature dependence of intrinsic KIEs is a sensitive probe of the nature of the H-transfer or, more specifically, the donor-acceptor distance (DAD) dynamics and distribution at the TRS2,14,15. It is fair to say that the temperature dependence of KIEs is a more meaningful probe of the nature of the catalyzed chemical step than are the rate constants or KIEs themselves2.\n\nIn most wild-type and well evolved enzymes, it has been found that the intrinsic KIEs for H-transfer reactions are temperature-independent14, suggesting the enzymes evolved to have short and narrowly distributed DADs (i.e., a well- reorganized TRS)16. Mutations that affect the chemical step, as well as unnatural substrates or non-physiological reaction conditions, often lead to more highly temperature-dependent KIEs (with poorly reorganized TRSs). The broader distribution of DADs means lower frequency of DAD sampling at the TRS, and this dynamic search for short DADs results in increased KIE temperature dependence.\n\n\nExperimental test of computed prediction\n\nA test case is presented below in which the temperature dependence of intrinsic KIEs was used to study a predicted network of coupled motions in the DHFR from E. coli17. Figure 4 presents residues that are predicted to be coupled to each other and be part of the reaction coordinate of the C–H→C hydride transfer steps. Some of these residues are in the active site and in direct contact with the reactants (e.g., I14), while others are far from the active site, and their coupling to the chemistry catalyzed by the active site is not trivial at all.\n\nDihydrofolate reductase (DHFR) structure (PDB ID 1rx2; 18) colored based on genetic coupling analysis as conserved (red), strongly coupled (pink), and weakly coupled (orange). The NADPH cofactor (dark blue) and folate (light blue) are highlighted as sticks, and an arrow is drawn at the path of the hydride transfer under study. The four coevolving residues that are discussed in the text are highlighted as spheres, and the insertion sites at N23 and G51 are highlighted as dark blue spheres. Reproduced from 15 with permission from the American Society of Biochemistry and Molecular Biology.\n\nVarious molecular calculations of DHFR from E. coli, along with bioinformatics statistics of DHFR from various organisms, predicted that several residues in the enzyme’s active site as well as several residues remote from the active site are dynamically and genetically coupled to the catalyzed reaction19–23. The term genetically coupled refers to residues that co-evolve, and thus while they are not highly conserved, their distribution differs from random statistical distribution22. One example from a molecular calculation and one from bioinformatics are presented in Figure 5. Interestingly, both methods predicted that several residues are coupled to the chemistry (e.g., G121, M42, and F125), while others were implicated only by bioinformatics (e.g., W133). To test these predictions, we measured the intrinsic KIEs for the wild-type enzyme and for its single and double mutants, testing residues predicted to be coupled by one of these methods or both.\n\nLeft Panel: QM/MM calculations. A map of all Escherichia coli dihydrofolate reductase (ecDHFR) residues that are coupled to each other and to the reaction coordinate. The two axes are identical and represent the atoms of the enzyme in sequential order. Residues under study here are marked by arrows (M42, G121, F125). Right Panel: Bioinformatics calculations. The evolutionary coupling network of amino acid residues in DHFR, with highly conserved residues in red boxes and lines connecting residues that co-evolve (i.e., are genetically coupled). Reprinted from 21 and 22 with permission from National Academy of Science and Annual Reviews, respectively.\n\nFigure 6 summarizes the isotope effects on the activation parameters for the intrinsic KIEs, where ΔEa (H/T) represents the slope of the temperature dependence of the H/T KIEs (the larger the value, the more temperature-dependent are the KIEs), and AH/AT is the isotope effect on the Arrhenius pre-exponential factors. The green data points are for single mutations at the active site (I14 to V, A, and G), each of which is designed to generate a poorly reorganized TRS by decreasing the size of an enzymatic side-chain holding the H-donor close to the acceptor24. For these mutants, the smaller the side chain is, the larger is ΔEa (H/T) and smaller AH/AT. This observation is in accordance with the fact that longer DADs, with broader distributions, are associated with greater temperature dependence of intrinsic KIEs. The effect of active site mutations on the catalyzed reaction and its DAD is more obvious than that of remote mutants. Studies of mutants far from the active site indicate that double mutants have a non-additive effect that is much larger than their respective single mutants (ΔEa (H/T) double mutant A&B > ΔEa (H/T) mutant A +ΔEa (H/T) mutant B). This finding supports the prediction that those residues are coupled to each other along the reaction coordinate for the hydride transfer in the wild-type enzyme. Interestingly, I14 is also found to be coupled to remote residues like G121, offering a path through which the remote residues can affect the H-transfer step25.\n\nPresented are data for wild-type (WT) (black), distal (red), and local (green) mutants of Escherichia coli dihydrofolate reductase (ecDHFR). The double mutant bridging local and distal is in blue. Error bars represent standard deviation. The yellow block represents the semi-classical range of the Arrhenius pre-exponential factor (0.3–1.7). Reproduced from 26 with permission of the American Chemical Society.\n\nIn accordance with predictions made by the calculations presented above, these findings indicate that several residues across the whole protein, including some far from the active site, are involved in a network of coupled motions that affect the chemical step catalyzed by DHFR from E. coli. The fact that the bioinformatics calculation alone predicted that W133 is coupled, yet it had no visible effect on the chemical step, suggests that there might be more than one functional network. In addition to a network that affects activation of the chemical bond, there could be networks that are important for proper folding or for other biological functions. Notably, a very different type of calculation did not predict “protein promoting vibrations” to be part of the chemical step in this enzyme27, but it is not clear that these calculated vibrations address the same phenomena and motions on the same timescale as those examined by the studies19–23 and experiments presented above24–26,28.\n\n\nConcluding remarks\n\nAn important take-home message from the above studies is that observed rate constants and KIEs should not be taken as a probe for a single kinetic step. These observed values often represent a complex kinetic expression rather than the chemical step per se. The observed rate constants and KIEs, and especially their temperature dependence (and thus their activation parameters) often reflect complex phenomena involving many microscopic rate constants. This is unfortunate, as most calculations address only the barrier that is the chemical step, but not other steps affecting the experimental measurement. In many cases the bond cleavage step of interest occurs at the picosecond to femtosecond timescale and is a fleeting event within catalytic turnover, which occurs at the millisecond timescale. Not many experimental methods are available that report on the time scale of the bond activation, yet the ability to probe intrinsic parameters that probe the chemical step, such as those presented in Figure 6, is critical when experimentally assessing any molecular calculation that focuses on the single kinetic step in which the chemical conversion occurs.",
"appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThis work is supported by a grant from the National Science Foundation (CHE-1149023) and the National Institutes of Health (RO1 GM65368).\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\nWolfenden R: Degrees of difficulty of water-consuming reactions in the absence of enzymes. Chem Rev. 2006; 106(8): 3379–96. PubMed Abstract | Publisher Full Text\n\nKohen A: Role of dynamics in enzyme catalysis: substantial versus semantic controversies. Acc Chem Res. 2015; 48(2): 466–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu CT, Francis K, Layfield JP, et al.: Escherichia coli dihydrofolate reductase catalyzed proton and hydride transfers: temporal order and the roles of Asp27 and Tyr100. Proc Natl Acad Sci U S A. 2014; 111(51): 18231–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFierke CA, Johnson KA, Benkovic SJ: Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli. Biochemistry. 1987; 26(13): 4085–92. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMaglia G, Allemann RK: Evidence for environmentally coupled hydrogen tunneling during dihydrofolate reductase catalysis. J Am Chem Soc. 2003; 125(44): 13372–3. PubMed Abstract | Publisher Full Text\n\nMiller GP, Benkovic SJ: Stretching exercises--flexibility in dihydrofolate reductase catalysis. Chem Biol. 1998; 5(5): R105–13. PubMed Abstract | Publisher Full Text\n\nCook PF: Enzyme Mechanism from Isotope Effects. In Enzyme Mechanism from Isotope Effects. Edited by Cook PF. Florida: CRC Press, Boca Raton, 1991; 181–202. Reference Source\n\nCook PF, Cleland WW: Enzyme Kinetics and Mechanism. New York: Garland Publishing Inc; 2007. Reference Source\n\nKohen A, Limbach H: Isotope effects in chemistry and biology. Boca Raton: Taylor & Francis; 2006. Reference Source\n\nSen A, Yahashiri A, Kohen A: Triple isotopic labeling and kinetic isotope effects: exposing H-transfer steps in enzymatic systems. Biochemistry. 2011; 50(29): 6462–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Z, Roston D, Kohen A: Experimental and theoretical studies of enzyme-catalyzed hydrogen-transfer reactions. In Structural and Mechanistic Enzymology - Bringing Together Experiments and Computing. Elsevier, Adv Protein Chem Struct Biol. 2012; 87: 155–80. PubMed Abstract | Publisher Full Text\n\nNorthrop DB: Intrinsic isotope effects in enzyme catalyzed reactions. In Enzyme mechanism from isotope effects. Edited by Cook PF. Florida: CRC Press, Boca Raton; 1991: 181–202. Reference Source\n\nNorthrop DB: Determining the absolute magnitude of hydrogen isotope effects. In Isotope effects on enzyme-catalyzed reactions. Edited by Cleland WW, O'Leary MH, Northrop DB: Baltimore: University Park Press; 1977; 122–52. Reference Source\n\nKlinman JP, Kohen A: Hydrogen tunneling links protein dynamics to enzyme catalysis. Annu Rev Biochem. 2013; 82: 471–96. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKlinman JP, Kohen A: Evolutionary aspects of enzyme dynamics. J Biol Chem. 2014; 289(44): 30205–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrancis K, Stojkovic V, Kohen A: Preservation of protein dynamics in dihydrofolate reductase evolution. J Biol Chem. 2013; 288(50): 35961–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgarwal PK, Billeter SR, Rajagopalan PT, et al.: Network of coupled promoting motions in enzyme catalysis. Proc Natl Acad Sci U S A. 2002; 99(5): 2794–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSawaya MR, Kraut J: Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence. Biochemistry. 1997; 36(3): 586–603. PubMed Abstract | Publisher Full Text\n\nRod TH, Brooks CL 3rd: How dihydrofolate reductase facilitates protonation of dihydrofolate. J Am Chem Soc. 2003; 125(29): 8718–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRadkiewicz JL, Brooks CL: Protein Dynamics in Enzymatic Catalysis: Exploration of Dihydrofolate Reductase. J Am Chem Soc. 2000; 122(2): 225–31. Publisher Full Text\n\nWong KF, Selzer T, Benkovic SJ, et al.: Impact of distal mutations on the network of coupled motions correlated to hydride transfer in dihydrofolate reductase. Proc Natl Acad Sci U S A. 2005; 102(19): 6807–12. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHammes-Schiffer S, Benkovic SJ: Relating protein motion to catalysis. Annu Rev Biochem. 2006; 75: 519–41. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHammes-Schiffer S: Hydrogen tunneling and protein motion in enzyme reactions. Acc Chem Res. 2006; 39(2): 93–100. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nStojković V, Perissinotti LL, Willmer D, et al.: Effects of the donor-acceptor distance and dynamics on hydride tunneling in the dihydrofolate reductase catalyzed reaction. J Am Chem Soc. 2012; 134(3): 1738–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh P, Francis K, Kohen A: Network of Remote and Local Protein Dynamics in Dihydrofolate Reductase Catalysis. ACS Catal. 2015; 5(5): 3067–73. Publisher Full Text\n\nSingh P, Sen A, Francis K, et al.: Extension and limits of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase. J Am Chem Soc. 2014; 136(6): 2575–82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDametto M, Antoniou D, Schwartz SD: Barrier Crossing in Dihydrofolate Reductase does not involve a rate-promoting vibration. Mol Phys. 2012; 110(9–10): 531–6. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWang L, Goodey NM, Benkovic SJ, et al.: Coordinated effects of distal mutations on environmentally coupled tunneling in dihydrofolate reductase. Proc Natl Acad Sci U S A. 2006; 103(43): 15753–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11607",
"date": "17 Dec 2015",
"name": "Giovanni Gadda",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11608",
"date": "17 Dec 2015",
"name": "Liz Howell",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1464
|
https://f1000research.com/articles/4-1462/v1
|
16 Dec 15
|
{
"type": "Review",
"title": "Primordial Germ Cell Specification and Migration",
"authors": [
"Florence Marlow"
],
"abstract": "Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration.",
"keywords": [
"Primordial Germ Cell",
"Primordial Germ Cell Specification",
"Migration",
"gametes",
"germ plasm",
"Germ Plasm Assemblers",
"Germ Granules",
"Germline Identity"
],
"content": "Introduction\n\nIn 1892, August Weismann challenged the notion that the germline (reproductive cells) was derived from the soma (cells of the body). Instead, Weismann proposed that the germ cells possessed a special immortal substance called “ancestral germ plasm” that was inherited from germ cells of one generation to the next in his “theory of the continuity of the germ plasm”1,2. Germ plasm is a maternally supplied substance comprised of RNAs, proteins, and organelles that are amassed in oocytes and later is sequestered during the first embryonic cleavages within a few cells that will become the primordial germ cells (PGCs). In the next century, the identification of conserved germline-specific markers and the germ cell accumulation of alkaline phosphatase made it possible to trace the origins of the germline from its earliest emergence through PGC migration to the presumptive gonad where they differentiate as male or female gametes. Such lineage tracing revealed that indeed some animals establish their germline by inheritance of maternal factors and post-transcriptional regulation in the context of a silenced genome but that others do not. In the latter case, these animals lack detectable maternal germ plasm and induce their germ cells by zygotic transcription factors. Genetic and overexpression screens to identify germ cell inducers have uncovered only a few factors with the capacity to generate ectopic PGCs. Oskar3 and bucky ball4 are “drivers” of germline fate among animals that use a maternal inheritance mode of PGC specification, but these genes are specific to different subsets of species5–7, and it remains to be determined whether their mechanisms of action and specific activities are conserved (Figure 1). In humans, which use an inductive mode of specification, sox17 is sufficient to specify human primordial germ cell-like cells (hPGCLCs)8, but in mouse no germ cell inducer has been identified. This suggests that only a few genes possess germ cell-inducing activity or that the coordinated action of multiple genes is required to establish the germline or both. Consistent with this notion, many factors involved in germ cell development are involved in RNA regulation, including Vasa, a universal marker of and regulator of germ cell development9–26. Recently, investigators proposed a “last cell standing model”, whereby early PGC determination, as occurs in maternal germ plasm inducers, is not an innovation to protect germline traits. Instead, they proposed that germ plasm provides a means to specify the germline lineage earlier, before gastrulation, and thereby liberate the somatic cells of the embryo to more rapidly evolve27. According to this model, innovation in animals that do not specify the germline early—prior to or in early gastrulation either by maternal germ plasm or inductive modes—but instead specify PGCs late in gastrulation by zygotic inductive modes are constrained by signaling and morphogenesis requirements associated with germ layer specification and gastrulation27. Significantly, many of the same or related genes regulate PGC development independently of specification mode. This review focuses on the earliest events of PGC specification in the zygote, the mechanisms that induce early PGC-like cells in culture.\n\nThe left column lists activities, localization, or other properties. Those attributed to Drosophila oskar or zebrafish bucky ball or both are indicated in the relevant columns. PGC, primordial germ cell.\n\n\nPerinuclear accumulations in germ cells\n\nWeismann recognized the germ plasm as a peculiar and complicated structure, but at that time the molecular components were not known. Since then, germ cell-specific accumulations have been detected in germ cells at all stages of the germline cycle. Further adding to the complexity, germ cell substances that have been identified throughout the germline cycle have been referred to by a variety of terms—nuage, germ plasm, and germ granules, or P-granules—at different developmental stages and in different animals. In recent years, molecular identification of the components of these germ cell substances has revealed that the molecular components overlap somewhat and thus nuage, germ plasm, and germ granules have at times been treated as largely equivalent. This assumption has made it challenging to decipher the functions of these germ cell manifestations, particularly when attempting to compare studies of these germ cell substances conducted at different developmental stages and in different organisms. To facilitate comparisons of functional studies between species, definitions of nuage, germ plasm, and germ granules in the context of this review are provided below.\n\nNuage is the perinuclear granulo-fibrillar electron-dense material that has been identified through histological and ultrastructural examination of oocytes from invertebrates through vertebrates and that is present in various conformations in cells of the male and female gametes28. Although nuage occupies a distinct subcellular space, it is generally not asymmetric in its distribution. In some animals, a subset of nuage components become asymetrically localized to a specific subcellular location in oocytes where the germ plasm that is transmitted from oocytes to the embryo forms (Figure 1). This substance is present prior to zygotic genome activation and contains germ cell-inducing activity (Figure 1). After germ cell specification and zygotic genome activation, germline-specific aggregates of proteins and RNA-binding proteins called germ granules form next to the nucleus (Figure 1). The functions of each of these germ cell-specific substances and their distinguishing features are active areas of study.\n\nAs an initial step toward defining nuage function, the identity of nuage molecules has been sought. The products of conserved germline-specific genes that are necessary for germ cell development, such as Vasa protein, are enriched in perinuclear regions where nuage is found16,18,29,30. Nuage has been postulated to be involved in the maternal germ plasm pathway; however, nuage is present even in animals that do not use inheritance of maternal germ plasm to specify their germline, indicating that nuage components may have other functions. It is also possible that nuage in animals that use inductive modes also contains germ plasm precursor materials, but that a nucleator or assembly/scaffold factor that can assemble nuage components into active germ plasm is lacking. Notably, evidence suggests that specification by zygotic induction is the ancestral mode of germline determination in insects and vertebrates6,7,29,31. Maternal specification by germ plasm in some insects has been associated with the presence of the germ plasm inducer oskar, discussed in the following section. Oskar is thought to have been co-opted from an ancestral neural role and to have facilitated the transition from zygotic germ cell induction to maternal specification via germ plasm6,7. Based on the conserved presence and perinuclear localization of nuage and the nature of the molecules that localize there, nuage functions in processes other than maternal germ plasm assembly have been proposed.\n\nAmong molecules that are enriched in nuage are proteins involved in genesis of piwi-interacting RNAs (piRNAs). piRNAs are components of a gonad-specific RNA silencing pathway that is thought to protect genome integrity by counteracting transposable or selfish genetic elements that promote their own transmission at the expense of other elements (reviewed in 32,33). piRNAs are small RNAs that are produced from the cleavage of precursor RNAs by endonuclease activity of germline-specific members of the Argonaut family, called Piwi proteins, and secondary amplification34,35. Argonaut family members, including Piwi and Aubergine in flies, and human Argonaut homologs have been implicated in transcriptional and post-transcriptional regulation of gene expression36. Like Vasa protein, Piwi protein homologs reside adjacent to the nucleus within the nuage in some species (reviewed in 37). Based on the conserved localization of piRNA pathway components as well as genetic and other functional data, a conserved nuage role as the site of piRNA amplification has been proposed38–56. While this may indeed be the case, not all piRNA components localize to the nuage (reviewed in 37). Moreover, the phenotypes of some piRNA pathway components suggest additional functions in diverse processes, including nuclear functions in chromosome rearrangements, chromosome dynamics, roles in RNA metabolism and storage, stem cell maintenance, regulation of cell divisions at stages before germ plasm would assemble in oocytes, or later roles in PGC maintenance; these roles have generated models whereby nuage and later germ granules serve to extend the nuclear environment and have been reviewed elsewhere57. Owing to its dynamic nature, its varied composition at different stages, phenotypic differences between genders and species, and multiple stage-specific activities indicated by nuage component mutants, including piRNA pathway molecules, the developmental functions of nuage are not fully understood.\n\n\nGerm plasm assemblers\n\nIn primary oocytes of some animals, maternal germ plasm first assembles within an ancient perinuclear oocyte structure known as the Balbiani body28,58,59 and later is found at the oocyte cortex, the posterior pole in some insects or the vegetal pole of some vertebrates28,58,59 (Figure 1). Expression-based screens have identified germ plasm components and candidate regulators on the basis of their localization to sites of germ plasm assembly in oocytes, such as the Balbiani body of early oocytes or the cortex of late-stage oocytes (reviewed in 15,59,60). However, localization to the germ plasm is only suggestive of potential function as not all molecules that localize to germ plasm are essential for its assembly or activity16–64. Functional assays to define the component(s) of germ plasm that can impart germ cell identity, the germ plasm nucleators or assemblers, have included isolation and transplantation of cytoplasm from oocytes to embryos to identify the substance with PGC-inducing activity in model systems such as Drosophila65, zebrafish66, and Xenopus67. To date, only a limited number of factors that can induce germ cells have been identified, and how the germ plasm assembles remains a key question in the field.\n\nThe molecular constituents of the germ plasm are best understood in Drosophila because of the powerful genetic screens which led to the identification of key factors of germline assembly and germ cell development68–70, including the germ plasm assembler, oskar71. Alternative translation generates two forms of Oskar with distinct activities72. The short form of Oskar is required for germ plasm assembly and function, whereas long Oskar lacks the assembly activity and instead is required to anchor germ plasm72–74. Until recently, Oskar (Osk) was viewed as a scaffolding protein that gathered germ plasm via interactions with a myriad of partners25,74–94. The regions of Oskar mediating its association with partners such as Vasa defined Oskar functional domains73. The recently elucidated Oskar crystal structure revealed that both Oskar dimerization and interaction with Vasa are mediated via the Osk N-terminal LOTUS domain, named after Limkain, Oskar and Tudor domain-containing proteins 5 and 783. LOTUS is a globular domain present in several germ plasm/granule components95, including the conserved Tudor family, first discovered for Tudor’s function in germ plasm assembly in Drosophila68. This finding is in contrast to previous work that mapped the Vasa interaction to Oskar’s C-terminus73. In the new study, Vasa-Oskar interactions were tested without RNAs to exclude RNA-mediated association and this may explain the different binding sites. These findings further support interaction between Vasa and Oskar and raise new questions and models to explain how these different Oskar complexes promote germ plasm formation and activity.\n\nAnother exciting aspect of the recent study is the evidence that short, but not long, Oskar associates directly with RNA. Surprisingly, this binding interaction is not via a canonical RNA-binding motif, but instead through a domain that resembles an enzymatically inactive SGNH hydrolase domain83. SGNH hydrolases are a large enzyme family with thousands of members that are found in all life forms. An interesting property among some bacterial SGNH domain-containing proteins that may be of relevance to germ plasm assembly is their propensity to oligomerize to form amorphous aggregates or amyloid-like fibrils96,97. In addition, proteins encoded by some LINE (long interspersed nuclear elements) mobile genetic elements contain SGNH-like domains and mediate RNP assembly98,99. For example, the zebrafish LINE protein ZfL2-1 ORF1p forms multimers, binds nucleotides, and like Oskar possesses an SGNH-like domain that lacks overt RNA-binding domain structure98. In addition, ORF1ps, including ZfL2-1, have been shown to function as chaperones. In this context, ORF1ps interact with the LINE RNA and are postulated to mediate rearrangement of the RNA into a stable conformation that protects the RNA from degradation, but later can be reversed to facilitate reverse transcription98. Based on in vitro structure function studies, ZfL2-1 is postulated to mediate RNP assembly via interactions with positively charged peptides that bind RNA structural elements99. It remains to be determined whether Oskar has similar chaperone functions, but it is easy to imagine how such an activity could apply to germ plasm RNAs, which must be translationally silent and protected from degradation during transport but later are translated in a specific96 subcellular location. The unique functional domains and distinct interaction properties of Oskar isoforms discussed above provide new models to test the mechanism by which Oskar could promote RNP and germ plasm assembly either directly via its RNA-binding domain or directly or indirectly via its disordered LOTUS and interaction with Vasa.\n\nVertebrates, even those that use maternal inheritance to specify their germline, lack oskar and instead have a vertebrate-specific gene called bucky ball (buc) or vegetally localized 1 (velo1)4,100. Buc shares features with Oskar in that both are localized in oocytes as RNAs and proteins4,81,101–103, both are required for and can organize germ plasm3,4, both have been viewed as unstructured proteins, both display complex post-transcriptional regulation at the level of splicing81,104 and RNA localization72,81,105, and both have properties of self-assembling aggregates and interact with RNA-binding proteins and other factors at the RNA and protein levels73,75–90,92,94. Whether or not Buc protein, like Osk, can directly bind RNAs is unknown. Based on homology searches, Buc lacks identifiable functional domains and thus the mechanisms by which it promotes Balbiani body formation, germ plasm assembly, and oocyte axis specification are not clear and have relied on identifying Buc interaction partners and mapping their interaction sites on Buc78,81,106. However, Bucky ball and Oskar germ plasm factors also have some differences (Figure 1). Specifically, Buc protein is present in early-stage oocytes81,101, whereas Osk protein is detected in late-stage oocytes72, and the few reported Buc interaction partners are vertebrate-specific78. Finally, osk RNA has functions independent of its protein-coding role84,107, whereas no evidence of protein-independent RNA functions of buc have been reported. These differences support convergent evolution or co-option of these genes as germ plasm assemblers; however, further analysis, including cross-species comparisons and rescue experiments, are required to determine the extent to which the activities of these germ plasm assemblers overlap.\n\nMuch less is understood about the molecular regulators of maternal germ plasm specification in vertebrates; however, recently, endogenous Buc protein was shown to localize to the cleavage furrows of early embryos101,106 by a maternal Kinesin 1 (Mkif5Ba)-dependent mechanism106. Moreover, the ability of Buc to induce ectopic PGCs requires Mkif5Ba and Buc protein localization to the cleavage furrows106. Similarly, formation of germ plasm aggregates in Xenopus requires the Kinesin-like protein Xklp2108, indicating a conserved role for microtubule motor-dependent assembly of germ plasm in these vertebrates. Because germ plasm is present in zebrafish Mkif5Ba mutant embryos but germ cells are absent, this mutant provides evidence that inheritance of maternal germ plasm from oocytes alone is not sufficient to specify PGCs. Moreover, germline establishment requires proper spatiotemporal localization of PGC determinants.\n\n\nGerm plasm, germ granules, and germline identity\n\nThe capacity of germ plasm to specify PGC fate has clear support from studies in Drosophila65, zebrafish66, and Xenopus67, in which transplantation of the germ plasm can induce PGCs. In contrast, when germ granules are not properly segregated in Caenorhabditis elegans mutants, excess PGCs do not form nor do all cells adopt germline fates; instead, the cells take somatic fates109. This example suggests that germ granules are not sufficient for PGC fate. These differences in germ cell induction capacity between granules and plasm raise the question of whether germ plasm and granules are based on their shared enrichment of RNA-binding proteins (RNAbps), RNAs, and other overlapping component4,73,78,101,106,110 manifestations of the same mechanism/structure operating at different stages or instead are distinct entities with unique properties. Alternatively, not all granules are equivalent. The maternal germ plasm associated with the maternal inheritance specification mode is continuous from oocyte to embryo, whereas perinuclear germ granules assemble only in PGCs, even in zygotically induced/discontinuous modes of germline specification. Thus, maybe these germline entities are not functionally equivalent. If this is the case, germ plasm from oocytes should contain germ cell-inducing factors, which germ granules of PGCs lack. Consistent with this notion, the perinuclear nuage in Drosophila oocytes is distinct from the polar granules nucleated by Oskar111 during specification of pole cells/PGCs65. Moreover, recent studies in Drosophila, C. elegans, and zebrafish provide evidence that germ granules are heterogeneous. In Drosophila, single RNA fluorescent in situ hybridization (FISH) analyses of granules revealed their organized architecture of “core” germ plasm proteins (e.g., Vasa and Oskar) distributed throughout granules and specific RNAs spatially arranged within granules112. Significantly, although Oskar protein is present in granules, oskar RNA is not112,113 (Figure 2). When chimeric osk with a nanos 3’ untranslated region (UTR) is directed to granules, germ cell numbers and Vasa expression are reduced113, indicating that osk segregation from granules is needed for normal PGC development. Taken together, these findings are consistent with plasm and granules being distinct entities in Drosophila. Similarly, endogenous Buc protein localizes to germ plasm of oocytes and early embryos but is not detected in germ granules of PGCs106. Moreover, buc RNA is a component of germ plasm of oocytes but not embryos, and even when overexpressed, Buc protein, but not buc RNA, localizes to germ granules of zebrafish PGCs4,101,106 (Figure 2). In addition, in zebrafish, vasa RNA but not Vasa protein is a component of the Balbiani body, where germ plasm localizes in oocytes18. In contrast, Vasa protein is a PGC granule component in zebrafish11,18,114 and other animals examined (reviewed in 15,115,116). In zebrafish, as in Drosophila, analysis of MS2-tagged and endogenous vasa and nanos RNAs indicates that granules are not equivalent117,118; therefore, it is likely that some but not all have the capacity to induce the germ cell fate or that these granules have another function.\n\nThe first column depicts the localization of germ plasm components (listed beneath the cartoon) at the posterior pole of mid-stage oocytes of Drosophila (top) and in the Balbiani body of early-stage oocytes of zebrafish. After fertilization, maternal germ plasm components (listed beneath the schematic) localize to specific membranes within the syncitial blastoderm of Drosophila and meroblastic cleavage (cells are incompletely separated and connected to the yolk) stage zebrafish embryos. The cells that receive these membranes develop as the PGCs. White rectangles depict maternal germ plasm components. The last column (grey rectangle) depicts PGCs of Drosophila and zebrafish after their migration to the gonad and indicates components of embryonic PGCs and germ granules. Dazap2, deleted in azoospermia-associated protein 2; Dazl, deleted in azoospermia-like; Elav, HuC; gcl, germ cell-less; Rbpms2, ribonuclear-binding protein with multiple splice isoforms 2.\n\nIn addition to their varied composition discussed above, other evidence indicates that germ plasm and germ granules may not be functionally equivalent. For example, the germ plasm and germ granules occupy distinct subcellar locations. In zebrafish and Xenopus, germ plasm associates with the endoplasmic reticulum in oocytes28. In zebrafish embryos, germ plasm first accumulates at furrows of cleavage-stage embryos by a mechanism that involves RNA recruitment and clearance106,119, whereas germ granules are perinuclear and form after genome activation120. In addition, the period when cells are competent to develop as PGCs, regardless of specification mode, precedes granule assembly and is developmentally restricted and brief, indicating that germ cell-inducing activity is tightly regulated. In Drosophila, rescue of osk mutants and the number of excess PGCs in overexpression contexts depend on Osk levels3,71. In zebrafish, expression of exogenous Buc after fertilization produces only a few additional PGCs, indicating that levels or timing may be limiting4,106. Similarly, in mice, high levels of the zinc finger transcriptional protein Blimp/Prdm1 are required to drive the epigenetic and cellular features of germ cells in vivo121, and BLIMP1 specifies hPGCLCs in a similar dosage-dependent manner122. These observations suggest that a limiting threshold or additional non-mutually exclusive factors (molecular, spatial, or temporal), or both, are essential for PGC identity. Accordingly, splitting determinants among sister cells, as occurs in C. elegans maternal-effect sterile (mes-1) mutants109,123, would produce two cells lacking sufficient factors for germline fate. Notably, granules are also heterogeneous and dynamic structures in C. elegans124,125. Finally, other evidence from C. elegans shows that specific germ granule factors, including PGL-1 and PGL-3, induce aggregate/granule formation in non-germline cells without converting those cells to PGCs, further indicating that granules alone are not sufficient for germ cell identity/specification126,127.\n\nGerm granules are enriched for RNA-binding proteins and proteins with other roles in post-transcriptional regulation; therefore, if germ granules do impart germline identity, it is possible that post-transcriptional regulation of other factors that may or may not be granule components is involved. If so, granules could impart germline fate only to cells that already express that factor; for example, Osk recruits components that regulate localization and translation of two germ granule components nanos and pgc, which promote patterning and pole cell development in flies110,112,128–130, and Buc must be localized to induce PGCs in zebrafish106. Consistent with an essential role for granules in germ cells, depletion of one or more germ granule components in C. elegans causes sterility131–133. Recently, maternal dazap2 was shown to maintain germ granules of zebrafish PGCs by acting epistatic to Tudor-7 and antagonistic to Dynein activity78. Because PGCs are specified, and granules form but later are not maintained in PGCs lacking maternal Dazap2, Mdazap2 mutant germ cells provide an opportunity to explore potential roles of granules in maintenance of vertebrate PGCs. The historical view of nuage, germ plasm, and germ granules was that each of these entities would promote the germ cell fate beginning with their specification to maintenance of germline identity. Efforts to gain a deeper understanding of the components of germ granules and functional assessment of these conserved elements of PGCs have provided strong evidence for mechanisms to preserve or protect germline identity; therefore, it is worth considering the possibility that the germ granules of specified embryonic PGCs contribute to a mechanism that preserves germline identity rather than specification of PGC fate. Understanding the contribution of granules to germ cell development and fertility remains an active area of investigation.\n\n\nGermline specification in mammals\n\nIn mammals, the germ cells are not specified by inheritance of maternal cytoplasm (germ plasm), but instead are specified later by inductive signals. In the mouse embryo just before gastrulation, signals from extra-embryonic ectoderm and visceral endoderm are necessary to specify germ cells within the posterior epiblast that is adjacent to the forming primitive streak (Figure 3). Unlike in flies and fish, in mice, no single factor that is necessary and sufficient to specify germ cells has been discovered. The earliest markers of mouse PGCs, including Blimp1 (Prdm1) and Prdm14, two critical factors that suppress somatic gene expression (thus promoting germ cell-specific gene programs)133–137 and developmental pluripotency-associated 3 (DPP3/Stella), are required to maintain rather than specify the PGCs138. Strikingly, nearly all cells of the mouse pregastrula epiblast can express Prdm1 or Prdm14 when induced by ubiquitous expression of the bone morphogenetic protein ligands Bmp4139 and Bmp8b140. Importantly, induced cells in culture can reconstitute functional sperm to germ cell-depleted neonates141. However, in vivo, only a few cells within the posterior epiblast that are positioned proximal to the extra-embryonic ectoderm become PGCs142. A Wnt3 signal from the epiblast primes these cells to respond to BMP produced by the extra-embryonic ectoderm141. The mechanisms that limit germ cell induction in anterior regions are not fully understood. However, PGC formation involves anterior endoderm factors and additional BMP family members that repress Blimp1 and thus promote acquisition of pluripotency and PGC development specifically in the posterior epiblast141. Other factors, such as microRNAs and their antagonists, contribute to PGC development (for example, by regulating Prdm1 expression)143. However, the functions of these molecules are not confined to PGCs and so they do not represent germline determinants. Significant unresolved questions are how and what factors limit selection of just a few cells within this region to become PGCs. Moreover, the identity of the factor or factors that specify them remains unknown.\n\n(A) The early stages of PGC emergence in mouse and humans. In mouse, cues from extra-embryonic ectoderm, including bone morphogentic proteins (BMPs), induce transcriptional regulators that promote PGC identity in the cells of the adjacent posterior epiblast. In humans, PGCs are first detected around the onset of gastrulation within the endodermal yolk sac wall. The signals that induce human PGCs in vivo are not known. (B, C) The molecular players involved in PGC specification from stem cells in culture. (B) For mouse cells, the program to induce primordial germ cell-like cells (PGCLCs) from embryonic stem cells (ESCs) in culture requires transforming growth factor-beta (TGF-β) family members (Activin) and basic fibroblast growth factors (bFGFs) to promote an epiblast-like cell (EpiLC) state. Exposure of the EpiLCs to BMPs (BMP4) and stem cell factor (SCF) and leukemia inhibitory factor (LIF) converts the EpiLCs to cells with mouse PGC-specific gene expression profiles. (C) For human cells, the program to induce PGCLCs from human-induced pluripotency cells (iPSCs) in culture like mouse requires TGF-β family members (Activin) and CHIRON to inhibit glycogen synthase kinase 3, an inhibitor of Wnt activity. This combination of factors promotes an induced mesoderm-like cell (iMLC) fate, which upon exposure to BMP4, LIF, SCF, plus epidermal growth factor (EGF) generates cells with human PGC-specific gene expression profiles. Notably, the transcription factor Sox2 is required in mouse cells, whereas Sox17 is necessary for human cells. In addition, PRDM14 is highly expressed in mouse PGCs and has been reported to be low or not expressed in human PGCs. AVE, anterior visceral endoderm; ExM, extra-embryonic mesoderm; ICM, inner cell mass; VE, visceral endoderm.\n\nPGCs in humans, as in mice, are specified in extragonadal regions around the time of gastrulation onset. However, there are important differences in the timing and development of extra-embryonic tissues and likely PGC specification between human and mouse. During the second week of development, the human embryo is composed of epiblast and primitive endoderm, which gives rise to the yolk sac (Figure 3). Owing to technical and ethical boundaries, lineage tracing to capture the first emergence of human PGCs prior to gastrulation has not been feasible. Nonetheless, human PGCs were identified in extragonadal regions more than 100 years ago and have been detected in human embryos in the Carnegie collection as early as stage 6 (around 2-week-old embryos) in the yolk sac endoderm in the vicinity of the developing allantois, an amniote structure involved in nutrition and waste removal (Figure 3)144. Functional studies of the earliest stages of PGC specification in humans would require manipulation and analysis of embryos within the first month following fertilization and this is not feasible. As an alternative to in vivo mammalian contexts, more tractable in vitro systems to study specification of mouse and human PGCs have been sought. In mice, key advances in PGC reprogramming paradigms have been facilitated by the development of germline reporters to identify and select for the lineage, including a mouse Vasa homolog (MVH) transgenic embryonic stem (ES) cell line, wherein the MVH promoter drives GFP reporter expression145 and a germ cell-specific gcOct4-GFP146. These tools enabled identification of germ cell-like cells in culture within just a few days. With markers in hand and using the germline-promoting factors defined from developmental studies, it was not long before mouse sperm147 and egg146 cells were derived from long-term ES cells. As germline stem cells (GSCs), PGCs are expected to generate cells that ultimately develop as functional gametes, sperm, or eggs that can produce a normal healthy fertile animal. The first animals generated from PGC-like cells (PGCLCs) were abnormal148, a limitation to using such cells to study normal germline development or infertility. However, in 2011, healthy offspring were produced from male-derived PGCLCs building on the observation that nearly all of the pregastrula cells in the mouse could express key PGC factors Blimp1 (Prdm1) and Prdm14 in response to BMP4149. In that study, the authors recapitulated gametogenesis in vitro by first reprogramming cells to a pregastrula epiblast state before exposure to germ cell-differentiation cues (Figure 3A,B). Later, the same group generated female PGCLCs with meiotic potential in reconstituted ovaries that produced fertile progeny after in vitro fertilization150, indicating that the in vitro produced cells could develop as functional female gametes in the correct environment.\n\nEncouraged by the success in establishing in vitro models of mouse PGC generation, several groups pursued in vitro models of human PGC development. With the mouse methods based on developmental paradigms in hand, the opportunity to discover the molecular programs responsible for producing the human germline seemed imminent. However, initial attempts to derive human PGCs quickly revealed that the mouse programming strategies were not effective, suggesting that despite conserved germ cell factors there must be differences in their mechanisms to generate germline cells (Figure 3A). Thus, several groups sought and recently reported conditions to efficiently generate hPGCLCs8,122,151 (Figure 3C). Similar transcriptional programs associated with successful generation of hPGCLCs were discovered in two independent studies; one study used transcription activator-like effector nucleases to engineer human cell lines that express fluorescent reporters for BLIMP1 and TFAP2C to select for germ cells122, and the second8 imparted pluripotency by using a combination of four inhibitors and selected for germline cells by using a reporter for the conserved PGC-specific protein Nanos62,63,152–154. Interestingly, these studies indicate that PGC specification is somewhat more direct in human cells compared with mouse cells. In human cells, Sox17 induces and is necessary to specify hPGCLCs, with Blimp1 acting downstream to promote PGC gene expression and repress expression of mesendodermal, neuronal, and epigenetic reprogramming genes as the cells differentiate into hPGCLCs8,122. In mice, ES cells first transition to an epiblast state, and Blimp1, but not Sox17155,156, represses somatic programs in nascent mPGCLCs149 (Figure 3). In both, epigenetic reprogramming is associated with PGCLC differentiation. However, the observed overlap in transcriptional programs between the human and mouse PGCLCs was surprisingly limited122, indicating distinct PGC specification programs between mouse and human despite their reliance on shared signaling molecules. Notably, the hPGCLCs generated so far resemble early hPGCs and thus provide an unprecedented opportunity to study the earliest events in hPGC specification. However, late-stage hPGCLCs have not been obtained on the basis of the absence of markers that are normally expressed after PGCs migrate to the gonad, indicating that further differentiation or refinement of the protocols is required to obtain and study development of these later stages. Recapitulating development from stem cell to mature germ cell remains a challenge and an important step to realize the full potential of hPGCLCs to provide insight into human diseases, including infertility with a genetic basis or environmental/toxicological basis, as well as developmental disorders.\n\nOnce specified, PGCs in flies, fish, and mammals, but not C. elegans, must travel from their extragonadal site of specification to the presumptive gonad where the PGCs will differentiate into sperm in males or oocytes in females (reviewed in 157,158). Successful migration to the gonad anlagen is essential for further PGC development and survival. With PGC markers and molecular and genetic approaches, key steps in PGC migration and the underlying cell behaviors have been defined and have been comprehensively reviewed elsewhere (for detailed reviews, see 157,158). The RNA-binding protein Nanos regulates PGC migration and survival in Drosophila63,159, C. elegans160, and zebrafish62,161, and nanos orthologs have a conserved role from invertebrates to humans in GSC maintenance62–64,152,159,160,162–166. Despite conserved requirements for nanos orthologs, the relevant RNA targets of nanos genes and downstream mechanisms promoting germ cell survival are not fully understood. In non-mammalian animals, GSCs persist into adulthood and continue to produce new gametes throughout the reproductive life of the animal. In mammals, GSCs maintain spermatogenesis throughout the lifetime of adult males, but whether or not female mammals have the capacity to generate new oocytes after birth has been highly controversial and an area of active study (reviewed in 167). Coming back to August Weismann’s words, “The importance of such a theory lies primarily in its suggestiveness, by which alone it becomes a step towards the ideal at which we aim, namely, the formulation of the true and complete theory”1,2. Clearly, the existence of GSCs, either naturally occurring in adult mammalian females or in vitro produced, has important therapeutic implications for the field of reproductive medicine; however, further development of tools and studies of the mechanisms to specify and maintain stem cell niches and ovarian reserve in diverse systems are essential for an improved understanding of germline development and reproductive health.",
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PubMed Abstract | Publisher Full Text | Free Full Text\n\nZimyanin V, Lowe N, St Johnston D: An oskar-dependent positive feedback loop maintains the polarity of the Drosophila oocyte. Curr Biol. 2007; 17(4): 353–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCallebaut I, Mornon JP: LOTUS, a new domain associated with small RNA pathways in the germline. Bioinformatics. 2010; 26(9): 1140–4. PubMed Abstract | Publisher Full Text\n\nHwang H, Kim S, Yoon S, et al.: Characterization of a novel oligomeric SGNH-arylesterase from Sinorhizobium meliloti 1021. Int J Biol Macromol. 2010; 46(2): 145–52. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKim S, Bae SY, Kim SJ, et al.: Characterization, amyloid formation, and immobilization of a novel SGNH hydrolase from Listeria innocua 11262. Int J Biol Macromol. 2012; 50(1): 103–11. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNakamura M, Okada N, Kajikawa M: Self-interaction, nucleic acid binding, and nucleic acid chaperone activities are unexpectedly retained in the unique ORF1p of zebrafish LINE. Mol Cell Biol. 2012; 32(2): 458–69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchneider AM, Schmidt S, Jonas S, et al.: Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition. Nucleic Acids Res. 2013; 41(22): 10563–72. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClaussen M, Pieler T: Xvelo1 uses a novel 75-nucleotide signal sequence that drives vegetal localization along the late pathway in Xenopus oocytes. Dev Biol. 2004; 266(2): 270–84. PubMed Abstract | Publisher Full Text\n\nRiemer S, Bontems F, Krishnakumar P, et al.: A functional Bucky ball-GFP transgene visualizes germ plasm in living zebrafish. Gene Expr Patterns. 2015; 18(1–2): 44–52. 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PubMed Abstract\n\nCampbell PD, Heim AE, Smith MZ, et al.: Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis. Development. 2015b; 142(17): 2996–3008. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJenny A, Hachet O, Závorszky P, et al.: A translation-independent role of oskar RNA in early Drosophila oogenesis. Development. 2006; 133(15): 2827–33. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRobb DL, Heasman J, Raats J, et al.: A kinesin-like protein is required for germ plasm aggregation in Xenopus. Cell. 1996; 87(5): 823–31. PubMed Abstract | Publisher Full Text\n\nStrome S, Martin P, Schierenberg E, et al.: Transformation of the germ line into muscle in mes-1 mutant embryos of C. elegans. Development. 1995; 121(9): 2961–72. PubMed Abstract\n\nEphrussi A, Dickinson LK, Lehmann R: Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 1991; 66(1): 37–50. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSnee MJ, Macdonald PM: Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components. J Cell Sci. 2004; 117(Pt 10): 2109–20. PubMed Abstract | Publisher Full Text\n\nTrcek T, Grosch M, York A, et al.: Drosophila germ granules are structured and contain homotypic mRNA clusters. Nat Commun. 2015; 6: 7962. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLittle SC, Sinsimer KS, Lee JJ, et al.: Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nat Cell Biol. 2015; 17(5): 558–68. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKnaut H, Steinbeisser H, Schwarz H, et al.: An evolutionary conserved region in the vasa 3'UTR targets RNA translation to the germ cells in the zebrafish. Curr Biol. 2002; 12(6): 454–66. PubMed Abstract | Publisher Full Text\n\nRaz E: The function and regulation of vasa-like genes in germ-cell development. Genome Biol. 2000; 1(3): REVIEWS1017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYajima M, Wessel GM: The multiple hats of Vasa: its functions in the germline and in cell cycle progression. Mol Reprod Dev. 2011; 78(10–11): 861–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCampbell PD, Chao JA, Singer RH, et al.: Dynamic visualization of transcription and RNA subcellular localization in zebrafish. Development. 2015a; 142(7): 1368–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGross-Thebing T, Paksa A, Raz E: Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos. BMC Biol. 2014; 12: 55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEno C, Pelegri F: Gradual recruitment and selective clearing generate germ plasm aggregates in the zebrafish embryo. Bioarchitecture. 2013; 3(4): 125–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrasser MJ, Mackenzie NC, Dumstrei K, et al.: Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development. BMC Dev Biol. 2008; 8: 58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKurimoto K, Yamaji M, Seki Y, et al.: Specification of the germ cell lineage in mice: a process orchestrated by the PR-domain proteins, Blimp1 and Prdm14. Cell Cycle. 2008b; 7(22): 3514–8. PubMed Abstract | Publisher Full Text\n\nSasaki K, Yokobayashi S, Nakamura T, et al.: Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells. Cell Stem Cell. 2015; 17(2): 178–94. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBerkowitz LA, Strome S: MES-1, a protein required for unequal divisions of the germline in early C. elegans embryos, resembles receptor tyrosine kinases and is localized to the boundary between the germline and gut cells. Development. 2000; 127(20): 4419–31. PubMed Abstract | Free Full Text\n\nBrangwynne CP, Eckmann CR, Courson DS, et al.: Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science. 2009; 324(5935): 1729–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWang JT, Smith J, Chen BC, et al.: Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans. eLife. 2014; 3: e04591. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHanazawa M, Yonetani M, Sugimoto A: PGL proteins self associate and bind RNPs to mediate germ granule assembly in C. elegans. J Cell Biol. 2011; 192(6): 929–37. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nUpdike DL, Hachey SJ, Kreher J, et al.: P granules extend the nuclear pore complex environment in the C. elegans germ line. J Cell Biol. 2011; 192(6): 939–48. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRangan P, DeGennaro M, Jaime-Bustamante K, et al.: Temporal and spatial control of germ-plasm RNAs. Curr Biol. 2009; 19(1): 72–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSmith JL, Wilson JE, Macdonald PM: Overexpression of oskar directs ectopic activation of nanos and presumptive pole cell formation in Drosophila embryos. Cell. 1992; 70(5): 849–59. PubMed Abstract | Publisher Full Text\n\nZaessinger S, Busseau I, Simonelig M: Oskar allows nanos mRNA translation in Drosophila embryos by preventing its deadenylation by Smaug/CCR4. Development. 2006; 133(22): 4573–83. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKawasaki I, Amiri A, Fan Y, et al.: The PGL family proteins associate with germ granules and function redundantly in Caenorhabditis elegans germline development. Genetics. 2004; 167(2): 645–61. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKawasaki I, Shim YH, Kirchner J, et al.: PGL-1, a predicted RNA-binding component of germ granules, is essential for fertility in C. elegans. Cell. 1998; 94(5): 635–45. PubMed Abstract | Publisher Full Text\n\nUpdike DL, Knutson AK, Egelhofer TA, et al.: Germ-granule components prevent somatic development in the C. elegans germline. Curr Biol. 2014; 24(9): 970–5. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKurimoto K, Yabuta Y, Ohinata Y, et al.: Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice. Genes Dev. 2008a; 22(12): 1617–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhinata Y, Payer B, O'Carroll D, et al.: Blimp1 is a critical determinant of the germ cell lineage in mice. Nature. 2005; 436(7048): 207–13. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nVincent SD, Dunn NR, Sciammas R, et al.: The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development. 2005; 132(6): 1315–25. PubMed Abstract | Publisher Full Text\n\nYamaji M, Seki Y, Kurimoto K, et al.: Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat Genet. 2008; 40(8): 1016–22. PubMed Abstract | Publisher Full Text\n\nBortvin A, Goodheart M, Liao M, et al.: Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice. BMC Dev Biol. 2004; 4: 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLawson KA, Dunn NR, Roelen BA, et al.: Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 1999; 13(4): 424–36. PubMed Abstract | Free Full Text\n\nYing Y, Liu XM, Marble A, et al.: Requirement of Bmp8b for the generation of primordial germ cells in the mouse. Mol Endocrinol. 2000; 14(7): 1053–63. PubMed Abstract | Publisher Full Text\n\nOhinata Y, Ohta H, Shigeta M, et al.: A signaling principle for the specification of the germ cell lineage in mice. Cell. 2009; 137(3): 571–84. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLawson KA, Hage WJ: Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found Symp. 1994; 182: 68–84; discussion 84–91. PubMed Abstract | Publisher Full Text\n\nWest JA, Viswanathan SR, Yabuuchi A, et al.: A role for Lin28 in primordial germ-cell development and germ-cell malignancy. Nature. 2009; 460(7257): 909–13. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWitschi, E: Migration of germ cells of human embryos from the yolk sac to the primitive gonadal folds. Contr Embryol Carnegie Inst. 1948; 209: 67–80.\n\nToyooka Y, Tsunekawa N, Akasu R, et al.: Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A. 2003; 100(20): 11457–62. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHübner K, Fuhrmann G, Christenson LK, et al.: Derivation of oocytes from mouse embryonic stem cells. Science. 2003; 300(5623): 1251–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGeijsen N, Horoschak M, Kim K, et al.: Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature. 2004; 427(6970): 148–54. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNayernia K, Nolte J, Michelmann HW, et al.: In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell. 2006; 11(1): 125–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHayashi K, Ohta H, Kurimoto K, et al.: Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell. 2011; 146(4): 519–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHayashi K, Ogushi S, Kurimoto K, et al.: Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science. 2012; 338(6109): 971–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSugawa F, Araúzo-Bravo MJ, Yoon J, et al.: Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile. EMBO J. 2015; 34(8): 1009–24. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBeer RL, Draper BW: nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary. Dev Biol. 2013; 374(2): 308–18. PubMed Abstract | Publisher Full Text\n\nGkountela S, Li Z, Vincent JJ, et al.: The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat Cell Biol. 2013; 15(1): 113–22. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJulaton VT, Reijo Pera RA: NANOS3 function in human germ cell development. Hum Mol Genet. 2011; 20(11): 2238–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHara K, Kanai-Azuma M, Uemura M, et al.: Evidence for crucial role of hindgut expansion in directing proper migration of primordial germ cells in mouse early embryogenesis. Dev Biol. 2009; 330(2): 427–39. PubMed Abstract | Publisher Full Text\n\nKanai-Azuma M, Kanai Y, Gad JM, et al.: Depletion of definitive gut endoderm in Sox17-null mutant mice. Development. 2002; 129(10): 2367–79. PubMed Abstract\n\nPaksa A, Raz E: Zebrafish germ cells: motility and guided migration. Curr Opin Cell Biol. 2015; 36: 80–5. PubMed Abstract | Publisher Full Text\n\nRichardson BE, Lehmann R: Mechanisms guiding primordial germ cell migration: strategies from different organisms. Nat Rev Mol Cell Biol. 2010; 11(1): 37–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKobayashi S, Yamada M, Asaoka M, et al.: Essential role of the posterior morphogen nanos for germline development in Drosophila. Nature. 1996; 380(6576): 708–11. PubMed Abstract | Publisher Full Text\n\nSubramaniam K, Seydoux G: nos-1 and nos-2, two genes related to Drosophila nanos, regulate primordial germ cell development and survival in Caenorhabditis elegans. Development. 1999; 126(21): 4861–71. PubMed Abstract\n\nKöprunner M, Thisse C, Thisse B, et al.: A zebrafish nanos-related gene is essential for the development of primordial germ cells. Genes Dev. 2001; 15(21): 2877–85. PubMed Abstract | Free Full Text\n\nKusz K, Tomczyk L, Spik A, et al.: NANOS3 gene mutations in men with isolated sterility phenotype. Mol Reprod Dev. 2009; 76(9): 804. PubMed Abstract | Publisher Full Text\n\nQin Y, Zhao H, Kovanci E, et al.: Mutation analysis of NANOS3 in 80 Chinese and 88 Caucasian women with premature ovarian failure. Fertil Steril. 2007; 88(5): 1465–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantos MG, Machado AZ, Martins CN, et al.: Homozygous inactivating mutation in NANOS3 in two sisters with primary ovarian insufficiency. Biomed Res Int. 2014; 2014: 787465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuzuki H, Tsuda M, Kiso M, et al.: Nanos3 maintains the germ cell lineage in the mouse by suppressing both Bax-dependent and -independent apoptotic pathways. Dev Biol. 2008; 318(1): 133–42. PubMed Abstract | Publisher Full Text\n\nWu X, Wang B, Dong Z, et al.: A NANOS3 mutation linked to protein degradation causes premature ovarian insufficiency. Cell Death Dis. 2013; 4: e825. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrieve KM, McLaughlin M, Dunlop CE, et al.: The controversial existence and functional potential of oogonial stem cells. Maturitas. 2015; 82(3): 278–81. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11598",
"date": "16 Dec 2015",
"name": "Ruth Lehmann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11599",
"date": "16 Dec 2015",
"name": "Prasanth Rangan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11600",
"date": "16 Dec 2015",
"name": "Bruce Draper",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1462
|
https://f1000research.com/articles/4-1460/v1
|
16 Dec 15
|
{
"type": "Review",
"title": "Drug-Induced Metabolic Acidosis",
"authors": [
"Amy Quynh Trang Pham",
"Li Hao Richie Xu",
"Orson W. Moe",
"Amy Quynh Trang Pham",
"Li Hao Richie Xu"
],
"abstract": "Metabolic acidosis could emerge from diseases disrupting acid-base equilibrium or from drugs that induce similar derangements. Occurrences are usually accompanied by comorbid conditions of drug-induced metabolic acidosis, and clinical outcomes may range from mild to fatal. It is imperative that clinicians not only are fully aware of the list of drugs that may lead to metabolic acidosis but also understand the underlying pathogenic mechanisms. In this review, we categorized drug-induced metabolic acidosis in terms of pathophysiological mechanisms, as well as individual drugs’ characteristics.",
"keywords": [
"metabolic",
"acidosis",
"drug-induced",
"MALA"
],
"content": "Introduction\n\nMetabolic acidosis is defined as an excessive accumulation of non-volatile acid manifested as a primary reduction in serum bicarbonate concentration in the body associated with low plasma pH. Certain conditions may exist with other acid-base disorders such as metabolic alkalosis and respiratory acidosis/alkalosis1.\n\nHumans possess homeostatic mechanisms that maintain acid-base balance (Figure 1). One utilizes both bicarbonate and non-bicarbonate buffers in both the intracellular and the extracellular milieu in the immediate defense against volatile (mainly CO2) and non-volatile (organic and inorganic) acids before excretion by the lungs and kidneys, respectively. Renal excretion of non-volatile acid is the definitive solution after temporary buffering. This is an intricate and highly efficient homeostatic system. Derangements in over-production, under-excretion, or both can potentially lead to accumulation of excess acid resulting in metabolic acidosis (Figure 1).\n\n1. A strong non-volatile acid HA dissociates to release H+ and poses an immediate threat to plasma pH.\n\n2. Bicarbonate buffers the H+ and generates CO2, which is expelled in the lungs and results in depletion of body HCO3-. Non-bicarbonate buffers (collectively referred to as B) carry the H+ until the kidneys excrete it.\n\n3. The kidneys split CO2 into H+ and HCO3- and selectively secrete H+ into the lumen and HCO3- into the blood. In addition, any excess H+ from the body fluid is also excreted.\n\n4. Most H+ excreted in the urine is carried by urinary buffers (UBs).\n\n5. Some organic anions (A) (e.g. lactate, ketoanions) can be metabolized to regenerate the HCO3-. If A is not metabolizable (e.g. phosphate or sulfate), it is excreted in the urine.\n\n* Two possible ways by which metabolic acidosis can occur.\n\nDrug-induced metabolic acidosis is often mild, but in rare cases it can be severe or even fatal. Not only should physicians be keenly aware of this potential iatrogenic complication but they should also be fully engaged in understanding the pathophysiological mechanisms. Metabolic acidosis resulting from drugs and/or ingestion of toxic chemicals can be grouped into four general categories (Figure 2):\n\n1. Drugs as exogenous acid loads\n\n2. Drugs leading to loss of bicarbonate in the gastrointestinal (GI) tract or kidney\n\n3. Drugs causing increased endogenous acid production\n\n4. Drugs that decrease renal acid excretion\n\nSome medications cannot be placed into one single category, as they possess multiple mechanisms that can cause metabolic acidosis.\n\n1. Increased exogenous ingestion of acidic precursors that are converted into strong acids.\n\n2. Loss of alkali from kidney or GI tract.\n\n3. Increased endogenous production of strong organic acids.\n\n4. Compromised renal net acid excretion by inhibition of the renin-angiotensin-aldosterone system (RAAS), impaired proximal tubule (PT), or distal tubule (DT) H+ secretion.\n\nIn suspected drug-induced metabolic acidosis, clinicians should establish the biochemical diagnosis of metabolic acidosis along with the evaluation of respiratory compensation and whether there is presence of mixed acid-based disorders2, then convert the biochemical diagnosis into a clinical diagnosis with identification of the invading acid/drug3. Next is to review the list of medications by history and record to determine whether any of the drugs are culprits in either the generation or the exacerbation of the disorder. Note that just because a patient has, for example, lactic acidosis and is on a drug that can potentially cause lactic acidosis does not mean that the two are causally related. Finally, if a drug is indeed causing some degree of metabolic acidosis, the clinician should make an appraisal of the benefits from the drug weighed against the severity of the metabolic complication to determine whether cessation of therapy is justified. For example, if a patient with problematic seizures is effectively controlled by topiramate, a mild degree of metabolic acidosis can be more tolerable than seizures.\n\n\nDrugs resulting in exogenous acid precursors\n\nMethanol4, ethylene glycol5, diethylene glycol6, and isopropanol7 are volatile alcohols that produce a high plasma osmolar gap (the alcohol itself and the aldehyde metabolite), pure high anion gap metabolic acidosis from their metabolism into strong carboxylic acids such as formic acid (from methanol), and a combination of oxalic, glyoxylic, and glycolic acid (from ethylene/diethylene glycol). Isopropanol alcohol, due to the absence of an alpha-carbon, could only be metabolized to a keto- group and contributes to an osmolar gap but not high anion gap metabolic acidosis in poisoning encounters. Toluene abuse with glue or paint thinner sniffing can cause hippuric metabolic acidosis that presents with a normal plasma anion gap but elevated urinary osmolar gap because of the rapid clearance of hippurate8. Note that the time at which blood is sampled may reveal variable osmolar and anion gap. When the hydroxyl group is metabolized to carboxyl with a low pKa, there will not be an osmolar gap due to the contemporaneous consumption of bicarbonate; however, the metabolite between hydroxyl and carboxyl is an aldehyde, which still contributes to an osmolar gap but not an anion gap.\n\nAmmonium chloride is not usually abused but is used extensively by investigators to study overproduction acidosis and used outside the laboratory9. There is a rise in acid excretion and a fall in serum HCO3- concentration that remains constant after initial drop10,11.\n\nThe excessive use of amino acids with a net positive charge would result in liberation of H+ during metabolism (arginine and lysine) in parenteral alimentation with inadequate concomitant administration of alkali12. Another example in this category is propylene glycol (1,2-propanediol [PG]), a common hygroscopic and emulsifying agent that is metabolized to lactate13. The U.S. Food and Drug Administration classified PG as GRAS (generally recognized as safe). The recommended maximum daily intake of PG should be less than 25 mg/kg/day (equivalent to 21 mmol/day for a 70 kg person)14. Each drug injection may have very different amounts of PG. Clinically significant toxicity is seen only in rapid, massive, and protracted parenteral administration of high quantities, especially in patients with renal impairment. PG intoxication from intravenous vitamin therapy was reported in pediatric patients who developed stupor15. Intoxication with lactic acidosis and hyperosmolality were found during treatment of schizophrenia16, with the use of intravenous benzodiazepines13,17, etomidate18, nitroglycerin19, and barbiturates20, all with PG as a vehicle. Approximately 55% of PG undergoes oxidation to propionaldehyde and pyruvic, acetic, and lactic acid, while the remainder is excreted unchanged in the urine21,22. Some studies have demonstrated PG-injured proximal tubular cells, leading to impaired renal acidification20,23. Patients with hepatic dysfunction, renal insufficiency, and diabetic ketoacidosis are more susceptible to PG toxicity and development of lactic acidosis24.\n\n\nDrugs causing external base loss\n\nCarbonic anhydrases (CAs) are critical enzymes for bicarbonate reabsorption. Acetazolamide is a commonly used CA inhibitor in the treatment of ocular and convulsive disorders. It causes bicarbonaturia and a mild degree of hyperchloremic metabolic acidosis25. There have also been reports of symptomatic anion gap metabolic acidosis associated with acetazolamide therapy in elderly patients26 and in those with impaired renal function26,27 and diabetes mellitus28. Severe metabolic acidosis may result from inhibition of pyruvate carboxylase and mitochondrial damage29. Ocular solution of CA inhibitor-induced acidosis is rare but has been reported30.\n\nTopiramate is approved for the treatment of seizure, as a migraine headache prophylaxis, and for weight loss, with off-label use for bipolar disorder, obesity, neuropathic pain, and smoking cessation31. It inhibits CA II, IV, and XII31. Topiramate generates a mild hyperchloremic metabolic acidosis32,33 but increases urinary pH and drastically lowers urinary citrate excretion, thus increasing the risk for calcium phosphate urolithiasis34,35.\n\nThe sulfonamide class of drugs also has CA inhibitory activity. Topical application and absorption over large areas in burn patients can cause extremely high blood levels and systemic CA inhibition36. This results in more than mere renal bicarbonate loss but rather a systemic disequilibrium syndrome.\n\nCholestyramine is an oral agent for treating hypertriglyceridemia and cholestasis by binding and sequestering bile acids from the entero-hepatic circulation; the non-absorbable complexes are eventually excreted in the stool37. In the GI tract, cholestyramine also binds phosphate, sulfate, and bicarbonate, leading to potential loss of bicarbonate from the body. Under normal conditions, this is easily corrected by renal regeneration of bicarbonate. However, patients with impaired renal function are at risk of hyperchloremic acidosis37–39.\n\nSevelamer hydrochloride is a non-reabsorbable phosphate binder. Dialysis patients on sevelamer hydrochloride have lower mean serum bicarbonate concentration during and at the end of therapy compared to those treated with calcium acetate40,41. The chloride released upon phosphate stimulates bicarbonate secretion by the gut via chloride-bicarbonate exchange40. This secretion coupled with defective ability to regenerate bicarbonate in renal patients leads to hyperchloremic acidosis. This complication is avoided by using sevelamer carbonate, which binds phosphate and releases carbonate instead42, or by bixalomer, which contains no chloride, and seems to demonstrate equal efficacy of phosphate binding with no evidence of acidosis in clinical trials43,44. Laxative abuse, calcium chloride, and magnesium sulfate could also cause hyperchloremic acidosis because the secreted bicarbonate from the pancreas is trapped by calcium and magnesium45–47 and then excreted in stools.\n\n\nDrugs causing increased endogenous acid production\n\nLactic acid is produced under basal metabolic conditions and H+ ions are released. Normally, an equivalent amount of H+ ions is consumed when the liver and renal cortex utilize lactate for gluconeogenesis or oxidize it to water and CO2 so that acid-base balance remains undisturbed (Figure 3). Lactic acidosis is arbitrarily classified into overproduction of lactate (type A), underutilization of lactate (type B), or both48. Type A lactic acidosis is associated with generalized or regional tissue hypoxia, while type B is seen in patients with metabolic abnormalities in malignancy, hepatic dysfunction, diabetes mellitus, congenital enzymatic deficiency, and drugs or toxins45,49. In 1995, metformin replaced phenformin, a notorious inducer of lactic acidosis, and became the primary biguanide used today50. Post marketing safety surveillance revealed no cases of fatal lactic acidosis51. There are still reports of metformin-associated lactic acidosis (MALA)52,53 with proposed mechanisms shown in Figure 3.\n\n1. Metformin inhibits pyruvate carboxylase (PC) → inhibits hepatic gluconeogenesis146 → excess lactate84. Metformin also inhibits complex I of the mitochondrial electron transport chain (ETC)84 → increases NADH/NAD+ ratio → blocks the entry of pyruvate into the tricarboxylic acid (TCA) cycle147. LDH = lactate dehydrogenase\n\n2. In vitro, nucleoside reverse transcriptase inhibitors (NRTIs) inhibit β-oxidation, the tricarboxylic acid (Krebs) cycle, and DNA γ-polymerase → mitochondrial dysfunction and loss of transcription of essential enzymes → hepatic steatosis (increased triglycerides), myopathy, pancreatitis, nephrotoxicity, and lactic acidosis68.\n\n3. Linezolid may cross-react with mammalian cellular processes → disrupts mitochondrial protein synthesis involved in ETC75,148.\n\n4. Propofol may inhibit coenzyme Q and cytochrome C at Complex IV in ETC, and also inhibit mitochondrial fatty acid metabolism88.\n\n5. Isoniazid inhibits metabolism of lactate to pyruvate82.\n\nMost cases of MALA were associated with some underlying conditions such as acute renal failure induced by volume depletion, other potential nephrotoxic agents and concurrent use of radio-contrast media, or hepatic insufficiency54–58. Blood pH and lactate levels are not prognostic in MALA59. Although the incidence of MALA is low, once developed, the mortality can be staggeringly high52, particularly in the critical care setting, so discontinuation is advised in a patient with impending renal and multi-organ failure. Recently, a less restrictive guideline is proposed on metformin usage in patients of stable chronic kidney disease60–63. In general, the mortality of MALA decreased from 50% to 25% from the 1960s to the present64.\n\nHighly active antiretroviral therapy (HAART) has led to dramatic reductions in HIV-associated morbidity and mortality65 (Figure 3). However, lactic acidosis complicated this therapy, especially with the nucleoside and nucleotide reverse transcriptase inhibitor (NRTI)-based regimens: didanosine, stavudine, lamivudine, zidovudine, and abacavir66–71. Combined use of these drugs further increases the risk of lactic acidosis72. Moreover, didanosine73, cidofovir74, lamivudine, and stavudine75 could cause Fanconi syndrome with pan-proximal tubular dysfunction leading to exacerbation of the acidosis and reduction of the plasma anion gap. The mortality of HAART-induced lactic acidosis can be as high as 50%76.\n\nLinezolid is a long-term antibiotic against serious resistant Gram-positive organisms77,78 with adverse effects including bone marrow toxicity, optic/peripheral neuropathy, and lactic acidosis77,79,80. Concurrent use of selective serotonin uptake inhibitors such as citalopram and sertraline may predispose patients to lactic acidosis81,82. The vast majority of lactic acidosis occurred in the elderly and those receiving prolonged treatment, and most resolved upon cessation of linezolid80. Children receiving linezolid appeared to suffer lactic acidosis earlier during treatment83 (Figure 3).\n\nIsoniazid is commonly used to treat tuberculosis84. Dosing more than 300 mg/day can lead to refractory grand mal or localized seizure, coma, and lactic acidosis84–86. Some suggested acidosis stems from excessive muscle activity during refractory seizure86,87, and slow reversal was observed in the postictal period. One proposed mechanism is inhibition of conversion of lactate to pyruvate84,87–89 (Figure 3).\n\nPropofol is commonly used for induction and maintenance of anesthesia, sedation, and interventional procedures. Two cases were reported on propofol-associated severe metabolic acidosis90,91. Risk factors include severe head injury, critical illness, prolonged administration (>48 hours) of large doses (>4 mg/kg/hour, equivalent to 1.6 mmol/hour for a 70 kg person), and inborn errors of fatty acid oxidation90,92 (Figure 3).\n\nKetosis develops when metabolism of fatty acid exceeds the removal of ketoacids (acetoacetic and β-hydroxybutyric). Typically there is insulin deficiency and/or resistance coupled with elevated glucagon and catecholamine. Glucose utilization is impaired and lipolysis is increased, augmenting the delivery of glycerol, alanine, and fatty acids for ketoacid generation45,93.\n\nOverdose with salicylates in children commonly produces high anion gap acidosis, while adults exhibit a mixed respiratory alkalosis and metabolic acidosis. Metabolic acidosis occurs during salicylate toxicity due to uncoupling of oxidative phosphorylation and interfering with the Krebs cycle45,86, resulting in accumulation of lactic acid and ketoacids in as many as 40% of adult patients with salicylate poisoning45,94,95. The anion gap is mainly composed of ketoanions and lactate, while salicylate anion seldom exceeds 3 mEq/L.\n\nAlcoholic ketoacidosis occurs when ethanol is abused chronically in the setting of poor carbohydrate intake and volume contraction. Ketosis resolves when the ethanol intake is interrupted and the patient is provided with nutrients and fluid, which stimulates insulin secretion and promotes the regeneration of bicarbonate from the metabolism of ketoacid anions45.\n\nThe γ-glutamyl cycle produces glutathione, which is involved in the inactivation of free radicals, detoxification of many compounds, and amino acid transport45,96,97. Acetaminophen can deplete glutathione, leading to increased formation of γ-glutamyl cysteine, which is converted and accumulated as pyroglutamic acid (5-oxoproline)45,97. Patients at risk include those with malnutrition, sepsis, alcohol abuse, underlying liver disease, and/or renal insufficiency96. Acetaminophen ingestion alone may not cause pyroglutamic acidosis, but synergistic interaction between acetaminophen and the other factors as noted above96 can. Concomitant use of other drugs such as aminoglycoside and β-lactam penicillin is reported to increase the risk98.\n\n\nDrugs causing decreased renal acid excretion\n\nBoth angiotensin II and aldosterone are stimulators of the H+-ATPase α-intercalated cells in the cortical collecting tubule99,100, adding H+ into the urinary luminal. Inhibition of the renin-angiotensin-aldosterone system (RAAS), which leads to secondary inhibition of the H+-ATPase, can lead to decreased H+ secretion and metabolic acidosis. Additionally, inhibition of the RAAS reduces Na+ reabsorption, which reduces the luminal electronegativity and reduces H+ excretion by the H+-ATPase100. The same mechanisms can cause hyperkalemia, which can in turn reduce stimulation of the H+/K+-ATPase101. Hyperkalemia suppresses ammoniagenesis in the proximal tubule, impairs NH4+ transport in the medullary thick ascending limb, and reduces medullary interstitial ammonium concentration, all of which can lower urine acid excretion45,102. Therefore, any drug that affects the RAAS or causes hyperkalemia can increase the risk of metabolic acidosis. These drugs include the following (Figure 4):\n\n- Cyclooxygenase (COX) inhibitors45,103\n\n- β-adrenergic receptor blockers45,104\n\n- Angiotensin-converting enzyme inhibitors (ACEIs), angiotensin II receptor blockers (ARBs), and direct renin inhibitors104–106\n\n- Heparin107 and ketoconazole108,109\n\n- Spironolactone and eplerenone45,110\n\n- Potassium-sparing diuretics: amiloride and triamterene45,110\n\n- Pentamidine and trimethoprim111–113\n\n- Calcineurin inhibitors: cyclosporine and tacrolimus114,115\n\n1. Cyclooxygenase (COX) inhibitors and β-blockers interfere with release of renin, leading to hyperkalemia with metabolic acidosis43,101.\n\n2. Angiotensin-converting enzyme inhibitors (ACEIs), aldosterone receptor blockers (ARBs), and renin inhibitors all interfere with the renin-angiotensin-aldosterone system (RAAS), causing hyperkalemia with hyperchloremic metabolic acidosis102–104.\n\n3. Heparin105 and ketoconazole106,107 interfere with aldosterone synthesis.\n\n4. Spironolactone and eplerenone block aldosterone receptors43,108.\n\n5. Na+ channel blockers lead to reduced net negative charge in lumen in cortical collecting ducts (CCD), which reduces K+ and H+ excretion and causes hyperkalemia and acidosis43,108–111.\n\n6. Calcineurin inhibitors interfere with Na, K-ATPase in the principal cell decreasing transepithelial K secretion and H+ secretion, cause vasoconstriction of afferent glomerular arterioles, and decrease glomerular filtration rate and alter filtration fraction112,113.\n\n7. Lithium causes a voltage-dependent defect for H+ secretion and decreases H+-ATPase activity114–116.\n\n8. Amphotericin B binds to sterol in mammalian cell membranes106,107 forming intramembranous pores which increase permeability and back diffusion of H+.\n\nWhen these drugs are administered in combination, there is increased risk for hyperkalemia and metabolic acidosis, especially in patients with diabetes, chronic kidney disease, and liver disease45.\n\nIn contrast, patients with classic distal renal tubular acidosis (dRTA) generally have hypokalemic hyperchloremic metabolic acidosis. The metabolic acidosis results from the inability to acidify urine in the distal nephron and impaired excretion of NH4+100. Inherited forms of dRTA have defects in the basolateral HCO3-/Cl- exchanger, B1 or A4 subunits of the H+-ATPase, or CA. Some medications can mimic these defects by altering membrane permeability and causing leaky pathways45. Amphotericin B108,109, lithium116–118, and foscarnet119 are known to cause leak and lead to hypokalemic hyperchloremic metabolic acidosis (Figure 4).\n\nThe proximal tubule is the initial step in renal acidification and is essential in maintaining acid-base homeostasis by reclaiming 80% of filtered bicarbonate (HCO3-) (Figure 5). Bicarbonate reabsorption occurs by luminal H+ excretion and HCO3- extrusion back into the blood at the basolateral membrane100. CAs catalyze the reaction: CO2 + H2O → HCO3-+ H+. If proximal HCO3- reclamation is impaired, more HCO3- is delivered to the distal tubule, which has limited capacity for HCO3- reabsorption. Bicarbonaturia ensues and net acid excretion decreases, which eventually leads to metabolic acidosis45,120. Generalized proximal tubule dysfunction is termed Fanconi syndrome. Potential drugs that could induce Fanconi syndrome include the following (Figure 5):\n\n- CA inhibitors (e.g. acetazolamide)25.\n\n- Anti-viral/HIV drugs (e.g. lamivudine, stavudine75 and tenofovir121–124). Most tenofovir-induced cases are subclinical125\n\n- Platinum-containing agents (e.g. cisplatin126,127) and DNA alkylating agents (e.g. ifosfamide128–130) are common proximal tubule toxins. Note that cyclophosphamide, structural isomer of ifosfamide, can also cause hemorrhagic cystitis but is not nephrotoxic by producing less chloroacetaldehyde128\n\n- Valproic acids (VPAs)131–133\n\n- Outdated tetracycline134–136\n\n- Aminoglycoside137 accumulation in proximal tubule would lead to nephrotoxicity with an unclear mechanism; however, incidence decreased recently due to a better monitoring strategy138\n\n- Deferasirox139–143\n\nMany other agents such as fumaric acid144, suramin145, and imatinib146 have also been associated with Fanconi syndrome in case reports. This field remained to be further explored as proximal tubule toxicity is common due to the existence of multiple drug transporters at the surface membrane, leading to very high uptake of drugs by this segment147.\n\n1. CA inhibitors25 cause bicarbonaturia and hyperchloremic metabolic acidosis in the elderly26 and patients with renal failure27 and diabetes28.\n\n2. Antineoplastic platinum-containing agents126,127 and DNA-alkylating agents128–130 damage proximal tubule cells through accumulation and induced cell apoptosis.\n\n3. Anti-viral/HIV drugs75,121–124, valproic acid (VPA)131–133, and outdated tetracycline134–136 interfere with mitochondrial function within proximal tubule cells, leading to tubular dysfunction.\n\n4. Aminoglycosides137,148,149 induce acidosis with unclear mechanisms150.\n\n5. Deferasirox139–143 increases hemodynamic iron removal, causes vacuolization of proximal tubular epithelial cells142, and elevates iron absorption in various organs. All could lead to acidosis.\n\n\nConclusion\n\nIn summary, metabolic acidosis can occur as a side effect of therapy. Instead of memorizing the catalogue of drugs, clinicians should classify these agents based on their pathophysiologic mechanisms to facilitate the recognition of potential causal relationships. Some of these side effects are inferred from empirical observations, but some have undergone extensive studies to determine the pathogenesis of metabolic acidosis. We hope that this review will intrigue our readers to experience that eureka moment identifying unrecognized explanations for metabolic acidosis in patients or to partake in extending clinical observations to clinical investigations.\n\n\nAbbreviations\n\nACEI, Angiotensin-converting enzyme inhibitor; ARB, Aldosterone receptor blocker; CA, Carbonic anhydrase; COX, Cyclooxygenase; dRTA, Distal renal tubular acidosis; FDA, Food & Drug Administration; GRAS, Generally recognized as safe; GI, Gastrointestinal; HAART, Highly active antiretroviral therapy; MALA, Metformin-associated lactic acidosis; NAD, Nicotinamide Adenine Dinucleotide; PG, Propylene glycol; RAAS, Renin-angiotensin-aldosterone system; VPA, Valproic acid.",
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Acid-Base and Electrolyte Disorders: A Companion to Brenner and Rector's The Kidney. Philadelphia: WB Saunders; 2002; 1–21.\n\nArena FP, Dugowson C, Saudek CD: Salicylate-induced hypoglycemia and ketoacidosis in a nondiabetic adult. Arch Intern Med. 1978; 138(7): 1153–4. PubMed Abstract | Publisher Full Text\n\nProudfoot AT, Krenzelok EP, Brent J, et al.: Does urine alkalinization increase salicylate elimination? If so, why? Toxicol Rev. 2003; 22(3): 129–36. PubMed Abstract | Publisher Full Text\n\nFenves AZ, Kirkpatrick HM 3rd, Patel VV, et al.: Increased anion gap metabolic acidosis as a result of 5-oxoproline (pyroglutamic acid): a role for acetaminophen. Clin J Am Soc Nephrol. 2006; 1(3): 441–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nEmmett M: Acetaminophen toxicity and 5-oxoproline (pyroglutamic acid): a tale of two cycles, one an ATP-depleting futile cycle and the other a useful cycle. Clin J Am Soc Nephrol. 2014; 9(1): 191–200. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCroal BL, Glen AC, Kelly CJ, et al.: Transient 5-oxoprolinuria (pyroglutamic aciduria) with systemic acidosis in an adult receiving antibiotic therapy. Clin Chem. 1998; 44(2): 336–40. PubMed Abstract\n\nWagner CA, Devuyst O, Bourgeois S, et al.: Regulated acid-base transport in the collecting duct. Pflugers Arch. 2009; 458(1): 137–56. PubMed Abstract | Publisher Full Text\n\nRodríguez-Soriano J: New insights into the pathogenesis of renal tubular acidosis--from functional to molecular studies. Pediatr Nephrol. 2000; 14(12): 1121–36. PubMed Abstract | Publisher Full Text\n\nEiam-Ong S, Kurtzman NA, Sabatini S: Regulation of collecting tubule adenosine triphosphatases by aldosterone and potassium. J Clin Invest. 1993; 91(6): 2385–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDuBose TD Jr: Molecular and pathophysiologic mechanisms of hyperkalemic metabolic acidosis. Trans Am Clin Climatol Assoc. 2000; 111: 122–33; discussion 133–4. PubMed Abstract | Free Full Text\n\nCheng HF, Harris RC: Cyclooxygenases, the kidney, and hypertension. Hypertension. 2004; 43(3): 525–30. PubMed Abstract | Publisher Full Text\n\nWeinberg JM, Appel LJ, Bakris G, et al.: Risk of hyperkalemia in nondiabetic patients with chronic kidney disease receiving antihypertensive therapy. Arch Intern Med. 2009; 169(17): 1587–94. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWhite WB, Bresalier R, Kaplan AP, et al.: Safety and tolerability of the direct renin inhibitor aliskiren in combination with angiotensin receptor blockers and thiazide diuretics: a pooled analysis of clinical experience of 12,942 patients. J Clin Hypertens (Greenwich). 2011; 13(7): 506–16. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSakallı H, Baskın E, Bayrakcı US, et al.: Acidosis and hyperkalemia caused by losartan and enalapril in pediatric kidney transplant recipients. Exp Clin Transplant. 2014; 12(4): 310–3. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPreston RA, Hirsh MJ MD, Oster MD Jr, et al.: University of Miami Division of Clinical Pharmacology therapeutic rounds: drug-induced hyperkalemia. Am J Ther. 1998; 5(2): 125–32. PubMed Abstract | Publisher Full Text\n\nAyub M, Levell MJ: Inhibition of human adrenal steroidogenic enzymes in vitro by imidazole drugs including ketoconazole. J Steroid Biochem. 1989; 32(4): 515–24. PubMed Abstract | Publisher Full Text\n\nOhlsson A, Cedergreen N, Oskarsson A, et al.: Mixture effects of imidazole fungicides on cortisol and aldosterone secretion in human adrenocortical H295R cells. Toxicology. 2010; 275(1–3): 21–8. PubMed Abstract | Publisher Full Text\n\nDavies DL, Wilson GM: Diuretics: mechanism of action and clinical application. Drugs. 1975; 9(3): 178–226. PubMed Abstract | Publisher Full Text\n\nKleyman TR, Roberts C, Ling BN: A mechanism for pentamidine-induced hyperkalemia: inhibition of distal nephron sodium transport. Ann Intern Med. 1995; 122(2): 103–6. PubMed Abstract | Publisher Full Text\n\nSchlanger LE, Kleyman TR, Ling BN: K+-sparing diuretic actions of trimethoprim: inhibition of Na+ channels in A6 distal nephron cells. Kidney Int. 1994; 45(4): 1070–6. PubMed Abstract | Publisher Full Text\n\nVelázquez H, Perazella MA, Wright FS, et al.: Renal mechanism of trimethoprim-induced hyperkalemia. Ann Intern Med. 1993; 119(4): 296–301. PubMed Abstract | Publisher Full Text\n\nCaliskan Y, Kalayoglu-Besisik S, Sargin D, et al.: Cyclosporine-associated hyperkalemia: report of four allogeneic blood stem-cell transplant cases. Transplantation. 2003; 75(7): 1069–72. PubMed Abstract | Publisher Full Text\n\nLea JP, Sands JM, McMahon SJ, et al.: Evidence that the inhibition of Na+/K+-ATPase activity by FK506 involves calcineurin. Kidney Int. 1994; 46(3): 647–52. PubMed Abstract | Publisher Full Text\n\nKim YH, Kwon TH, Christensen BM, et al.: Altered expression of renal acid-base transporters in rats with lithium-induced NDI. Am J Physiol Renal Physiol. 2003; 285(6): F1244–57. PubMed Abstract | Publisher Full Text\n\nRoscoe JM, Goldstein MB, Halperin ML, et al.: Lithium-induced impairment of urine acidification. Kidney Int. 1976; 9(4): 344–50. PubMed Abstract | Publisher Full Text\n\nGrünfeld JP, Rossier BC: Lithium nephrotoxicity revisited. Nat Rev Nephrol. 2009; 5(5): 270–6. PubMed Abstract | Publisher Full Text\n\nNavarro JF, Quereda C, Quereda C, et al.: Nephrogenic diabetes insipidus and renal tubular acidosis secondary to foscarnet therapy. Am J Kidney Dis. 1996; 27(3): 431–4. PubMed Abstract | Publisher Full Text\n\nHamm LL, Nakhoul N: Renal Acidification. In: Brenner BM, editor. Brenner and Rector's The Kidney. Philadelphia: Saunders; 2007; 248–69.\n\nMalik A, Abraham P, Malik N: Acute renal failure and Fanconi syndrome in an AIDS patient on tenofovir treatment--case report and review of literature. J Infect. 2005; 51(2): E61–5. PubMed Abstract | Publisher Full Text\n\nHall AM, Hendry BM, Nitsch D, et al.: Tenofovir-associated kidney toxicity in HIV-infected patients: a review of the evidence. Am J Kidney Dis. 2011; 57(5): 773–80. PubMed Abstract | Publisher Full Text\n\nHerlitz LC, Mohan S, Stokes MB, et al.: Tenofovir nephrotoxicity: acute tubular necrosis with distinctive clinical, pathological, and mitochondrial abnormalities. Kidney Int. 2010; 78(11): 1171–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLucey JM, Hsu P, Ziegler JB: Tenofovir-related Fanconi's syndrome and osteomalacia in a teenager with HIV. BMJ Case Rep. 2013; 2013: pii: bcr2013008674. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHall AM, Edwards SG, Lapsley M, et al.: Subclinical tubular injury in HIV-infected individuals on antiretroviral therapy: a cross-sectional analysis. Am J Kidney Dis. 2009; 54(6): 1034–42. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nCachat F, Nenadov-Beck M, Guignard JP: Occurrence of an acute Fanconi syndrome following cisplatin chemotherapy. Med Pediatr Oncol. 1998; 31(1): 40–1. PubMed Abstract | Publisher Full Text\n\nSahni V, Choudhury D, Ahmed Z: Chemotherapy-associated renal dysfunction. Nat Rev Nephrol. 2009; 5(8): 450–62. PubMed Abstract | Publisher Full Text\n\nZamlauski-Tucker MJ, Morris ME, Springate JE: Ifosfamide metabolite chloroacetaldehyde causes Fanconi syndrome in the perfused rat kidney. Toxicol Appl Pharmacol. 1994; 129(1): 170–5. PubMed Abstract | Publisher Full Text\n\nStöhr W, Paulides M, Bielack S, et al.: Ifosfamide-induced nephrotoxicity in 593 sarcoma patients: a report from the Late Effects Surveillance System. Pediatr Blood Cancer. 2007; 48(4): 447–52. PubMed Abstract | Publisher Full Text\n\nLeem AY, Kim HS, Yoo BW, et al.: Ifosfamide-induced Fanconi syndrome with diabetes insipidus. Korean J Intern Med. 2014; 29(2): 246–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWatanabe T, Yoshikawa H, Yamazaki S, et al.: Secondary renal Fanconi syndrome caused by valproate therapy. Pediatr Nephrol. 2005; 20(6): 814–7. PubMed Abstract | Publisher Full Text\n\nKnorr M, Schaper J, Harjes M, et al.: Fanconi syndrome caused by antiepileptic therapy with valproic Acid. Epilepsia. 2004; 45(7): 868–71. PubMed Abstract | Publisher Full Text\n\nEndo A, Fujita Y, Fuchigami T, et al.: Fanconi syndrome caused by valproic acid. Pediatr Neurol. 2010; 42(4): 287–90. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWegienka LC, Weller JM: Renal tubular acidosis caused by degraded tetracycline. Arch Intern Med. 1964; 114(2): 232–5. PubMed Abstract | Publisher Full Text\n\nCleveland WW, Adams WC, Mann JB, et al.: Acquired Fanconi syndrome following degraded tetracycline. J Pediatr. 1965; 66(2): 333–42. PubMed Abstract | Publisher Full Text\n\nMontoliu J, Carrera M, Darnell A, et al.: Lactic acidosis and Fanconi's syndrome due to degraded tetracycline. Br Med J (Clin Res Ed). 1981; 283(6306): 1576–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIzzedine H, Launay-Vacher V, Isnard-Bagnis C, et al.: Drug-induced Fanconi's syndrome. Am J Kidney Dis. 2003; 41(2): 292–309. PubMed Abstract | Publisher Full Text\n\nBanerjee S, Narayanan M, Gould K: Monitoring aminoglycoside level. BMJ. 2012; 345: e6354. 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PubMed Abstract | Faculty Opinions Recommendation\n\nDell'Orto VG, Bianchetti MG, Brazzola P: Hyperchloraemic metabolic acidosis induced by the iron chelator deferasirox: a case report and review of the literature. J Clin Pharm Ther. 2013; 38(6): 526–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHäring N, Mähr HS, Mündle M, et al.: Early detection of renal damage caused by fumaric acid ester therapy by determination of urinary β2-microglobulin. Br J Dermatol. 2011; 164(3): 648–51. PubMed Abstract | Publisher Full Text\n\nRago RP, Miles JM, Sufit RL, et al.: Suramin-induced weakness from hypophosphatemia and mitochondrial myopathy. Association of suramin with mitochondrial toxicity in humans. Cancer. 1994; 73(7): 1954–9. PubMed Abstract | Publisher Full Text\n\nFrançois H, Coppo P, Hayman JP, et al.: Partial fanconi syndrome induced by imatinib therapy: a novel cause of urinary phosphate loss. Am J Kidney Dis. 2008; 51(2): 298–301. PubMed Abstract | Publisher Full Text\n\nLaunay-Vacher V, Izzedine H, Karie S, et al.: Renal tubular drug transporters. Nephron Physiol. 2006; 103(3): p97–106. PubMed Abstract | Publisher Full Text\n\nGhiculescu RA, Kubler PA: Aminoglycoside-associated Fanconi syndrome. Am J Kidney Dis. 2006; 48(6): e89–93. PubMed Abstract | Publisher Full Text\n\nGainza FJ, Minguela JI, Lampreabe I: Aminoglycoside-associated Fanconi's syndrome: an underrecognized entity. Nephron. 1997; 77(2): 205–11. PubMed Abstract | Publisher Full Text\n\nLopez-Novoa JM, Quiros Y, Vicente L, et al.: New insights into the mechanism of aminoglycoside nephrotoxicity: an integrative point of view. Kidney Int. 2011; 79(1): 33–45. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11589",
"date": "16 Dec 2015",
"name": "Jeffrey Kraut",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11590",
"date": "16 Dec 2015",
"name": "Mitchell Halperin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11593",
"date": "16 Dec 2015",
"name": "Pierluigi Brazzola",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1460
|
https://f1000research.com/articles/4-1459/v1
|
16 Dec 15
|
{
"type": "Review",
"title": "Recent advances in treatment of severe primary immunodeficiencies",
"authors": [
"Andrew R. Gennery"
],
"abstract": "Primary immunodeficiencies are rare, inborn errors that result in impaired, disordered or uncontrolled immune responses. Whilst symptomatic and prophylactic treatment is available, hematopoietic stem cell transplantation is an option for many diseases, leading to cure of the immunodeficiency and establishing normal physical and psychological health. Newborn screening for some diseases, whilst improving outcomes, is focusing research on safer and less toxic treatment strategies, which result in durable and sustainable immune function without adverse effects. New conditioning regimens have reduced the risk of hematopoietic stem cell transplantation, and new methods of manipulating stem cell sources should guarantee a donor for almost all patients. Whilst incremental enhancements in transplantation technique have gradually improved survival outcomes over time, some of these new applications are likely to radically alter our approach to treating primary immunodeficiencies.",
"keywords": [
"immunodeficiency",
"Primary immunodeficiency",
"hematopoietic stem cell",
"hematopoietic stem cell transplantation",
"Haematopoietic Stem Cell",
"Haematopoietic Stem Cell Transplantation",
"Severe combined immunodeficiencies",
"Chemotherapy conditioning"
],
"content": "Introduction\n\nGenetically inherited inborn errors of immunity impair immune function, which leaves affected individuals exposed to increased risks of infection, inflammation and autoimmunity. To date, over 300 diseases with X-linked, autosomal recessive and autosomal dominant inheritance have been identified1. The majority of described diseases result from complete or partial loss of function of the gene product, but more recently, increasing numbers of diseases in which the gene mutation leads to a gain-of-function effect have been described. For an increasing number of diseases, replacement of the defective recipient immune system with a functioning system from a healthy donor, by hematopoietic stem cell transplantation (HSCT), can lead to a permanent cure.\n\nThe first HSCTs for primary immunodeficiencies (PIDs) were performed in 19682,3, and so nearly 50 years of experience has led to many significant improvements in technique and outcome. Severe combined immunodeficiencies (SCIDs) are the most profound defects, and HSCT, until recently, has been the only approach to treatment (with the exception of adenosine deaminase [ADA] deficiency, for which enzyme replacement is possible). Other PIDs have had conservative or HSCT approaches to management, although HSCT is now becoming a more widely accepted modality of treatment, as long-term outcomes of conservative management are investigated, and outcomes improve through earlier diagnosis and safer approaches to transplantation.\n\nTreatment of PIDs has resulted in the recognition of better outcomes with early or pre-emptive treatment, development of newborn screening programs for PID, development of gene therapy, and is now driving the development of gene editing as well as the search for minimally toxic conditioning regimens. This article will outline recent developments in the field.\n\n\nSevere combined immunodeficiencies\n\nSCIDs are heterogeneous PIDs that are characterized by the absence of thymopoiesis, T-lymphocyte maturation and function, and which affect cellular and humoral acquired immunity; without definitive treatment within the first 12 to 18 months of life, the condition is invariably fatal. Classic presentation is with persistent viral respiratory or gastrointestinal infection in infancy and with failure to clear virus and persistent and deteriorating symptoms4. Multiple pathogens may co-exist, and opportunistic infection—for example, with Pneumocystis jiroveci—is common. Immunization with live Bacillus Calmette-Guerin or rotavirus vaccine can cause persistent and disseminated infection5–7. The genetic bases of 75% to 80% of SCID types are now understood. Definitive treatment is predominantly by allogeneic HSCT, although gene therapy and enzyme replacement therapy are available for some specific genetic sub-types. Depending on the genetic defect, recipient B-lymphocyte or natural killer (NK) cells or both may be present. In contradistinction to the treatment of hematological malignancies, in which eradication of malignancy is required, the objective of HSCT in patients with SCID is to provide normal HSCs, facilitating correction of the immune defect. Therefore, it is critical to minimize potential sequelae of treatment but to establish effective long-term immune function. The outcome of HSCT for SCID is related to a number of different factors, including genotype, pre-existing morbidities at time of HSCT, and in particular pre-existing viral infection, as well as the type (and degree of human leukocyte antigen [HLA] match) of the donor8–10. Current issues of interest to address include early detection of infants with SCID, so that referral for treatment may be initiated before the onset of infection, and approach to conditioning.\n\n\nNewborn screening for severe combined immunodeficiency\n\nDuring T-lymphocyte receptor development, redundant DNA is excised but remains within the cell and can be used as a marker of thymopoiesis. Patients with SCID (and some other PIDs) lack thymopoiesis and subsequently the redundant DNA (known as a T-cell receptor excision circle, or TREC) is not present. With the blood taken during routine neonatal screening, it is possible to detect TRECs by polymerase chain reaction and thus identify infants with SCID before symptoms develop11,12. Alternately, for two related PIDs in which a DNA salvage enzyme is deficient, leading to absent lymphocyte development (ADA and purine nucleoside phosphorylase deficiency), metabolic by-products can be detected by mass spectrometry on blood eluted from the neonatal blood spot13,14. It has previously been demonstrated that the outcome of HSCT for newborn patients with SCID is significantly superior to that of patients presenting with infection8–10. The introduction of newborn screening enables the detection of infants with SCID before they become symptomatic, allowing definitive treatment before they acquire infection. Many states in the USA have now implemented newborn screening for SCID15, enabling early detection and treatment. Other countries are considering implementation of newborn screening programs for SCID and other PIDs16,17.\n\n\nChemotherapy conditioning\n\nFollowing appropriate diagnosis, there remains debate about the best approach to treatment. Infusion of unfractionated donor HSC inoculum without preparative chemotherapy leads to T-lymphocyte immune reconstitution18. However, without HSC engraftment, the establishment of thymopoiesis and the durability of T-lymphocyte function are variable and depend on the phenotype and hence genotype. Infants with NK cell-negative SCID are more likely to survive than those who have recipient NK cells, and also develop high-level donor T-lymphocyte chimerism with superior long-term persistence of CD4+ T-lymphocyte immunity without preconditioning chemotherapy. The presence of recipient NK cells is a strong indicator that preparative chemotherapy conditioning will be required for engraftment of T-lymphocyte precursors capable of supporting robust and durable T-lymphocyte reconstitution19. However, the type of donor used is also important, as use of an unrelated HLA-matched donor, rather than an HLA-matched sibling donor, significantly increases the risk of graft-versus-host disease (GvHD)20.\n\nThere are a number of issues regarding the use of chemotherapy preconditioning. Acute toxicities are frequently observed, and in the presence of active infection, mortality is increased unless a matched sibling donor is available10. Whilst durability and sustainability of thymopoiesis and consistency of B-lymphocyte function are more likely in most forms of SCID following chemotherapy and, in particular, those genotypes with recipient NK cells19,21,22, there are concerns about the effects, albeit short courses, of chemotherapy on young infants. There are currently no good multicenter studies looking at the long-term (>20 year) immunological, general health or quality-of-life outcomes of HSCT in SCID, either using chemotherapy preconditioning or just infusing the donor inoculum. A joint European Inborn Errors Working Party/North American Primary Immune Deficiency Treatment Consortium study looking at these outcomes is in progress. However, for a subset of patients with radiosensitive SCID, alkylating agents are associated with more significant long-term co-morbidities, even when compared with other NK cell-positive SCID phenotypes23. New conditioning regimens with analogues of busulfan appear safer and have fewer short-term toxicities, but long-term outcomes are uncertain24,25. The problems of administering chemotherapy will become more focused only as the majority of infants begin to be diagnosed in the newborn period through newborn screening programs. Whilst the mortality risk associated with chemotherapy, particularly in well patients with no co-morbidities, is low, it is not absent, and the concerns over administering such toxic drugs to newborns are driving the search for alternative strategies. Minimally intensive regimens using monoclonal antibodies have been successfully employed in treating SCID, even with significant co-morbidities, but these still employ low-dose chemotherapeutic agents26. Unfortunately, to date, neither treatment with alemtuzumab monotherapy nor plerixafor in conjunction with granulocyte-colony-stimulating factor appears to facilitate donor stem engraftment in patients27,28. In utero animal models have demonstrated some beneficial effect of administering an anti-c-Kit receptor antibody, which interrupts an important signaling pathway in homing, adhesion, maintenance, and survival of HSCs in the hematopoietic niche, and transplanting pre-treated animals on the first day of life; some gain in donor stem cell engraftment was observed29. Whilst these results are encouraging, further work is required before patient benefit can be demonstrated. However, clinical trials using therapeutic-grade antibodies are being planned.\n\n\nGene therapy\n\nGene therapy for SCID has been successful at curing patients. Random integration of a viral vector containing the corrected gene into the genome of harvested autologous HSCs and re-infusion of the transduced product have demonstrated clinical benefit and cure of patients with X-linked and ADA-deficient SCID30,31. Early trials were complicated by graft failure and in some cases insertional mutagenesis, leading to lymphoproliferation and leukemia, at least in X-linked SCID32,33. Modifications of the retroviral vector with the addition of self-inactivating gamma-retroviral vectors, with enhancer-deleted U3 regions34,35, and adoption of lenti-viral vectors should reduce or eliminate the risk of insertional mutagenesis36,37. New methodologies of gene editing use highly specific, targeted double-stranded DNA cleavage nucleases to remove the defective gene and replace it with a corrected copy at the appropriate genomic locus through the use of homologous recombination of corrected gene sequences by cellular DNA repair pathways38–40. These techniques are a more physiologically sound method of genetic correction as the appropriate regulatory control of gene expression is maintained.\n\nPre-clinical studies have demonstrated efficacy of this technique in cell lines, although correction in primary HSCs has been limited. Current gene therapy protocols do not consistently result in full correction of the defect, and in some trials, low-dose chemotherapy has been employed to improve autologous stem cell engraftment and give a competitive advantage over non-transduced cells. Thus, in an approaching era of hopefully universal screening for SCID, a chemotherapy-free approach for either conventional HSCT or gene-targeted therapy may be possible, eliminating concerns about long-term toxicity and ensuring durable and sustainable immune reconstitution.\n\n\nAlternative treatments for severe combined immunodeficiencies\n\nFor a few SCID genotypes, alternative therapies are available and, though not curative, may improve the physical condition of the patient pending curative treatment. For patients with ADA-deficient SCID, polyethylene-glycosylated adenosine deaminase (PEG-ADA) can be given as an infusion, thus partially reversing the enzyme deficiency. This can rapidly reverse some of the toxicity associated with ADA deficiency (for example, ADA deficiency-related pulmonary alveolar proteinosis41) and improve the clinical condition of the patient to facilitate successful HSCT42. Long-term treatment with PEG-ADA leads to poorer immunoreconstitution than following HSCT43 and gene therapy and may induce PEG-ADA-specific antibody formation, compromising further immunoreconstitution44.\n\nDefects in the folate and cobalamin pathway can impact immune development45–47. Recently, a patient with SCID immunophenotype has been described48,49, in whom a mutation in MTHFD1, which encodes a protein essential for folate metabolism, was found. Treatment with folate and hydroxocobalamin improved but did not fully correct lymphocyte counts and proliferation responses, although no benefit to the neurological impairment was observed48–50.\n\nPhosphoglucomutase 3 (PGM3) is a hexose phosphate mutase, a key enzyme in many glycosylation pathways. Mutations in PGM3 are associated with neutropenia, B and T lymphocytopenia and bone marrow failure51,52, although extra-immune manifestations, including facial dysmorphism, skeletal anomalies and intellectual impairment, are also apparent. Conventional HSCT cures the immunological features but not the other features. N-acetyl-galactosamine supplementation may have a role in bypassing the metabolic defect and stabilising these patients prior to definitive treatment. Thymic stromal defects—for example, complete DiGeorge or CHARGE (coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies/deafness) syndrome or FOXN1 deficiency—can be successfully treated by thymic transplant and with better outcomes than HSCT53–55.\n\n\nOther primary immunodeficiencies\n\nAn increasing number of non-SCID PIDs are successfully treated by HSCT. A patient with Wiskott-Aldrich syndrome was among the first to undergo HSCT2. Subsequently, HSCT has been performed for many different PID diseases. HSCT initially was restricted to patients with combined immunodeficiencies or severe T-lymphocyte defects, but now there is an expanding list of appropriate indications. Specifically, cytotoxicity defects (e.g., familial hemophagocytic syndromes), defects of phagocytes (e.g., chronic granulomatous disease, leukocyte adhesion deficiency, and GATA2 deficiency), and defects in cytokine signaling pathways are now indications for HSCT. As the definition of immunodeficiency broadens, so do the indications for HSCT56. Patients with autoimmune disease or autoimmune enteropathy, including those with immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, defects in interleukin-10 (IL-10) signaling pathways, gain-of-function STAT-3 disease, and CTLA-4 deficiency, have received curative transplants. Often patients may achieve a molecular diagnosis following successful transplantation, so proof of concept of treatment may precede genetic diagnosis.\n\nPatients with non-SCID PID have residual T lymphocyte-mediated immunity which is able to mediate rejection of allogeneic grafts, necessitating pre-transplant chemotherapy conditioning to facilitate donor stem cell engraftment and immune reconstitution. The decision to refer for HSCT can be difficult, as many diseases have alternative medical therapies, which if taken regularly may prevent many complications from developing. However, careful cohort studies indicate that, even with regular medication, life-threatening complications occur, and quality of life may be significantly and adversely affected. With modern HSCT techniques, the survival outcome following HSCT may be equivalent to “conventional” treatment57. In addition, successful HSCT for non-SCID PID may lead to an improved, and even normal, quality of life, as well as abolition of the risk of disease-associated sequelae, and removal of necessity to take life-long medication58. Unfortunately, as for patients with SCID, outcomes are best for those with no pre-existing co-morbidities59,60. Parents may be left with the choice of transplanting a young healthy child, and accepting the small but finite risk of failure and likely death, or waiting until the child is more sick with established co-morbidities but with a diminished chance of successful transplantation. For some patients with non-SCID PID, immunodeficiency is part of a wider syndrome. Successful HSCT can correct the hemato-immunological defect, but extra-immune manifestations are generally not modified61 and may present many years after successful HSCT62.\n\n\nSafer chemotherapy conditioning regimens\n\nSeveral recent advances are changing the landscape for these patients. Firstly, the development of low-toxicity conditioning regimens targeting sub-myeloablative busulfan levels has enabled successful transplantation, even in older patients with significant pre-existing co-morbidities63, giving survival of more than 95%. Furthermore, some of the concerns about long-term sequelae of chemotherapy may be partially resolved, particularly with regard to fertility64,65. Long-term sequelae of treosulfan-containing regimens are less certain, but short-term results in survival and establishment of immune function are encouraging24,25. For many patients with PID, partial donor chimerism is sufficient to induce cure if the affected recipient cell lineage is replaced completely or partially by donor cells, although complete donor chimerism is best in some diseases (Table 1).\n\nIL7Ra, interleukin 7 receptor alpha; RAG, recombination-activating gene; SCID, severe combined immunodeficiency.\n\n\nTransplantation of patients with no matched donor\n\nIn contrast to patients with SCID, there is usually sufficient time to seek alternative non-family adult matched donors or cord blood stem cell units from national and international registries. Owing to the following, T lymphocyte-depleted haplo-identical donors have not been widely used in non-SCID PID conditions:\n\n• The increased risk of non-engraftment or rejection.\n\n• Patients often harbor pre-existing viral infection. T-lymphocyte depletion of the allograft to prevent GvHD prolongs the time to immune reconstitution, thus increasing the risk of death from disseminated viral infection.\n\n• Most non-SCID disorders can be managed in the medium term with supportive care, including prophylactic antimicrobials, immunoglobulin replacement, immunosuppressive agents, and careful nutritional and respiratory support.\n\nNew methods of depleting allografts of T lymphocytes that cause GvHD, but retaining those with an anti-viral effect, are demonstrating efficacy for patients with non-SCID PID. The most widely used technique involves depletion of CD3+ T lymphocytes bearing the αβ T-lymphocyte receptor, as well as CD19+ lymphocytes, by using magnetic bead technology. Two groups have published results with excellent outcomes of 97% survival in 37 patients with different PIDs65, and 91% in 23 patients with non-malignant disease, of which 13 had PID66, results which compare favorably with historic data56. Although some patients developed mild GvHD, the few deaths were from viral infection. Fewer data are available for the other methods of T-lymphocyte depletion. Naïve (CD45+), rather than memory, T lymphocytes are predominantly associated with allo-reactivity and mediate GvHD67. A pilot study of depletion of CD45RA+ T lymphocytes, using magnetic bead technology, in five patients with combined immunodeficiencies showed promising results, with four patients engrafting and clearing viral infection within 2 months of transplantation, and no development of GvHD68. Cyclophosphamide is non-toxic to pluripotent HSC but selectively toxic to recently activated lymphocytes and so administration a few days after HSCT would target newly activated previous naïve cells stimulated by recipient allo-antigens, but preserving anti-viral competence. To date, there are no published data on the infusion of replete HLA haplo-identical grafts followed by administration of cyclophosphamide after stem cell infusion for patients with PID. However, the technique has been shown to be successful for malignant conditions69 and there are early reports of its use in non-malignant conditions70,71.\n\nIn conjunction with the less toxic conditioning regimens, the transplant outlook for PID patients without a good HLA-matched donor is now significantly improved. For patients with Wiskott-Aldrich syndrome, an alternative approach is the use of gene-transduced autologous cells. Initial clinical trials using retro-viral vectors resulted in insertional mutagenesis, giving rise to lymphoproliferation and leukemia72. The use of lenti-viral vectors does not seem to induce in vivo clonal selection with vector integrations near oncogenes, and partially reverses the Wiskott-Aldrich phenotype with significant clinical effect73.\n\n\nManagement of post-transplant sequelae\n\nThree major complications of HSCT are GvHD, overwhelming viral infection and sinusoidal obstruction syndrome (SOS). New developments in tackling these sequelae are beginning to reduce significant complications associated with them. T-lymphocyte depletion of donor stem cell sources and less toxic chemotherapy regimens will reduce the risk of GvHD. First-line treatment of acute GvHD is systemic corticosteroids, but for steroid-recalcitrant or steroid-resistant GvHD, treatment options that do not cause further profound immunosuppression and are not associated with significant adverse events are limited. Extracorporeal photopheresis has long been recognized as a treatment for chronic GvHD, but use in acute GvHD is now being explored74. Published data have demonstrated efficacy, and with experienced operators, treatment of low-body weight (<40 kg) patients is possible. Clinical trials investigating rapid and early intervention with extracorporeal photochemotherapy (ECP) in patients with acute GvHD are in progress.\n\nSystemic viral infections, particularly with cytomegalovirus, Epstein-Barr virus and adenovirus, remain a major cause of morbidity and mortality in all HSCT patients. Available anti-viral pharmacotherapy has limited efficacy and major associated toxicities, particularly myelosuppression and nephrotoxicity. Effective anti-viral immunity is established only when viral-specific T lymphocytes develop. Particularly in the context of T lymphocyte-depleted stem cell sources, this may take several months. Infusion of ex vivo expanded donor- or third party-derived T lymphocytes with activity against one or more viruses whilst excluding allo-reactive GvHD-causing T lymphocytes has been used successfully in preventing and treating viral infections following HSCT. A recent retrospective study of 36 patients with PID treated with viral-specific T lymphocytes before or after HSCT showed an overall survival of 80% with mild, self-limiting GvHD in some patients only (Michael Keller, personal communication). Previous studies have focused on the use of virus-exposed donors as a source of viral-specific T lymphocytes, but trials are now focusing on cells that have been generated from healthy donors and banked for use as “off the shelf” therapy for viral infections, which eliminates the time and expense required for custom-produced products75.\n\nSOS is a severe and potentially life-threatening complication occurring after HSCT and secondary to sinusoidal endothelial cell damage. Endothelial cell damage in other organs can lead to associated syndromes, including capillary leak syndrome, engraftment syndrome, transplant-associated microangiopathy or diffuse alveolar hemorrhage. Risk factors for the development of SOS include allogeneic HSCT, use of unrelated or HLA-mismatched donor, young age (<2 years), myeloablative conditioning, particularly when busulphan or irradiation is used, and previous or current hepatic damage. Disease-specific risk factors include hemophagocytic lymphohistiocytosis and osteopetrosis. Mortality for severe SOS with multi-organ failure is high (>80%). Numerous treatments have been used to treat SOS, including ursodiol, glutamine, vitamin E, low-molecular-weight heparin, recombinant tissue plasminogen activator, and prostaglandin E1. Most show dubious efficacy and are associated with significant toxicities and, in particular, hemorrhage. Supportive therapy includes early and meticulous fluid and electrolyte balance and judicious use of diuretics. Respiratory support, peritoneo-centesis and hemodialysis or hemofiltration to support renal impairment and fluid balance may be required in severe SOS. Recently, the European Medicines Agency approved defibrotide in European countries as the only curative treatment of severe SOS after HSCT. Defibrotide, a polydisperse oligonucleotide, exhibits local anti-thrombotic, anti-ischemic and anti-inflammatory properties and seems to protect endothelial cells and restore the disrupted thrombotic-fibrinolytic homeostasis. Remission from SOS and survival seem better in children than adults when receiving defibrotide76. Defibrotide has also demonstrated benefit when used as prophylaxis to prevent SOS in pediatric patients, and interestingly patients receiving defibrotide also developed significantly less GvHD77.\n\nIn conclusion, steady improvements in the outcome of HSCT for SCID and other PIDs mean that, for the majority of patients born with these conditions today, curative treatment is to be expected. Advances in tackling the recognized complications of HSCT have enabled survival today to approach 90%, even for patients with significant disease-related sequelae. As new diseases are described, the challenge is to determine the best therapeutic option, but HSCT is a realistic treatment for many patients, including adults with late-diagnosed or late-onset disease. Important questions remain, however; the three most pressing are how to achieve earlier diagnosis, how to develop non-toxic conditioning regimens to achieve durable and sustained immune reconstitution, and an evaluation of the long-term outcomes of HSCT for these conditions, including immune function and general and psychological health. Continued collaborations between physicians caring for these patients and scientific societies dedicated to the study of diseases and their treatments are likely to further these aspirations. As we learn more about the biology of these conditions and their treatments, the information may also benefit patients receiving HSCT for other diseases as well as those taking part in gene therapy trials and patients with autoimmune diseases or those receiving solid organ transplants.\n\n\nAbbreviations\n\nADA, adenosine deaminase; GvHD, graft-versus-host disease; HLA, human leukocyte antigen; HSC(T), hematopoietic stem cell (transplantation); NK, natural killer; PEG-ADA, polyethylene-glycosylated adenosine deaminase; PGM3, phosphoglucomutase 3; PID, primary immunodeficiency; SCID, severe combined immunodeficiency; SOS, sinusoidal obstruction syndrome; TREC, T-cell receptor excision circle.",
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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarkert ML, Devlin BH, Alexieff MJ, et al.: Review of 54 patients with complete DiGeorge anomaly enrolled in protocols for thymus transplantation: outcome of 44 consecutive transplants. Blood. 2007; 109(10): 4539–47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarkert ML, Marques JG, Neven B, et al.: First use of thymus transplantation therapy for FOXN1 deficiency (nude/SCID): a report of 2 cases. Blood. 2011; 117(2): 688–96. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nJanda A, Sedlacek P, Hönig M, et al.: Multicenter survey on the outcome of transplantation of hematopoietic cells in patients with the complete form of DiGeorge anomaly. Blood. 2010; 116(13): 2229–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGennery AR, Slatter MA, Grandin L, et al.: Transplantation of hematopoietic stem cells and long-term survival for primary immunodeficiencies in Europe: entering a new century, do we do better? J Allergy Clin Immunol. 2010; 126(3): 602–10.e1–11. PubMed Abstract | Publisher Full Text\n\nCole T, Pearce MS, Cant AJ, et al.: Clinical outcome in children with chronic granulomatous disease managed conservatively or with hematopoietic stem cell transplantation. J Allergy Clin Immunol. 2013; 132(5): 1150–5. PubMed Abstract | Publisher Full Text\n\nCole T, McKendrick F, Titman P, et al.: Health related quality of life and emotional health in children with chronic granulomatous disease: a comparison of those managed conservatively with those that have undergone haematopoietic stem cell transplant. J Clin Immunol. 2013; 33(1): 8–13. PubMed Abstract | Publisher Full Text\n\nSeger RA, Gungor T, Belohradsky BH, et al.: Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985–2000. Blood. 2002; 100(13): 4344–50. PubMed Abstract | Publisher Full Text\n\nGennery AR, Khawaja K, Veys P, et al.: Treatment of CD40 ligand deficiency by hematopoietic stem cell transplantation: a survey of the European experience, 1993–2002. Blood. 2004; 103(3): 1152–7. PubMed Abstract | Publisher Full Text\n\nChen R, Giliani S, Lanzi G, et al.: Whole-exome sequencing identifies tetratricopeptide repeat domain 7A (TTC7A) mutations for combined immunodeficiency with intestinal atresias. J Allergy Clin Immunol. 2013; 132(3): 656–664.e17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPachlopnik Schmid J, Moshous D, Boddaert N, et al.: Hematopoietic stem cell transplantation in Griscelli syndrome type 2: a single-center report on 10 patients. Blood. 2009; 114(1): 211–8. PubMed Abstract | Publisher Full Text\n\nGüngör T, Teira P, Slatter M, et al.: Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study. Lancet. 2014; 383(9915): 436–48. PubMed Abstract | Publisher Full Text\n\nPanasiuk A, Nussey S, Veys P, et al.: Gonadal function and fertility after stem cell transplantation in childhood: comparison of a reduced intensity conditioning regimen containing melphalan with a myeloablative regimen containing busulfan. Br J Haematol. 2015; 170(5): 719–26. PubMed Abstract | Publisher Full Text\n\nBalashov D, Shcherbina A, Maschan M, et al.: Single-Center Experience of Unrelated and Haploidentical Stem Cell Transplantation with TCRαβ and CD19 Depletion in Children with Primary Immunodeficiency Syndromes. Biol Blood Marrow Transplant. 2015; 21(11): 1955–62. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBertaina A, Merli P, Rutella S, et al.: HLA-haploidentical stem cell transplantation after removal of αβ+ T and B cells in children with nonmalignant disorders. Blood. 2014; 124(5): 822–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChérel M, Choufi B, Trauet J, et al.: Naïve subset develops the most important alloreactive response among human CD4+ T lymphocytes in human leukocyte antigen-identical related setting. Eur J Haematol. 2014; 92(6): 491–6. PubMed Abstract | Publisher Full Text\n\nTouzot F, Neven B, Dal-Cortivo L, et al.: CD45RA depletion in HLA-mismatched allogeneic hematopoietic stem cell transplantation for primary combined immunodeficiency: A preliminary study. J Allergy Clin Immunol. 2015; 135(5): 1303–9.e1–3. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nO'Donnell PV, Luznik L, Jones RJ, et al.: Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. 2002; 8(7): 377–86. PubMed Abstract | Publisher Full Text\n\nde la Fuente J, Harrington Y, Bradshaw A, et al.: Parental Haploidentical HSCT with a Post-Infusion of Stem Cells Cyclophosphamide Approach is Feasible and Leads to a High Rate of Donor Engraftment in Haemoglobinopathies Allowing Universal Application of Transplantation. Bone Marrow Transplant. 2015; 50(Suppl 1): S45. F1000 Recommendation\n\nHongeng S, Anurathapan U, Pakakasama S, et al.: Haploidentical Hematopoietic Stem Cell Transplantation (Haplo-SCT) with Pre-Transplant Immunosuppression (PTIS) Followed by Reduced Toxicity Regimen and Post- Transplant Cyclophosphamide (Post-Cy) in Severe Thalassemia. Bone Marrow Transplant. 2015; 50(Suppl 1): S88. F1000 Recommendation\n\nBraun CJ, Boztug K, Paruzynski A, et al.: Gene therapy for Wiskott-Aldrich syndrome--long-term efficacy and genotoxicity. Sci Transl Med. 2014; 6(227): 227ra33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAiuti A, Biasco L, Scaramuzza S, et al.: Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science. 2013; 341(6148): 1233151. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDas-Gupta E, Dignan F, Shaw B, et al.: Extracorporeal photopheresis for treatment of adults and children with acute GVHD: UK consensus statement and review of published literature. Bone Marrow Transplant. 2014; 49(10): 1251–8. PubMed Abstract | Publisher Full Text\n\nLi Pira G, Ivaldi F, Starc N, et al.: A registry of HLA-typed donors for production of virus-specific CD4 and CD8 T lymphocytes for adoptive reconstitution of immune-compromised patients. Transfusion. 2014; 54(12): 3145–54. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRichardson P, Tomblyn M, Kernan N, et al.: Results of a Phase 3 study utilizing a historical control. Defibrotide (DF) in the treatment of severe hepatic veno-occlusive disease (VOD) with multi-organ failure (MOF) following stem cell transplantation (SCT). ASH Annual Meeting Abstracts. 2009; 114: 654. Reference Source\n\nCorbacioglu S, Cesaro S, Faraci M, et al.: Defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial. Lancet. 2012; 379(9823): 1301–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11587",
"date": "16 Dec 2015",
"name": "Alain 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": []
},
{
"id": "11588",
"date": "16 Dec 2015",
"name": "Joshua D. Milner",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1459
|
https://f1000research.com/articles/4-1458/v1
|
16 Dec 15
|
{
"type": "Research Article",
"title": "High pre-transplant TREC levels indicate good prognosis after hematopoietic stem cell transplantation",
"authors": [
"Adriano de Moraes Arantes",
"Kharen Kawemura",
"Adriana Seber",
"José Salvador Rodrigues de Oliveira",
"Maria Gerbase-DeLima",
"Natalia Shulzhenko",
"Andrey Morgun",
"Kharen Kawemura",
"Adriana Seber",
"José Salvador Rodrigues de Oliveira",
"Maria Gerbase-DeLima"
],
"abstract": "BackgroundThymus-dependent T-cell reconstitution plays a role in immune recovery after stem cell transplantation (HSCT). High pre-HCST thymic function has been associated with higher survival, lower incidence of acute and chronic graft versus host disease (GVHD) and lower incidence of infections. The aim of this study was to analyze the relationship between pre-HSCT peripheral blood levels of T-cell receptor excision circles (TREC) and post-HSCT clinical events in recipients of HLA-identical hematopoietic stem cell transplants.MethodDelta deletion signal joint TRECs (sjTRECs) formed by the dREC-yJa rearrangement were quantified by real time PCR in peripheral blood lymphocytes of 62 HSCT recipients.ResultsUnivariate analysis revealed an association between low TREC levels and a higher incidence of grade II-IV acute GVHD (p=0.026), bacterial infection (p=0.005) and cytomegalovirus infection (p=0.033), whereas high TREC levels were associated with higher overall survival (p=0.028). In the multivariate analysis, low pre-HSCT TREC levels remained independently associated with lower survival (p=0.032; RR 2.6), occurrence of grade II-IV acute GVHD (p=0.031; RR: 2.5), bacterial infection (p=0.006, RR: 6.6) and cytomegalovirus infection (p=0.039; RR:2.8).ConclusionOur results corroborate the concept that pre-HSCT recipient´s thymic function is an important predictor of risk for acute grade II-IV GVHD and infection.",
"keywords": [
"T cell receptor excision circle",
"thymic function",
"stem cell transplantation"
],
"content": "Introduction\n\nAlthough a hematopoietic stem cell transplant (HSCT) is an effective treatment for several types of malignant blood diseases, it can cause a number of different side effects, mainly related to opportunistic infections and graft versus host disease (GVHD). The immediate post-transplant period is followed by severe immunodeficiency resulting in persistent susceptibility to infection1–5. Even more prolonged immunodeficiency arises from inadequacy in effective CD4+ T-cell and B cell reconstitution6–9. Regeneration of adequate T-cell numbers and repertoire are key elements in the recovery of immunity, required for protection against infection10.\n\nPost-transplant T-cells are derived from mature T-cells present in the donor graft, and T-cells developing de novo in the recipient from donor stem cells. It is likely that the latter pathway of differentiation leads to long-term immune reconstitution11,12. It has been suggested that the reconstitution and maintenance of effective T-cell immunity after HSCT is dependent on education about T-cell precursors in the thymus, as shown by the correlation between increased thymic output and an increment in naïve T-cells, and broader TCR repertoires after transplantation13–15. Factors that can delay thymic function include age, cell source, histocompatibility leukocyte antigen mismatches, GVHD and direct thymic damage from chemoradiotherapy11,16–19.\n\nThe production of a functional T-cell receptor requires the deletion of the TCRδ gene, which is positioned within the TCRα- locus20. The deleted TCRδ-genes remain present as extrachromosomal circular products called signal joints T-cell receptor excision circles (sjTRECs). These products are stable21, are not duplicated during mitosis, and are therefore diluted with each cellular division22. Thus, sjTRECs are markers of developmental proximity to the thymus, and their concentrations in peripheral blood can be used to estimate thymic output and evaluate thymic function in patients after stem cell transplantation23.\n\nIt has been reported that TREC levels correlate to post-transplant outcomes18,24,25. Several factors affecting thymic output have been suggested, but there is still some controversy11,26–28. Herein we tested whether pre-transplant thymic function, assessed by the quantification of TRECs in the peripheral blood, may predict a prognosis in the setting of HLA-identical HSCT with a large prevalence of high risk disease. Furthermore, we also assessed TREC levels before and during acute GVHD.\n\n\nMaterials and methods\n\nOver a consecutive 54-month period, 62 allogeneic stem cell transplant recipients from the UNIFESP (Bone Marrow Transplant Unit of Sao Paulo Hospital and Pediatric Bone Marrow Transplant Unit of the Pediatric Institute of Oncology) and Santa Marcelina Hospital, Brazil, between 2003 and 2007, were included. The group was comprised of children and adults who received a myeloablative preparative regimen and unmanipulated bone marrow or peripheral blood stem cell graft from an HLA-matched related donor. Patients submitted a stem cell transplant with a T-cell depleted graft and children with severe combined immunodeficiency were excluded. The risk of disease was classified based on the International Bone Marrow Transplant Register criteria (www.cibmtr.org). Patients’ characteristics, treatment and complications after transplantation are summarized in Table 1. This study was approved by the Institutional Medical Ethics Committee (number CEP 0532/02) and all patients or guardians gave informed consent before their enrollment.\n\nCSA – cyclosporine, MTX – methotrexate, TBI – total body irradiation, TNC – total nucleated cell, GVHD – graft versus host disease.\n\nGVHD prophylaxis consisted predominantly of cyclosporine or cyclosporine and short course methotrexate. Cyclosporine levels were monitored weekly and kept at the therapeutic range of 150-300 ng/dl during the first 60 days, and then the drug dose was continuously tapered through the next 180 days, until a complete suspension, depending on disease status at the time of transplantation and the presence or absence of GVHD. Antibacterial, antifungical and antiviral prophylaxis consisted of 160mg of trimethoprim-sulfamethoxazol 3 times/week on alternative days for Pneumocistis jiroveci, fluconazole 150mg once/day for fungical prophylaxis and acyclovir 400mg twice/day or valcyclovir 500mg twice/day for Herpes simplex Surveillance cytomegalovirus (CMV) antigenemia testing was performed for all patients. Diagnosis and clinical grading of acute and chronic GVHD were performed according to established criteria29–31. Bacterial, cytomegalovirus and fungical infection, sepsis and septic shock were defined according to previously established criteria32–34.\n\nBuffy coat samples were obtained and frozen before the conditioning regimen for the 62 patients. Thirty-one patients developed acute GVHD, and we were able to collect samples within 2 weeks before acute GVHD, and in ongoing GVHD from 16 of these patients. A control group was composed of 27 healthy donors from whom peripheral blood or bone marrow was collected before G-CSF stimulation.\n\nSixty-two patients received a myeloablative conditioning regimen followed by allogeneic HCT (Table 1). During the follow-up period of 18 months, among all patients, there were 14 severe bacterial infections, 22 CMV infections, six relapses and three fungical infections. Acute GVHD (aGVHD) occurred in 31 patients, and the incidence of grade I, II and III-IV was 13%, 18% and 19%, respectively. Among the 52(83.8%) who survived for at least 100 days after transplantation, 34(65%) developed chronic GVHD (cGVHD). As the number of relapses and fungal infection were low in our cohort, we focused the analysis on overall survival and occurrence of GVHD and severe bacterial infections.\n\nTo perform the TREC assay, total DNA was extracted from buffy-coat by the DTAB/CTAB method (dodecyl trimethyl-ammonium bromide/cetyl trimethyl-ammonium bromide)35, and DNA concentrations were adjusted to 100ng/ul. One μl of DNA was used in all reactions. Delta deletion sjTRECs formed by δREC- ψJα rearrangement were amplified and quantified using SYBR-Green PCR Master Mix and an ABI 7000 instrument (Applied Biosystems, Foster City, CA). The standard curve was generated as previously described36. Primers for sjTRECs were: forward primer (5’CATCCCTTTCAACCATGC’3) and reverse primer (5’CGTGCCTAAACCCTGCAGC’3), which produced an approximately 102 base-pair product. To improve TREC detection, two steps of amplification were performed. The first PCR conditions were: 50°C for 2 minutes and 95°C for 10 minutes, followed by 10 cycles of 95°C for 15 seconds and 63°C for 1 minute. Afterwards, 1ul was utilized in second-step amplification. The second PCR conditions were: 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 63°C for 1 minute. TREC values obtained by RT-PCR were normalized by the proportion of lymphocytes in the blood cell count. This count was performed in the same peripheral blood sample that was utilized for DNA isolation. All DNA samples were amplified in triplicate and universal PCR precautions were taken to avoid amplicon contaminations along with negative control reactions.\n\nA receiver-operating-characteristic (ROC) curve was used to obtain the most discriminating overall survival cut-off for TREC values. Correlation between Gaussian distribution variables was calculated with Spearman correlation. Survival analysis was calculated using Kaplan-Meier plot and log rank test. Cumulative incidence analysis was calculated using death as a competing event for infections, acute or chronic GVHD. Cox proportional hazard model analysis was used to identify independent risk factors for death, infection, and acute or chronic GVHD, reviewed in the context of hematopoietic cell transplantation37. The variables included in the model were those that were marginally significant, p-value ≤ 0.10, in the univariate analyses. Non-parametric (Wilcoxon and Mann-Whitney) tests were utilized to compare TREC levels between patients with high and low disease risk, and before and during ongoing acute GVHD. All p values were two tailed and a value <0.05 was considered to indicate statistical significance. The analysis was done using the SPSS v10.0 Statistical Software (Chicago, Illinois, 1992).\n\n\nResults\n\nBecause we used a modified assay for TREC detection, we first wanted to confirm that it gives similar results to previous studies38,39. To this end, we analyzed the relationship between TREC levels and age and, as expected, observed a highly significant negative correlation in healthy control individuals as well as in patients before transplantation (r=-0.46 and r=-0.70, respectively; p≤0.0001).\n\nAlthough the pre-transplant TREC levels have been previously associated with post-transplant prognosis24, we sought to repeat this analysis in our population of patients because it had a much bigger proportion of malignant high-risk diseases (27% vs 1% in 24). Indeed, we observed higher levels of TRECs in patients with no high-risk disease (nonmalignant, low and intermediate risk) compared to high-risk disease patients in both adult and children populations (p<0.001, Figure 1A and Figure 1B), thus making critical the inclusion of this factor in further analysis.\n\nA) Comparison of TRECs levels between high-risk disease (n=7) and others (nonmalignant, low and intermediated risk disease; n=24) in children before stem cell transplantation (p<.001). B) Comparison of TRECs levels between high risk disease (n=10) and others (nonmalignant, low and intermediated risk disease; n=21) and in adults before stem cell transplantation (p<.001).\n\nUsing the receiver-operating-characteristic (ROC) curve, we found a cut-off value of 134 TREC/µg of lymphocyte DNA that discriminated between patients that survived or not for at least 6 months after transplantation (65% of sensitivity and 60% of specificity). Based on this cut-off, patients were divided into “high” and “low” TREC groups. We observed higher survival in “high” TREC group (p=0.028) (Figure 2) with almost twice the survivors in the “high” TREC group vs the “low” TREC group at 18 months after transplantation (65% vs. 37%, respectively). Applying a multivariate analysis including TREC level, cell source (peripheral blood), recipient age (>25 years), ABO incompatibility, sex mismatch, and high risk disease, we found that worse survival was independently associated (p<0.05) to low TREC values before transplantation (RR: 2.6) and high risk disease (RR: 2.2) (Table 2).\n\nPatients with high TREC (n=35) are shown as a solid line, while the patients with low TREC (n=27) are plotted as a dashed line. Log rank p=.028.\n\n(F→M) denotes female donor into male recipient, RR indicates relative ratio\n\n95% CI, 95% confidence interval\n\nWe also observed a higher incidence of grades II-IV aGVHD (Figure 3) among low TREC patients (p=0.02), with the incidence of aGVHD grade II-IV in high and low TREC groups of 25.7% and 57.8%, respectively. Multivariate analysis showed that low TREC levels (RR 2.5, p<0.03) and the antecedent of high risk disease (RR: 5.2 p<0.025) were independently associated with an increased incidence of aGVHD grades II-IV (Table 2).\n\nPatients with high TREC are shown as a solid line, while the patients with low TREC are plotted as a dashed line. Log rank p=.026.\n\nAlthough we did not find an association between TREC levels and cGVHD, the expected associations of cGVHD with age40–42 and source of cells for transplant43–45 were detected (Table 2).\n\nNext we performed the same analysis for bacterial and cytomegalovirus infection. There was a higher incidence of severe bacterial infection in the low TREC (42%) compared to the high TREC (8.5%) group (p=0.005) (Figure 4). Considering other variables, only low TREC level and peripheral blood as a source of transplant independently associated to bacterial infections (RRs: 6.6 and 4.2, respectively) (Table 2). CMV infection was also more frequently observed in the low TREC patients (63% vs. 22%, p<0.03) (Figure 5). Furthermore, low TREC values before transplantation and high risk disease had independent impact on the incidence of CMV infection (Table 2).\n\nPatients with high TREC are shown as a solid line, while the patients with low TREC are plotted as s dashed line Log rank p=.005.\n\nPatients with high TREC are shown as a solid line, while the patients with low TREC are plotted as a dashed line. Log rank p=.027.\n\nWith the result of pre-transplant thymus function associated with aGVHD, we wondered if thymus output after transplantation (i.e. TREC levels) would also be related to aGVHD. For this purpose we assessed TRECs levels in blood samples collected before (pre-aGVHD) and during aGVHD from 16 patients. The time period between pre-aGVHD and aGVHD samples was 11.5±8 days. Comparing TREC levels between pre-aGVHD and aGVHD samples, we observed increased levels during aGVHD (147 vs. 480 TRECs/µg of lymphocyte DNA, p=0.015). One could suggest that this difference may be explained by the gradual increase in TREC levels after transplantation. In which case, the more time between pre-aGVHD and GVHD samples, the larger the increment in TREC levels. The analysis, however, showed no correlation between distance in time and TREC levels (Dataset 1). In addition, there was no correlation between the number of days after transplantation (17 to 49 days) and TREC levels in this time period. This indicates that we have observed an increase in TREC levels associated with aGVHD independently on the overall increase in TREC levels after transplantation reported previously25,46.\n\n\nDiscussion\n\nIt has been postulated that a good immune reconstitution after HSCT means a better prognosis for the patients under stem cell transplantation47,48. The evaluation of immune reconstitution assessed by TREC quantification has been a matter of research. Here we analyzed the impact of pre-transplant thymic function in patients undergoing an HSCT. Although it has been reported that TREC levels correlate to post-transplant outcome5,18,24,25,49 it was not clear whether these observations are results of association of TREC levels with the severity of disease risk, especially considering a strong relation between disease risk and TREC levels (Figure 1A and 1B). In contrast to previously published work24, we analyzed a cohort of patients with a much higher proportion of malignant high-risk diseases, which allowed us to address the above mentioned concern. Thus, our results suggest that TREC levels is an independent marker for complications after HSCT.\n\nInterestingly, we found that low TREC levels were associated with a higher probability of bacterial infection and aGVHD. This suggests that TREC levels are not just associated with high or low T-cell response after transplantation, but rather reflect an overall response to damage as well as potential capacity to establish immune homeostasis after transplantation. Indeed, in the setting of high-risk disease, patients were treated prior to transplantation with multiples cycles of chemotherapy and/or radiotherapy, potential contributors to thymic injury. Cortical and medullary thymic epithelial cells are dramatically altered after radiotherapy and chemotherapy14,50 as damage of thymic stroma precludes normal thymocyte development51 and probably the generation of TRECs.\n\nDiminished levels of TRECs have been reported during chronic GVHD, or with a previous history of acute or chronic GVHD10,11. However, others studies have failed to demonstrate a direct association between TRECs levels and acute or chronic GVHD46,52. In a mouse model of aGVHD, TRECs levels were analyzed in the spleen and thymus, but not in blood, making difficult the direct comparison of these experiments in mice with our results53. TREC levels during acute GVHD have not been reported in patients. We, however, observed an increase of TREC levels during ongoing acute GVHD in comparing to the TREC levels two weeks before aGVHD. Interestingly, herein observed increases in TREC levels during acute GVHD are very similar to our previous observation of increased TREC levels during acute cardiac rejection38 suggesting that similar events may be occuring in these two pathological processes in the blood. The explanation for these results, on one hand, could be an increased output of TREC positive naïve T-cells into circulation, stimulated by the ongoing immune response25. On the other hand, this result might indicate the exit of activated/effector cells from the circulation into target tissues and a consequent increase in a naïve T-cell pool in the blood. Keeping in mind the limitations of our analysis (a small number of patients and an indirect comparison), additional research is necessary in order to draw definitive conclusions about changes in post-transplant TREC levels in relation to aGVHD.\n\nIn this work we demonstrated a relatively easy way of normalizing TREC values determined by RT-PCR, without the concomitant need for flow cytometry to estimate the numbers of CD3+ cells. Notably, a cut-off established by ROC-curve analysis, which could discriminate between a good or bad prognosis, was similar to the value found at the 60th percentile, after TREC values distribution in percentiles from published works24,25. Using our cut-off, we observed very similar results when compared to published results in relation to survival, acute GVHD, bacterial and CMV infections24. Finally, we have shown disease risk as one the most important sources of TREC level variability in patients before HSCT. Further studies should consider this factor in order to establish how TREC levels can be used in diagnostics.\n\nIn conclusion, our results further support the idea that the thymic competence before transplantation is a critical factor for a good prognosis after hematopoietic stem cell transplantation.\n\n\nData availability\n\nF1000Research: Dataset 1. sjTREC before transplant - patient dataset, 10.5256/f1000research.7330.d108555",
"appendix": "Author contributions\n\n\n\nAMA- designed the study, participated in patients enrolment, performed experiments, analyzed results, wrote manuscript; KK- performed experiments, analyzed results; AS- participated in patients enrolment and study design; JSRO- designed the study, participated in patients enrolment, contributed to analyses of results; MGL- designed the study, contributed to analyses of results, contributed to guidance of the laboratory part of the project; NS and AM- designed the study, analyzed results, wrote manuscript, provided overall leadership of the project. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThe study was supported by AFIP’s internal funding. The sponsor of the study had no role in study design, data collection, data analysis, interpretation or writing of the report.\n\n\nAcknowledgements\n\nWe thank Anatoliy Yambarstev for discussion of statistical analysis.\n\n\nReferences\n\nMarks DI, Cullis JO, Ward KN, et al.: Allogeneic bone marrow transplantation for chronic myeloid leukemia using sibling and volunteer unrelated donors. A comparison of complications in the first 2 years. Ann Intern Med. 1993; 119(3): 207–214. PubMed Abstract | Publisher Full Text\n\nHansen JA, Gooley TA, Martin PJ, et al.: Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Engl J Med. 1998; 338(14): 962–968. PubMed Abstract | Publisher Full Text\n\nHongeng S, Krance RA, Bowman LC, et al.: Outcomes of transplantation with matched-sibling and unrelated-donor bone marrow in children with leukaemia. Lancet. 1997; 350(9080): 767–771. 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Biol Blood Marrow Transplant. 1997; 3(2): 65–75. PubMed Abstract\n\nStorek J, Gooley T, Witherspoon RP, et al.: Infectious morbidity in long-term survivors of allogeneic marrow transplantation is associated with low CD4 T cell counts. Am J Hematol. 1997; 54(2): 131–138. PubMed Abstract | Publisher Full Text\n\nStorek J, Espino G, Dawson MA, et al.: Low B-cell and monocyte counts on day 80 are associated with high infection rates between days 100 and 365 after allogeneic marrow transplantation. Blood. 2000; 96(9): 3290–3293. PubMed Abstract\n\nTalvensaari K, Clave E, Douay C, et al.: A broad T-cell repertoire diversity and an efficient thymic function indicate a favorable long-term immune reconstitution after cord blood stem cell transplantation. Blood. 2002; 99(4): 1458–1464. PubMed Abstract | Publisher Full Text\n\nWeinberg K, Blazar BR, Wagner JE, et al.: Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation. Blood. 2001; 97(5): 1458–1466. PubMed Abstract | Publisher Full Text\n\nDouek DC, Vescio RA, Betts MR, et al.: Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution. Lancet. 2000; 355(9218): 1875–1881. PubMed Abstract | Publisher Full Text\n\nDumont-Girard F, Roux E, van Lier RA, et al.: Reconstitution of the T-cell compartment after bone marrow transplantation: restoration of the repertoire by thymic emigrants. Blood. 1998; 92(11): 4464–4471. PubMed Abstract\n\nMackall CL, Fleisher TA, Brown MR, et al.: Age, thymopoiesis, and CD4+ T-lymphocyte regeneration after intensive chemotherapy. N Engl J Med. 1995; 332(3): 143–149. PubMed Abstract | Publisher Full Text\n\nRoux E, Dumont-Girard F, Starobinski M, et al.: Recovery of immune reactivity after T-cell-depleted bone marrow transplantation depends on thymic activity. Blood. 2000; 96(6): 2299–2303. PubMed Abstract\n\nDouek DC, McFarland RD, Keiser PH, et al.: Changes in thymic function with age and during the treatment of HIV infection. Nature. 1998; 396(6712): 690–695. PubMed Abstract | Publisher Full Text\n\nFallen PR, McGreavey L, Madrigal JA, et al.: Factors affecting reconstitution of the T cell compartment in allogeneic haematopoietic cell transplant recipients. Bone Marrow Transplant. 2003; 32(10): 1001–1014. PubMed Abstract | Publisher Full Text\n\nSairafi D, Mattsson J, Uhlin M, et al.: Thymic function after allogeneic stem cell transplantation is dependent on graft source and predictive of long term survival. Clin Immunol. 2012; 142(3): 343–350. PubMed Abstract | Publisher Full Text\n\nPolitikos I, Boussiotis VA: The role of the thymus in T-cell immune reconstitution after umbilical cord blood transplantation. Blood. 2014; 124(22): 3201–3211. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLivak F, Schatz DG: Identification of V(D)J recombination coding end intermediates in normal thymocytes. J Mol Biol. 1997; 267(1): 1–9. PubMed Abstract | Publisher Full Text\n\nLivak F, Schatz DG: T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells. Mol Cell Biol. 1996; 16(2): 609–618. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTakeshita S, Toda M, Yamagishi H: Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus. EMBO J. 1989; 8(11): 3261–3270. PubMed Abstract | Free Full Text\n\nWu X, Zhu K, Du X, et al.: Frequency analysis of TRBV subfamily sjTRECs to characterize T-cell reconstitution in acute leukemia patients after allogeneic hematopoietic stem cell transplantation. J Hematol Oncol. 2011; 4: 19. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClave E, Rocha V, Talvensaari K, et al.: Prognostic value of pretransplantation host thymic function in HLA-identical sibling hematopoietic stem cell transplantation. Blood. 2005; 105(6): 2608–2613. PubMed Abstract | Publisher Full Text\n\nChen X, Barfield R, Benaim E, et al.: Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients. Blood. 2005; 105(2): 886–893. PubMed Abstract | Publisher Full Text\n\nPrzybylski GK, Kreuzer KA, Siegert W, et al.: No recovery of T-cell receptor excision circles (TRECs) after non-myeloablative allogeneic hematopoietic stem cell transplantation is correlated with the onset of GvHD. J Appl Genet. 2007; 48(4): 397–404. PubMed Abstract | Publisher Full Text\n\nClave E, Busson M, Douay C, et al.: Acute graft-versus-host disease transiently impairs thymic output in young patients after allogeneic hematopoietic stem cell transplantation. Blood. 2009; 113(25): 6477–6484. PubMed Abstract | Publisher Full Text\n\nCastermans E, Hannon M, Dutrieux J, et al.: Thymic recovery after allogeneic hematopoietic cell transplantation with non-myeloablative conditioning is limited to patients younger than 60 years of age. Haematologica. 2011; 96(2): 298–306. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGlucksberg H, Storb R, Fefer A, et al.: Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors. Transplantation. 1974; 18(4): 295–304. PubMed Abstract | Publisher Full Text\n\nSullivan KM, Agura E, Anasetti C, et al.: Chronic graft-versus-host disease and other late complications of bone marrow transplantation. Semin Hematol. 1991; 28(3): 250–259. PubMed Abstract\n\nPrzepiorka D, Weisdorf D, Martin P, et al.: 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant. 1995; 15(6): 825–828. PubMed Abstract\n\nLjungman P, De Bock R, Cordonnier C, et al.: Practices for cytomegalovirus diagnosis, prophylaxis and treatment in allogeneic bone marrow transplant recipients: a report from the Working Party for Infectious Diseases of the EBMT. Bone Marrow Transplant. 1993; 12(4): 399–403. PubMed Abstract\n\nAmerican College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 1992; 20(6): 864–874. PubMed Abstract\n\nLossos IS, Breuer R, Or R, et al.: Bacterial pneumonia in recipients of bone marrow transplantation. A five-year prospective study. Transplantation. 1995; 60(7): 672–678. PubMed Abstract\n\nGustincich S, Manfioletti G, Del Sal G, et al.: A fast method for high-quality genomic DNA extraction from whole human blood. Biotechniques. 1991; 11(3): 298–300, 302. PubMed Abstract\n\nShulzhenko N, Morgun A, Rampim GF, et al.: Monitoring of intragraft and peripheral blood TIRC7 expression as a diagnostic tool for acute cardiac rejection in humans. Hum Immunol. 2001; 62(4): 342–347. PubMed Abstract | Publisher Full Text\n\nKlein JP, Rizzo JD, Zhang MJ, et al.: Statistical methods for the analysis and presentation of the results of bone marrow transplants. Part I: unadjusted analysis. Bone Marrow Transplant. 2001; 28(10): 909–915. PubMed Abstract | Publisher Full Text\n\nMorgun A, Shulzhenko N, Socorro-Silva A, et al.: T cell receptor excision circles (TRECs) in relation to acute cardiac allograft rejection. J Clin Immunol. 2004; 24(6): 612–616. PubMed Abstract | Publisher Full Text\n\nSvaldi M, Lanthaler AJ, Dugas M, et al.: T-cell receptor excision circles: a novel prognostic parameter for the outcome of transplantation in multiple myeloma patients. Br J Haematol. 2003; 122(5): 795–801. PubMed Abstract | Publisher Full Text\n\nRingdén O, Paulin T, Lönnqvist B, et al.: An analysis of factors predisposing to chronic graft-versus-host disease. Exp Hematol. 1985; 13(10): 1062–1067. PubMed Abstract\n\nAtkinson K, Horowitz MM, Gale RP, et al.: Risk factors for chronic graft-versus-host disease after HLA-identical sibling bone marrow transplantation. Blood. 1990; 75(12): 2459–2464. PubMed Abstract\n\nKondo M, Kojima S, Horibe K, et al.: Risk factors for chronic graft-versus-host disease after allogeneic stem cell transplantation in children. Bone Marrow Transplant. 2001; 27(7): 727–730. PubMed Abstract | Publisher Full Text\n\nMohty M, Kuentz M, Michallet M, et al.: Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood. 2002; 100(9): 3128–3134. PubMed Abstract | Publisher Full Text\n\nAnderson D, DeFor T, Burns L, et al.: A comparison of related donor peripheral blood and bone marrow transplants: importance of late-onset chronic graft-versus-host disease and infections. Biol Blood Marrow Transplant. 2003; 9(1): 52–59. PubMed Abstract | Publisher Full Text\n\nCutler C, Giri S, Jeyapalan S, et al.: Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol. 2001; 19(16): 3685–3691. PubMed Abstract\n\nHochberg EP, Chillemi AC, Wu CJ, et al.: Quantitation of T-cell neogenesis in vivo after allogeneic bone marrow transplantation in adults. Blood. 2001; 98(4): 1116–1121. PubMed Abstract | Publisher Full Text\n\nParkman R, Cohen G, Carter SL, et al.: Successful immune reconstitution decreases leukemic relapse and improves survival in recipients of unrelated cord blood transplantation. Biol Blood Marrow Transplant. 2006; 12(9): 919–927. PubMed Abstract | Publisher Full Text\n\nKim DH, Sohn SK, Won DI, et al.: Rapid helper T-cell recovery above 200 × 106/l at 3 months correlates to successful transplant outcomes after allogeneic stem cell transplantation. Bone Marrow Transplant. 2006; 37(12): 1119–1128. PubMed Abstract | Publisher Full Text\n\nWils EJ, Rombouts EJ, van Mourik I, et al.: Stem cell factor consistently improves thymopoiesis after experimental transplantation of murine or human hematopoietic stem cells in immunodeficient mice. J Immunol. 2011; 187(6): 2974–2981. PubMed Abstract | Publisher Full Text\n\nAdkins B, Gandour D, Strober S, et al.: Total lymphoid irradiation leads to transient depletion of the mouse thymic medulla and persistent abnormalities among medullary stromal cells. J Immunol. 1988; 140(10): 3373–3379. PubMed Abstract\n\nHollander GA, Wang B, Nichogiannopoulou A, et al.: Developmental control point in induction of thymic cortex regulated by a subpopulation of prothymocytes. Nature. 1995; 373(6512): 350–353. PubMed Abstract | Publisher Full Text\n\nStorek J, Joseph A, Espino G, et al.: Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation. Blood. 2001; 98(13): 3505–3512. PubMed Abstract | Publisher Full Text\n\nKrenger W, Schmidlin H, Cavadini G, et al.: On the relevance of TCR rearrangement circles as molecular markers for thymic output during experimental graft-versus-host disease. J Immunol. 2004; 172(12): 7359–7367. PubMed Abstract | Publisher Full Text\n\nde Moraes Arantes A, Seber A, Kawemura K, et al.: Dataset 1 in: High pre-transplant TREC levels indicate good prognosis after hematopoietic stem cell transplantation. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11691",
"date": "30 Dec 2015",
"name": "Richard Champlin",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 examine the effect of pre transplant TREC levels and outcome of hematopoietic transplantation. TRECs are new thymic emigrants and are related to thymic function. CritiqueThe authors confirm the findings reported by Clave et al ten years ago (their reference 24), that higher pre transplant TREC levels are associated with less GVHD, fewer infections and better survival.Clave E, Rocha V, Talvensaari K, et al.: Prognostic value of pretransplantation host thymic function in HLA-identical sibling hematopoietic stem cell transplantation. Blood. 2005; 105(6): 2608–2613.These findings are not surprising in that improved thymic function would enhance immune reconstitution, which would be expected to be protective against infection. Thymic dysregulation occurs with GVHD.",
"responses": []
},
{
"id": "11689",
"date": "11 Jan 2016",
"name": "John Wagner",
"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 presented by de Moraes Arantes et al. corroborates those of others that suggest the potential significance of pretransplant TREC levels on, infection, GVHD and survival outcomes after allogeneic transplant. While very interesting, it is difficult to make firm conclusions considering the fact that there are numerous potential interactions that could influence outcome. For example, age, use of TBI and cell source have themselves been associated with risks of acute and/or chronic GVHD. An older patient might be expected to have lower pretransplant TRECs because of age. A younger with a non malignant patient might be expected to have higher TREC's because of age and lack of prior treatment. Although not shown, use of bone marrow is more likely in children than in adults. As many of these issues are not random, it is possible that these intriguing findings are not related to pretransplant TRECs but to some other factor or group of factors. Still, the data argues for larger studies as the relevant technologies are more readily available today.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1458
|
https://f1000research.com/articles/4-1457/v1
|
16 Dec 15
|
{
"type": "Review",
"title": "Advances in upper gastrointestinal endoscopy",
"authors": [
"David G. Graham",
"Matthew R. Banks"
],
"abstract": "The rapidly moving technological advances in gastrointestinal endoscopy have enhanced an endoscopist’s ability to diagnose and treat lesions within the gastrointestinal tract. The improvement in image quality created by the advent of high-definition and magnification endoscopy, alongside image enhancement, produces images of superb quality and detail that empower the endoscopist to identify important lesions that have previously been undetectable. Additionally, we are now seeing technologies emerge, such as optical coherence tomography and confocal laser endomicroscopy, that allow the endoscopist to visualize individual cells on a microscopic level and provide a real time, in vivo histological assessment. Within this article we discuss these technologies, as well as some of the results from their early use in clinical studies.",
"keywords": [
"gastrointestinal endoscopy",
"gastrointestinal tract",
"optical coherence tomography",
"confocal laser endomicroscopy",
"endoscopy",
"endomicroscopy"
],
"content": "Introduction\n\nWhilst the intricate system of mirrors and lens, with a lamp flame as a light source, that was the early endoscope provided a unique opportunity to visualize the gastrointestinal tract, it was not clinically practical until the flexibility of fiber-optics was introduced and a new era of endoscopy, pioneered by Curtiss and Hirschowitz, began1. Further advances, in the development of a charge couple device that produced electronic images and linked the endoscope to the computer, prompted a surge in technological endoscopic advancements and revolutionized gastroenterology2.\n\nThe ability to visualize and sample the gastrointestinal tract is now an accepted norm for gastroenterologists and, as such, the focus is now on enhancing the diagnostic yield further by enabling endoscopists to accurately diagnose microscopic pathology, particularly high-risk pre-malignant lesions or early stage cancers, where their detection would significantly alter the prognosis. Furthermore, the promising developments in optical biopsy techniques aim to address the issues of variability in histopathological assessment of biopsy samples and the ever-increasing demand on these services3.\n\nThis article aims to explore some of the exciting advancements in endoscopic upper gastrointestinal imaging that are presently being used within healthcare, as well as those that are currently being developed which provide great potential.\n\n\nAdvances in digital imaging\n\nThe well recognized rapid advances in television technology that provide the viewer with a more engaging, immersive experience have also occurred in endoscopic imaging. Endoscopists are becoming increasingly able to detect subtle, minute mucosal changes that were previously indistinguishable from normal tissue. This is due to improving standards in resolution and magnification, combined with image enhancement techniques available to the endoscopist at a touch of a button.\n\nSimilar to the changes in television, the endoscope has switched from “standard definition” to digital, high-definition white light imaging. Three companies (Pentax, Olympus, and Fujinon) are presently leading the field in endoscopic imaging, and currently have advanced high-definition endoscopic systems available for clinical use. They offer the improved ability to distinguish subtle mucosal differences in tissue in close proximity to each other, which is partly due to improved resolution (defined by pixel density). Standard definition endoscopes offered images of approximately 300,000 pixels whereas the new high-definition endoscopes, when combined with the latest processors, can achieve image quality of over 2 million pixels. Furthermore, all three of these companies offer endoscopes with high magnification to further enhance imaging. A standard endoscope magnifies an image by 30–35 times normal. However, these companies produce high-definition endoscopes that can optically magnify images by up to 150 times (Pentax MagniView, Olympus near-focus imaging, and Fujinon optical magnification)4.\n\nIn addition to high-definition white light imaging, current market-leading endoscopes also provide further image enhancement by offering the endoscopist the ability to filter certain wavelengths of light. iScan (Pentax), OE (Pentax), narrow-band imaging (NBI, Olympus) and Fuji Intelligent Chromo Endoscopy (FICE, Fujinon) are all post-processing optical technologies that are designed to enhance subtle architectural or vascular patterns on the mucosal surface, and therefore enhance an endoscopist’s ability to detect subtle lesions within the gastrointestinal tract. However, these techniques do have their limitations. For example, NBI is excellent at demonstrating the changes in microvasculature seen in early gastric and esophageal lesions. However, it is not so effective at identifying some of the associated mucosal changes seen in these conditions, an area in which FICE excels5. Similarly, the viewing of lesions at a distance is problematic with these techniques5. Blue laser imaging (BLI, Fujinon) has been developed to overcome these issues, through combining narrow-band laser light with high-definition white light.\n\nWhilst these techniques aim to offer an improved visualization of the upper gastrointestinal tract (Figure 1), they still require the endoscopist to be suitably trained and engaged in utilizing them. There are no data on how often these techniques are actually applied in clinical practice, however, within our center we offer training for endoscopists from around the UK on these techniques and have anecdotally found their uptake is variable. Similarly, we have found the ability of these techniques to aid in detecting lesions varies according to the endoscopist’s interest. Our unpublished data found that specialist upper gastrointestinal endoscopists using iScan could detect dysplasia within a Barrett’s segment with 75% sensitivity. However, when general endoscopists were asked to detect dysplasia using iScan, this dropped to 55%. There are obviously other mitigating factors in this unpublished study, but this does demonstrate the need for these technologies to be combined with an endoscopist’s expertise and experience.\n\nThere have been many studies exploring the benefits of high-definition, high-magnification endoscopes. This work has mainly focused on Barrett’s esophagus, which is a pre-malignant condition of the esophagus. There is limited work investigating the benefits of these endoscopes in the stomach and small bowel. However, the work within the esophagus has shown these technologies to be useful. An example of this was demonstrated by Wolfsen et al., focusing on dysplasia detection in Barrett’s esophagus (a high risk form of this disease which carries an increased cancer risk)6. They compared high definition and NBI endoscopy (HD-NBI) with targeted biopsies to standard white light endoscopy and random biopsies. The study demonstrated that HD-NBI techniques provided a greater dysplasia yield (57% vs 43%) and required fewer biopsies6.\n\nStudies using these techniques in the stomach have had varying success. A study using iScan for the detection of pre-cancerous or early cancerous lesions within the stomach found that, although imaging quality was improved, there was little additional diagnostic benefit4. However, recently Matsuo et al. found that using magnifying endoscopy with NBI (enhanced by the application of acetic acid) aided the diagnosis of early gastric cancers7. Similarly, a study by Dohi et al. found that using BLI increased the detection of early gastric cancer with an accuracy, sensitivity, and specificity of 90.7, 84.6 and 92.4%, respectively, compared to 72.9, 30.8 and 84.8% using high-definition white light alone8.\n\nWork within the small bowel has also shown promise. Cammarota et al. performed a study of 191 patients that demonstrated that high definition magnification endoscopy had 95% sensitivity, 99% specificity, 95% positive predictive value, and 99% negative predictive value to detect the presence of any villous abnormality, and thus make a diagnosis within the small bowel without the need to biopsy9.\n\nThis is a rapidly growing discipline in medical imaging, utilizing unique molecular signatures for targeted imaging of pathology. This technique relies upon the development of exogenous molecular probes that specifically locate and highlight desired pathology. The potential for molecular imaging goes beyond that of just aiding in the detection of lesions. Other possible applications of this technology are within the field of therapy, where molecular imaging could enhance drug delivery and monitor drug response10,11.\n\nAutofluorescence is an area of molecular imaging that is based upon the detection of natural tissue fluorescence emitted by endogenous molecules (fluorophores), such as collagen, flavins and porphyrins, producing a virtual chromoendoscopy technique12. Studies demonstrated that dysplastic or cancerous tissue emitted a different autofluorescence spectrum compared to normal tissue. Consequently wide-field autofluorescence imaging was integrated with high-definition white light endoscopy and NBI to produce “trimodal endoscopic imaging”13,14. This technique is not yet in clinical use and there is little data from large-scale clinical trials, although the data from smaller studies appear promising (Figure 2 below depicting trimodal imaging of an early cancerous lesion). Similarly, the use of near-infrared endoscopy and fluorescent activatable probes has limited clinical data, but early work demonstrates promise15. Using autofluorescence, early esophageal squamous neoplasia was visualized more reliably than using white light endoscopy alone (79% vs 51%). Dysplasia within Barrett’s esophagus was detected more often using autofluorescence than white light endoscopy alone (90% vs 53%), but this was at the expense of a high false-positive rate of 81%. Finally the detection of early gastric cancers increased by 13% using autofluorescence, but again at the expense of a poor specificity12.\n\n\nOptical biopsies\n\nThe need for effective optical biopsy techniques is apparent. As the field of endoscopy widens and lesions are more readily identified and removed, the workload for the already stretched histopathology and endoscopy departments increases. The cost of processing and analyzing tissue specimens removed endoscopically and then bringing the patients back for further follow-up procedures is spiraling. In addition to this, the existing random biopsy techniques utilized in areas such as Barrett’s esophagus surveillance are time-consuming, plagued with issues of missed cancers, and only sample around 5% of the mucosa16,17. It is clear that an effective “bed-side” immediate diagnostic technique would prove to be invaluable.\n\nOCT has produced some promising data in the field of optical biopsy techniques. It relies upon the backscattering of light to provide cross-sectional images of tissue with high resolution and a scanning depth of 1–3 mm. The construction of an image through reflected light is similar to the use of acoustic waves in ultrasound, although, unlike ultrasound, neither a water or tissue apposition is required. However, the use of OCT in clinical practice has been limited due to imaging speed, and attempts to increase this have resulted in an unwanted loss of sensitivity18. Optical Frequency Domain Imaging (OFDI) was developed to overcome the issue of image acquisition speed whilst maintaining sensitivity.\n\nIn 2013, an OCT system for esophageal imaging became commercially available (NvisionVLE, NinePoint Medical). This device uses a through-the-scope balloon that can acquire a 6 cm circumferential image in an automated scan. Clinical studies are underway exploring the device’s efficacy19. Recently, Gora et al. published on their development of a tethered capsule providing OFDI imaging of the esophagus which potentially provides a simple, quick, and effective means for imaging the esophagus, however, clinical data are awaited20.\n\nThe majority of published data focuses on esophageal pathology, as OCT images within the stomach are characterized by low tissue contrast and poor visualization. However, within the esophagus there have been some promising data. A study using 177 biopsy correlated OCT images in patients with Barrett’s esophagus found that this modality was capable of identifying dysplasia with a sensitivity of 83% and specificity of 75%. OCT has also been effectively used for the staging of esophageal tumors. In a study of 123 patients with esophageal squamous cell carcinomas, OCT correctly staged 95% of the lesions limited to the epithelium/lamina propria with higher accuracy than the existing endoscopic ultrasonographic technique21.\n\nThis provides real-time histological assessment through high-resolution imaging, used as an adjunct to high-definition endoscopy. The key principal behind CLE is its ability to provide an in-focus image from a selected depth, whilst light from the out of focus planes are inefficiently collected. Unlike OCT, this technique relies upon fluorescent dye, most commonly injected fluorescein, prior to laser illumination. Whilst injection of fluorescein has an excellent safety profile, having been used extensively in ophthalmology, its use does add an additional process to the technique. However, this must be weighed up alongside the opportunity to obtain greater image contrast than in OCT22,23.\n\nThere are currently two CLE platforms; probe based (pCLE) and endoscope based (eCLE). The probe based system (Cellvizio confocal miniprobes, Mauna Kea Technologies, Paris, France) can only scan in a single plane, due to a fixed focal length, and provide an imaging depth between 40 and 70 mm depending on the probe used. For the upper gastrointestinal probe the maximal field view is 240 µm with a resolution of 1 μm. The AQ-Flex 19 Miniprobe has recently been developed for use through an EUS FNA (endoscopic ultrasound and fine needle aspiration) needle, however, there is little data on its clinical application23,24. The integration of a confocal microscope (Optiscan, Victoria, Australia) into an endoscope (Pentax, Tokyo, Japan) allowed for high-definition endoscopy to be performed simultaneously to eCLE with images displayed on dual monitors. eCLE provided a greater field view (475 mm) and was otherwise comparable to the performance of pCLE. The largest study comparing these two platforms in gastrointestinal disease found that pCLE offered shorter procedure times and comparable diagnostic yields to eCLE apart from in esophageal disease, where eCLE was found to offer better image quality and therefore improved examination25.\n\nThe clinical application of CLE in the upper gastrointestinal tract has provided promising results. Studies with the largest numbers have explored its use in detecting dysplasia within Barrett’s esophagus and gastric lesions, however, there are some data showing that CLE can be used to accurately diagnose Celiac disease26. In Barrett’s esophagus, a recent randomized trial recruited 192 patients on routine Barrett’s esophagus surveillance, or those referred for confirmation of suspected dysplasia within their Barrett’s segment. This trial compared high definition endoscopy plus eCLE and targeted biopsy with high-definition endoscopy alone with random biopsies, and demonstrated that the use of CLE significantly improved the ability to detect dysplasia and early cancers within Barrett’s esophagus (34% vs 7%) whilst requiring fewer biopsies27. The use of CLE would have eliminated the need for a biopsy in 65% of patients, which would have a potentially huge impact on healthcare resource provision27. Similarly, CLE has been demonstrated to enhance diagnostic yield in pre-cancerous or early cancerous lesions of the stomach and can detect small intestinal pathology (Figure 3), such as Celiac disease, with a 94% sensitivity and 92% specificity, which again would greatly reduce the burden of biopsies on the histopathology department23. Due to the high cost of equipment, additional endoscopist expertise, and longer duration of the endoscopic procedure, CLE utilization was initially limited to enthusiasts and centers of excellence, but its use is now growing more widespread.\n\nThese images are taken from Barrett’s esophagus.\n\nThe principles behind spectroscopy rely upon how objects of differing size and structure interact with light, typically light in the backward direction (towards the incident light). This provides insights into the molecular composition of the target tissue. Unlike other forms of optical biopsy techniques, the spectroscopic output is quantitative rather than being reliant upon user interpretation of an image, which potentially lends itself to inter-observer variation. Whilst this may be seen as an advantage to some, it requires a shift in the established approach to reaching a diagnosis, and consequently may be met with some skepticism16.\n\nWhilst there has been work looking at various properties of spectroscopic methods, an area that has a great deal of published data is that of inelastic (Raman) scattering spectroscopy. This measures signals obtained when the incident light undergoes wavelength shifts caused by energy transfer in the tissue. The different molecular composition and therefore molecular bonds of various tissue types (e.g., cancerous, dysplastic, normal) respond to energy uniquely and produce distinct shifts in light wavelength. Ex vivo studies assessing the ability of Raman spectroscopy to detect dysplasia in Barrett’s esophagus has shown sensitivities ranging from 73–100% and specificities of 90–100%19, whereas a recent in vivo study demonstrated sensitivity of 87% and specificity of 84.7%28. Within the stomach, Raman spectroscopy has demonstrated similar success in identifying gastric lesions with a sensitivity of 90% and specificity of 73.3%29.\n\nAn important issue of these studies and an aspect of spectroscopy that has proven to be a stumbling block is that the majority rely upon a probe-based system. Accurately studying large areas of tissue with a probe is impractical in clinical practice and will likely continue to lead to missed lesions. Recent work addressing this issue has been published whereby an endoscopic polarized scanning spectroscopy system (EPSS) has been developed that is capable of performing rapid optical scanning and multispectral imaging of the entire esophageal surface and provide a real-time diagnosis. Early data on a small number of patients demonstrate a sensitivity of 92% and specificity of 96%, however, further studies with a larger cohort of patients are required30.\n\n\nSummary\n\nThis article highlights just some of the many advancements in upper gastrointestinal endoscopy. As the science behind these technologies improves, so does the ability of the endoscopist to visualize and treat lesions within the gastrointestinal tract. It is without doubt that the improved ability to identify pre-cancerous or early cancerous lesions that have previously evaded even the most diligent endoscopist will have a significant impact on prognosis. Alongside this, the impact these technologies could have in reducing the need for biopsies and therefore the number of procedures will significantly alter the management of strained healthcare resources.\n\nHowever, one must acknowledge that much of this technology is still in its early stages and is yet to be fully tested in clinical practice. Similarly, although these technologies provide an exciting platform for the endoscopist, one must remember that they are still reliant on the diligence and expertise of their user. These techniques are only as good as the quality of training and the acquisition of knowledge that precedes their use. We believe, however, that as with high-definition endoscopy, some of these technologies will emerge into mainstream use providing significant benefit to patient outcomes.",
"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\nHirschowitz BI, Curtiss LE, Peters CW, et al.: Demonstration of a new gastroscope, the fiberscope. Gastroenterology. 1958; 35(1): 50; discussion 51–3. PubMed Abstract | Faculty Opinions Recommendation\n\nSivak MV: Gastrointestinal endoscopy: past and future. Gut. 2006; 55(8): 1061–4. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKerkhof M, van Dekken H, Steyerberg EW, et al.: Grading of dysplasia in Barrett’s oesophagus: substantial interobserver variation between general and gastrointestinal pathologists. Histopathology. 2007; 50(7): 920–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nASGE Technology Committee: High-definition and high-magnification endoscopes. Gastrointest Endosc. 2014; 80(6): 919–27. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOsawa H, Yamamoto H: Present and future status of flexible spectral imaging color enhancement and blue laser imaging technology. Dig Endosc. 2014; 26(Suppl 1): 105–15. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWolfsen HC, Crook JE, Krishna M, et al.: Prospective, controlled tandem endoscopy study of narrow band imaging for dysplasia detection in Barrett’s Esophagus. Gastroenterology. 2008; 135(1): 24–31. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMatsuo K, Takedatsu H, Mukasa M, et al.: Diagnosis of early gastric cancer using narrow band imaging and acetic acid. World J Gastroenterol. 2015; 21(4): 1268–74. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDohi O, Majima A, Onozawa Y, et al.: Blue LASER Imaging-Bright Improves Real-Time Detection Rate of Early Gastric Cancer. Gastrointest Endosc. 2015; 81(5): AB132. Reference Source\n\nCammarota G, Martino A, Pirozzi GA, et al.: Direct visualization of intestinal villi by high-resolution magnifying upper endoscopy: a validation study. Gastrointest Endosc. 2004; 60(5): 732–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWang MS, Luo Z, Nitin N: Rapid assessment of drug response in cancer cells using microwell array and molecular imaging. Anal Bioanal Chem. 2014; 406(17): 4195–206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCho YW, Kim K, Park K, et al.: Molecular imaging in the aid of drug delivery technology. Macromol Res. 2014; 22(9): 926–931. Publisher Full Text\n\nASGE Technology Committee, Song LM, Banerjee S, et al.: Autofluorescence imaging. Gastrointest Endosc. 2011; 73(4): 647–50. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\ndi Pietro M, Boerwinkel DF, Shariff MK, et al.: The combination of autofluorescence endoscopy and molecular biomarkers is a novel diagnostic tool for dysplasia in Barrett’s oesophagus. Gut. 2015; 64(1): 49–56. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBoerwinkel DF, Holz JA, Hawkins DM, et al.: Fluorescence spectroscopy incorporated in an Optical Biopsy System for the detection of early neoplasia in Barrett’s esophagus. Dis Esophagus. 2015; 28(4): 345–51. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDing S, Eric Blue R, Chen Y, et al.: Molecular imaging of gastric neoplasia with near-infrared fluorescent activatable probes. Mol Imaging. 2012; 11(6): 507–15. PubMed Abstract | Free Full Text | Faculty Opinions Recommendation\n\nRoy HK, Backman V: Spectroscopic applications in gastrointestinal endoscopy. Clin Gastroenterol Hepatol. 2012; 10(12): 1335–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarud SS, Keilin S, Cai Q, et al.: Diagnosis and management of Barrett’s esophagus for the endoscopist. Therap Adv Gastroenterol. 2010; 3(4): 227–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYun S, Tearney G, de Boer J, et al.: High-speed optical frequency-domain imaging. Opt Express. 2003; 11(22): 2953–63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nASGE Technology Committee: Enhanced imaging in the GI tract: spectroscopy and optical coherence tomography. Gastrointest Endosc. 2013; 78(4): 568–73. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGora MJ, Sauk JS, Carruth RW, et al.: Tethered capsule endomicroscopy enables less invasive imaging of gastrointestinal tract microstructure. Nat Med. 2013; 19(2): 238–40. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHatta W, Uno K, Koike T, et al.: A prospective comparative study of optical coherence tomography and EUS for tumor staging of superficial esophageal squamous cell carcinoma. Gastrointest Endosc. 2012; 76(3): 548–55. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNeumann H, Kiesslich R, Wallace MB, et al.: Confocal laser endomicroscopy: technical advances and clinical applications. Gastroenterology. 2010; 139(2): 388–92, 392.e1–2. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nASGE Technology Committee: Confocal laser endomicroscopy. Gastrointest Endosc. 2014; 80(6): 928–38. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWallace MB, Fockens P: Probe-based confocal laser endomicroscopy. Gastroenterology. 2009; 136(5): 1509–13. PubMed Abstract | Publisher Full Text\n\nLi CQ, Zuo XL, Guo J, et al.: Comparison between two types of confocal laser endomicroscopy in gastrointestinal tract. J Dig Dis. 2015; 16(5): 279–85. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFort Gasia M, Gui SX, Poon T, et al.: Accurate diagnosis of villous atrophy in celiac disease using confocal laser endomicroscopy. Can J Gastroenterol Hepatol. 2014; 28(3): 123–4. PubMed Abstract | Free Full Text | Faculty Opinions Recommendation\n\nCanto MI, Anandasabapathy S, Brugge W, et al.: In vivo endomicroscopy improves detection of Barrett’s esophagus-related neoplasia: a multicenter international randomized controlled trial (with video). Gastrointest Endosc. 2014; 79(2): 211–21. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBergholt MS, Zheng W, Ho KY, et al.: Fiberoptic confocal Raman spectroscopy for real-time in vivo diagnosis of dysplasia in Barrett’s esophagus. Gastroenterology. 2014; 146(1): 27–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDuraipandian S, Sylvest Bergholt M, Zheng W, et al.: Real-time Raman spectroscopy for in vivo, online gastric cancer diagnosis during clinical endoscopic examination. J Biomed Opt. 2012; 17(8): 081418. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nQiu L, Pleskow DK, Chuttani R, et al.: Multispectral scanning during endoscopy guides biopsy of dysplasia in Barrett’s esophagus. Nat Med. 2010; 16(5): 603–6, 1p following 606. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11585",
"date": "16 Dec 2015",
"name": "Michael B. Wallace",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11586",
"date": "16 Dec 2015",
"name": "Yan-Qing Li",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1457
|
https://f1000research.com/articles/4-880/v1
|
21 Sep 15
|
{
"type": "Research Article",
"title": "Variable effects of nicotine and anabasine on parasitized bumble bees",
"authors": [
"Lukas P. Thorburn",
"Lynn S. Adler",
"Rebecca E. Irwin",
"Evan C. Palmer-Young",
"Lukas P. Thorburn",
"Lynn S. Adler",
"Rebecca E. Irwin"
],
"abstract": "Secondary metabolites in floral nectar have been shown to reduce parasite load in two common bumble bee species. Previous studies on the effects of nectar secondary metabolites on parasitized bees have focused on single compounds in isolation; however, in nature, bees are simultaneously exposed to multiple compounds. We tested for synergistic effects of two alkaloids found in the nectar of Nicotiana spp. plants, nicotine and anabasine, on parasite load and mortality in bumble bees (Bombus impatiens) infected with the intestinal parasite Crithidia bombi. Adult worker bees inoculated with C. bombi were fed nicotine and anabasine diet treatments in a factorial design, resulting in four nectar treatment combinations: 2 ppm nicotine, 5 ppm anabasine, 2ppm nicotine and 5 ppm anabasine together, or a control alkaloid-free solution. We conducted the experiment twice: first, with bees incubated under variable environmental conditions (‘Variable’; temperatures varied from 10-35°C); and second, under carefully controlled environmental conditions (‘Controlled’; 27°C incubator, constant darkness). In ‘Variable’, each alkaloid alone significantly decreased parasite loads, but this effect was not realized with the alkaloids in combination, suggesting an antagonistic interaction. Nicotine but not anabasine significantly increased mortality, and the two compounds had no interactive effects on mortality. In ‘Controlled’, nicotine significantly increased parasite loads, the opposite of its effect in ‘Variable’. While not significant, the relationship between anabasine and parasite loads was also positive. Interactive effects between the two alkaloids on parasite load were non-significant, but the pattern of antagonistic interaction was similar to that in the variable experiment. Neither alkaloid, nor their interaction, significantly affected mortality under controlled conditions. Our results do not indicate synergy between Nicotiana nectar alkaloids; however, they do suggest a complex interaction between secondary metabolites, parasites, and environmental variables, in which secondary metabolites can be either toxic or medicinal depending on context.",
"keywords": [
"Bumble bee",
"Bombus impatiens",
"parasites",
"Crithidia bombi",
"plant secondary metabolites",
"nicotine",
"alkaloids",
"tritrophic interactions"
],
"content": "Introduction\n\nThroughout the past two decades, many wild and managed bee species have experienced severe declines (Allen-Wardell et al., 1998; Cameron et al., 2011; Potts et al., 2010). In many cases of bee decline, parasitism has been implicated as a potential cause (reviewed in Goulson et al., 2015 and Potts et al., 2010). The parasitic mite Varroa destructor has been linked to honey bee declines in Ontario, Canada (Guzmán-Novoa et al., 2010). Infection with the microsporidian pathogen Nosema ceranae may be responsible for cases of honey bee colony collapse in Spain (Higes et al., 2009). Bumble bee species that have experienced recent declines had significantly higher levels of Nosema bombi than the species whose populations were stable (Cameron et al., 2011). Finding means of mitigating the effects of parasitism on bees would therefore be beneficial to the agricultural community as well as natural ecosystems.\n\nSecondary metabolites – plant compounds that do not play a role in the plant’s primary metabolism – frequently have antimicrobial properties (Schmidt et al., 2012), and could offer a means of natural parasite control. Secondary metabolites are found in the floral nectar of many plant species (Heil, 2011). The effects of secondary metabolites on insects, including bees and other pollinators, are context-dependent. A wide range of secondary metabolites, including terpenes, alkaloids, and phenolics, are toxic to insects (Detzel & Wink, 1993; Kumrungsee et al., 2014; Raffa et al., 1985; Singaravelan et al., 2006; Wink & Theile, 2002). Interaction with other stressors, such as infection or climatic stress, can exacerbate these toxic effects (Goulson et al., 2015; Holmstrup et al., 2010; Köhler et al., 2012a). However, under some circumstances, the antimicrobial properties of secondary metabolites can provide health benefits to infected insects. Insects have been shown to self-medicate with secondary metabolites in response to parasite infection (reviewed in Abbott, 2014). For example, Grammia incorrupta (wooly bear) caterpillars exhibited self-medication behavior in response to tachinid fly parasitism by increasing their consumption of pyrrolizidine alkaloids, which decreased the survival of unparasitized caterpillars but increased the survival of parasitized caterpillars (Singer et al., 2009). Several recent studies have indicated that plant secondary metabolites, including those found in nectar, can benefit infected pollinators as well. Honey bees self-medicated in response to parasitism through increased foraging for resins, which are used in hive construction and have antimicrobial properties (Simone-Finstrom & Spivak, 2012), and through preferentially feeding on certain types of honey, such as sunflower honey, which reduced pathogen load (Gherman et al., 2014). Consumption of the alkaloid gelsemine significantly reduced infection intensity in bumble bees (Bombus impatiens) infected with the intestinal parasite Crithidia bombi (Manson et al., 2010), and four other nectar secondary compounds had significant medicinal effects in the same bee-parasite system, with an additional four compounds causing non-significant decreases in infection severity (Richardson et al., 2015a).\n\nPrevious studies of the effects of nectar secondary metabolites on pollinators have focused primarily on single compounds in isolation. Under natural conditions, however, pollinators would likely encounter several compounds at once, since many plant species produce multiple secondary metabolites. For example, many Nicotiana species contain both nicotine and anabasine in nectar (Adler et al., 2012), and Chelone glabra contains the iridoid glycocides aucubin and catalpol in nectar (Richardson et al., 2015b). This raises the possibility of interactions between secondary metabolites in nectar. Synergistic interactions between secondary metabolites from other plant tissues are well established. The iridoid glycosides aucubin and catalpol had synergistic effects on the survival of common buckeye (Junonia coenia Hübner) caterpillars that specialize on plants with these compounds; caterpillars that consumed both iridoid glycosides had an increased rate of survival relative to caterpillars that consumed either glycoside alone (Richards et al., 2012). Amides in plants in the Piper genus had synergistic deterrent effects on herbivorous ants, while the same compounds were neutral or attractive in isolation (Dyer et al., 2003). Synergy between secondary metabolites can also alter antimicrobial effects. Carvacrol and thymol, for example, inhibited the growth of the bacterium Listeria innocua more effectively in combination than alone (García-García et al., 2011). Carvacrol was also more effective against the bacterium Vibrio cholerae when combined with cymene, although cymene alone had no antimicrobial activity (Rattanachaikunsopon & Phumkhachorn, 2010). Antagonism between secondary metabolites has also been demonstrated. The deterrent effect of the amide piperine on the hemipteran Sibaria englemani is significantly reduced when piperine is combined with the amide piplartine, although piplartine alone had no effect on S. englemani feeding preference (Whitehead & Bowers, 2014). The linear furanocoumarins psoralen, bergapten, and xanthotoxin exhibited antagonistic interactions in their effects on insect mortality; the toxicity of psoralen combined with either or both of the other two compounds was significantly lower than would be predicted based on their toxicities in isolation (Diawara et al., 1993). If similar interactions, either synergistic or antagonistic, are present between secondary metabolites in nectar, they could exacerbate or ameliorate the effects of single compounds found in previous studies.\n\nTo evaluate interactions between secondary metabolites from the nectar of a single plant, we tested the effects of nicotine and anabasine alone and in combination on bumble bee resistance to the gut parasite Crithidia bombi. Nicotine and anabasine co-occur in the nectar of several species in the genus Nicotiana, which includes cultivated tobacco (Nicotiana tabacum) as well as several ornamental species (Adler et al., 2012). The effects of nicotine and anabasine in combination on bee disease have not previously been studied.\n\nWe tested the effects of these compounds in two environmental contexts, variable and controlled conditions. Bumble bees in the wild encounter a wide range of environmental conditions, which could alter the effects of diet and parasitism. In general, temperature can decrease tolerance to environmental toxins, including secondary metabolites (Holmstrup et al., 2010), and exert unpredictable effects on insect-parasite interactions through modulation of host survival, host immune function, and parasite viability (Thomas & Blanford, 2003). Variable temperatures impose exceptional energetic costs on bumble bees by forcing them to actively regulate body temperature in order to fly (Heinrich, 1972). These costs might create caloric deficits that increase parasite virulence in Bomubs (Brown et al., 2000). Alternatively, heightened energy needs could lead to increased consumption of plant foods, thereby elevating exposure to secondary metabolites. Globally, responses to environmental variability have implications for conservation: Bumble bee species with narrow climatic ranges are particularly vulnerable to decline (Williams et al., 2007; Williams et al., 2009), and projected climate change may further restrict these species’ distributions through increases in mean temperature and the frequency of extreme events (Diffenbaugh & Field, 2013).\n\n\nMethods\n\nBombus impatiens is the most common bumble bee species in eastern North America, with a range extending from Ontario and Maine to southern Florida (Balaban et al., 2014). It is an important pollinator in agriculture, and commercial distribution of B. impatiens is becoming increasingly common (Colla et al., 2006).\n\nCrithidia bombi is a common trypanosome parasite of bumble bees in Europe and North America (Colla et al., 2006; Lipa & Triggiani, 1988). Its range has been expanding within North America and into parts of South America, potentially due to spillover from commercial to wild bumble bee populations (Colla et al., 2006; Schmid-Hempel et al., 2014; but see Whitehorn et al., 2013). C. bombi is known to increase mortality in bumble bees under food stress conditions (Brown et al., 2000), and to reduce bumble bee foraging rate (Otterstatter & Thomson, 2006).\n\nNicotine is an agonist of the nicotinic acetylcholine receptor (nAChR), and therefore acts as both a stimulant drug and a toxin to many organisms (Benowitz, 1998). Nicotine is toxic to many insects, and has been historically used as an insecticide (Ujváry, 1999). Honey bees are deterred by nicotine in nectar (Köhler et al., 2012b), and both honey bees (Köhler et al., 2012b; Singaravelan et al., 2006) and bumble bees (Baracchi et al., 2015) are adversely affected by nicotine consumption when they are not infected by parasites. However, nicotine also has antimicrobial properties (Pavia et al., 2000), and recent studies have suggested that it can reduce parasite load in bumble bees infected with C. bombi (Baracchi et al., 2015; Richardson et al., 2015a), and may improve survival of diseased honey bee colonies (Köhler et al., 2012b). Anabasine, like nicotine, is a nAChR agonist, and has been used as an insecticide (MacBean, 2012). The behavioral effects of anabasine are similar to those of nicotine, although anabasine, unlike nicotine, does not have addictive effects (Caine et al., 2014). Anabasine in nectar has been found to deter honey bees (Singaravelan et al., 2005), and reduced C. bombi load in infected bumble bees (Richardson et al., 2015a).\n\nWe inoculated bumble bees with C. bombi, and assessed the differences in pathogen load and mortality between adult bees fed nicotine (yes/no) and anabasine (yes/no) in a factorial design, resulting in four diet treatments: 2 ppm nicotine, 5 ppm anabasine, 2 ppm nicotine and 5 ppm anabasine together, or a control alkaloid-free solution. All diet treatments also contained 30% sucrose in distilled water. Chemicals ((-)-nicotine, cat. no. N3876; (+/-)-anabasine, cat. no. 284599) were purchased from Sigma-Aldrich (St. Louis, MO). Alkaloid concentrations were chosen to mimic the highest concentrations that would be found in Nicotiana nectar under natural conditions (Adler et al., 2006; Tadmor-Melamed et al., 2004).\n\nWe conducted two experiments. The first experiment (‘Variable’, conducted 26 February 2014 to 20 March 2014, Dataset 1) had a smaller sample size (n = 178 bees) and less strictly controlled environmental conditions, while the second experiment (‘Controlled’, conducted 20 May 2014 to 14 July 2014, Dataset 2) had a larger sample size (n = 339 bees) and carefully controlled environmental conditions (see sample sizes in Table S1). In ‘Variable’, experimental bees were kept on the lab bench (temperature range 10–35°C due to a steam leak, approximately 12 h photoperiod). In ‘Controlled’, experimental bees were incubated at 27°C in constant darkness to more closely mimic conditions in a bumble bee hive.\n\nExperimental bees were obtained from pupal clumps of commercial B. impatiens (Biobest, Leamington, Ontario, Canada). Pupal clumps were removed from colonies weekly and kept in 500 mL plastic containers, with each container containing the pupal clumps from a single colony that were collected on a specific date. In ‘Variable’, pupal clumps were initially incubated on the lab bench, but were later incubated at 30°C in an incubator (Percival Scientific, Perry, IA) due to excessive pre-experiment mortality under the variable lab conditions. In ‘Controlled’, pupal clumps were incubated at 27°C throughout the experiment. Callow bees (newly emerged worker bees less than one day old) were collected upon emergence from pupal clumps. They were weighed and their mass at emergence, date of emergence, and colony of origin were recorded. Bees were assigned systematically to diet treatments in blocks of four, such that each block contained a bee in each treatment. Bees were then isolated in individual 20 mL vials. The lid of each vial was equipped with a 2 mL microcentrifuge tube with a cotton wick containing 500 μL artificial nectar (30% sucrose solution). Each day, bees were transferred to clean vials and given 500 μL fresh artificial nectar and a 10 mg piece of multifloral pollen (Koppert Biological Systems, Howell, MI) on which they fed ad libitum. For two days, bees were fed pollen and control nectar (30% sucrose solution). Bees were inoculated with C. bombi two days after emergence. They were starved for several hours to ensure that they would consume the inoculum, and then fed 10 μL of C. bombi inoculum containing 6,000 C. bombi cells (see below). Bees were then fed pollen and the appropriate nectar treatment ad libitum for 7 days.\n\nTo inoculate experimental bees, inoculum (C. bombi cells in sucrose solution) was prepared from the gut tracts of bees taken from colonies infected with C. bombi. These colonies were obtained from the same supplier as the experimental colonies, and were infected with C. bombi from wild bees collected in Amherst, Massachusetts (September 2013). Infected bees were dissected and their gut tracts were macerated with a plastic pestle in microcentrifuge tubes containing 300 μL distilled water. Samples were incubated for 5 hours at room temperature to allow gut tissue to settle. C. bombi cell density was then assessed using a hemocytometer, and inoculum was prepared from the supernatant of the samples with sufficient concentrations of C. bombi cells. The supernatant was diluted to a concentration of 1200 cells/μL and had an equal volume of 50% sucrose solution added to result in a 25% sucrose solution. Each bee was fed 10 μL of inoculum, containing 6,000 C. bombi cells, using a 20 μL micropipette.\n\nSeven days after inoculation, bees were dissected to assess parasite loads. Gut tracts were extracted and crushed with a pestle in microcentrifuge tubes containing 300 μL distilled water. Samples were allowed to sit for 5 hours to allow gut tissue to settle. C. bombi cell concentrations in the gut extract were measured using a hemocytometer. C. bombi cells were counted in five cells of the hemocytometer and summed (0.004 µL each; 0.02 µL total).\n\nData were analyzed using R version 3.2.1 for Windows (R Core Team, 2014).\n\nFor ‘Variable’, for which exact dates of death were not recorded, mortality was analyzed using a generalized linear mixed model with binomial error distribution (Pinheiro et al., 2015). Probability of death was used as the response variable with nicotine treatment, anabasine treatment, and their interaction as predictor variables. Bee colony was included as a fixed predictor, and date of inoculation was included as a random factor. Wald tests (Lesnoff & Lancelot, 2012) were used to test the marginal significance of individual predictor variables (see Supplementary material script 1). Mortality data for ‘Controlled’, in which we recorded time from inoculation to death to the nearest day, were analyzed using a Cox proportional hazards mixed-effects model (Therneau, 2015). Death hazard rate was used as the response variable; nicotine, anabasine, and their interaction as predictor variables; colony as a fixed predictor; and date of inoculation as a random factor (see Supplementary material script 3).\n\nParasite counts were found to best fit the log-normal distribution and were analyzed using generalized linear mixed models (Bates et al., 2015) with penalized quasi-likelihood parameter estimation (Venables & Ripley, 2002). Parasite counts were (x+1)-transformed for use as the response variable. Nicotine, anabasine, and their interaction were used as predictor variables. Bee colony was included as a fixed predictor, mass as a model covariate, and date of inoculation as a random factor. Marginal significance of individual terms was evaluated using Wald tests (Lesnoff & Lancelot, 2012). Code for analysis is given in Supplementary material script 2 (‘Variable’ experiment) and Supplementary material script 4 (‘Controlled’ experiment).\n\n\nResults\n\nIn variable temperature conditions, the nicotine treatment significantly increased mortality (Table 1). Nearly half of bees fed nicotine-containing nectar died within 7 days of inoculation, which was nearly double the frequency of death in treatments without nicotine (Figure 1). Anabasine did not affect mortality, and there was no significant interaction between the two alkaloid treatments (Figure 1, Table 1).\n\nPoints show adjusted mean probability of death in each treatment group. Error bars represent ±1 standard error.\n\nTable shows binomial mixed model results of χ2 tests for effects of predictor variables on probability of death during the 7 d experiment.\n\nNicotine (linear model β = -1.01 ± 0.295 standard error) and anabasine (β = -0.94 ± 0.31 S.E.) each significantly decreased parasite loads. However, nicotine and anabasine displayed antagonistic effects (Nicotine * Anabasine β = 1.96 ± 0.44 S.E.), such that bees consuming both alkaloids did not realize the medicinal effects of either compound (Figure 2, Table 2).\n\nPoints show adjusted mean parasite count in each treatment group. Error bars represent ±1 standard error.\n\nResults of Wald tests for marginal significance of terms in a generalized linear mixed model with penalized quasi-likelihood parameter estimation.\n\nUnder controlled conditions (27°C with constant darkness), neither alkaloid nor their interaction significantly affected mortality (Figure 3, Table 3). However, nicotine significantly increased parasite loads (β = 0.28 ± 0.12 S.E., Table 4), while the effects of anabasine (β = 0.20 ± 0.12 S.E.) were also positive but not significant (Figure 4, Table 4). This was the opposite result of that observed in ‘Variable’, in which alkaloid ingestion decreased the severity of Crithidia infection. Although much weaker than in ‘Variable’, we found the same pattern of antagonistic interaction between the two alkaloids (Nicotine * Anabasine β = -0.26 ± 0.16 S.E., Figure 4), indicating that the deleterious effects of each compound were reduced in bees consuming the nicotine-anabasine combination (Figure 4). However, this interaction was not statistically significant (Table 4). Overall parasite loads in ‘Controlled’ were much higher, with median parasite loads more than double those observed in ‘Variable’ (compare Figure 2 and Figure 4).\n\nLines show survival curves for bees each treatment group. There were no significant effects of diet treatments on survival.\n\nTable shows marginal significance of individual terms in Cox proportional hazards test for effects of predictor variables on mortality hazard rate.\n\nResults of Wald tests for marginal significance of terms in a generalized linear mixed model with penalized quasi-likelihood parameter estimation.\n\nPoints show adjusted mean parasite count in each treatment group. Error bars represent ±1 standard error.\n\n\nDiscussion\n\nNicotine consumption increased mortality in ‘Variable’, but did not affect mortality in ‘Controlled’. The difference in temperature between the two experiments may be responsible for this context-dependent response. In ‘Variable’, the incubation temperature of the experimental bees was not controlled. Bees were kept on lab benches, in a room with temperatures that ranged from 10 to 35°C. In ‘Controlled’, by contrast, bees were incubated at a constant temperature of 27°C. One hypothesis to explain these divergent responses is that bees in the first experiment may have experienced heat stress that could have exacerbated toxic effects of nicotine, and may have consumed larger quantities of the alkaloid-rich artificial nectar to compensate for evaporative water loss. However, we did not measure nectar consumption, and so cannot be certain that consumption increased under heat stress. Interaction between heat stress and secondary metabolites has been documented in several other species (reviewed in Holmstrup et al., 2010), including some insects and related arthropods. For example, Li et al. (2014) found synergistic interaction between heat stress and avermectin toxicity in the western flower thrips (Frankliniella occidentalis), which led to reduced survival and increased upregulation of heat shock proteins. Mercury exposure reduced heat tolerance in springtails (Folmosia candidia) (Slotsbo et al., 2009), and high temperature increased uptake and toxicity of organophosphate insecticides to the midge Chironomus tentans (Lydy et al., 1999). Our results suggest that interaction between heat stress and toxins may occur in B. impatiens as well. An experiment in which temperature and secondary metabolite consumption are manipulated in a factorial design would more definitively test for such interaction.\n\nOur results indicate that nicotine can be toxic to bees even at very low concentrations when bees are parasitized. This contrasts with previous studies, which did not find significant toxic effects of nicotine at natural concentrations on mortality in bees of unknown parasite status. Detzel & Wink (1993) determined the honey bee LD50 for nicotine to be 2000 ppm, far higher than any concentration that occurs in nectar. Singaravelan et al. (2006) found that larval survival of honey bees was not affected by naturally occurring concentrations of nicotine (up to 5 ppm), even when consumed consistently for several days, although a much higher concentration of nicotine (50 ppm) did significantly reduce survival. These studies focused on honey bees, while our study used bumble bees, so the discrepancy between their results and ours may be due to bumble bees having a greater sensitivity to nicotine than honey bees. However, our results suggest that the toxic effects of nicotine are greater under temperature-stressed conditions; use of optimal incubation conditions could account for the lack of toxic effects observed in these previous studies. Drastic temperature variation similar to that experienced by bees in ‘Variable’ is common in continental climates. For example, in Amherst, MA, where this study was conducted, daily temperature swings of over 15°C are common, and temperatures as low as 10°C and as high as 30°C are frequently experienced within a few days of each other, or even within a single day (Menne et al., 2012a; Menne et al., 2012b). Wild bees, therefore, are likely to experience temperature conditions under which nicotine could be significantly toxic.\n\nIn ‘Variable’, bees that consumed either alkaloid alone had significantly lower parasite counts than control bees, but this effect was not present in bees that consumed both alkaloids. This is consistent with the results of recent studies that found reduced parasite loads under nicotine and anabasine consumption (Baracchi et al., 2015; Richardson et al., 2015a). The reduction in parasite load may be due to alkaloid-induced increases in gut motility. Both nicotine and anabasine have been demonstrated to reduce gut transit time in the Palestinian sunbird Nectarinia osea (Tadmor-Melamed et al., 2004). Although their effect on gut transit time in insects has not been studied, rapid excretion is known to be part of some insects’ physiological response to alkaloids (Wink & Theile, 2002). It is therefore plausible that consumption of nicotine and anabasine could cause an increased rate of excretion in bees, thus clearing C. bombi cells from the gut and leading to the observed reduction in parasite load.\n\nThe lack of effect of the combined alkaloids on parasite load is more puzzling. The concentrations of the individual alkaloids may have been within the medicinal window of concentration at which antiparasitic effects were dominant. However, the combined effects of both alkaloids may have weakened bees’ ability to fight infection through excessive stimulatory, laxative, and/or immunosuppressive effects. These combined toxic effects could have offset the medicinal effects realized at lower concentrations in the single-alkaloid treatments.\n\nIn ‘Controlled’, nicotine consumption significantly increased parasite counts, while anabasine also increased parasite loads, although not significantly. This result is consistent with a growing body of research demonstrating that neonicotinoids, a class of insecticides chemically similar to nicotine, have immunosuppressant effects on bees (reviewed in Goulson et al., 2015). While the effects of nicotine are not necessarily the same as those of neonicotinoids, both nicotine and neonicotinoids function as nAChR agonists, (Jeschke et al., 2011), suggesting similar pharmacological activity. The immunosuppressant effects of neonicotinoids have been most well studied in honey bees. Sub-lethal colony-level exposure to the neonicotinoid imidacloprid has been shown to lead to increased levels of Nosema infection in honey bees (Pettis et al., 2012). A field study by Alburaki et al. (2015) found significantly higher levels of infection by both brood queen cell virus and Varroa mites in honey bee colonies that had foraged on corn treated with the neonicotinoid thiabendazole than in colonies that had foraged on untreated corn. The neonicotinoid pesticides clothianidin and imidacloprid induced increased transcription of a gene coding for a negative modulator of NF-Kβ immune signaling in honey bees, causing decreased immune function and increased viral replication (Di Prisco et al., 2013).\n\nThe apparent contradiction between the results of our first and second experiments may be due to a complex multi-directional interaction between alkaloid consumption, heat stress, and immunity. Under the variable conditions of ‘Variable’, bees may have consumed more liquid, causing them to ingest greater amounts of alkaloids. This increased alkaloid consumption could lead to stronger effects of the alkaloids, both in the form of increased toxicity and increased gut motility, accounting for both the higher mortality and decreased C. bombi counts in ‘Variable’. Bees in ‘Variable’ were also exposed to external stimuli in the lab environment, including light and vibration, which may have further promoted alkaloid consumption by increasing energetic requirements and synergized with stimulatory effects of the alkaloids to promote intestinal peristalsis.\n\nThe higher temperatures of ‘Variable’ may have additionally functioned as an externally imposed fever that reversed the immunosuppressive effects of nicotine. Febrile amelioration of infection has been shown in many animals (reviewed in Kluger, 1978), including honey bees (Campbell et al., 2010) and other insects (Stahlschmidt & Adamo, 2013). The lower absolute parasite counts relative to ‘Controlled’ may reflect heat-related inhibition of C. bombi, which grows best at 27°C (Salathé et al., 2012). Stimulatory effects of nicotine and anabasine, enhanced by exposure to everyday disturbance in ‘Variable’, could have increased activity level and metabolic rate, thereby further raising body temperature and slowing parasite growth. The effects of a given increase in body temperature would have been more pronounced under the hot conditions of ‘Variable’, which may have approached the parasite’s thermal tolerance limit.\n\nOur results contrast with the results of a recent study by Richardson et al. (2015a). Richardson et al., found that both nicotine and anabasine significantly reduced C. bombi parasite load in bumble bees without affecting mortality. In ‘Variable’, we similarly found that both alkaloids reduced pathogen load, but we also found that nicotine increased mortality. This discrepancy may be due to a simple difference in study design: we used the (-)-enantiomer of nicotine, whereas Richardson et al. (2015a) used a +/- enantiomeric mixture (Sigma N0267, personal communications). (-)-Nicotine is far more common in nature, comprising between 99.77% and 99.83% of the nicotine in tobacco (Armstrong et al., 1998), and is more pharmacologically active than (+)-nicotine. In vertebrates, (-)-nicotine was 2.4–3.1 times more toxic to vertebrates than was (+)-nicotine (Gause & Smaragdova, 1939) and had stronger effects on the peripheral nervous system, particularly on muscle contraction (Barlow & Hamilton, 1965). In aquatic invertebrates known to use acetylcholine as a neurotransmitter, (-)-nicotine was again on average 2.6-fold more toxic that the (+)-enantiomer (Gause & Smaragdova, 1939). In insects, the (-)-enantiomer had stronger affinity for the nAChR in housefly and honey bee head membranes (Tomizawa & Yamamoto, 1992). Interestingly, Gause & Smaragdova (1939) found the two enantiomers to be isotoxic to the protozoans they tested. If this is the case for C. bombi, it suggests an explanation for our difference in results. If (-)-nicotine is more toxic to bees than (+)-nicotine, but both enantiomers are equally toxic to C. bombi, than (+)-nicotine could reduce parasite count without significantly affecting bee mortality, while (-)-nicotine could reduce parasite count but also be toxic to bees.\n\nAnother possible explanation for our differing results is a difference in the C. bombi itself. C. bombi is known to be genetically diverse; Salathé & Schmid-Hempel (2011) identified 213 strains infecting bumble bees in Switzerland. Multiple strains are often present in a single host. Tognazzo et al. (2012) found that 67% of infected workers and 54% of infected queens carried mixed-genotype infections, with queens harboring up to 29 different genotypes. In addition, it is possible that not all supposed C. bombi infections in fact represent a single Crithidia species. Schmid-Hempel & Tognazzo (2010) identified two genetically and morphologically distinct lineages within the C. bombi complex, which they classified as cryptic species. They retained the name C. bombi for the lineage which more closely matches Lipa & Triggiani’s (1988) original description of C. bombi, and proposed the name C. expoeki for the other lineage. Both lineages are present in both Europe and North America, suggesting an old divergence. If our C. bombi cultures and those used by Richardson et al. (2015a) represent different strains, or different species, it is possible that they vary in their alkaloid tolerance.\n\nOur results represent an important first step towards understanding the interactive effects of multiple secondary metabolites on pollinators. We did not find evidence for synergy between Nicotiana nectar alkaloids, although we did find some evidence for antagonism. To elucidate the potential role of interactions between compounds in the plant-pollinator-parasite system, it will be necessary to test for interactions between other sets of compounds. Within Nicotiana, the wild tobacco N. attenuata contains at least 35 nectar secondary compounds, including sesquiterpenes (Kessler & Baldwin, 2007); many terpenoids have potent trypanocidal activity, yet are relatively benign to animal cells (Otoguro et al., 2011). Among other plant families, Asclepias species are pollinated by bumble bees and contain several cardenolides in their nectar (Manson et al., 2012) that could be tested for interactive effects. Another plant species to investigate is Chelone glabra, which has high concentrations of the iridoid glycosides aucubin and catalpol in its nectar (Richardson et al., 2015b). Synergy between these glycosides has been demonstrated in their effect on Junonia coenia caterpillars (Richards et al., 2012).\n\nThe effect of nectar alkaloids on parasitized pollinators may represent a tradeoff between toxicity to the parasite and toxicity to the host. In the case of nicotine, bees appear to be more sensitive to alkaloid toxicity than parasites are. While nicotine inhibits the growth of many microbial pathogens, significant antimicrobial effects require concentrations between 100 and 250 ppm (Pavia et al., 2000). By contrast, Singaravelan et al. (2006) found that nicotine was toxic to bees at 50 ppm, and our own results suggest that nicotine can have toxic effects at concentrations as low as 2 ppm. This suggests that bees are less tolerant of nicotine than microbes are. Despite their significant toxicity, nectar secondary metabolites such as nicotine are unlikely to pose a health risk to bees in the wild. The studies establishing toxicity of nicotine in bees have all focused on chronic consumption of a diet high in nicotine. This is an artificial condition that bees would be unlikely to encounter in nature. Bumble bees are generalist pollinators, and are known to forage on several plant species within a narrow time frame and even within a single foraging trip (Free, 1970). They would therefore be unlikely to consume enough nicotine from nectar to experience toxic effects, although exposure to pharmacologically similar, agriculturally ubiquitous neonicotinoid insecticides appears concerning (Goulson et al., 2015).\n\n\nConclusion\n\nOur results emphasize the importance of interactions between stressors in pollinator health, and demonstrate that the effect of any single factor can vary greatly depending on the other factors involved. Research on pollinator health often focuses on single factors in isolation; however, in natural conditions, pollinators are often exposed to several stressors simultaneously (Goulson et al., 2015). Previous research has demonstrated both medicinal and toxic effects of secondary metabolites such as nicotine and anabasine. Our results suggest that the predominant effect can vary with environmental context. In order to better elucidate the role of secondary metabolites in pollinator health, future research should explicitly address the role of these complex interactions.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for ‘Variable’ experiment, 10.5256/f1000research.6870.d101937 (Thorburn et al., 2015a).\n\nF1000Research: Dataset 2. Data for ‘Controlled’ experiment, 10.5256/f1000research.6870.d101940 (Thorburn et al., 2015b).",
"appendix": "Author contributions\n\n\n\nAll authors conceived the study. ECPY and LPT designed and conducted the experiments using methods developed by REI. ECPY analyzed the data. LPT and ECPY prepared the first draft of the manuscript. All authors revised the manuscript and have agreed upon the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by the National Science Foundation under NSFDEB-1258096, NSF GRFP (Grant DGE-0907995 to ECPY), and NSF DDIG (Grant NSFDEB-1501907 to ECPY and LSA); by the National Research Initiative (NRI) Arthropod and Nematode Biology and Management Program of the USDA Cooperative State Research, Education, and Extension Service (CSREES) Grant no. USDA-AFRI 2013-02536; and by the Garden Club of America Centennial Pollinator Fellowship (ECPY).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors are grateful to Biobest for supplying bee colonies; and to Ali Hogeboom, Melissa Ha, Caitlin McAllister, and lab assistants for help with ‘Controlled’ experiment.\n\n\nSupplementary material\n\nScript 1. R script for analysis of C. bombi parasite loads in ‘Variable’ experiment.\n\nClick here to access the data.\n\nhttp://dx.doi.org/10.5256/f1000research.6870.s101933\n\nScript 2. R script for analysis of mortality in ‘Variable’ experiment.\n\nClick here to access the data.\n\nhttp://dx.doi.org/10.5256/f1000research.6870.s101934\n\nScript 3. R script for analysis of C. bombi parasite loads in ‘Controlled’ experiment.\n\nClick here to access the data.\n\nhttp://dx.doi.org/10.5256/f1000research.6870.s101935\n\nScript 4. R script for analysis of mortality in ‘Controlled’ experiment.\n\nClick here to access the data.\n\nhttp://dx.doi.org/10.5256/f1000research.6870.s101936\n\n\nReferences\n\nAbbott J: Self-medication in insects: current evidence and future perspectives. Ecol Entomol. 2014; 39(3): 273–280. Publisher Full Text\n\nAdler LS, Seifert MG, Wink M, et al.: Reliance on pollinators predicts defensive chemistry across tobacco species. Ecol Lett. 2012; 15(10): 1140–1148. PubMed Abstract | Publisher Full Text\n\nAdler LS, Wink M, Distl M, et al.: Leaf herbivory and nutrients increase nectar alkaloids. Ecol Lett. 2006; 9(8): 960–967. 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Publisher Full Text\n\nWhitehorn PR, Tinsley MC, Brown MJF, et al.: Investigating the impact of deploying commercial Bombus terrestris for crop pollination on pathogen dynamics in wild bumble bees. J Apicult Res. 2013; 52(3): 149–157. Publisher Full Text\n\nWilliams PH, Araújo MB, Rasmont P: Can vulnerability among British bumblebee (Bombus) species be explained by niche position and breadth? Biol Conserv. 2007; 138(3–4): 493–505. Publisher Full Text\n\nWilliams P, Colla S, Xie Z: Bumblebee vulnerability: common correlates of winners and losers across three continents. Conserv Biol. 2009; 23(4): 931–940. PubMed Abstract | Publisher Full Text\n\nWink M, Theile V: Alkaloid tolerance in Manduca sexta and phylogenetically related sphingids (Lepidoptera: Sphingidae). Chemoecology. 2002; 12(1): 29–46. Publisher Full Text"
}
|
[
{
"id": "10403",
"date": "07 Oct 2015",
"name": "Michael Simone-Finstrom",
"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\nThorburn et al. describe an experiment where they test the dual effect of two secondary plant compounds on bumble bee mortality and parasite load after infections with the pathogen Crithidia bombi. This paper addresses an interesting question that follows up recent findings about the potential use of secondary metabolites of nectar as a self-medication mechanism to reduce parasite infections in bumble bees. However, we found several major points that the authors should address to clarify the results of the study and to better frame it within the context of previous findings.Major concerns:We found multiple points of the statistical analysis that were unclear:For the ‘controlled’ experiment, the authors say they used a Cox proportional hazards mixed-effect model. However, the risk ratio of death is not reported, which is one of the most informative parameters to quantitatively estimate the difference in survival between the four groups. It is also surprising that there was no significant difference between the survival of the groups. From Fig. 3, it seems that the group in the Nicotine+Anabasine had lower probability of survival.naWouldn’t a model that allows for interactions of treatment, colony and mass be more appropriate?Regarding mass and colony, since both terms were highly significant, how does that affect the interpretation of the results? Was there no relationship with mass and treatment? Also did the masses of bees from the 'Variable' experiment differ from those in the 'Controlled' experiment due to the stress they experienced during pupation? The authors report a high pre-experiment mortality rate in the “controlled” treatment. Is it possible that the pupae were stressed before starting the experiment and that this condition had a significant effect in the bee survival during the experiment? Even in the control bees, a 60% mortality after 7 days was reported. This numbers seem high compared to other recent studies (Baracchi et al. 2015, Richardson et al 2015). It was interesting to find out that the ‘variable’ treatment generated the most interesting results. However, the conflicting findings of the ‘controlled’ and ‘variable treatments need to better reconcile in the discussion. There also needs to be more information reported regarding how the temperature fluctuated. Why do authors think that the results of the ‘variable treatment’ were the product of heat stress and not cold stress, or just stress in general? Similarly the fact that the ‘variable treatment’ was during pupation and not just during the treatment period needs to be addressed along with the implications of that possible effect. This is an overall comment about the structure of the introduction and discussion. Even though the text is well written, the structure of these sections does not connect ideas very well. It reads as a list of ideas, rather than one story with a beginning and an end.Minor comments:In the abstract, authors state that they tested for ‘synergistic’ effect between two alkaloid compounds. However, results show antagonistic effects between them. Would it be better to just broadly state that the authors are testing for possible interactions between these compounds? The 2nd to last paragraph in the introduction. The authors should make the point that this is the first study to look at a combination of any alkaloids, and not just nicotine and anabasine, if that’s the case. It makes this study more significant and should be highlighted. In M&M, the subheading ‘diet treatment’ could be confused with a treatment where variable amounts of protein, carbohydrates and lipids were used. Would it be better to call this section ‘Secondary Compound Treatments’? On a similar note, the authors may consider changing the names of the treatment groups to “Variable” and “Stable” or something to that effect so there are not two control treatments. Was the inoculation done before or after the ‘diet treatment’? If it was done before, please mention this section of the methods in the order in which the experiment took place. This will help the reader with clarity about the process. The sample sizes for the parasite load analysis could be in the text or figure, rather than in a supplementary file. 2nd paragraph of the discussion. Baracchi et al. 2015 found the same result of low levels of nicotine causing mortality, which is left out here. The section entitled “In a controlled temperature environment…” starts by seemingly contradicting the previous section without any discussion of that. Also these results need to be reconciled with Baracchi et al. 2015. Should be black queen cell virus (instead of brood queen cell virus) The paragraph explaining the difference in chemical composition between the nicotine in Richardson et al and this manuscript is too long and it still doesn’t explain why there was no effect on the parasite loads. The last sentence of the discussion states that ‘bees would be unlikely to consume enough nicotine from nectar to experience toxic effects’. There is a large body of literature on the effects toxic nectar on bees, how can the authors be certain about that? Particularly given that some nectar is stored within the nest and combined with other nectars.",
"responses": [
{
"c_id": "1723",
"date": "16 Dec 2015",
"name": "Evan Palmer-Young",
"role": "Author Response",
"response": "Dear Dr.’s Simone-Finstrom and Lopez-Uribe,Many thanks for your careful review of our research. We have revised the manuscript to incorporate your suggestions, and hope that you find the new version improved in content and clarity. We respond to each of your points below: Major concerns: Reviewer Comment: We found multiple points of the statistical analysis that were unclear:For the ‘controlled’ experiment, the authors say they used a Cox proportional hazards mixed-effect model. However, the risk ratio of death is not reported, which is one of the most informative parameters to quantitatively estimate the difference in survival between the four groups. It is also surprising that there was no significant difference between the survival of the groups. From Fig. 3, it seems that the group in the Nicotine+Anabasine had lower probability of survival. Author Response: The death hazard ratios are now reported in the text, along with results of the pairwise comparison between the Control and Nicotine + Anabasine groups. Although the Nicotine + Anabasine group did have a higher estimated death hazard ratio (1.27 ± 0.22 SE) compared to the control group (0.82 ± 0.16 SE), this difference was not statistically significant (p=0.446 for pairwise comparison).Reviewer Comment: Wouldn’t a model that allows for interactions of treatment, colony and mass be more appropriate? Author Response: We agree that it would have been interesting to investigate a more complex model. However, we were concerned that adding additional terms would prevent meaningful tests of the effects of alkaloid consumption, particularly in the ‘Variable’ experiment, which had a smaller sample size and an unbalanced design due to the high mortality among bees that consumed nicotine. Although the ‘Stable’ experiment had a larger sample size, we chose to analyze it with the same model used for the ‘Variable’ experiment, to facilitate comparison between the two results. Therefore, we formulated our models a priori without interaction terms. In previous studies testing the effects of secondary metabolite consumption on Crithidia infection, interaction terms were found to be non-significant (e.g. Baracchi et al. (2015)) or were not reported (e.g., Richardson et al. 2015 Proc Biol Sci. 22;282(1803):20142471. doi: 10.1098/rspb.2014.2471; Manson et al. 2010, Oecologia.62(1):81-9. doi: 10.1007/s00442-009-1431-9).Reviewer Comment: Regarding mass and colony, since both terms were highly significant, how does that affect the interpretation of the results?Author Response: This significance shows that Crithidia infection intensity varies by mass and also by colony, which is consistent with the results of previous studies (e.g., Otterstatter & Thompson 2006 Parasitology 133(Pt 6):749–61. http://doi.org/10.1017/S003118200600120X), but does not invalidate our conclusions about the effects of treatment. We have emphasized the effects of mass and colony genotype in the new paragraph of the discussion section about the differences between our study and that of Baracchi and colleagues. Reviewer Comment: Was there no relationship with mass and treatment?Author Response: We apologize for this confusion. Bee mass refers to mass at time of emergence from pupation, before treatments were applied. We now clarify this in Methods: Statistical analysis and in the legends of Tables 2 and 4.Reviewer Comment: Also did the masses of bees from the 'Variable' experiment differ from those in the 'Controlled' experiment due to the stress they experienced during pupation?Author Response: Yes—somewhat surprisingly, bees in the ‘Variable’ experiment were significantly larger (mean = 0.160 g) than in the ‘Stable’ (formerly ‘Controlled’) experiment (mean = 0.121 g). We expect that this reflects high mortality of the smaller bees in ‘Variable’, which might have been more likely to die during pupation or during the two days between emergence and inoculation. This finding has been added to the section of the Results describing the ‘Stable’ experiment, and is mentioned again in the first paragraph of the Discussion.Reviewer Comment: The authors report a high pre-experiment mortality rate in the “controlled” treatment. Is it possible that the pupae were stressed before starting the experiment and that this condition had a significant effect in the bee survival during the experiment?Author Response: In the ‘Variable’ experiment, pre-experiment mortality was indeed high, and yes, likely impacted survival during the experiment as well. The chronic exposure of colonies, pupae, and adult bees to variable temperatures is now noted in the third paragraph of “Methods: Rearing conditions” and in the first paragraph of the discussion.Reviewer Comment: Even in the control bees, a 60% mortality after 7 days was reported. This numbers seem high compared to other recent studies (Baracchi et al. 2015, Richardson et al 2015).Author Response: It is correct that our study found much higher rates of mortality, even under ‘Stable’ conditions (up to 35% over 7 d), than these two prior papers. However, both of these studies employed different rearing conditions from ours in their experiments measuring survival. Richardson et al. (2015) measured mortality in microcolonies (3-bee communities) incubated in 500 mL plastic deli containers, as opposed to the solitary bees in 30 mL snap-cap vials used in our experiments. Baracchi et al. used individual bees, but they were housed in petri dishes rather than vials; in addition, whereas Baracchi et al. kept bees in the same petri dish throughout the experiment (D. Baracchi, personal communication), we moved our bees to clean vials daily, which may have imposed additional stress resulting from handling. Finally, Baracchi et al. used a different strain of Crithidia, and also a different bee species (B. terrestris) from the one we used (B. impatiens). Any of these social, environmental and genetic factors may have contributed to the observed differences in mortality. These differences are described in the new Discussion paragraph contrasting our study with that of Baracchi et al. Reviewer Comment: It was interesting to find out that the ‘variable’ treatment generated the most interesting results. However, the conflicting findings of the ‘controlled’ and ‘variable treatments need to better reconcile in the discussion.Author Response: We have added and clarified speculation about the reasons for these differences and their implications in “Discussion: Interactive effects of abiotic conditions, alkaloids, and parasites on bees”, wherein we consider a number of non-exclusive possible explanations for differences in the effects of alkaloids’ effects on mortality and parasitism between ‘Variable’ and ‘Stable’:(1) Increased consumption: We hypothesized that the stronger effects of alkaloids in ‘Variable’ may have resulted from increased liquid (nectar) intake under hot conditions. We tested this idea in the ‘Consumption experiment’. To our surprise, bees drank substantially less at high temperatures similar to those in ‘Variable’. These results are now reported (Figure 5, Table 5). (2) Interactions between multiple stressors:(a) Mortality: Stronger effects of alkaloid intake on mortality in ‘Variable’ may reflect increased susceptibility to alkaloid toxicity in the presence of multiple stressors, including temperature fluctuation and external disturbance. Given the results of the ‘Consumption experiment’, we now speculate that nicotine may have increased mortality in ‘Variable’ by increasing fecal moisture losses. Because the warmer temperatures of ‘Variable’ appear to have increased evaporative moisture loss while reducing nectar intake, any additional nicotine-induced moisture loss would have increased the risk of lethal dehydration.(b) Parasitism: By the same logic, stronger effects of alkaloid intake on parasitism in variable temperatures may reflect synergistic effects of alkaloids and external stimuli on locomotor and gut activity. In the new paragraph describing the differences between our study and that of Baracchi et al. (2015), we have noted that we both we and Baracchi et al. showed antiparasitic effects of nicotine consumption when bees were kept on the lab bench (as they were in ‘Variable’), but not when bees were housed in constant darkness (as in ‘Stable’). (3) Inhibitory effects of high temperatures on Crithidia compensating for nicotine-induced immunosuppression: We suggest that in ‘Stable’, immunosuppressive effects of nicotine contributed to increased parasite loads. In contrast, in ‘Variable’, stimulatory effects of alkaloid consumption on locomotor activity may have caused bee body temperature to reach or exceed the thermal tolerance limits of the parasite. These febrile effects may have offset any immunosuppressive effects of nicotine. (4) In the paragraph describing differences between Crithidia strains, we have incorporated an additional suggestion from the other reviewer. Dr. Baracchi suggested that nicotine and anabasine may have their greatest medicinal value at lower levels of infection, such as those observed in ‘Variable’, where median Crithidia counts were lower than in ‘Stable’.Reviewer Comment: There also needs to be more information reported regarding how the temperature fluctuated. Why do authors think that the results of the ‘variable treatment’ were the product of heat stress and not cold stress, or just stress in general?Author Response: We agree that the effects seen in ‘Variable’ could reflect both heat and cold stress. However, my personal recollection (ECPY) was that most of the time the lab was very hot, around 35 °C, at least during the day when the experimenters were present. Cold stress seemed to be more occasional, with sporadic morning and weekend periods when the temperature decreased to 10-15 °C due to building steam being shut off for repairs. We would have liked to have kept more careful records, ideally from data loggers, but the extent and duration of the variability in temperature was entirely unexpected, and we did not consistently note the precise conditions. Additional detail about the temperature fluctuation has been added to the first paragraph of Methods: Rearing conditions.Reviewer Comment: Similarly the fact that the ‘variable treatment’ was during pupation and not just during the treatment period needs to be addressed along with the implications of that possible effect.Author Response: We have now noted this additional stress throughout the developmental period in 3 places: in the third paragraph of “Methods: Rearing conditions”, at the end of “Results: ‘Stable’”, and in the first paragraph of the discussion.Reviewer Comment: This is an overall comment about the structure of the introduction and discussion. Even though the text is well written, the structure of these sections does not connect ideas very well. It reads as a list of ideas, rather than one story with a beginning and an end.Author Response: We appreciate this gracious compliment, and have attempted to improve the organization of the introduction, where we have shortened or separated some paragraphs. In the discussion, we have shortened the paragraph contrasting our results with those of Richardson et al. (2015), and have elaborated on the differences between ‘Stable’ and ‘Variable’ as well as between our study and previous ones.Minor comments:Reviewer Comment: In the abstract, authors state that they tested for ‘synergistic’ effect between two alkaloid compounds. However, results show antagonistic effects between them. Would it be better to just broadly state that the authors are testing for possible interactions between these compounds?Author Response: We agree and have rephrased the abstract. Reviewer Comment: The 2nd to last paragraph in the introduction. The authors should make the point that this is the first study to look at a combination of any alkaloids, and not just nicotine and anabasine, if that’s the case. It makes this study more significant and should be highlighted.Author Response: Thank you. We now highlight this unique contribution in the aforementioned paragraph. Reviewer Comment: In M(aterials&Methods), the subheading ‘diet treatment’ could be confused with a treatment where variable amounts of protein, carbohydrates and lipids were used. Would it be better to call this section ‘Secondary Compound Treatments’?Author Response: We have implemented this suggestion. Reviewer Comment: On a similar note, the authors may consider changing the names of the treatment groups to “Variable” and “Stable” or something to that effect so there are not two control treatments.Author Response: We apologize that the naming of the “Controlled” experiment was confusing. We have implemented this suggestion and renamed the “Controlled” experiment to “Stable”.Reviewer Comment: Was the inoculation done before or after the ‘diet treatment’? If it was done before, please mention this section of the methods in the order in which the experiment took place. This will help the reader with clarity about the process.Author Response: We apologize for the confusion. The inoculation was performed before the secondary compound treatment began. Bees were fed 30% sucrose nectar without secondary metabolites for 2 days after emergence, then inoculated with Crithidia. Diet treatments were implemented immediately post-inoculation. We have reordered this description in the methods section (“Rearing conditions”: final paragraph).Reviewer Comment: The sample sizes for the parasite load analysis could be in the text or figure, rather than in a supplementary file.Author response: We have now listed the sample sizes for each analysis in the legends of the relevant figures.Reviewer Comment: 2nd paragraph of the discussion. Baracchi et al. 2015 found the same result of low levels of nicotine causing mortality, which is left out here.Author response: This result is now cited (third paragraph of Discussion).Reviewer Comment: The section entitled “In a controlled temperature environment…” starts by seemingly contradicting the previous section without any discussion of that.Author response: We have added two transition sentences at the beginning of this paragraph.Reviewer Comment: Also these results need to be reconciled with Baracchi et al. 2015.Author response: We have added a full paragraph to the discussion section outlining the differences between our experiments and those of Baracchi et al. (2015).Reviewer Comment: Should be black queen cell virus (instead of brood queen cell virus)Author response: Thank you for pointing out this error. It is now corrected. Reviewer Comment: The paragraph explaining the difference in chemical composition between the nicotine in Richardson et al and this manuscript is too long and it still doesn’t explain why there was no effect on the parasite loads.Author response: This paragraph has been substantially reduced, but is retained in a shortened form; the editors encouraged us to include this paragraph in order to reconcile our results with those of Richardson et al., which included experiments done in our own laboratory.Reviewer Comment: The last sentence of the discussion states that ‘bees would be unlikely to consume enough nicotine from nectar to experience toxic effects’. There is a large body of literature on the effects toxic nectar on bees, how can the authors be certain about that? Particularly given that some nectar is stored within the nest and combined with other nectars.Author response: We have now reduced this speculation to say that in varied landscapes, bees might be able to reduce or avoid toxicity by exploiting different, lower-nicotine flower species.Thanks you again for your critical review and thoughtful suggestions.Sincerely,Evan Palmer-Young on behalf of the authors"
}
]
},
{
"id": "10860",
"date": "19 Oct 2015",
"name": "David Baracchi",
"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 investigated how the consumption of nectar alkaloids, either in isolation or combination, affect survival and pathogen load in a pollinator species. The main goal of the paper is to test the synergistic effects of two alkaloids on bee survival and parasite loads. To address this question the authors used the pollinator Bombus impatiens, its common midgut parasite, Crithidia bombi, and the natural alkaloids anabasine and nicotine, which are two nectar secondary metabolites produced by Nicotiana species. They provided evidence that both alkaloids decreased the parasite loads when bumble bees were kept under variable laboratory conditions. By contrast, they showed that nicotine but not anabasine increased the parasite load when bees were kept under controlled laboratory conditions. The consumption of the alkaloids in combinations did not have any synergistic effect, but rather an antagonistic effect especially under variable rearing conditions.The authors concluded that the interaction between secondary metabolites are complex and that environmental variables may play an important role in determining the positive or negative impact of the diet on pollinator health. The study addresses an important question and provides useful results. There are, however, some points that need clarification and some issues that should be addressed. Major concerns: In \"Methods\" the authors stated that \" In ‘Variable’, pupal clumps were initially incubated on the lab bench, but were later incubated at 30°C in an incubator....\". It is not clear if they used the bees obtained from these initial pupal clumps for the experiments or not. If they excluded them, then there is no need to report this information. If not, authors should discuss the possible implications of this \"extra\" stress. \"The overall parasite loads in ‘Controlled’ were much higher, with median parasite loads more than double those observed in \"Variable\". In my view this point is very important and neglected by the authors and it might partially account for the apparent contradiction between the results of \"Variable\" and \"Controlled\" experiments. There are evidences for the existence of a trade-off between alkaloid toxicity to the parasite and toxicity to the host. If the parasite load is too high and bumblebee weakened, the toxicity to the bees might overcome that to the parasite, resulting in even higher parasite loads. On the other hand, if the parasite load is lower, the costs in consuming the alkaloids might have been counterbalanced by the advantages in slowing the progression of the infection. Authors should discuss this point. Do authors have any explanation why bumble bees exposed to 2 ppm nicotine had a so high mortality rate after only 7 days since exposure? For instance, Baracchi et al 2015 also found a toxic effect of nicotine, but the detrimental effect on bee lifespan started to be evident only about 20-30 days after the start of the experiment. \"Variable\" and \"Controlled\" experiments also differed in time of year (the experiments were carried out in February-March and May-July respectively). Season is thus an existing confounding factor that authors should seriously take into account in the discussion. Authors suggested that under variable conditions, bees may have consumed more liquid, causing ingestions of more alkaloids accounting for both the higher mortality and decreased parasite load in ‘Variable’. Here I respectfully disagree since I would expect that bees constantly exposed to high temperatures would consume more liquid. It is also possible that bees exposed to high temperatures might have less water in the faeces or might defecate less for suppressing water loss, which may results in higher parasite load. Unfortunately, authors did not provide any information about the overall quantity of sucrose consumed by bumblebees over the experiments. I think that authors should address this point with a simple additional experiment. Measuring for few days the quantity of liquid consumed, and possibly the excretion rate of bees exposed to different temperatures, might really help them to better discuss and clarify their findings. Minor points: The most novel aspect of the manuscript is the fact that authors investigated the possible interactions between two natural alkaloids. I suggest to mention this aspect in the title. In my opinion the first paragraph of the introduction is not necessary. The second paragraph of the introduction discusses about the topic of self-medication and preferential feeding on alkaloid enriched nectars by pollinators and Baracchi et al. 2015 should be included in the paragraph since it is really pertinent here. The authors dedicated an entire long paragraph on the chemical nature on nicotine used by Richardson et al. 2015 and the present manuscript. The paragraph is too long. On top of that, Richarson et al. 2015 and Baracchi et al 2015 (we amended this information in the paper) used different types of nicotine (the former used a +/- enantiomeric mixture, whereas the latter used (-)-enantiomer of nicotine, as the authors of the present work also did) obtaining the same results. Thus, it is improbable that the chemical composition of the alkaloid is responsible for the differences found. I suggest to reduce or remove this paragraph.\n\nFor the \"Variable\" data set authors used the GLMM while for the \"Controlled\" data set authors used a Cox proportional hazards mixed-effects model. Given that the \"Variable\" data set does not allow to use a Cox prop hazards mixed-effects model, I agree with this statistical approach, but nonetheless, it might be useful if the authors analyze the \"Controlled\" data also with the GLMM, so that a direct comparison of the two situations would be possible. \"Variable\" and \"Controlled\" experiments also differed in the fact that only bees in the \"Controlled\" were kept with constant darkness, which could have an effect on bee locomotor activity, energy consumption etc.. Authors should mention this in the discussion.",
"responses": [
{
"c_id": "1722",
"date": "16 Dec 2015",
"name": "Evan Palmer-Young",
"role": "Author Response",
"response": "Dear Dr. Baracchi,Many thanks for your thorough critical review of our article. We feel that the revised version is now improved in response to your comments. We address each point below:Reviewer comment: The study addresses an important question and provides useful results.Author Response: We thank the reviewer for this gracious comment.Major concerns:Reviewer Comment: In \"Methods\" the authors stated that \" In ‘Variable’, pupal clumps were initially incubated on the lab bench, but were later incubated at 30°C in an incubator....\". It is not clear if they used the bees obtained from these initial pupal clumps for the experiments or not. If they excluded them, then there is no need to report this information. If not, authors should discuss the possible implications of this \"extra\" stress.Author Response: We now clarify in “Methods: Rearing Conditions” that we used the bees from all of these pupal clumps. The pupal clumps were only moved to the incubator during the last week of inoculations, 2 days before the emergence of the last bees to be inoculated. Hence, all of the bees in the “Variable” experiment would have been exposed to variable temperatures during pupation. We agree that this additional stress could have affected both survival and immunity, and now mention both of these possibilities in the discussion.Reviewer Comment: \"The overall parasite loads in ‘Controlled’ were much higher, with median parasite loads more than double those observed in \"Variable\". In my view this point is very important and neglected by the authors and it might partially account for the apparent contradiction between the results of \"Variable\" and \"Controlled\" experiments. There are evidences for the existence of a trade-off between alkaloid toxicity to the parasite and toxicity to the host. If the parasite load is too high and bumblebee weakened, the toxicity to the bees might overcome that to the parasite, resulting in even higher parasite loads. On the other hand, if the parasite load is lower, the costs in consuming the alkaloids might have been counterbalanced by the advantages in slowing the progression of the infection. Author Response: Thank you for this suggestion. We have added the idea that alkaloids might be most medicinal against mild infections to the final paragraph of “Discussion: Interactive effects of abiotic conditions, alkaloids, and parasites on bees”.Reviewer Comment: Do authors have any explanation why bumble bees exposed to 2 ppm nicotine had a so high mortality rate after only 7 days since exposure? For instance, Baracchi et al 2015 also found a toxic effect of nicotine, but the detrimental effect on bee lifespan started to be evident only about 20-30 days after the start of the experiment.Author Response: In the ‘Variable’ experiment, we believe that the stress from fluctuating temperatures contributed to overall higher levels of mortality and greater susceptibility to alkaloid toxicity than under the “standard laboratory conditions” employed in Baracchi et al (2015).The other reviewers, Dr.’s Simone-Finstrom and Lopez-Uribe, had a similar comment about our high reported rates of mortality in ‘Stable’ in comparison with Richardson et al. 2015 and Baracchi et al. 2015. We duplicate that response here:It is correct that our study found much higher rates of mortality, even under ‘Stable’ conditions (up to 35% over 7 d), than these two prior papers. However, both of these studies employed different rearing conditions from ours in their experiments measuring survival. Richardson et al. (2015) measured mortality in microcolonies (3-bee communities) incubated in 500 mL plastic deli containers, as opposed to the solitary bees in 30 mL snap-cap vials used in our experiments. Baracchi et al. used individual bees, but they were housed in petri dishes rather than vials; in addition, whereas Baracchi et al. kept bees in the same petri dish throughout the experiment (D. Baracchi, personal communication), we moved our bees to clean vials daily, which may have imposed additional stress resulting from handling. Finally, Baracchi et al. used a different strain of Crithidia, and also a different bee species (B. terrestris) from the one we used (B. impatiens). Any of these social, environmental and genetic factors may have contributed to the observed differences in mortality.Reviewer comment: \"Variable\" and \"Controlled\" experiments also differed in time of year (the experiments were carried out in February-March and May-July respectively). Season is thus an existing confounding factor that authors should seriously take into account in the discussion.Author Response: We are hesitant to speculate on the effects of season, because bee colonies were kept indoors in cardboard boxes and inside closed cabinets, which shielded them from circadian cues. In addition, we do not know enough about the rearing conditions employed by our commercial supplier—e.g. how long bees had been raised in captivity, to what seasonal cues they were exposed-- to offer an informed discussion of this point. However, we do feel that exposure to environmental cues was an important factor within the experiment itself, and have emphasized the possible interactive effects of alkaloid consumption together with daylight and lab stimuli, as astutely pointed out in Dr. Baracchi’s final comment. We mention these interactive effects in the first paragraph of “Discussion: Interactive effects of abiotic conditions, alkaloids, and parasites on bees”, and reiterate their possible importance in the new Discussion paragraph contrasting our results with those of Baracchi et al. Reviewer comment: Authors suggested that under variable conditions, bees may have consumed more liquid, causing ingestions of more alkaloids accounting for both the higher mortality and decreased parasite load in ‘Variable’. Here I respectfully disagree since I would expect that bees constantly exposed to high temperatures would consume more liquid. It is also possible that bees exposed to high temperatures might have less water in the faeces or might defecate less for suppressing water loss, which may results in higher parasite load. Unfortunately, authors did not provide any information about the overall quantity of sucrose consumed by bumblebees over the experiments. I think that authors should address this point with a simple additional experiment. Measuring for few days the quantity of liquid consumed, and possibly the excretion rate of bees exposed to different temperatures, might really help them to better discuss and clarify their findings.Author response: We conducted a follow-up experiment (‘Consumption experiment’, Figure 5) in which we measured nectar consumption at 2 different temperatures, 27 °C and 33 °C. The lower temperature matched that used in the ‘Stable’ experiment (formerly ‘Controlled’), whereas the higher temperature was designed to simulate typical daytime temperatures during ‘Variable’. To our surprise, nectar consumption was reduced by over 50% at the higher temperature. Accordingly, we have removed speculation that bees in the generally warmer ‘Variable’ experiment may have consumed greater quantities of nectar and alkaloids, and instead suggest that nicotine might have exacerbated stress from dehydration.Bees in both temperature treatments were housed in an incubator under dark conditions, similar to those in the ‘Stable’ experiment but lacking the light and other stimuli to which bees were exposed in the ‘Variable’ experiment. The interactive effects of temperature and external stimuli offer an intriguing area for further study, and we encourage such investigation in the Discussion. We thank Dr. Baracchi for suggestions and encouragement with the Consumption experiment.Minor points:Reviewer comment: The most novel aspect of the manuscript is the fact that authors investigated the possible interactions between two natural alkaloids. I suggest to mention this aspect in the title.Author response: We agree. We now mention compound interactions in the title.Reviewer comment: In my opinion the first paragraph of the introduction is not necessary.Author response: This paragraph has been shortened and combined with former second paragraph.Reviewer comment: The second paragraph of the introduction discusses about the topic of self-medication and preferential feeding on alkaloid enriched nectars by pollinators and Baracchi et al. 2015 should be included in the paragraph since it is really pertinent here.Author response: Baracchi et al. 2015 is now cited in the third paragraph of the introduction that documents medicinal effects of secondary metabolites on pollinators. Reviewer comment: The authors dedicated an entire long paragraph on the chemical nature on nicotine used by Richardson et al.2015 and the present manuscript. The paragraph is too long. On top of that, Richarson et al. 2015 and Baracchi et al 2015 (we amended this information in the paper) used different types of nicotine (the former used a +/- enantiomeric mixture, whereas the latter used (-)-enantiomer of nicotine, as the authors of the present work also did) obtaining the same results. Thus, it is improbable that the chemical composition of the alkaloid is responsible for the differences found. I suggest to reduce or remove this paragraph. Author response: This paragraph has been reduced by half, but we felt that it was important to note this difference at some level, given the similarities between our study and that of Richardson et al. 2015 and the multiple studies demonstrating the disparate effects of the two enantiomers. Reviewer comment: For the \"Variable\" data set authors used the GLMM while for the \"Controlled\" data set authors used a Cox proportional hazards mixed-effects model. Given that the \"Variable\" data set does not allow to use a Cox prop hazards mixed-effects model, I agree with this statistical approach, but nonetheless, it might be useful if the authors analyze the \"Controlled\" data also with the GLMM, so that a direct comparison of the two situations would be possible. Author response: This analysis is now given in supplementary Table S2.Reviewer comment: \"Variable\" and \"Controlled\" experiments also differed in the fact that only bees in the \"Controlled\" were kept with constant darkness, which could have an effect on bee locomotor activity, energy consumption etc.. Authors should mention this in the discussion.Author response: We now comment on this difference in a new Discussion paragraph in which we contrast our results with those of Dr. Baracchi and colleagues.Thank you again for the thorough review and helpful suggestions.Sincerely,Evan Palmer-Young on behalf of the authors"
}
]
}
] | 1
|
https://f1000research.com/articles/4-880
|
https://f1000research.com/articles/4-1449/v1
|
15 Dec 15
|
{
"type": "Review",
"title": "Regional or general anesthesia for fast-track hip and knee replacement - what is the evidence?",
"authors": [
"Henrik Kehlet",
"Eske Kvanner Aasvang",
"Eske Kvanner Aasvang"
],
"abstract": "Regional anesthesia for knee and hip arthroplasty may have favorable outcome effects compared with general anesthesia by effectively blocking afferent input, providing initial postoperative analgesia, reducing endocrine metabolic responses, and providing sympathetic blockade with reduced bleeding and less risk of thromboembolic complications but with undesirable effects on lower limb motor and urinary bladder function. Old randomized studies supported the use of regional anesthesia with fewer postoperative pulmonary and thromboembolic complications, and this has been supported by recent large non-randomized epidemiological database cohort studies. In contrast, the data from newer randomized trials are conflicting, and recent studies using modern general anesthetic techniques may potentially support the use of general versus spinal anesthesia. In summary, the lack of properly designed large randomized controlled trials comparing modern general anesthesia and spinal anesthesia for knee and hip arthroplasty prevents final recommendations and calls for prospective detailed studies in this clinically important field.",
"keywords": [
"Regional anesthesia",
"general anesthesia",
"hip replacement",
"knee replacement"
],
"content": "Introduction and context\n\nThe discussion on the optimal anesthetic technique for most surgical procedures regarding the use of regional anesthetic versus general anesthetic techniques has been going on for decades. In hip and knee replacement, several randomized trials performed several decades ago were in favor of spinal or epidural analgesia1,2. This is probably explained by the positive physiological effects of the provided afferent blockade with better initial pain relief, a reduced endocrine metabolic response, and sympathetic blockade with less blood loss and increased leg blood flow, all resulting in reduced cardiopulmonary and thromboembolic morbidity, but at the potential cost of reduced capability for early postoperative mobilization, urinary bladder dysfunction, and rare but potentially severe neurological complications.\n\nIn recent years, several large epidemiological studies based on the large US databases (Premier and National Surgical Quality Improvement Program) have supported the old studies by demonstrating less postoperative morbidity when using regional anesthetic techniques2–7. However, these large-scale studies have little or no information on the type of general anesthesia, perioperative pain management, or details on the provided regional anesthetic technique. Furthermore, information on the care principles regarding the use of the fast-track methodology8 has not been provided, and most importantly comparisons have not been made on a randomized basis, thereby introducing a potential large selection bias. More recent reviews from randomized studies, but again without exact data on care principles and pain management, have questioned the benefits of regional anesthesia versus general anesthesia9 or even a higher risk for cardiovascular complications with neuraxial anesthesia10. In conclusion, the jury is still out for conclusive evidence for the optimal choice of regional versus general anesthetic techniques for knee and hip arthroplasty.\n\nThe goal of this brief review is to update the literature and discuss the potential for a more balanced view regarding the choice of anesthesia for hip and knee arthroplasty within a fast-track setup8, in which length of stay (LOS) before going home is now usually between 1 and 3 days11–13 and in which previous data have not shown firm differences between the two anesthetic techniques or in selected patient groups2–7,9.\n\n\nRecent advances and topics of interest\n\nRecently, two relatively small randomized studies (n = 120 in each) have compared modern target-controlled infusion with propofol and remifentanil versus a conventional spinal anesthesia (without opioids), within a fast-track setup and expected LOS of around 2 days, and with additional multimodal oral opioid-sparing analgesia14,15. These two studies showed no clinically relevant differences in functional recovery outcomes, LOS, or side effects regarding urinary bladder dysfunction and mobilization. However, after the initial few postoperative hours with residual effects of the spinal anesthesia, there were minor but probably not clinically relevant advantages in analgesia and opioid requirements in the general anesthesia group. Though of interest because of the modern general anesthesia technique and fast-track setup, these studies obviously cannot answer the important question about safety issues and potential differences in postoperative morbidity between the two anesthetic techniques but merely serve as a stimulus to perform the required large comparative studies.\n\n\nType of regional anesthesia\n\nEpidural analgesia should not be used routinely in fast-track total hip arthroplasty (THA) or total knee arthroplasty (TKA) because of the limited analgesic effect, especially in comparison with local wound infiltration (local infiltration analgesia, or LIA) in TKA16 combined with the potential for adverse effects such as urinary retention, pruritus, hypotension, and motor blockade17,18, all of which delay recovery.\n\nSpinal anesthesia should be performed using only local anesthetics, as intrathecal opioids increase the risk of urinary retention, pruritus, and respiratory depression19 unless low doses (less than 200 µg) are used, and may not have superior analgesic efficacy compared with LIA in TKA16,20. Recommendations on the optimal dosage of the various types of local anesthetics are beyond the scope of this review. However, one of the challenges in spinal anesthesia for fast-track THA and TKA is optimal titration to provide sufficient analgesia during surgery without a recovery delay due to adverse effects, including impairment to motor function. This requires a strict focus on time spent for preparation and surgery, where a dosage of local anesthetic that is too low may result in the need for supplemental intravenous analgesics (opioids) or conversion to general anesthesia during surgery. However, doses as low as 5 mg bupivacaine have been proven sufficient for 60-minute procedures without the need for conversion to general anesthesia, but in combination with femoral and sciatic nerve blocks21 and their possible negative implications for motor function and recovery.\n\n\nComplications to the anesthesia per se\n\nGeneral anesthesia imposes various degrees of potential risks related mainly to airway management and respiration (dental and oral soft tissue injuries, vocal cord trauma, barotrauma from positive pressure ventilation, aspiration, and so on) and circulation (negative inotropic and chronotropic cardiac effects from anesthetics)4,6,7. Complications to spinal and epidural anesthesia also include hypotension due to the vasodilatory effect of the sympathetic blockade, in addition to the rare but potentially serious risk of compressive neuraxial hematoma. However, this occurs after spinal anesthesia in a maximum of 1 out of 775,000 procedures but in 1 out of 9000 to 1 out of 26,000 epidural procedures, again emphasizing that epidural should not be used22–24. The occurrence of neurological deficits from neuraxial blockade should be held against the overall risk of nerve injury after THA (0.08% to 1.7% in larger series) and TKA (0.3% to 0.9% in larger series)25. Comparison of the risk profiles for adverse events after general and spinal anesthesia needs to take into account that the choice of anesthesia and subsequent complications are affected mainly by patient characteristics. This is a main bias in the current large nationwide database studies reporting significantly higher complication rates after general anesthesia4,6,7. There is general agreement that neuraxial anesthesia may lead to bladder dysfunction in the perioperative period, even in patients undergoing THA and TKA26–29. So far, preoperative selection criteria, including preoperative urinary bladder function, have failed to solve the problem, but potentially postoperative catheterization may be avoided or reduced by using a lower-dose spinal anesthesia30. Furthermore, a higher ultrasound-verified bladder volume before catheterization may reduce catheterization, but the literature is inconclusive27. In the two recent fast-track modern anesthesia randomized series14,15, no differences in need for urinary catheterization with well-defined criteria were found.\n\nIn summary, there is a need for large-scale randomized studies with well-defined criteria for urinary bladder catheterization to demonstrate potential differences between the two anesthetic techniques. Importantly, such studies need to provide multimodal opioid-sparing analgesia since opioids may have a negative effect on urinary bladder function27.\n\n\nImplications within a fast-track setup\n\nImprovements in overall perioperative care regarding anesthesia, analgesia, fluid management, nursing care, and rehabilitation have led to a pronounced reduction of LOS to about 1 to 3 days with return to home11–15 and more recently even the potential to perform THA and TKA on an outpatient basis in selected patients31–33. A common feature of previous randomized studies as well as the large epidemiological studies2–7 is the lack of detailed information about the perioperative management and the two anesthetic techniques, including patient characteristics. Furthermore, the epidemiological studies rely on diagnostic codes, which may not always be exact. Although a balanced view of all available data from within a reasonably recent time frame may support the use of regional anesthesia, there is a severe need for large-scale prospective randomized controlled trials to compare general versus spinal anesthesia, knowing that the choice of anesthesia represents only one of the many factors that influence outcome. In this context, the focus must include potential identification of subgroups of patients who may or may not benefit from a given anesthetic procedure. Such studies must use an evidence-based approach when choosing the two anesthetic techniques, especially within the context of a fast-track setup with provision of an optimized multimodal, oral opioid-sparing analgesia to facilitate early mobilization and reduce adverse events, including the possibility for early mobilization and urinary bladder dysfunction34. Thus, most previous studies have not included gabapentinoids, which may be appropriate in hip replacement35 but not in knee replacement36,37, and preoperative high-dose glucocorticoid may provide major analgesic effects with reduced opioid use and side effects38,39. Furthermore, the use of high-volume LIA is evidence based in TKA but not in THA16. Also, future studies should include early (within a few hours) mobilization, which may be important to reduce thromboembolic complications40 that may be independent of anesthetic technique.\n\nIn summary, the recent development of optimized general and regional anesthetic techniques together with advances in multimodal opioid-sparing analgesia combined with the fast-track methodology may provide an opportunity in a large randomized study to answer the old question of whether regional anesthesia is better than general anesthesia41.",
"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\nRodgers A, Walker N, Schug S, et al.: Reduction of postoperative mortality and morbidity with epidural or spinal anaesthesia: results from overview of randomised trials. BMJ. 2000; 321(7275): 1493. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMemtsoudis SG, Sun X, Chiu YL, et al.: Perioperative comparative effectiveness of anesthetic technique in orthopedic patients. Anesthesiology. 2013; 118(5): 1046–58. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPugely AJ, Martin CT, Gao Y, et al.: Differences in short-term complications between spinal and general anesthesia for primary total knee arthroplasty. J Bone Joint Surg Am. 2013; 95(3): 193–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMemtsoudis SG, Rasul R, Suzuki S, et al.: Does the impact of the type of anesthesia on outcomes differ by patient age and comorbidity burden? Reg Anesth Pain Med. 2014; 39(2): 112–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHelwani MA, Avidan MS, Ben Abdallah A, et al.: Effects of regional versus general anesthesia on outcomes after total hip arthroplasty: a retrospective propensity-matched cohort study. J Bone Joint Surg Am. 2015; 97(3): 186–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nOpperer M, Danninger T, Stundner O, et al.: Perioperative outcomes and type of anesthesia in hip surgical patients: An evidence based review. World J Orthop. 2014; 5(3): 336–43. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBasques BA, Toy JO, Bohl DD, et al.: General compared with spinal anesthesia for total hip arthroplasty. J Bone Joint Surg Am. 2015; 97(6): 455–61. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKehlet H: Fast-track hip and knee arthroplasty. Lancet. 2013; 381(9878): 1600–2. PubMed Abstract | Publisher Full Text\n\nMacfarlane AJ, Prasad GA, Chan VW, et al.: Does regional anaesthesia improve outcome after total hip arthroplasty? A systematic review. Br J Anaesth. 2009; 103(3): 335–45. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLeslie K, Myles P, Devereaux P, et al.: Neuraxial block, death and serious cardiovascular morbidity in the POISE trial. Br J Anaesth. 2013; 111(3): 382–90. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKhan SK, Malviya A, Muller SD, et al.: Reduced short-term complications and mortality following Enhanced Recovery primary hip and knee arthroplasty: results from 6,000 consecutive procedures. Acta Orthop. 2014; 85(1): 26–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nden Hartog YM, Mathijssen NM, Vehmeijer SB: Reduced length of hospital stay after the introduction of a rapid recovery protocol for primary THA procedures. Acta Orthop. 2013; 84(5): 444–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJorgensen CC, Knop J, Nordentoft M, et al.: Psychiatric disorders and psychopharmacologic treatment as risk factors in elective fast-track total hip and knee arthroplasty. Anesthesiology. 2015; 123(6): 1281–91. Publisher Full Text\n\nHarsten A, Kehlet H, Toksvig-Larsen S: Recovery after total intravenous general anaesthesia or spinal anaesthesia for total knee arthroplasty: a randomized trial. Br J Anaesth. 2013; 111(3): 391–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHarsten A, Kehlet H, Ljung P, et al.: Total intravenous general anaesthesia vs. spinal anaesthesia for total hip arthroplasty: a randomised, controlled trial. Acta Anaesthesiol Scand. 2015; 59(3): 298–309. PubMed Abstract | Publisher Full Text\n\nAndersen LØ, Kehlet H: Analgesic efficacy of local infiltration analgesia in hip and knee arthroplasty: a systematic review. Br J Anaesth. 2014; 113(3): 360–74. PubMed Abstract | Publisher Full Text\n\nChoi PT, Bhandari M, Scott J, et al.: Epidural analgesia for pain relief following hip or knee replacement. Cochrane Database Syst Rev. 2003; (3): CD003071. PubMed Abstract | Publisher Full Text\n\nRawal N: Epidural technique for postoperative pain: gold standard no more? Reg Anesth Pain Med. 2012; 37(3): 310–7. PubMed Abstract | Publisher Full Text\n\nFernandez MA, Karthikeyan S, Wyse M, et al.: The incidence of postoperative urinary retention in patients undergoing elective hip and knee arthroplasty. Ann R Coll Surg Engl. 2014; 96(6): 462–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEssving P, Axelsson K, Åberg E, et al.: Local infiltration analgesia versus intrathecal morphine for postoperative pain management after total knee arthroplasty: a randomized controlled trial. Anesth Analg. 2011; 113(4): 926–33. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAwad IT, Cheung JJ, Al-Allaq Y, et al.: Low-dose spinal bupivacaine for total knee arthroplasty facilitates recovery room discharge: a randomized controlled trial. Can J Anaesth. 2013; 60(3): 259–65. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPitkänen MT, Aromaa U, Cozanitis DA, et al.: Serious complications associated with spinal and epidural anaesthesia in Finland from 2000 to 2009. Acta Anaesthesiol Scand. 2013; 57(5): 553–64. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPumberger M, Memtsoudis SG, Stundner O, et al.: An analysis of the safety of epidural and spinal neuraxial anesthesia in more than 100,000 consecutive major lower extremity joint replacements. Reg Anesth Pain Med. 2013; 38(6): 515–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nRosencher N, Llau JV, Mueck W, et al.: Incidence of neuraxial haematoma after total hip or knee surgery: RECORD programme (rivaroxaban vs. enoxaparin). Acta Anaesthesiol Scand. 2013; 57(5): 565–72. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDwyer T, Drexler M, Chan VW, et al.: Neurological Complications Related to Elective Orthopedic Surgery: Part 2: Common Hip and Knee Procedures. Reg Anesth Pain Med. 2015; 40(5): 443–54. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChoi S, Mahon P, Awad IT: Neuraxial anesthesia and bladder dysfunction in the perioperative period: a systematic review. Can J Anaesth. 2012; 59(7): 681–703. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBalderi T, Carli F: Urinary retention after total hip and knee arthroplasty. Minerva Anestesiol. 2010; 76(2): 120–30. PubMed Abstract | F1000 Recommendation\n\nBjerregaard LS, Bagi P, Kehlet H: Postoperative urinary retention (POUR) in fast-track total hip and knee arthroplasty. Acta Orthop. 2014; 85(1): 8–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHollman F, Wolterbeek N, Veen R: Risk Factors for Postoperative Urinary Retention in Men Undergoing Total Hip Arthroplasty. Orthopedics. 2015; 38(6): e507–11. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKarason S, Olafsson TA: Avoiding bladder catheterisation in total knee arthroplasty: patient selection criteria and low-dose spinal anaesthesia. Acta Anaesthesiol Scand. 2013; 57(5): 639–45. PubMed Abstract | Publisher Full Text\n\nHartog YM, Mathijssen NM, Vehmeijer SB: Total hip arthroplasty in an outpatient setting in 27 selected patients. Acta Orthop. 2015; 86(6): 667–70. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKort NP, Bemelmans YF, Schotanus MG: Outpatient surgery for unicompartmental knee arthroplasty is effective and safe. Knee Surg Sports Traumatol Arthrosc. 2015. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nThienpont E, Lavand'homme P, Kehlet H: The constraints on day-case total knee arthroplasty: the fastest fast track. Bone Joint J. 2015; 97-B(10 Suppl A): 40–4. PubMed Abstract | Publisher Full Text\n\nAasvang EK, Luna IE, Kehlet H: Challenges in postdischarge function and recovery: the case of fast-track hip and knee arthroplasty. Br J Anaesth. 2015; 115(6): 861–6. PubMed Abstract | Publisher Full Text\n\nHøjer Karlsen AP, Geisler A, Petersen PL, et al.: Postoperative pain treatment after total hip arthroplasty: a systematic review. Pain. 2015; 156(1): 8–30. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nYaDeau JT, Lin Y, Mayman DJ, et al.: Pregabalin and pain after total knee arthroplasty: a double-blind, randomized, placebo-controlled, multidose trial. Br J Anaesth. 2015; 115(2): 285–93. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLunn TH, Husted H, Laursen MB, et al.: Analgesic and sedative effects of perioperative gabapentin in total knee arthroplasty: a randomized, double-blind, placebo-controlled dose-finding study. Pain. 2015; 156(12): 2438–48. PubMed Abstract | Publisher Full Text\n\nLunn TH, Kristensen BB, Andersen LØ, et al.: Effect of high-dose preoperative methylprednisolone on pain and recovery after total knee arthroplasty: a randomized, placebo-controlled trial. Br J Anaesth. 2011; 106(2): 230–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLunn TH, Andersen LØ, Kristensen BB, et al.: Effect of high-dose preoperative methylprednisolone on recovery after total hip arthroplasty: a randomized, double-blind, placebo-controlled trial. Br J Anaesth. 2013; 110(1): 66–73. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJørgensen CC, Jacobsen MK, Soeballe K, et al.: Thromboprophylaxis only during hospitalisation in fast-track hip and knee arthroplasty, a prospective cohort study. BMJ Open. 2013; 3(12): e003965. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcCartney CJ, Choi S: Does anaesthetic technique really matter for total knee arthroplasty? Br J Anaesth. 2013; 111(3): 331–3. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11560",
"date": "15 Dec 2015",
"name": "Stavros Memtsoudis",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11561",
"date": "15 Dec 2015",
"name": "Jan Jakobsson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1449
|
https://f1000research.com/articles/4-1448/v1
|
15 Dec 15
|
{
"type": "Review",
"title": "Molecular chaperones: guardians of the proteome in normal and disease states",
"authors": [
"Wilson Jeng",
"Sukyeong Lee",
"Nuri Sung",
"Jungsoon Lee",
"Francis T.F. Tsai",
"Wilson Jeng",
"Sukyeong Lee",
"Nuri Sung",
"Jungsoon Lee"
],
"abstract": "Proteins must adopt a defined three-dimensional structure in order to gain functional activity, or must they? An ever-increasing number of intrinsically disordered proteins and amyloid-forming polypeptides challenge this dogma. While molecular chaperones and proteases are traditionally associated with protein quality control inside the cell, it is now apparent that molecular chaperones not only promote protein folding in the “forward” direction by facilitating folding and preventing misfolding and aggregation, but also facilitate protein unfolding and even disaggregation resulting in the recovery of functional protein from aggregates. Here, we review our current understanding of ATP-dependent molecular chaperones that harness the energy of ATP binding and hydrolysis to fuel their chaperone functions. An emerging theme is that most of these chaperones do not work alone, but instead function together with other chaperone systems to maintain the proteome. Hence, molecular chaperones are the major component of the proteostasis network that guards and protects the proteome from damage. Furthermore, while a decline of this network is detrimental to cell and organismal health, a controlled perturbation of the proteostasis network may offer new therapeutic avenues against human diseases.",
"keywords": [
"molecular chaperones",
"chaperones",
"proteases",
"protein folding",
"misfolding",
"aggregation",
"ATP-dependent molecular chaperones"
],
"content": "Introduction\n\nThe vast majority of proteins must fold correctly in order to gain functional activity. While the protein folding information is encoded within the nascent polypeptide chain, newly synthesized polypeptides (or those imported into organelles) are prone to misfolding, causing aggregation and formation of other toxic species1. Consequently, maintaining protein homeostasis (proteostasis) is essential for cell and organismal health2. To accomplish this, cells have evolved a sophisticated network of protein quality control machines, consisting of molecular chaperones and proteases, which monitor the folding of proteins and their assembly into functional complexes, and selectively remove excess and damaged proteins from the cell. Challenging the capacity of this proteostasis network increases the risk of human diseases associated with protein misfolding and aggregation1.\n\nWhile most proteins adopt a defined three-dimensional structure, several exceptions are known to exist. Notable examples include prions that can adopt multiple, distinct, three-dimensional structures3–5, and an ever-increasing number of intrinsically disordered proteins (IDPs), which feature large regions of random coil or lack a defined structure altogether6–8. At least in yeast, it is now widely accepted that molecular chaperones play an essential role in prion replication9,10 by governing the inheritance and maintenance of yeast prions, and in some cases their elimination by chaperone overexpression11–15. However, concrete evidence of an involvement of molecular chaperones in mammalian prion replication, although proposed16, is missing, and whether molecular chaperones play a role in the stabilization and/or protection of IDPs remains uncertain.\n\nWhat is a molecular chaperone? A molecular chaperone can be generally defined as any protein that assists other macromolecules in folding and/or assembling into higher order structures, without it being a component of these final structures17. Thus, while their main function inside the cell is to assist in the folding and maturation of unfolded or partially folded macromolecules and to prevent their misfolding and aggregation, it was widely assumed that molecular chaperones involved in de novo protein folding do not recover functional protein once aggregation has occurred. This concept was challenged by the discovery of a novel stress-inducible molecular chaperone known as Hsp10418, which functions as an ATP-dependent protein disaggregase that rescues stress-damaged proteins from a previously aggregated state19,20. The discovery of Hsp104 has since expanded our definition of molecular chaperones to include those that promote the forward folding or prevent the aggregation of proteins on one hand, and those that recover functional protein from aggregates on the other hand.\n\nAt the molecular level, molecular chaperones come in diverse shapes and sizes, and can be broadly separated into two groups: those that depend on metabolic energy to fuel their chaperone activity, and those that do not21. Examples of the former include all ATP-dependent molecular chaperones22, while the latter include small heat shock proteins23, protein disulfide isomerase24, ribosome-associated chaperones such as trigger factor25, and conditionally activated chaperones26.\n\nThe focus of this review is on ATP-dependent molecular chaperones that harness the energy from ATP binding and/or hydrolysis to assist protein folding and unfolding (i.e., disaggregation). Their cellular expression can be either constitutive in order to perform vital housekeeping functions, or inducible by short exposure to elevated temperatures or other forms of stress that cause protein denaturation. Those that are stress-inducible are also known as heat-shock proteins or HSPs, while those that are constitutively active are termed heat-shock cognates or HSCs. Different members of both groups are classified according to their molecular weight, for example, HSP of 60-kDa (Hsp60), 70-kDa (Hsp70), 90-kDa (Hsp90), and 100-kDa (Hsp100), although many are better known by their common name that is used to designate each chaperone homolog from eubacteria, for example, GroEL (Hsp60), DnaK (Hsp70), HtpG (Hsp90), and ClpB (Hsp100) (Figure 1). All of these aforementioned HSPs bind adenine nucleotide and hydrolyze ATP. Furthermore, another common feature is their cooperation with other proteins, termed co-chaperones, which regulate the ATPase and/or chaperone activity, or reset the functional cycle.\n\nProtein is shown as ribbon diagram with the bound nucleotide as red CPK model. For each chaperone, the domains of one subunit are shown in different colors in order of green, orange, and blue from N- to C-termini. Bound co-chaperones are colored cyan. (a) Hsp60/GroEL: Architecture and domain organization of the E. coli GroEL tetradecamer bound to ADP with a GroES heptamer capping the GroEL cis ring (PDB: 1AON)33. (b) Hsp70/DnaK: Architecture and domain organization of the E. coli DnaK monomer in the ATP-bound state (PDB: 4JNE)54. (c) Hsp90/HtpG: Architecture and domain organization of the ATP-bound yeast Hsp90 dimer in the closed-state conformation, and its stabilization by p23/Sba1 (PDB: 2CG9)81. (d) Hsp104/ClpB: Architecture and domain organization of a yeast Hsp104 hexamer bound to ATP (PDB: 1QVR; EMD-1631)97,99. The Hsp104 M-domain that mediates the species-specific interaction with Hsp70 is colored in magenta.\n\n\nThe Hsp60 family\n\nHsp60 chaperones are known as chaperonins27, and can be divided into two subgroups. Group I chaperonins are sevenfold symmetric and assemble into a barrel-like structure composed of two rings of seven identical subunits28. Notable examples include bacterial GroEL and Hsp60 from mitochondria and chloroplasts. Each GroEL subunit consists of an equatorial, intermediate, and apical domain (Figure 1a)29–31. ATP binding triggers a conformational rearrangement of the apical domains followed by GroES binding. The latter is a GroEL co-chaperone that assembles into a heptamer ring32, and caps one side of the GroEL barrel (the cis ring)33 to encapsulate the substrate34, and to promote protein folding35. The prevailing model suggests that GroEL-ES promotes folding through repetitive binding, encapsulation, and release of the substrate protein28,36. Group II chaperonins are homo- or hetero-oligomers forming an eightfold double barrel structure composed of sixteen subunits, and include the eukaryotic chaperonin containing TCP1 complex (CCT), also known as the TCP-1 Ring Complex (TRiC), and the thermosome and Methanococcus maripaludis chaperonin (Mm-Cpn) from Archaea37. Unlike Group I chaperonins, group II members do not function together with a GroES-like co-chaperone, but instead contain a built-in lid that undergoes an iris-like motion to promote protein folding38.\n\nMuch of our current understanding of chaperonin function comes from seminal work on Escherichia coli GroEL. E. coli GroEL is essential since many vital proteins, including metabolic enzymes and components of the transcription-translation machinery, depend on the GroE system for folding39. While GroEL’s essential housekeeping function is beginning to be understood, an emerging question is the recent appreciation of multiple copies of GroEL in some bacterial genomes40, as seen in actinobacteria, which includes Mycobacterium tuberculosis, the causative agent of Tuberculosis (TB). TB accounts for ~2 million deaths annually and is a major public health problem exacerbated by the emergence and rapid spread of new multidrug-resistant M. tuberculosis strains. M. tuberculosis encodes two copies of groEL in its genome40. While M. tuberculosis GroEL2 is essential for viability, the function of the non-essential GroEL1 paralog remains less clear. The crystal structures of M. tuberculosis GroEL2 and a GroEL1 fragment showed that the apical domains have nearly identical three-dimensional structures41,42. While GroEL2 is believed to be the housekeeping chaperonin similar to E. coli GroEL, GroEL1 may function as a specialized chaperonin with a more limited substrate spectrum. Consistent with this notion, it has been proposed that mycobacterial GroEL1 is a dedicated chaperone for biofilm formation43, which is presumed to confer the extraordinary starvation survival and resistance of M. tuberculosis to known antibiotics44.\n\n\nThe Hsp70 family\n\nMembers of the Hsp70 chaperone family are found in all three surviving domains of life45. At the molecular level, Hsp70 is a two-domain protein consisting of a nucleotide-binding domain connected by a long and flexible hydrophobic linker to the substrate-binding domain that can be subdivided into a β-sandwich domain and an α-helical domain (Figure 1b). Furthermore, cytosolic eukaryotic Hsp70s feature a Glu-Glu-Val-Asp or “EEVD” motif at the extreme C-terminus, which is required for the interaction with Hsp70 co-chaperones that regulate the Hsp70 ATPase activity and its ability to bind substrate46. It has been shown that bacterial Hsp70 recognizes diverse polypeptides mostly in an unfolded or partially unfolded form by binding to a four to five residue stretch of hydrophobic amino acids flanked by regions enriched in basic amino acids47, which occur on average every 30–40 residues in most proteins. Since Hsp70 binding motifs are typically buried within the correctly folded protein, it provides a means to selectively seek out and bind proteins that are in a non-native conformation. However, Hsp70 chaperones rarely, if ever, function on their own and require the assistance of co-chaperones, which include nucleotide exchange factors, such as bacterial GrpE and eukaryotic Hsp110, and the large family of J-domain-containing Hsp40 co-chaperones, which accelerate ATP hydrolysis, serve as substrate targeting factors, and stabilize Hsp70-substrate interaction21.\n\nOver the last decade, high-resolution structural information on full-length Hsp70 chaperones has become available48–54, providing new insight into the Hsp70 conformational cycle and its allosteric regulation by nucleotide55. Hsp70 function is controlled by nucleotide binding with ATP, promoting an open-conformation with low substrate-binding affinity, and ADP, promoting a closed-conformation required for tight binding of substrates21.\n\nIn addition to Hsp70’s known role in protein folding, Hsp70 also has other non-chaperone functions. For instance, it was recently shown that Hsp70 functions as an activator of the ring-forming Hsp104 protein disaggregase and is required to unleash the potent protein disaggregating activity56,57. While no Hsp104 homolog is known to exist in metazoans, the discovery of a mammalian protein disaggregase, composed of Hsp70, Hsp110, and Hsp40, is exciting and supports functional conservation of a protein disaggregating activity in animal cells58–61. However, despite its nomenclature, Hsp110 is not an Hsp100 homolog, but instead belongs to an Hsp70 subfamily that is activated by nucleotide62, shares structural49,63,64 and perhaps functional conservation with Hsp7065, and functions as an Hsp70 nucleotide exchange factor66,67.\n\n\nThe Hsp90 family\n\nHsp90 belongs to a conserved group of ATP-dependent molecular chaperones68–70 which, together with Hsp70 and a cohort of co-chaperones, facilitates the late-stage folding and maturation of proteins71,72. Since Hsp90 substrates are mostly substantially folded proteins, they are known as “client proteins”68 to distinguish them from other chaperone substrates that lack a defined structure. More than 400 different clients are known to depend on Hsp90 for folding or maturation, and include protein kinases, transcription factors, and E3 ubiquitin ligases73. The large number of signaling and tumor promoting proteins amongst Hsp90 clients has made Hsp90 a promising drug target74.\n\nApart from Hsp90 chaperones in the eukaryotic cytosol, Hsp90 homologs are found in bacteria (HtpG) and eukaryotic organelles, including the endoplasmic reticulum (Grp94), mitochondrion (TRAP1), and chloroplast75. Interestingly, Hsp90-like domains with chaperone activity have also been found in Sacsin, a 521-kDa protein associated with an autosomal recessive form of spastic ataxia76,77. However, an Hsp90 homolog has not been found in Archaea.\n\nHsp90 chaperones share a similar domain structure consisting of an N-terminal (N-) nucleotide-binding domain, a middle (M-) domain, and a C-terminal (C-) dimerization domain (Figure 1c). The N-domain is connected to the M-domain via a flexible linker that is often highly charged and, in human Hsp90, is over 60 residues in length. While important to cytosolic eukaryotic Hsp90 function78–80, the charged-linker is not universally conserved and is essentially absent in both bacterial and mitochondrial Hsp90s. Crystal structures are now available for full-length members of all Hsp90 subfamilies mostly with bound nucleotide81–84, including the recent structure of an asymmetric TRAP1 dimer in the ATP-bound state84. The latter lends supports for a sequential ATP hydrolysis mechanism85,86, although asymmetric binding of nucleotide was not observed84. Consistent with the prevailing notion, the available structures confirmed that all Hsp90 chaperones form homodimers with the N-domain mediating nucleotide binding. Strikingly, however, apo Hsp90 forms a wide-open, V-shaped dimer with the N-domains separated by over 100 Å82, while Hsp90 in the ATP-bound state adopts an intertwined, N-terminally closed dimer81,84. Since the N-domains are too far apart in the open-state to signal the nucleotide status between neighboring subunits, how ATP-binding induces the closed-state conformation remains an open question. One model suggests that Hsp90 chaperones sample different three-dimensional conformations with different adenine nucleotides stabilizing distinct Hsp90 dimer conformations87–89. While not mutually exclusive, the crystal structures of intact Grp94, which were determined in the ATP- and ADP-bound state, revealed a very similar Hsp90 dimer conformation irrespective of the nature of the bound nucleotide83. Hence, further in vitro and in vivo studies are needed to address the exact roles of ATP and ADP for Hsp90 chaperone function.\n\n\nThe Hsp100 family\n\nMembers of the Hsp100 family were first discovered as the protein-activated ATPase components of the protease Ti from E. coli90,91, now better known as the ClpAP protease. Members of the Hsp100/Clp family belong to the large superfamily of ATPases Associated with diverse cellular Activities (AAA+)92,93. Hsp100/Clp members form a hexameric ring structure and function as the protein-unfolding component of chambered proteases94,95. The discovery of yeast Hsp104 that facilitates protein disaggregation19, as opposed to targeting proteins for degradation, established Hsp104 as the founding member of a new family of ATP-dependent molecular chaperones. In addition to yeast Hsp104, Hsp104 homologs were found subsequently in bacteria (ClpB), plants (Hsp101), and most recently in Dictyostelium discoideum (Hsp101)96.\n\nLike all Hsp100/Clp proteins, Hsp104 forms an oligomer, with the homohexamer being the functionally active form97–100. Hsp104 features two canonical Walker-type ATP-binding domains, known as AAA domains, in addition to several other structural elements that define members of the AAA+ superfamily and include the so-called arginine-finger and the sensor 1 and 2 motifs101–106. While the Hsp104 hexamer is stabilized by nucleotide and is an active ATPase in vitro107,108, it requires the cooperation of the cognate Hsp70 chaperone system, consisting of Hsp70 and Hsp40 in yeast20 and DnaK, DnaJ, and GrpE in eubacteria109–111, to recover functional protein from aggregates.\n\nAt the molecular level, Hsp104 consists of an N-terminal domain, and two tandem AAA+ domains, termed AAA-1 and AAA-2 (Figure 1d)97,103. The AAA-1 domain features an 85-Å long coiled-coil insertion, known as the M-domain, which is located on the outside of the hexamer99,100,112 and distinguishes Hsp104 members from other Hsp100/Clp ATPases. The M-domain is essential for protein disaggregation by mediating the interaction between Hsp104 and Hsp70113–115, and may function as a molecular toggle to allosterically control the ATPase and mechanical activities of the Hsp104 motor116.\n\nHow Hsp104 facilitates protein disaggregation has been revealed by the combined efforts of several groups117. It is now widely accepted that, inside the cell, the Hsp70 system targets the Hsp104 motor to both amorphous and ordered aggregates15,118, from which Hsp104 extracts polypeptides using an ATP-driven power stroke involving pore loops present in the AAA-1 and AAA-2 domains119, and threading the polypeptide through the central channel of the Hsp104 hexamer120,121.\n\nWhile we are beginning to understand the function of the M- and AAA domains, the role of the N-domain is less clear. It was shown that the N-domain is dispensable for Hsp104 function in vitro and in vivo15,103,122–125. However, others found that the N-domain is essential for bacterial Hsp104126,127 and mediates substrate interaction126,128–131. Consistently, the N-domain of yeast Hsp104 enhances protein disaggregation in vitro114, mediates prion interaction in yeast132, and is essential for yeast prion dissolution112 and curing by Hsp104 overexpression124.\n\nIn addition to Hsp104’s role in yeast stress responses and yeast prion replication, new roles are emerging, including the asymmetric distribution of oxidative damaged proteins133,134, facilitating the sorting of tail-anchored proteins to the endoplasmic reticulum membrane135, and septin folding and assembly136. Hence, future studies will provide a more complete picture as to the extent of Hsp104’s cellular function.\n\n\nFuture perspectives\n\nIt is now widely appreciated that molecular chaperones are intimately linked to proteostasis maintenance and are essential to cell and organismal health. Perturbation of the proteostasis network, for instance by “chaperone overload”137 or polyglutamine expansion138, invariably disrupts the balance of the protein folding landscape triggering protein misfolding and the formation of aggregates that are hallmarks of neurodegenerative diseases, prion-mediated infections, and amyloidosis. At the same time, a controlled perturbation of the functional interaction between molecular chaperones and proteases could provide new avenues for therapeutic intervention. This could be achieved by using small molecule compounds, or by RNA interference, or restoring the proteostasis network in disease states, for instance with chemical chaperones or by induced chaperone expression139,140.",
"appendix": "Author contributions\n\n\n\nWilson Jeng, Sukyeong Lee, Nuri Sung, Jungsoon Lee, and Francis T.F. Tsai conceived the study. Wilson Jeng, Sukyeong Lee, and Francis T.F. Tsai 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\nThe authors declare there were no competing interests.\n\n\nGrant information\n\nResearch in the Francis T.F. Tsai and Sukyeong Lee laboratory is supported by grants from the National Institutes of Health (R01-GM111084 and R01-GM104980) and the Robert A. Welch Foundation (Q-1530).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe sincerely apologize to all those colleagues whose important work was not cited in this review. We also wish to thank all present and past members of the Francis T.F. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJahn M, Rehn A, Pelz B, et al.: The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function. Proc Natl Acad Sci U S A. 2014; 111(50): 17881–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAli MM, Roe SM, Vaughan CK, et al.: Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature. 2006; 440(7087): 1013–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nShiau AK, Harris SF, Southworth DR, et al.: Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements. Cell. 2006; 127(2): 329–40. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDollins DE, Warren JJ, Immormino RM, et al.: Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones. Mol Cell. 2007; 28(1): 41–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLavery LA, Partridge JR, Ramelot TA, et al.: Structural asymmetry in the closed state of mitochondrial Hsp90 (TRAP1) supports a two-step ATP hydrolysis mechanism. Mol Cell. 2014; 53(2): 330–43. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRichter K, Muschler P, Hainzl O, et al.: Coordinated ATP hydrolysis by the Hsp90 dimer. J Biol Chem. 2001; 276(36): 33689–96. PubMed Abstract | Publisher Full Text\n\nMishra P, Bolon DN: Designed Hsp90 heterodimers reveal an asymmetric ATPase-driven mechanism in vivo. Mol Cell. 2014; 53(2): 344–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSouthworth DR, Agard DA: Species-dependent ensembles of conserved conformational states define the Hsp90 chaperone ATPase cycle. Mol Cell. 2008; 32(5): 631–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKrukenberg KA, Förster F, Rice LM, et al.: Multiple conformations of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90. Structure. 2008; 16(5): 755–65. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPartridge JR, Lavery LA, Elnatan D, et al.: A novel N-terminal extension in mitochondrial TRAP1 serves as a thermal regulator of chaperone activity. eLife. 2014; 3: e03487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang BJ, Woo KM, Goldberg AL, et al.: Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits. J Biol Chem. 1988; 263(18): 8727–34. PubMed Abstract\n\nKatayama Y, Gottesman S, Pumphrey J, et al.: The two-component, ATP-dependent Clp protease of Escherichia coli. Purification, cloning, and mutational analysis of the ATP-binding component. J Biol Chem. 1988; 263(29): 15226–36. PubMed Abstract\n\nBeyer A: Sequence analysis of the AAA protein family. Protein Sci. 1997; 6(10): 2043–58. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeuwald AF, Aravind L, Spouge JL, et al.: AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 1999; 9(1): 27–43. PubMed Abstract\n\nSauer RT, Baker TA: AAA+ proteases: ATP-fueled machines of protein destruction. Annu Rev Biochem. 2011; 80: 587–612. PubMed Abstract | Publisher Full Text\n\nAlexopoulos JA, Guarné A, Ortega J: ClpP: a structurally dynamic protease regulated by AAA+ proteins. J Struct Biol. 2012; 179(2): 202–10. PubMed Abstract | Publisher Full Text\n\nMalinovska L, Palm S, Gibson K, et al.: Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation. Proc Natl Acad Sci U S A. 2015; 112(20): E2620–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLee S, Sowa ME, Watanabe YH, et al.: The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell. 2003; 115(2): 229–40. PubMed Abstract | Publisher Full Text\n\nLee S, Choi J, Tsai FT: Visualizing the ATPase cycle in a protein disaggregating machine: structural basis for substrate binding by ClpB. Mol Cell. 2007; 25(2): 261–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee S, Sielaff B, Lee J, et al.: CryoEM structure of Hsp104 and its mechanistic implication for protein disaggregation. Proc Natl Acad Sci U S A. 2010; 107(18): 8135–40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarroni M, Kummer E, Oguchi Y, et al.: Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation. eLife. 2014; 3: e02481. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHattendorf DA, Lindquist SL: Cooperative kinetics of both Hsp104 ATPase domains and interdomain communication revealed by AAA sensor-1 mutants. EMBO J. 2002; 21(1–2): 12–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHattendorf DA, Lindquist SL: Analysis of the AAA sensor-2 motif in the C-terminal ATPase domain of Hsp104 with a site-specific fluorescent probe of nucleotide binding. Proc Natl Acad Sci U S A. 2002; 99(5): 2732–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMogk A, Schlieker C, Strub C, et al.: Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization, ATP hydrolysis, and chaperone activity. J Biol Chem. 2003; 278(20): 17615–24. PubMed Abstract | Publisher Full Text\n\nYamasaki T, Nakazaki Y, Yoshida M, et al.: Roles of conserved arginines in ATP-binding domains of AAA+ chaperone ClpB from Thermus thermophilus. FEBS J. 2011; 278(13): 2395–403. PubMed Abstract | Publisher Full Text\n\nBiter AB, Lee J, Sung N, et al.: Functional analysis of conserved cis- and trans-elements in the Hsp104 protein disaggregating machine. J Struct Biol. 2012; 179(2): 172–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZeymer C, Fischer S, Reinstein J: trans-Acting arginine residues in the AAA+ chaperone ClpB allosterically regulate the activity through inter- and intradomain communication. J Biol Chem. 2014; 289(47): 32965–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParsell DA, Kowal AS, Lindquist S: Saccharomyces cerevisiae Hsp104 protein. Purification and characterization of ATP-induced structural changes. J Biol Chem. 1994; 269(6): 4480–7. PubMed Abstract\n\nWatanabe YH, Motohashi K, Yoshida M: Roles of the two ATP binding sites of ClpB from Thermus thermophilus. J Biol Chem. 2002; 277(8): 5804–9. PubMed Abstract | Publisher Full Text\n\nMotohashi K, Watanabe Y, Yohda M, et al.: Heat-inactivated proteins are rescued by the DnaK.J-GrpE set and ClpB chaperones. Proc Natl Acad Sci U S A. 1999; 96(13): 7184–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoloubinoff P, Mogk A, Zvi AP, et al.: Sequential mechanism of solubilization and refolding of stable protein aggregates by a bichaperone network. Proc Natl Acad Sci U S A. 1999; 96(24): 13732–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZolkiewski M: ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli. J Biol Chem. 1999; 274(40): 28083–6. PubMed Abstract | Publisher Full Text\n\nSweeny EA, Jackrel ME, Go MS, et al.: The Hsp104 N-terminal domain enables disaggregase plasticity and potentiation. Mol Cell. 2015; 57(5): 836–49. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLum R, Tkach JM, Vierling E, et al.: Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104. J Biol Chem. 2004; 279(28): 29139–46. PubMed Abstract | Publisher Full Text\n\nClarke AK, Eriksson MJ: The truncated form of the bacterial heat shock protein ClpB/HSP100 contributes to development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol. 2000; 182(24): 7092–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeinker P, Schlee S, Groemping Y, et al.: The N terminus of ClpB from Thermus thermophilus is not essential for the chaperone activity. J Biol Chem. 2002; 277(49): 47160–6. PubMed Abstract | Publisher Full Text\n\nHung GC, Masison DC: N-terminal domain of yeast Hsp104 chaperone is dispensable for thermotolerance and prion propagation but necessary for curing prions by Hsp104 overexpression. Genetics. 2006; 173(2): 611–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLum R, Niggemann M, Glover JR: Peptide and protein binding in the axial channel of Hsp104. Insights into the mechanism of protein unfolding. J Biol Chem. 2008; 283(44): 30139–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarnett ME, Zolkiewska A, Zolkiewski M: Structure and activity of ClpB from Escherichia coli. Role of the amino-and -carboxyl-terminal domains. J Biol Chem. 2000; 275(48): 37565–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarnett ME, Nagy M, Kedzierska S, et al.: The amino-terminal domain of ClpB supports binding to strongly aggregated proteins. J Biol Chem. 2005; 280(41): 34940–5. PubMed Abstract | Publisher Full Text\n\nPark SK, Kim KI, Woo KM, et al.: Site-directed mutagenesis of the dual translational initiation sites of the clpB gene of Escherichia coli and characterization of its gene products. J Biol Chem. 1993; 268(27): 20170–4. PubMed Abstract\n\nLi J, Sha B: Crystal structure of the E. coli Hsp100 ClpB N-terminal domain. Structure. 2003; 11(3): 323–8. PubMed Abstract | Publisher Full Text\n\nTanaka N, Tani Y, Hattori H, et al.: Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity. Protein Sci. 2004; 13(12): 3214–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChow IT, Barnett ME, Zolkiewski M, et al.: The N-terminal domain of Escherichia coli ClpB enhances chaperone function. FEBS Lett. 2005; 579(20): 4242–8. PubMed Abstract | Publisher Full Text\n\nWinkler J, Tyedmers J, Bukau B, et al.: Hsp70 targets Hsp100 chaperones to substrates for protein disaggregation and prion fragmentation. J Cell Biol. 2012; 198(3): 387–404. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErjavec N, Larsson L, Grantham J, et al.: Accelerated aging and failure to segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p. Genes Dev. 2007; 21(19): 2410–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTessarz P, Schwarz M, Mogk A, et al.: The yeast AAA+ chaperone Hsp104 is part of a network that links the actin cytoskeleton with the inheritance of damaged proteins. Mol Cell Biol. 2009; 29(13): 3738–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang F, Brown EC, Mak G, et al.: A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum. Mol Cell. 2010; 40(1): 159–71. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nJohnson CR, Weems AD, Brewer JM, et al.: Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast. Mol Biol Cell. 2015; 26(7): 1323–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCsermely P: Chaperone overload is a possible contributor to 'civilization diseases'. Trends Genet. 2001; 17(12): 701–4. PubMed Abstract | Publisher Full Text\n\nGidalevitz T, Ben-Zvi A, Ho KH, et al.: Progressive disruption of cellular protein folding in models of polyglutamine diseases. Science. 2006; 311(5766): 1471–4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBalch WE, Morimoto RI, Dillin A, et al.: Adapting proteostasis for disease intervention. Science. 2008; 319(5865): 916–9. 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}
|
[
{
"id": "11558",
"date": "15 Dec 2015",
"name": "Jose Barral",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11559",
"date": "15 Dec 2015",
"name": "Hays Rye",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1448
|
https://f1000research.com/articles/4-1447/v1
|
15 Dec 15
|
{
"type": "Research Note",
"title": "Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae",
"authors": [
"James Green",
"Simon Harvey",
"James Green"
],
"abstract": "The life cycles of many free-living nematodes contain developmental switches that allow individuals to either develop directly to adulthood, or to arrest development as a stress resistant and long-lived dauer larval stage. Here, in a panel of Caenorhabditis briggsae recombinant inbred lines derived from the isolates HK104 x AF16, we use methodologies developed for C.elegans to map quantitative trait loci (QTLs) affecting the number of dauer larvae present at the point of food patch exhaustion. These analyses provide strong support for three QTLs and are suggestive of a further two.",
"keywords": [
"dauer larvae",
"Caenorhabditis briggsae",
"QTL",
"nematode",
"natural variation"
],
"content": "Introduction\n\nCaenorhabditis elegans is an important model species, but there is a need to further understand its ecology and that of the other species with which it is associated1–3. Active, growing, populations of Caenorhabditis nematodes are normally associated with nutrient and bacteria-rich substrates such as rotten fruit or very fresh compost1,3,4. Growing populations can reach very large sizes, and worms would be expected to have optimized fitness (population growth and/or the production of dispersal stages) under such conditions. Appropriate development of growing worms as dauer larvae, the developmentally-arrested alternate third larval stage, is therefore likely to be central to genotype survival.\n\nWe have previously looked at dauer larvae development in growing populations of C. elegans5–7. These analyses identified both extensive variation between wild isolates in the number of dauer larvae present when a bacterial food patch is exhausted6 and large numbers of QTLs effecting this trait in introgression lines (ILs) produced from the isolates N2 and CB48566,7. Naturally, C. elegans has however been found to be associated with both C. remanei and C. briggsae1,3,8. Here, using the methodologies developed for C. elegans, we have investigated dauer larvae formation in growing populations in a panel of C. briggsae recombinant inbred lines (RILs) produced from the isolates AF16 and HK1049.\n\n\nMethods\n\nThe C. briggsae RILs were generated from reciprocal crosses between males and sperm-depleted hermaphrodites of isolates HK104 and AF169 and were obtained from Asher Cutter (University of Toronto). Worms were maintained using standard methods10 and all assays were conducted at 20°C. Assays were performed as previously described6, with populations initiated from single fourth larval stage worms (L4s) grown from synchronized, arrested, L1s with 100 μl of a 20% w/v suspension of Escherichia coli in water and monitored daily until food exhaustion. At this point, the population size and the number of dauer larvae were determined5,6. RILs were analyzed in three experimental blocks with AF16 and HK104 assayed in each block and five populations initiated per genotype. Treatments (genotypes) were randomized within blocks and plates were blind coded such that counts were performed without knowledge of worm genotype. Populations that failed to grow were discarded.\n\nThe RILs have previously been genotyped at 451 markers distributed across all six chromosome pairs9 and these data were used for this analysis. QTL mapping was performed by composite interval mapping (CIM) using QTL Cartographer v2.511. Genome-wide thresholds (0.05) were estimated based on 1000 permutations of the data and CIM analysis undertaken with a 1.0cM walk speed.\n\n\nResults\n\nPosition is the 1 LOD interval for the QTL. R2 denotes the proportion of the inter-RIL variance explained by the QTL, and effect indicates how the QTL alters dauer larvae numbers, with a positive value indicating that the AF16 allele increases the number of dauer larvae compared to the HK104 allele.\n\nComparing the AF16 and HK104 controls between the experimental blocks indicates that the number of dauer larvae at food exhaustion varies extensively between blocks (H = 11.06, p = 0.004 and H = 8.21, p = 0.007, for AF16 and HK104, respectively: Figure 1A). This variation is much greater than that seen for population size at food exhaustion (Figure 1B). Such variability between experimental blocks has been observed before with this assay, and is likely to be due to variation in either the actual or perceived food quality between batches of bacteria6.\n\nGiven the variation in dauer larvae formation between the experimental blocks we considered it unlikely that analysis of raw data would identify any QTLs, and this is what was observed (Figure 2A). Two approaches were therefore taken to determine if we could control for this variation. Firstly, we scaled the number of dauer larvae observed for each RIL either to the AF16 or HL104 controls within that experimental block (Figure 2A), e.g. the AF16 scaled value for a RIL was determined as (RIL mean - AF16 mean)/AF16 mean and this was used for mapping. Secondly, we analyzed each of the experimental blocks independently (Figure 2B).\n\nThe A) number of dauer larvae, and B) population size, at food exhaustion in AF16 and HK104 controls in the three experimental blocks. Values from each plate are shown, with red bars indicating the median values.\n\nA) CIM of the number of dauer larvae (solid line), and the number of dauer larvae scaled to the numbers in the AF16 (dashed line) or HK104 (dotted line) controls from that experimental block. B) CIM of the number of dauer larvae observed in each experimental block separately. Horizontal lines indicate thresholds.\n\nIn combination, these approaches identify three QTLs on chromosomes IV, V and the X, that affect the number of dauer larvae present at food exhaustion (co-localizing QTLs present in both Figure 2A and B) (Table 1). The analysis is also suggestive of additional QTLs on chromosomes II and V (Figure 2A and B; Table 1). A similar analysis of the population size at food exhaustion did not detect any QTLs.\n\n\nDiscussion\n\nFor a comparative analysis of dauer larvae development in growing populations, a panel of C. briggsae RILs were analyzed. These analyses indicate that the general properties of growing populations of C. briggsae are similar to those described for C. elegans5,6. This means these methodologies will be suitable for more direct comparisons between the species. The RIL analysis identifies a number of QTLs (Figure 2; Table 1) although these are not resolved to narrow genomic regions and therefore contain many hundreds of genes. They do however provide a starting point for attempts to identify causal loci.\n\n\nData availability\n\nF1000Research: Dataset 1. The number of dauer larvae and the population size at food exhaustion for replicate populations of the RILs and the respective AF16 and HK104 controls from three experimental blocks, 10.5256/f1000research.7546.d10908812\n\nF1000Research: Dataset 2. Marker distances (from Ross et al., 2011) for informative markers in the RILs analysed, 10.5256/f1000research.7546.d10908913\n\nF1000Research: Dataset 3. RIL genotypes (from Ross et al., 2011) and analysed trait values for the RILs, 10.5256/f1000research.7546.d10909014",
"appendix": "Author contributions\n\n\n\nJG and SH conceived the study and designed the experiments. JG carried out the research. SH and JG analyzed the data, wrote the paper 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 Asher Cutter for the C. briggsae RILs. We thank Helen Orbidans for her assistance with the assays.\n\n\nReferences\n\nFélix MA, Braendle C: The natural history of Caenorhabditis elegans. Curr Biol. 2010; 20(22): R965–R969. PubMed Abstract | Publisher Full Text\n\nPetersen C, Dirksen P, Schulenburg H: Why we need more ecology for genetic models such as C. elegans. Trends Genet. 2015; 31(3): 120–127. PubMed Abstract | Publisher Full Text\n\nFrézal L, Félix MA: C. elegans outside the Petri dish. eLife. 2015; 4: e05849. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKiontke KC, Félix MA, Ailion M, et al.: A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits. BMC Evol Biol. 2011; 11(1): 339. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreen JW, Harvey SC: Development of Caenorhabditis elegans dauer larvae in growing populations. Nematology. 2012; 14(2): 165–173. Publisher Full Text\n\nGreen JW, Snoek LB, Kammenga JE, et al.: Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans. Heredity (Edinb). 2013; 111(4): 306–313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreen JW, Stastna JJ, Orbidans HE, et al.: Highly polygenic variation in environmental perception determines dauer larvae formation in growing populations of Caenorhabditis elegans. PLoS One. 2014; 9(11): e112830. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFélix MA, Duveau F: Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae. BMC Biol. 2012; 10(1): 59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRoss JA, Koboldt DC, Staisch JE, et al.: Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination. PLoS Genet. 2011; 7(7): e1002174. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStiernagle T: Maintenance of C. elegans. (february 11, 2006), WormBook. ed. the C. elegans Research Community, WormBook, 2006. PubMed Abstract | Publisher Full Text\n\nWang SC, Basten CJ, Zeng ZB: Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC, 2007. Reference Source\n\nGreen J, Harvey S: Dataset 1 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015a. Data Source\n\nGreen J, Harvey S: Dataset 2 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015b. Data Source\n\nGreen J, Harvey S: Dataset 3 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015c. Data Source"
}
|
[
{
"id": "11792",
"date": "18 Jan 2016",
"name": "Erik C. Andersen",
"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\nUsing techniques published previously, the authors measure the number of dauer larvae present at food patch exhaustion for a collection of Caenorhabditis briggsae recombinant lines. The dauer phenotype results are very noisy (as observed previously), but after data transformation quantitative trait loci (QTL) are detected using composite interval mapping. These results suggest that some regions of the C. briggsae genome confer differences in dauer formation, but more analysis needs to be performed to know more definitively. I would soften the abstract language (e.g. \"strong support\") to reflect the uncertainty in the results.Given the variability of the phenotype measurements and the small number of recombinant lines, I am surprised about the QTL results. I would have expected no QTL to be detected, as observed in the untransformed data. It would be nice to see some more analyses, including the following:What is the broad-sense heritability of this trait for these strains? It would be good to know how much of the trait variance you might expect to map to genomic regions in this study. What are the results of a non-parametric linkage mapping? Composite interval mapping has many caveats. Are these QTL robust to mapping technique? The significance threshold seems low given the allele frequency skews present in this mapping population. Please explain more about how that threshold was determined and how the skews were controlled. Can you plot the RIL phenotypes before and after transformation so we can assess how well the transformation affected the high variability of the assay?If the detected QTL represent expected amounts of the genetic variance, are robust to mapping technique, and are significant with respect to the known allele frequency skews in this mapping population, then I believe this article offers preliminary evidence that some genomic regions confer differences in dauer larvae formation. These results suggest genomic regions to test for causality in the trait of interest, including regions that could lead to conserved interspecific variation between C. elegans and C. briggsae.",
"responses": []
},
{
"id": "11992",
"date": "20 Jan 2016",
"name": "Eric Haag",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this report, Green and Harvey address the genetic architecture governing variation in one of the best-studied examples of developmental plasticity in animas, the dauer larva Caneorhabditis nematodes.Dauer larvae are dispersive forms of nearly all terrestrial nematodes, and are typically induced by the onset of food limitation and/or pheromone cues that are correlated with it. Given that the correctness of the decision to form a dauer (and thus abandon the direct path to reproductive adulthood) is expected to be highly contingent upon the exact ecological context in which it occurs, this trait should be both subject to strong selection and be potentially variable. This may be especially true for C. briggsae, which has a greater degree of geographic population structure than does its more famous congener, C. elegans.The authors take advantage of a set recombinant inbred lines between genetically distinct strains (lines that my own lab played a role in characterizing in 2011; see the Ross et al. reference) to examine whether there is indeed heritable variation for dauer formation, and if so whether there are any loci of variation with effect size big enough to show significant association with dauer formation. I have no doubts that this project is addressing a significant issue in biology, and the authors make some headway.General enthusiasm aside, as Erik Andersen also notes in his review the main concern here is the noisiness of the assay that is mapped. In understanding how the dauer formation assays is done, I think I may see why it is so noisy. The authors are founding each plate with a single L4 larva and a fixed amount of food. This is easy to reproduce, so I see the appeal. However, to then reach starvation on the plate, we presumably are looking at the grandchildren of the founder worm. As an exponential process, tiny differences in the generation time or rate of egg laying in the first few hours would produce big variability in the total number of worms, and also in the increase of the signals that are stimulatory for dauer production.One could try to model the nature of this variation, but I don’t think this is author’s goal. An alternative, though admittedly requiring a whole new set of experiments, would be to seed plates with 20 or 200 L4 larvae, and score dauer formation in the first generation. This ought to be far less variable, yet should also be assessing the same basic phenotype, which could be seen as the threshold signal required to induce dauer development. Rather than repeating the entire scan, perhaps the authors could take their most discordant RILs and repeat this assay on them? It may produce a stronger signal, or at least confirm the initial screen.",
"responses": []
},
{
"id": "11575",
"date": "01 Feb 2016",
"name": "Christian Braendle",
"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 research note presents a very brief summary of a QTL analysis based on a phenotyping assay of dauer formation (at food exhaustion) in C. briggsae AI-RILs. The main issue here is the low reproducibility of the phenotyping assay across replicates and blocks. Given this problem, it appears rather surprising that some significant QTL were picked up. Further assays would be required to validate these, and this might be worthwhile doing (given that reproducible assays can be developed). If I understood correctly, adjusted or unadjusted numbers of dauers were used as trait values – why not rather use the proportion of dauers (or number of dauers adjusted by population size) for mapping? [higher densities are expected to increase dauer formation] What is the relationship between proportion of dauers and population size? Can the variability in dauer formation be explained by variability in population size? Does mapping of population size give the same QTLs? Figure 1 shows how strong replicate and block effects are, just by looking at the parental lines, AF16 and HK104. Does an ANOVA analysis of these data (or the RIL data) actually reveal a significant genotype effect (so far, only block effects are given)? What are the heritabilities? How does this QTL analysis in briggsae compare to the QTL analysis done in elegans (N2xCB4856) – number of QTLs, regions? In summary: it would be useful to add some more info and data analysis to this note. Additional commentsperhaps mention that the lines used are advanced-intercross recombinant inbred lines (AI-RILs)the authors mention 451 markers but I had the impression many more were generated in Ross et al. 2011",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1447
|
https://f1000research.com/articles/4-1443/v1
|
14 Dec 15
|
{
"type": "Software Tool Article",
"title": "BioShaDock: a community driven bioinformatics shared Docker-based tools registry",
"authors": [
"François Moreews",
"Olivier Sallou",
"Hervé Ménager",
"Yvan Le bras",
"Cyril Monjeaud",
"Christophe Blanchet",
"Olivier Collin",
"Olivier Sallou",
"Hervé Ménager",
"Yvan Le bras",
"Cyril Monjeaud",
"Christophe Blanchet",
"Olivier Collin"
],
"abstract": "Linux container technologies, as represented by Docker, provide an alternative to complex and time-consuming installation processes needed for scientific software. The ease of deployment and the process isolation they enable, as well as the reproducibility they permit across environments and versions, are among the qualities that make them interesting candidates for the construction of bioinformatic infrastructures, at any scale from single workstations to high throughput computing architectures. The Docker Hub is a public registry which can be used to distribute bioinformatic software as Docker images. However, its lack of curation and its genericity make it difficult for a bioinformatics user to find the most appropriate images needed. BioShaDock is a bioinformatics-focused Docker registry, which provides a local and fully controlled environment to build and publish bioinformatic software as portable Docker images. It provides a number of improvements over the base Docker registry on authentication and permissions management, that enable its integration in existing bioinformatic infrastructures such as computing platforms. The metadata associated with the registered images are domain-centric, including for instance concepts defined in the EDAM ontology, a shared and structured vocabulary of commonly used terms in bioinformatics. The registry also includes user defined tags to facilitate its discovery, as well as a link to the tool description in the ELIXIR registry if it already exists. If it does not, the BioShaDock registry will synchronize with the registry to create a new description in the Elixir registry, based on the BioShaDock entry metadata. This link will help users get more information on the tool such as its EDAM operations, input and output types. This allows integration with the ELIXIR Tools and Data Services Registry, thus providing the appropriate visibility of such images to the bioinformatics community.",
"keywords": [
"bioinformatics",
"docker",
"container",
"deployment",
"interoperability",
"maintainability",
"community driven registry"
],
"content": "Introduction\n\nThe life sciences are becoming more and more digital and nowadays data analysis methods represent a key factor of the discovery process. In the case of bioinformatics, software is widely provided by the research community. Developers favor open source approaches and many software tools are available online. It is commonly agreed that such a distributed and free creation process accelerates discoveries in the life sciences1,2. However, this view must be nuanced, as multiple factors still hinder the discovery, integration, and maintenance of these software tools.\n\nFirst, domains such as genomics, where technological innovation leads to a exponential growth of data to analyse, also generate an ever-increasing number of new software methods. However, the discovery of new interesting tools by potential users remains limited by unstructured descriptions, lack of metadata and deprecated source codes. In this context, dedicated search engines like the ELIXIR Tools and Data Services Registry3,4 (hereafter referred as the \"ELIXIR registry\") have emerged as a potential solution to search, find and locate available and maintained tools.\n\nSecondly, the implementation methods of bioinformatic software are heterogeneous and their deployment requires multiple technical skills. The installation process is therefore expensive, in terms of human resources. It is worth recalling that the cost in supporting operating systems and hardware diversity can be high, the code compilation process is error prone and the required software dependencies are often conflicting with installed libraries. Consequently, the audience of a software can be limited to highly motivated and technical users or large bioinformatics facilities. The recent development of user-friendly data analysis environments like Galaxy5 ease access for biologists and bio-analysts to bioinformatic tools. These software workbenches provide a generic web user interface for command line based scientific applications, but do not solve the tools’ deployment issue. Even if the task can be submitted inside a container, it is the tool designer’s responsibility to provide a readily deployable component6 and the proportion of container based components in repositories such as the Galaxy Toolsheds7 is currently low.\n\nFinally, traditional academic publishing and funding processes emphasize the production of software with short-term goals, these being the publication of the method and/or results. Such an environment does not favor a software engineering-oriented approach to software development8, and this affects directly the portability and maintainability of the software products9. This in turn impacts the reproducibility of analyses, experiments or benchmarks described in published articles. However, even if various emerging initiatives are developing frameworks10–12 to enable a new kind of \"executable format\" of scientific publication, few journals have an innovative publishing policy that includes the long term storage of the source codes on a dedicated public web platform.\n\nNevertheless, today containerization brings new pragmatic solutions. Linux containers are a mature technology that has the potential to dramatically facilitate scientific software deployment and analysis reproducibility. Docker, one of the most popular container solutions13,14, is now used in a variety of computation environments, from commercial clouds15 to clusters with dedicated middleware16. It has been positively evaluated for data intensive computation, a recent study showing that the performance of bioinformatic workflows composed by medium or long running tasks are only very slightly affected by containerization17.\n\nContainer technology has the potential to impact audiences, developers and end-users. In the scientific field, it can effectively improve reproducibility, ease deployment and facilitate the building of software collections and search engines dedicated to a specific scientific domain or topic.\n\nFor these reasons, we created the BioShaDock registry that promotes the use of container technologies in bioinformatics. The BioShaDock registry provides a web entry point to deploy, search and discover ready to use bioinformatics tools, encapsulated in Docker containers.\n\nFuture works will focus on better integration with domain-centric registries as well as bioinformatic integrated environments, to enable the seamless discovery, integration, and execution of the BioShaDock containers. Our project will also greatly benefit from discussions with other existing bioinformatic container initiatives.\n\n\nMethods\n\nBioShaDock is a web server based system that allows the description, registration and automated building of Docker images (Figure 1). These images are publicly available on the web server for search, download and execution. Users can authenticate using local LDAP or Google/GitHub credentials. LDAP users have the possibility to push new images. External users (Google, etc.) can request those privileges by contacting the support team. This mechanism allows non local users to have access to the registry to provide new tools while keeping a controlled access on the submission of new tools to the registry, where contributions are based on trust.\n\nThe interface enables the creation of Dockerfiles and allows to search the repository using full text queries.\n\nOnce authenticated, the user can proceed to the registration of a Docker container. The information required includes:\n\nthe set of instructions to build the image, i.e. the Dockerfile and the associated source code. These can be provided by pasting directly the Dockerfile contents in the web interface, by pointing to a Git repository that contains the Dockerfile and the source code, or by pointing to the source code repository and manually providing the Dockerfile. In the case of Git repository registration, it is also possible to configure the branch and location of the Dockerfile in the repository.\n\nadditional metadata which is required to describe the contents of the image in scientific terms to its potential users. Such metadata includes for instance free tags, as well as EDAM18 terms.\n\nFollowing the completion of container registration, the image construction and integration steps (Figure 2) are automatically run on a dedicated server. The trigger of a new build is based on Dockerfile update or via a link (URL with an API Key), shown in the web interface when the user is the owner of the tool (created it). The creation of a tag on the image uses the same link mechanism. Such a link can be used directly (copy/paste in the brower) or via external tools or hooks (GitHub web hooks for example). The API also provides the possibility to trigger it manually, or to tag a container (i.e. set a version).\n\nThe Docker images, once built and stored in BioShaDock, can be registered in the ELIXIR registry (using some LABEL metadata in the Dockerfile). It is also possible to add a link to an existing ELIXIR registry entry. By linking its contents to and from the ELIXIR registry, BioShaDock enables the discovery of Docker images from a more generic system where users might look for a given software without specifically searching for container solutions. It hence maximizes the visibility of its images and contributes to better software dissemination.\n\nListing 1. An example of Docker image command line invocation using BioShaDock. After an automatic download, the container is executed. Here, the program BWA is called by default.\n\n\n\nThe images provided by BioShaDock can be executed in various ways (Figure 3):\n\nThe Docker repository acts as a platform that facilitates the dissemination of bioinformatics tools by providing ready to use Docker images.\n\n• on a personal computer with a Linux system (Windows and Mac are supported with the Docker Toolbox), in a command line (Listing 1), directly using Docker14;\n\n• on a cluster integrating a Docker scheduler front-end like GO-DOCKER (v1.0)16;\n\n• in any software implementing the CWL (Common Workflow Language) specification (draft 3)19,20 such as Arvados21 or Rabix (v0.6.5)22;\n\n• in the D4 workflow portal23 (v0.6);\n\n• in the Galaxy environment6 (v15.10);\n\n• in the cloud of the French Institute of Bioinformatics with the help of the Docker virtual machine image24.\n\nAs an illustration, we created a set of Galaxy tool descriptors based on Docker images stored by BioShaDock25 available in our Toolshed26. Thus, the stacks RADSeq pipeline27 is available as a Galaxy tool xml descriptor28 that calls a container stored in BioShaDock29.\n\nListing 2. A container ’Dockerfile’ that defines the automated image build process. The LABEL instructions represent metadata.\n\n\n\nBioShaDock is a web application written in python (>=2.7). It manages the container’s build and metadata. It is also in charge of authenticating the user against a local Docker registry and authorizing the user to push or pull a container according to their role (admin, editor, etc.) or rights. A user can give other users access to their repository for collaborative work in the edition page of the tool. Collaborators can have read only (for private repositories) or read/write access to the tool. The backend is based on a local instance of a Docker registry.\n\nA script extracts the metadata written by the image’s maintainer (Listing 2).\n\nListing 3. An XML container metadata description generated from the LABEL instructions by BioShaDock and used to publish the container metadata in bio.tools, the ELIXIR registry.\n\n\n\nThen, an integrated REST python client (v1.0) manages the container indexation in bio.tools (Listing 3). The first version of the registry integrates 80 Docker images that are versioned and can be re-built when the sources are updated. A REST API enables programmatic interaction with the server. For example, it can be used by external tools to extract the list of available images for job submissions. GO-DOCKER (v1.0) and the D4 workflow portal (v0.6) integrate this feature. The access to the images is public. To ensure the quality of available images, BioShaDock manages the authentication and ACL (access control list) to restrict the creation and update of its images to identified trustful contributors. The current implementation (v1.0) enables authentication using LDAP, Google or GitHub.\n\n\nDiscussion\n\nThe aim of BioShaDock is to contribute to the aggregation and standardization of bioinformatic tools and utilities. Maintaining ready to use validated and versioned software is key in ensuring the reproducibility needed in an open science approach.\n\nThereby, the creation of a collection of tools embedded in Docker containers, as provided by BioShaDock, is a pragmatic solution to this major bottleneck.\n\nA number of other projects also focus on the provision of bioinformatic Docker images. BioDocker30 is a community based initiative to encourage the use of Docker images in bioinformatics. A GitHub repository stores a list of Dockerfiles that define the construction of images for the corresponding bioinformatic tool, with an open yet controlled contribution mechanism. Bioboxes31 is an open source project that defines guidelines to build bioinformatic tool images using compatible interfaces for images which perform the same task, independent of the underlying tool, hence favoring interoperability between tools. It is therefore, among other characteristics, very well suited to automate tool and pipeline benchmarks. It has been applied to the assessment of different types of NGS data processing methods that concern assembly software as well as metagenomics tool. Dockstore32 is an open platform that enables the registration of Docker images described using CWL. It integrates with a number of external services for source code and image hosting, and focuses on the provision of images that can be integrated in CWL-ready environments. BioShaDock shares with these existing efforts the use of Docker as a container technology to facilitate the distribution and integration of bioinformatic tools. However, none of these systems are designed to provide local image building and storage options. Furthermore, we believe the integration of BioShaDock with external domain-centric and platform-agnostic registries such as the ELIXIR registry will significantly raise the visibility of both the images provided and the container technology itself to the community of bioinformatic tool users. Because the files that describe the image building process (Dockerfiles) are usually freely available online, the interoperability issues between Docker registry initiatives are potentially very limited.\n\n\nConclusions\n\nComputer scientists and bioinformaticians can more easily disseminate their programs and find potential users using a dedicated domain-centric Docker registry. There is a wide range of perspective uses for container registries in bioinformatics: repositories managed at a community level, based on tools embedded in containers, promote the ability to exchange and replicate data analyses.\n\nIn addition, the association between workflow models, data references and containerized tools could lead to the creation of interoperable and ready to use analysis components and pipeline collections maintained by many contributors. The development of such specifications is already in progress as illustrated by the CWL (Common Workflow Language)20 and the A-SCDFM (Autonomous Semi-Concrete Data Flow Model)33 portable workflow formats that are natively compatible with containers. In this case, the integration of programs in a container registry like BioShaDock and the formalization of the data processing following one of these new portable workflow specifications could simplify the creation of reproducible benchmarks, teaching material, demos and the production of use case prototypes. It could also be used by article reviewers to quickly evaluate a software.\n\nThe spread of container usage in the bioinformatics community and their indexing in repositories can be a solution to capture and share a large collection of data analysis methods. A wide set of bioinformatics components available on demand could induce better data analysis by simplifying tests and benchmarks.\n\n\nSoftware availability\n\n• BioShaDock registry: https://docker-ui.genouest.org\n\n• BioShaDock home page: http://bioshadock.genouest.org\n\n• BioShaDock client and tools: https://github.com/fjrmoreews/bioshadock_client\n\n• BioShaDock local server: https://bitbucket.org/osallou/bioshadock\n\n• Archived source code at the time of publication (client): https://zenodo.org/record/3458834\n\n• Archived source code at the time of publication (server): https://zenodo.org/record/3458735\n\nApache 2.0",
"appendix": "Author contributions\n\n\n\nFM and OS conceived the software and developed the web interface and the build system. HM participated to the meta-data publishing feature design. YLB and CM designed some of the first Dockerfile and integrated Docker images in our Galaxy toolshed. OC and CB managed the deployment and infrastructure availability. All authors helped prepare the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nFunding was provided from the Western France e-science project supported by Brittany and Pays de la Loire regions (e-Biogenouest/052012).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the IFB/Genouest platform for hosting the software and the use of its cluster to build the images. We thank the IFB-CORE team, especially Marie Grosjean and Sandrine Perrin, for creating containers and for supporting the deployment of the registry coupled with the IFB cloud facility. Finally, we also thank the BioDocker core team, Felipe da Veiga Leprevost, Yasset Perez-Riverol and Saulo Alves Aflitos for collaborative efforts.\n\n\nSupplementary material\n\nBioShaDock API documentation:\n\nhttp://www.genouest.org/api/bioshadock-api\n\n\nReferences\n\nWoelfle M, Olliaro P, Todd MH: Open science is a research accelerator. Nat Chem. 2011; 3(10): 745–748. PubMed Abstract | Publisher Full Text\n\nStajich JE, Lapp H: Open source tools and toolkits for bioinformatics: significance, and where are we? Brief Bioinform. 2006; 7(3): 287–296. PubMed Abstract | Publisher Full Text\n\nIson J, Rapacki K, Ménager H, et al.: Tools and data services registry: a community effort to document bioinformatics resources. Nucleic Acids Res. 2015; pii: gkv1116. PubMed Abstract | Publisher Full Text\n\nConnor BO, Kartashov A, Yuen D, et al.: ELIXIR Tools and Data Services Registry. 2015. Reference Source\n\nGoecks J, Nekrutenko A, Taylor J, et al.: Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAranguren ME: Merging OpenLifeData with SADI services using Galaxy and Docker. BioRxiv, Cold Spring Harbor Labs. 2015. Publisher Full Text\n\nBlankenberg D, Von Kuster G, Bouvier E, et al.: Dissemination of scientific software with Galaxy ToolShed. Genome Biol. 2014; 15(2): 403. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLawlor B, Walsh P: Engineering bioinformatics: building reliability, performance and productivity into bioinformatics software. Bioengineered. 2015; 6(4): 193–203. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrins P, de Ligt J, Tarasov A, et al.: Toward effective software solutions for big biology. Nat Biotechnol. 2015; 33(7): 686–687. PubMed Abstract | Publisher Full Text\n\nVan Gorp P, Mazanek S: SHARE: a web portal for creating and sharing executable research papers. Procedia Comput Sci. 2011; 4: 589–597. Publisher Full Text\n\nGranger B, Avila D, Perez F, et al.: Jupyter: Open source, interactive data science and scientific computing across over 40 programming languages. 2015. Reference Source\n\nKanterakis A, Kuiper J, Potamias G, et al.: PyPedia: using the wiki paradigm as crowd sourcing environment for bioinformatics protocols. Source Code Biol Med. 2015; 10(1): 14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMerkel D: Docker: Lightweight Linux containers for consistent development and deployment. Linux J. 2014; (239). Reference Source\n\nDocker. 2013; [Online; accessed 16-Nov-2015]. Reference Source\n\ngoogle Inc: Google Container Engine. 2015. Reference Source\n\nSallou O, Monjeaud C: GO-Docker: Batch scheduling with containers. IEEE Cluster 2015. 2015. Reference Source\n\nDi Tommaso P, Palumbo E, Chatzou M, et al.: The impact of Docker containers on the performance of genomic pipelines. PeerJ. 2015; 3: e1273. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIson J, Kalas M, Jonassen I, et al.: EDAM: an ontology of bioinformatics operations, types of data and identifiers, topics and formats. Bioinformatics. 2013; 29(10): 1325–1332. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeter A, Nebojsa T, Stian SR, et al.: Beyond Galaxy: portable workflows and tool definitions with the CWL. In Galaxy Community Conference 2015. Norwich, United Kingdom, 2015. Reference Source\n\nAmstutz P, Chilton J, Crusoe MR, et al.: Common Workflow Language. 2015. Reference Source\n\nArvados. 2015. Reference Source\n\nRabix. 2015. Reference Source\n\nFrancois M: D4 Workflow Portal. 2015. Reference Source\n\nIFB cloud: The academic cloud of the French Institute of Bioinformatics. Online; accessed 2015-09-24. Reference Source\n\nMoreews F, Sallou O, Bras YL, et al.: A curated Domain centric shared Docker registry linked to the Galaxy toolshed. In Galaxy Community Conference 2015. Norwich, United Kingdom, 2015. Reference Source\n\nBras YL, Monjeau C: GUGGO Galaxy ToolShed. 2014. [Online; accessed 05-Nov-2015]. Reference Source\n\nCatchen J, Amores A, Hohenlohe P, et al.: STACKS, a software pipeline for building loci from short-read sequence. 2010. [Online; accessed 02-Dec-2015]. Reference Source\n\nBras YL, Monjeaud C: STACKS pipeline, galaxy tool descriptor. 2010. [Online; accessed 02-Dec-2015]. Reference Source\n\nBras YL, Monjeaud C: STACKS pipeline, docker container. 2010. [Online; accessed 02-Dec-2015]. Reference Source\n\nBioDocker. 2015. Reference Source\n\nBelmann P, Dröge J, Bremges A, et al.: Bioboxes: standardised containers for interchangeable bioinformatics software. Gigascience. 2015; 4: 47. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConnor BO, Kartashov A, Yuen D, et al.: DockStore. 2015. Reference Source\n\nMoreews F: Design and share data analysis workflows. Application to bioinformatics intensive treatments. Thesis, université de rennes 1. 2015.\n\nFrancois M, Olivier S: BioShaDock client. Zenodo. 2015. Data Source\n\nOlivier S, Francois M: BioShaDock server. Zenodo. 2015. Data Source"
}
|
[
{
"id": "11548",
"date": "15 Dec 2015",
"name": "Rodrigo Lopez",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Moreews et al. describes a registry of bioinformatic tools images that are portable using Docker technology. The manuscript is well written and describes well the aims of the BioShaDock registry and it's possible interactions with the ELIXIR Tools and Data Services Registry as the means to find Docker containers in the wild. As pointed out in the abstract, other Docker registries exists, such as Docket HUB, but lack of curation and user engagement hampers their progress. Furthermore,BioShaDock provides user management at a level required for ensuring that the interoperability between the registries, images and local environments is secure, auditable and effective.The article describes well the overheads associated with typical software installations and maintenance and presents a balanced view on the advantages of using Docker to manage this processes. Although not perhaps within the scope of this article, this reviewer feels it would be useful to inform the readership of other alternatives to Docker; e.g. Rocket, DrawBridge and LXD from Canonical and FlockPort, as it is clear that Docker is still maturing and it is certainly not the only container available today.",
"responses": []
},
{
"id": "11546",
"date": "01 Feb 2016",
"name": "Björn A. Grüning",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes very well the current state of bioinformatics Linux container adoption and arising problems. It offers solutions to these and also describes real-world use-cases with an existing integration into systems like Galaxy. Especially interesting is the rich annotation system, that involves ELIXIR ontologies as well as the ELIXIR registry.This is needed and a big step forward.Personally, I would like to see stronger collaborations between the mentioned other registry and Docker-build projects. I still feel we have a lot of redundant work inside of the bioinformatics community. For example I think it would be relatively easy to configure travis in biodocker to push automatically into BioShaDock, if biodocker counts as trusted partner. On the other hand biodocker can profit largely by the rich annotation system.The manuscript is well written and I would encourage everyone to participate in this project. I certainly will.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1443
|
https://f1000research.com/articles/4-1431/v1
|
11 Dec 15
|
{
"type": "Review",
"title": "The therapeutic potential of genome editing for β-thalassemia",
"authors": [
"Astrid Glaser",
"Bradley McColl",
"Jim Vadolas",
"Astrid Glaser",
"Bradley McColl"
],
"abstract": "The rapid advances in the field of genome editing using targeted endonucleases have called considerable attention to the potential of this technology for human gene therapy. Targeted correction of disease-causing mutations could ensure lifelong, tissue-specific expression of the relevant gene, thereby alleviating or resolving a specific disease phenotype. In this review, we aim to explore the potential of this technology for the therapy of β-thalassemia. This blood disorder is caused by mutations in the gene encoding the β-globin chain of hemoglobin, leading to severe anemia in affected patients. Curative allogeneic bone marrow transplantation is available only to a small subset of patients, leaving the majority of patients dependent on regular blood transfusions and iron chelation therapy. The transfer of gene-corrected autologous hematopoietic stem cells could provide a therapeutic alternative, as recent results from gene therapy trials using a lentiviral gene addition approach have demonstrated. Genome editing has the potential to further advance this approach as it eliminates the need for semi-randomly integrating viral vectors and their associated risk of insertional mutagenesis. In the following pages we will highlight the advantages and risks of genome editing compared to standard therapy for β-thalassemia and elaborate on lessons learned from recent gene therapy trials.",
"keywords": [
"thalassemia",
"genome",
"gene therapy"
],
"content": "β-Thalassemia\n\nβ-Thalassemia is a common congenital blood disorder caused by mutations in the β-globin gene. Reduced or absent β-globin expression leads to an imbalance of the α-globin and β-globin subunits that form the hemoglobin tetramer. The toxic accumulation of excess α-globin chains in developing erythrocytes results in severe anemia due to ineffective erythropoiesis1. In its most serious form, β-thalassemia major, the condition is fatal if left untreated2. Currently, allogenic bone marrow transplantation (BMT) is the only curative therapeutic option. However, due to the rarity of suitable donors, this treatment is available only to a small subset of patients and the procedure itself entails a risk of potentially life-threatening immunological complications and graft failure, especially for patients over 3 years of age3,4.\n\nThe majority of β-thalassemia patients depend on regular blood transfusions combined with iron chelation therapy for their survival5. Even under optimal care, this treatment regimen provides a suboptimal quality of life and leaves patients at an increased risk of death from cardiomyopathies and infection6,7. New therapeutic strategies are therefore needed to better manage β-thalassemia.\n\n\nGene therapy for β-Thalassemia\n\nIn the past 25 years, the field of gene therapy has made considerable progress. Gene therapy aims at the functional cure of disorders through modification of a patient’s genome. Depending on the nature of the causative mutation, this could be achieved through the introduction of a therapeutic gene, correction of the disease-causing mutation, or the elimination of deleterious gene products (reviewed by Kay et al., 2011)8.\n\nThe major obstacle all gene therapy approaches face is safe and efficient gene delivery to the affected tissue or cell type. In vivo delivery is particularly difficult due to poor tissue accessibility, vector immunogenicity, and limited target cell specificity9. Monogenic blood disorders such as severe combined immune deficiency (SCID), sickle-cell anemia, and β-thalassemia are remarkably attractive targets for gene therapy due to the unique accessibility of hematopoietic progenitor cells, which can be isolated from patient bone marrow10. The ex vivo correction and re-introduction of autologous hematopoietic stem cells (HSCs) has no associated risk of graft-versus-host disease, the major adverse effect of allogenic BMT. Eliminating the necessity of a matched donor potentially makes this approach applicable to all patients. Gene therapy could therefore provide a safer and more generally available curative treatment for blood disorders than allogenic BMT.\n\nPast and ongoing gene therapy trials are mostly focused on the delivery of a therapeutic gene using integrating viral vectors. This gene addition approach has been successfully applied in severe combined immunodeficiencies11–13, retinal disorders14–16, and hemophilia17. The first successful gene therapy trial for β-thalassemia was reported in 201018. The trial employed a lentiviral vector for ex vivo delivery of a β-globin transgene into patient HSCs, which were subsequently returned to the patient. The treatment was successful in one patient who remained transfusion-independent for up to 7 years19,20. A second trial was subsequently initiated using a modified vector. Although long-term results are yet to be released, promising preliminary data describe two patients remaining transfusion-independent for 14 and 16 months, respectively21. These trials demonstrate that gene therapy has the potential to provide effective long-term therapy following a single treatment.\n\nThe greatest caveat in the use of integrating lentiviral and retroviral vectors lies in the inability to control for target site selection, which can result in considerable genotoxicity from the transactivation of nearby proto-oncogenes22,23. This was tragically confirmed when four out of nine children treated in the first gene therapy trial for SCID-X1 developed leukemia as a result of gamma-retrovirus vector integration, causing the death of one patient24. Following this setback, vector design was improved by the development of self-inactivating lentiviruses, insulator elements, and tissue-specific promoters25–27. Nonetheless, insertional mutagenesis still remains the major concern with retroviral and lentiviral gene therapy approaches28. The importance of understanding and managing this risk was again demonstrated by the appearance of a dominant clone with a transactivating insertion event near the HMGA2 gene in the HSCs of the first successfully treated β-thalassemia gene therapy patient18. This event, though only transient, has again emphasized the necessity for careful monitoring of patients following treatment with integrating vectors.\n\nThis issue has driven the search for safer gene therapy approaches. One possible solution is the targeted integration of a therapeutic gene into a genomic “safe harbor” site that supports long-term transgene expression without affecting transcriptional activity at endogenous loci. The natural preference of adeno-associated viruses (AAVs) for integration at the AAVS1 site on chromosome 19 could potentially provide an alternative to the semi-random integration profile of lentiviral and retroviral vectors29. However, their small transgene capacity limits the usefulness of AAVs as gene therapy vectors30–32. Hybrid strategies combining the site-selective recombinase activity of the AAV rep protein with larger vectors have the potential to overcome this limitation. Based on this principle, we have previously achieved targeted integration of a bacterial artificial chromosome carrying the whole human β-globin locus into the AAVS1 site in K562 cells33. Another approach, gene repair through homologous recombination, has been proposed already in the 1980s34,35. In 1985, Smithies et al. demonstrated the introduction of heterologous DNA sequences into the β-globin locus of human cell lines using homologous recombination36. These results led to the first speculation that targeted genome modification via homologous recombination in HSCs could provide a cure for β-hemoglobinopathies. However, before the emergence of targeted endonucleases, this approach remained limited by low efficiency.\n\n\nGenome editing\n\nThe discovery and development of targetable endonucleases has kindled a new enthusiasm for the previously niche area of genome modification through homologous repair. These enzymes can be engineered to introduce a site-specific double-strand break (DSB) into a target genome, which can subsequently be repaired by endogenous DNA repair mechanisms (Figure 1). Mammalian cells possess two major DSB repair pathways: non-homologous end-joining (NHEJ) and homology-directed repair (HDR)37,38. NHEJ is error prone and leads to the creation of small insertions or deletions at the DSB site. This has been used very efficiently for targeted gene knockout in a variety of cell types and for the generation of knockout animal models39–44. HDR uses a homologous DNA template to repair the broken strand with high fidelity. Fusion of a reporter to a gene of interest and gene insertion, as well as targeted gene correction, have been demonstrated using this approach45–47.\n\nZFNs, TALENs, and CRISPR/Cas9 are used to introduce site-specific DSBs into a target genome. Subsequently, cellular repair mechanisms can be harnessed to introduce precise genetic modifications. Small insertions and deletions generated by NHEJ can be used for gene knockout. In the presence of a homologous repair template, new sequences can be incorporated via HDR, allowing for gene repair, transgene insertion, and gene replacement.\n\nThere are three different types of programmable endonucleases. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are generated by the fusion of repetitive arrays of specific DNA-binding amino acid motifs to a FokI endonuclease domain (Figure 1)48–50. Binding of a pair of ZFNs or TALENs on opposite DNA strands allows their FokI domains to dimerize and become catalytically active, introducing a DSB with 5’ overhangs into the target site. ZFNs and TALENs both require complicated cloning approaches to achieve the arrangement of repetitive motifs, imposing penalties of time and expense upon their development. The recent adaptation of clustered-interspaced short palindromic repeats (CRISPRs) and CRISPR-associated protein 9 (Cas9) for use in mammalian cells has greatly facilitated genome editing applications. The Cas9 endonuclease is targeted to a specific DNA sequence by a complementary 20-nucleotide sequence in a guide RNA (gRNA) bound to the Cas9 protein. New gRNAs can be generated quickly and at low cost using standard cloning techniques51,52. Due to the ease of use of the Cas9 system, its use has rapidly surpassed that of ZFNs and TALENs in the past years (Figure 2).\n\nData obtained from Medline trend using the search terms “CRISPR Cas9”, “Zinc-finger nuclease”, and “TALEN” show an increase in the use of programmable endonucleases during this period99.\n\nSimilar to lentiviral gene therapy, genome editing could be used to correct patient HSCs ex vivo for the gene therapy of β-thalassemia. Ideally, scarless correction of the β-globin gene in HSCs could be achieved through HDR, resulting in the production of healthy erythrocytes. Several studies have shown that the human β-globin locus is amenable to genome editing (Table 1)53–61. However, technical limitations and safety concerns need to be overcome for this novel approach to become clinically applicable.\n\n(iPSCs: induced pluripotent stem cells).\n\nIn contrast to viral gene addition approaches, genome editing does not require the use of integrating vectors, as transient expression of a targeted endonuclease is sufficient to achieve the necessary DNA cleavage. This eliminates the issue of insertional mutagenesis. However, off-target cleavage at sites other than that intended is a major concern with genome editing approaches63–65. For the CRISPR/Cas9 system, strategies have been developed to reduce the relatively high off-target cleavage associated with wild-type Cas9. A mutated Cas9 protein that introduces a single-stranded nick rather than a DSB can be used to increase cleavage specificity. Consequently, two gRNAs designed to mediate nicking on opposite strands at the target site are required to form a DSB63. However, a single gRNA is still sufficient to introduce a DNA nick at off-target sites, which may have adverse effects in the target cell. Alternatively, an inactive Cas9 protein can be fused to FokI, which only becomes enzymatically active upon dimerization. With this approach, two Cas9/FokI hybrid units need to be brought together by specific gRNAs to allow cleavage at the target site66,67. The target specificity of Cas9 can be further increased through the use of a truncated guide sequence of 17 instead of 20 nucleotides68. However, off-target activity of any nuclease type still varies between different genomic targets and cell types69,70. Therefore, as with all gene therapy strategies, careful vector design and thorough evaluation of risks is necessary.\n\nOff-target site prediction tools that rank potential unintended cleavage sites based on similarity scores were developed to facilitate the evaluation of cleavage stringency for different nuclease platforms. It remains to be determined if the targeted analysis of selected putative off-target sites is sufficient for the determination of nuclease-associated risks. Further validation of the reliability of these prediction tools via unbiased genome-wide detection of off-target cleavage is therefore required. Approaches taking advantage of the occasional capture of foreign sequences in genomic DSBs show promise to close this information gap71–73. Many studies report minimal to no detectable off-target activity across a variety of nuclease platforms and target sites60,66,67,74–77. A 2015 publication using ZFNs to correct the sickle-cell mutation in primary patient HSCs indicates that therapeutic genome editing of the β-globin gene can be achieved without producing deleterious unintended mutations. The only off-target events detected in a genome-wide analysis were located in the highly homologous δ-globin gene, which is non-essential60. Also, the first clinical phase I human genome editing trial using ZFNs to disrupt the CCR5 co-receptor for HIV entry in autologous CD4 T cells has not produced any adverse events that could be attributed to the use of ZFNs78. While further confirmation is still required, these findings suggest that off-target effects will not restrict genome editing from clinical applications.\n\nGene therapy trials for SCID are simplified due to the selective advantage of gene corrected cells over unmodified HSCs11. In the case of the β-hemoglobinopathies, β-globin expression does not convey an advantage for HSCs. Consequently, a substantial fraction of HSCs needs to be modified to achieve a therapeutic effect. Lentiviral or retroviral delivery and nucleofection of DNA or mRNA can achieve transfection rates greater than 80% in primary human HSCs79–81. These methods are also suitable for the delivery of genome editing tools. A high transduction efficiency, leading to a high frequency of target cleavage, is essential for efficient genome editing. However, low HDR frequency in naïve HSCs, accompanied by a background of disruptive NHEJ, currently impedes the generation of therapeutic levels of edited cells60,82. Although NHEJ is unlikely to produce adverse effects in an already non-functional gene, it will be crucial to increase the fraction of cells that undergo HDR genome editing to be successful in the clinic. Several groups have developed screening methods that permit simultaneous quantification of NHEJ and HDR83–87. These can be used for the identification of conditions that favor HDR. Most notably, inhibition of DNA ligase 4, which is required for the NHEJ pathway, has been shown to not only decrease NHEJ but also increase HDR frequencies in cell lines and mouse embryos88,89. As the repair pathway choice in a cell is largely dependent on the cell cycle stage, cell synchronization and timed nuclease delivery could also bias cells towards HDR90. Increasing the frequency of gene correction in HSCs will be crucial in determining the feasibility of therapeutic genome editing in the hematopoietic system.\n\n\nA future in the clinic\n\nAlthough low HDR efficiency and safety concerns regarding off-target effects are currently obstructing the therapeutic application of genome editing, strategies to resolve these limitations are rapidly progressing. As with all novel therapeutics, every custom genome editing vector will be subject to careful clinical trials. It is therefore crucial to design therapeutic genome editing strategies to be as inclusive as possible, i.e. to minimize the number of different vectors required to treat the maximum number of patients. While over 200 mutations are known to cause β-thalassemia, a relatively small number of mutations account for the majority of cases91. Therefore, a small number of Cas9/gRNA vectors could be sufficient to address the majority of patients. Alternatively, the introduction of two DSBs at either side of the β-globin gene could allow for gene replacement without the need for allele-specific vectors, thus placing a therapeutic β-globin under the control of endogenous regulatory elements at the β-globin locus53,92. Like lentiviral gene therapy, genome editing can also be applied to gene addition. A single genome editing vector targeting a safe harbor site could be combined with a separate HDR template containing a therapeutic β-globin gene. This approach has the potential to provide a universally applicable strategy, as a single genome editing vector could be used for a large range of monogenic disorders by simply exchanging the HDR template. Genome editing also has the potential to introduce mutations that modify the severity of β-thalassemia. It is known from individuals with hereditary persistence of fetal hemoglobin that elevated expression of γ-globin, a developmentally silenced β-globin-like gene, can be protective of the pathologic effects associated with the absence of β-globin expression93,94. Replication of this phenotype through genome editing could therefore alleviate the symptoms in β-thalassemic patients. A recent study employed TALENs to introduce a single point mutation within the β-globin locus to increase the expression of γ-globin59. Interference with the expression of BCL11A, a major regulator of β-globin gene expression, has also been shown to promote the expression of γ-globin95,96. An erythroid-specific enhancer for BCL11A expression was recently identified by Bauer et al.97. Targeted elimination of this enhancer in patient-derived HSCs could allow the induction of γ-globin expression in erythroid cells without affecting BCL11A-dependent processes in other lineages98. This could be achieved through an NHEJ approach, unimpeded by the low frequency that currently limits strategies depending on HDR. However, in the future, the latter could be applied to the correction of patient-derived iPSCs, thus circumventing the issue of HDR efficiency, since a large number of cells can be generated from a few corrected clones. With this range of possibilities, genome editing is diversifying gene therapy research with the potential to greatly relieve the global health burden of the β-hemoglobinopathies.\n\n\nAbbreviations\n\nAAV, adeno-associated virus; BMT, bone marrow transplantation; Cas9, CRISPR-associated protein 9; CRISPRs, clustered interspaced palindromic repeats; DSB, double strand break; gRNA, guide RNA; HDR, homology-directed repair; HSCs, hematopoietic stem cells; iPSCs induced pluripotent stem cells; NHEJ, non-homologous end-joining; TALENs, transcription activator-like effector nucleases; SCID, severe combined immune deficiency; ZFNs, zinc-finger nucleases",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no disclosures.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe National Health and Medical Research Council, the Murdoch Childrens Research Institute, the Victorian Government’s Operational Infrastructure Support Program, and Thalassaemia Australia.\n\n\nReferences\n\nRibeil JA, Arlet JB, Dussiot M, et al.: Ineffective erythropoiesis in β -thalassemia. ScientificWorldJournal. 2013; 2013: 394295. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLanzkowsky P: Manual of pediatric hematology and oncology. 2011; 5. Reference Source\n\nKing A, Shenoy S: Evidence-based focused review of the status of hematopoietic stem cell transplantation as treatment of sickle cell disease and thalassemia. Blood. 2014; 123(20): 3089–94; quiz 3210. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nFu Y, Foden JA, Khayter C, et al.: High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31(9): 822–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCradick TJ, Fine EJ, Antico CJ, et al.: CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 2013; 41(20): 9584–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRan FA, Hsu PD, Lin CY, et al.: Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013; 154(6): 1380–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTsai SQ, Wyvekens N, Khayter C, et al.: Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014; 32(6): 569–76. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFu Y, Sander JD, Reyon D, et al.: Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014; 32(3): 279–84. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSmith C, Gore A, Yan W, et al.: Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell. 2014; 15(1): 12–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeres A, Gosis BS, Ding Q, et al.: Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell. 2014; 15(1): 27–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang X, Wang Y, Wu X, et al.: Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol. 2015; 33(2): 175–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nO'Geen H, Henry IM, Bhakta MS, et al.: A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 2015; 43(6): 3389–404. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCorrigan-Curay J, O'Reilly M, Kohn DB, et al.: Genome editing technologies: defining a path to clinic. Mol Ther. 2015; 23(5): 796–806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi L, Krymskaya L, Wang J, et al.: Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases. Mol Ther. 2013; 21(6): 1259–69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMussolino C, Alzubi J, Fine EJ, et al.: TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. 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Stem Cells Dev. 2006; 15(2): 278–85. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWiehe JM, Ponsaerts P, Rojewski MT, et al.: mRNA-mediated gene delivery into human progenitor cells promotes highly efficient protein expression. J Cell Mol Med. 2007; 11(3): 521–30. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLeurs C, Jansen M, Pollok KE, et al.: Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells. Hum Gene Ther. 2003; 14(6): 509–19. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGenovese P, Schiroli G, Escobar G, et al.: Targeted genome editing in human repopulating haematopoietic stem cells. Nature. 2014; 510(7504): 235–40. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMaruyama T, Dougan SK, Truttmann MC, et al.: Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015; 33(5): 538–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLin S, Staahl BT, Alla RK, et al.: Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife. 2014; 3: e04766. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWeatherall DJ, Clegg JB: The Thalassaemia Syndromes. Blackwell Science Ltd., 2001; 4. Publisher Full Text\n\nByrne SM, Ortiz L, Mali P, et al.: Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res. 2015; 43(3): e21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThein SL: Genetic modifiers of beta-thalassemia. Haematologica. 2005; 90(5): 649–60. PubMed Abstract\n\nGalanello R, Cao A: Relationship between genotype and phenotype. Thalassemia intermedia. Ann N Y Acad Sci. 1998; 850: 325–33. PubMed Abstract | Publisher Full Text\n\nSankaran VG, Menne TF, Xu J, et al.: Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science. 2008; 322(5909): 1839–42. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSankaran VG, Xu J, Ragoczy T, et al.: Developmental and species-divergent globin switching are driven by BCL11A. Nature. 2009; 460(7259): 1093–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBauer DE, Kamran SC, Lessard S, et al.: An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science. 2013; 342(6155): 253–7. 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}
|
[
{
"id": "11529",
"date": "11 Dec 2015",
"name": "Alan Schechter",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11530",
"date": "11 Dec 2015",
"name": "Donald Kohn",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1431
|
https://f1000research.com/articles/4-1427/v1
|
11 Dec 15
|
{
"type": "Review",
"title": "The lighthouse at the end of the chromosome*",
"authors": [
"Yahya Benslimane",
"Lea Harrington",
"Yahya Benslimane"
],
"abstract": "Fluorescence microscopy can be used to assess the dynamic localization and intensity of single entities in vitro or in living cells. It has been applied with aplomb to many different cellular processes and has significantly enlightened our understanding of the heterogeneity and complexity of biological systems. Recently, high-resolution fluorescence microscopy has been brought to bear on telomeres, leading to new insights into telomere spatial organization and accessibility, and into the mechanistic nuances of telomere elongation. We provide a snapshot of some of these recent advances with a focus on mammalian systems, and show how three-dimensional, time-lapse microscopy and single-molecule fluorescence shine a new light on the end of the chromosome.",
"keywords": [
"Fluorescence microscopy",
"telomeres",
"telomere elongation",
"telomere spatial organization",
"time-lapse microscopy",
"single-molecule fluorescence",
"chromosome"
],
"content": "Introduction\n\nOskar Heimstädt, who built the first fluorescence microscope, ended his 1911 paper with the following perspective: “If and to what degree fluorescence microscopy will widen the possibilities of microscopic imaging only the future will show”1. More than a century later, fluorescence microscopy has proven transformative in our ability to illuminate almost all aspects of cellular biology. One of the more recent frontiers in fluorescence microscopy is the resolution of biological phenomena at the single molecule level, called single molecule fluorescence2. Biological processes have evolved to be inherently heterogeneous, transient and dynamic3, and therefore difficult to track. Single molecule microscopy often permits a birds-eye view of ephemeral and complex mechanisms. This review will focus on selected recent advances in high-resolution microscopy including, but not limited to, single-molecule fluorescence microscopy, that have enlightened our understanding of chromosome ends and the enzyme that replenishes them.\n\nMany organisms must contend with the vulnerability of linear chromosome ends to enzymes that degrade, rearrange, or incompletely replicate DNA. This susceptibility to breakage, and their distinct “knob-like” appearance under the light microscope, made them an early target of study by scientists such as Barbara McClintock and Hermann Müller4. We now appreciate that telomeres are a highly specialized nucleoprotein structure whose maintenance is critical to genome stability (Figure 1A–C)5,6. Replenishment of the G-rich sequences that comprise the telomeres is carried out by telomerase, whose core components are a reverse transcriptase and an integral RNA that provides the telomere template (Figure 1D)7.\n\n(A) In many organisms, chromosome ends terminate in a single-stranded, G-rich overhang preceded by up to several kilobase pairs of double-stranded G-rich DNA. (B) Telomeres are capped by a six-subunit complex called shelterin. (C) Shelterin (particularly TRF2) promotes the formation of a higher order telomeric loop (T-loop) structure that serves to mask telomeres from the deleterious fates associated with a free DNA end5,6. (D) The catalytic cycle of the core telomerase enzyme, comprised of a protein (TERT) and RNA (hTR)7.\n\n\nZooming in on telomeres\n\nFluorescence microscopy has revolutionized our ability to probe the length, location, and recombination of telomeres in vivo through the application of fluorescently labeled peptide nucleic acids that bind tightly and specifically to telomeric DNA8–10. These techniques have uncovered interesting distinctions in the three-dimensional (3D) localization of mammalian telomeres in normal cells versus cancer cells11,12. Time-lapse microscopy has also revealed increased mobility of telomeres upon induction of a DNA break13 as well as unexpected long-range telomere associations in telomerase-negative cells after DNA breakage14. In the budding yeast Saccharomyces cerevisiae, fluorescent tagging of several components of telomerase have also provided considerable insight into the temporal and spatial dynamics of telomerase recruitment in living cells15–18.\n\nFluorescence microscopy is also being combined with other leading-edge technologies to directly target specific genomic regions, including the telomere. Using the CRISPR/Cas system in which Cas9 can be guided to specific genomic loci using a small RNA19, Chen and colleagues targeted an enhanced green fluorescent protein (EGFP)-tagged catalytically inert Cas9 specifically to telomeres20. Telomere-recruited Cas9 demonstrated a punctate pattern that spatially overlapped with that of the telomeric DNA binding protein, telomeric-repeat binding factor 2 (TRF2). Furthermore, EGFP intensity correlated linearly with telomere intensities obtained using fluorescence in situ hybridization (FISH) (Figure 2A)20. This method could also be employed to visualize Cas9 directed to a single genomic locus20. Technological advances such as these may soon permit the ability to explore the spatial and dynamic localization of telomeres and telomerase in living mammalian cells to a similar extent as has been explored in budding yeast.\n\n(A) The use of a fluorescently tagged Cas9 and a guide RNA specific to telomeres to measure telomere dynamics and length in live cells20. (B) The ability of single-molecule Förster resonance energy transfer (smFRET) to measure the dynamics of G-quadruplex folding27,30–36,38. (C) The application of fluorescent probes complementary to telomeric substrates to measure the elongation properties of telomerase47. (D) The application of FRET to assess the intermolecular proximity of the DNA substrate and the RNA subunit of telomerase during telomere synthesis48.\n\nThe telomere terminus ends in a 3’ G-rich overhang of variable length that can invade the upstream double-stranded telomeric DNA to form a structure called a T-loop21. Super-resolution microscopy, specifically stochastic optical reconstruction microscopy (STORM), has recently enabled an unprecedented visualization of T-loops that are crosslinked and purified from murine cells22 (Figure 1B,C). The loss of T-loops occurred specifically upon loss of the shelterin subunit TRF2, and not other shelterin components, which provides an elegant demonstration that it is the T-loop structure that protects ends from the deleterious molecular events observed when TRF2 is removed from telomeres22,23.\n\nTelomeric DNA sequences can also form in vitro intra- or inter-parallel structures called G-quadruplexes, and evidence is accruing to suggest their existence at telomeres and at interstitial G-rich regions24. Single-molecule Förster resonance energy transfer (smFRET) has been applied to the molecular dynamics of G-quadruplex formation in vitro25–28 (Figure 2B). The smFRET approach has several advantages that permit a high-resolution view of G-quadruplex dynamics. Firstly, this technique can resolve different conformations using FRET signal intensities and Gaussian curve fitting29. Secondly, individual single-molecule traces reveal dynamic switching between states in real time. Hidden Markov modeling can then extract the dwell time of each molecule, which is a reflection of the stability of each state29. Applying smFRET to the analysis of shelterin binding to telomeric substrates in vitro, several groups have investigated the intricate relationship between G-quadruplexes and POT1/TPP1 (protection of telomere 1/Pot1-interacting protein TINT1-PTOP-PIP1)30,31. Furthermore, this technique also revealed the binding and unfolding of G-quadruplexes by proteins such as RAD51, WRN, BLM, RecQ and RPA32–36. Recently, the use of smFRET combined with magnetic tweezers spectroscopy37 has been used to measure the thermodynamic properties of G-quadruplex folding38. These findings provide important insights into the dynamics of telomeric DNA structure in a more native nucleoprotein context.\n\n\nShining a light on telomerase\n\nThe telomerase reverse transcriptase, TERT, is able to synthesize new telomeric DNA, one nucleotide at a time, by virtue of an integral telomerase RNA that contains a short telomere-complementary sequence (Figure 1D). Although TERT shares several features in common with other viral reverse transcriptases, one unique aspect is its ability to repeatedly copy the same template for many cycles in an iterative process termed repeat addition processivity (RAP)39,40. Although much information has been gleaned using standard biochemical techniques regarding the complex DNA-protein, RNA-protein and protein-protein interactions that contribute to telomerase RAP (for a few current examples, see 41–46), it is only recently that single-molecule fluorescence has been brought to bear on the elongation properties of telomerase in vitro47. Hwang and colleagues immobilized immunopurified telomerase from cell extracts on a surface and employed a fluorescently labeled probe complementary to telomeric DNA to obtain a digital readout of telomerase activity in real time (Figure 2C). Their results suggested that active telomerase can exist in two dynamic states defined as an initial activation period followed by an extension period in which telomere elongation was visualized as a step-wise increase in fluorescence47. In addition, they found that TPP1-POT1 stimulated the elongation rate and overall product length47, consistent with previously described properties of TPP1-POT1. Although the nature of the fluorescent probe binding (which comprises the sequence 5′-CCCTAACCCTAACCC-3′) precludes single base resolution, this technique promises to provide an unprecedented, single-molecule view of the intricacies of the telomerase elongation cycle.\n\nIn another smFRET approach, the distance between the region 5’ of the RNA template of telomerase and a DNA substrate was followed during telomeric repeat synthesis (Figure 2D)48. Using different combinations of regular and chain-terminating nucleotides, information about the position of the DNA oligonucleotide substrate was registered at single-base pair resolution during elongation. While the substrate appears in a compact conformation after initial binding by telomerase, after a round of DNA synthesis the substrate realigns with the template via Watson-Crick base pair interactions. The aforementioned study by Parks and colleagues suggests that DNA dissociation and realignment is not the limiting step, and that an additional conformational adjustment is necessary after DNA:RNA repositioning to re-acquire a catalytically competent state48. This study illustrates the power of non-linear Gaussian curve fitting and hidden Markov modeling to extract the dwell time of the different FRET states from individual traces, which in turn permits a very precise dissection of the catalytic mechanism, albeit only over the short distances in which FRET can be observed.\n\nsmFRET has also been used to evince the real-time dynamics of folding of the pseudoknot domain with the telomerase RNA49–51. It has also been applied to examine the assembly and activity of the Tetrahymena thermophila telomerase RNP51–53 and, more recently, the role of the N-terminal domain of T. thermophila TERT in the stabilization of short RNA:DNA hybrids during telomerase catalysis54. In addition to valuable insights into telomerase catalysis, these techniques might also permit a precise elucidation of the mechanism-of-action of chemical modulators of telomerase activity, as well as an in vivo determination of human telomerase component stoichiometry, as was recently demonstrated for budding yeast telomerase18.\n\n\nFuture perspectives\n\nIn the year in which fluorescence microscopy was first described, Arthur Brisbane offered the sage advice: “Use a picture. It’s worth a thousand words.”55. We have reached a technological watershed in biology that will enable an unprecedented single-molecule and high-resolution view of the inner workings of many cellular machines. As we have illustrated here with but a few selected examples, fluorescence microscopy can be applied in many different ways to different problems, but key advances are the ability to dissect individual events instead of ensemble, population-based outputs, and to permit dynamic measurements in living cells, in real time. How far we have come from seeing telomeres as a cytogenetic “knob”, and how far we have yet 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\nAcknowledgments\n\nWe thank Paul Maddox, Tracy Bryan, Michael Stone, and Raymund Wellinger for comments and constructive input on the review.\n\n\nReferences\n\nHeimstadt O: Das Fluoreszenzmikroskop. Z Wiss Mikrosk. 1911; 28: 330–7.\n\nHaustein E, Schwille P: Single-molecule spectroscopic methods. Curr Opin Struct Biol. 2004; 14(5): 531–40. PubMed Abstract | Publisher Full Text\n\nPross A: On the emergence of biological complexity: life as a kinetic state of matter. Orig Life Evol Biosph. 2005; 35(2): 151–66. PubMed Abstract | Publisher Full Text\n\nBlackburn EH: A History of Telomere Biology. In: de Lange T, Lundblad V, Blackburn EH, editors. Telomeres. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 2006; 1–19. 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PubMed Abstract | Publisher Full Text\n\nOkumus B, Ha T: Real-time observation of G-quadruplex dynamics using single-molecule FRET microscopy. Methods Mol Biol. 2010; 608: 81–96. PubMed Abstract | Publisher Full Text\n\nLong X, Stone MD: Kinetic partitioning modulates human telomere DNA G-quadruplex structural polymorphism. PLoS One. 2013; 8(12): e83420. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDhakal S, Cui Y, Koirala D, et al.: Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Res. 2013; 41(6): 3915–23. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBlanco M, Walter NG: Analysis of complex single-molecule FRET time trajectories. Methods Enzymol. 2010; 472: 153–78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang H, Buncher N, Opresko PL, et al.: POT1-TPP1 regulates telomeric overhang structural dynamics. Structure. 2012; 20(11): 1872–80. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRay S, Bandaria JN, Qureshi MH, et al.: G-quadruplex formation in telomeres enhances POT1/TPP1 protection against RPA binding. Proc Natl Acad Sci U S A. 2014; 111(8): 2990–5. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHwang H, Kreig A, Calvert J, et al.: Telomeric overhang length determines structural dynamics and accessibility to telomerase and ALT-associated proteins. Structure. 2014; 22(6): 842–53. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBudhathoki JB, Ray S, Urban V, et al.: RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP. Nucleic Acids Res. 2014; 42(18): 11528–45. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBudhathoki JB, Stafford EJ, Yodh JG, et al.: ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity. Nucleic Acids Res. 2015; 43(12): 5961–70. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nQureshi MH, Ray S, Sewell AL, et al.: Replication protein A unfolds G-quadruplex structures with varying degrees of efficiency. J Phys Chem B. 2012; 116(19): 5588–94. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRay S, Qureshi MH, Malcolm DW, et al.: RPA-mediated unfolding of systematically varying G-quadruplex structures. Biophys J. 2013; 104(10): 2235–45. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWoodside MT, Block SM: Reconstructing folding energy landscapes by single-molecule force spectroscopy. Annu Rev Biophys. 2014; 43: 19–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLong X, Parks JW, Bagshaw CR, et al.: Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy. Nucleic Acids Res. 2013; 41(4): 2746–55. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGreider CW: Telomerase is processive. Mol Cell Biol. 1991; 11(9): 4572–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeng Y, Mian IS, Lue NF: Analysis of telomerase processivity: mechanistic similarity to HIV-1 reverse transcriptase and role in telomere maintenance. Mol Cell. 2001; 7(6): 1201–11. PubMed Abstract | Publisher Full Text\n\nBerman AJ, Akiyama BM, Stone MD, et al.: The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis. Nat Struct Mol Biol. 2011; 18(12): 1371–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQi X, Xie M, Brown AF, et al.: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency. EMBO J. 2012; 31(1): 150–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteczkiewicz K, Zimmermann MT, Kurcinski M, et al.: Human telomerase model shows the role of the TEN domain in advancing the double helix for the next polymerization step. Proc Natl Acad Sci U S A. 2011; 108(23): 9443–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTomlinson CG, Moye AL, Holien JK, et al.: Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding. Biochem J. 2015; 465(2): 347–57. PubMed Abstract | Publisher Full Text\n\nWu RA, Collins K: Human telomerase specialization for repeat synthesis by unique handling of primer-template duplex. EMBO J. 2014; 33(8): 921–35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDalby AB, Hofr C, Cech TR: Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase. J Mol Biol. 2015; 427(6 Pt B): 1291–303. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang H, Opresko P, Myong S: Single-molecule real-time detection of telomerase extension activity. Sci Rep. 2014; 4: 6391. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nParks JW, Stone MD: Coordinated DNA dynamics during the human telomerase catalytic cycle. Nat Commun. 2014; 5: 4146. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHolmstrom ED, Nesbitt DJ: Single-molecule fluorescence resonance energy transfer studies of the human telomerase RNA pseudoknot: temperature-/urea-dependent folding kinetics and thermodynamics. J Phys Chem B. 2014; 118(14): 3853–63. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHengesbach M, Kim NK, Feigon J, et al.: Single-molecule FRET reveals the folding dynamics of the human telomerase RNA pseudoknot domain. Angew Chem Int Ed Engl. 2012; 51(24): 5876–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMihalusova M, Wu JY, Zhuang X: Functional importance of telomerase pseudoknot revealed by single-molecule analysis. Proc Natl Acad Sci U S A. 2011; 108(51): 20339–44. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nStone MD, Mihalusova M, O'connor CM, et al.: Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein. Nature. 2007; 446(7134): 458–61. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWu JY, Stone MD, Zhuang X: A single-molecule assay for telomerase structure-function analysis. Nucleic Acids Res. 2010; 38(3): e16. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nAkiyama BM, Parks JW, Stone MD: The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA-DNA hybrids. Nucleic Acids Res. 2015; 43(11): 5537–49. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBrisbane A: Speakers Give Sound Advice. Syracuse Post Standard. 1911."
}
|
[
{
"id": "11519",
"date": "11 Dec 2015",
"name": "Raymund Wellinger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11520",
"date": "11 Dec 2015",
"name": "Tracy Bryan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11521",
"date": "11 Dec 2015",
"name": "Michael Stone",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1427
|
https://f1000research.com/articles/4-1426/v1
|
11 Dec 15
|
{
"type": "Review",
"title": "Recent advances in the management of acromegaly",
"authors": [
"Georgia Ntali",
"Niki Karavitaki",
"Georgia Ntali"
],
"abstract": "Acromegaly is a rare condition of GH excess associated with significant morbidities (e.g. hypertension, glucose intolerance or diabetes mellitus, cardiac, cerebrovascular, respiratory disease and arthritis) and, when uncontrolled, high mortality. Surgery, medical treatment and radiotherapy remain our therapeutic tools. Advances in these options during the last years have offered further perspectives in the management of patients and particularly those with challenging tumours; the impact of these on the long-term morbidity and mortality remains to be assessed.",
"keywords": [
"Acromegaly",
"management",
"somatostatin analogues",
"adenomectomy",
"radiotherapy"
],
"content": "Introduction\n\nAcromegaly is a rare condition characterized by growth hormone (GH) excess and elevated insulin growth factor 1 (IGF-1) levels attributed in the vast majority of cases to a pituitary adenoma. Mortality is high in uncontrolled disease (standardized mortality ratio [SMR]: 0.94–2.5)1–3, but adequate biochemical control can restore it to normal (SMR: 0.44–1.13)1–3. Early diagnosis and treatment, close monitoring, and control of co-morbidities play a major role in the optimal management of these patients. Based on the recently published Endocrine Society Clinical Practice Guideline on acromegaly, therapeutic goals include an age-normalized serum IGF-1 value and a random GH level <1 μg/L4. Surgery, drugs, and radiotherapy remain the mainstay of treatment, and this review will focus on recent advances in the management of this challenging condition.\n\n\nSurgery\n\nIn the last decade, endoscopic endonasal surgery, by offering improved visualization and less nasal trauma, is more widely utilized, replacing the traditional microscopic technique. Based on the 2010 consensus criteria, remission rates after endoscopic transsphenoidal surgery range between 63 and 100% for microadenomas5–10 and between 40 and 72% for macroadenomas5–10; these are comparable to those reported with microsurgical approaches. Peri-operative complications are also similar, apart from sinusitis and alterations in taste or smell, which are described more often in patients managed endoscopically7,11.\n\n\nMedical therapy\n\nSomatostatin downregulates GH secretion and induces cell cycle arrest and apoptosis through binding to five subtypes (somatostatin receptors 1–5 [SSTR1-SSTR5]) of G-protein-coupled receptors expressed on the somatotroph cells. Somatotroph adenomas express somatostatin receptor subtype 2 (SSTR2) and SSTR5 at high levels, and somatostatin analogues (SSAs) with greater specificity for these receptors and a longer half-life (than the 2–3 minutes of the natural somatostatin molecule) have been developed (octreotide and lanreotide).\n\nSomatostatin analogues as pre-operative medical therapy. Acromegalic patients present a challenge peri-operatively, as their airway anatomy and cardiovascular co-morbidities increase the risk of anesthetic complications12,13. Pre-operative treatment with SSAs is not recommended routinely4, but in selected cases it may rapidly reduce soft tissue swelling, improve sleep apnea and cardiac function, and reduce intubation-related complications13–16. A recent meta-analysis of five retrospective controlled, two prospective non-randomized, and three prospective randomized controlled studies explored the role of pre-operative use of SSAs in improving biochemical cure rate after surgery; the results were consistent with a borderline significant effect (pooled odds ratio [OR] 1.62; 95% confidence interval [CI] 0.93–2.82). When only the three randomized prospective controlled trials were analyzed, a significant benefit was found with a pooled OR of 3.62 (95% CI 1.88–6.96)17.\n\nIn a group of 30 newly diagnosed acromegalics, pre-surgical treatment with lanreotide autogel for 24 weeks induced tumor shrinkage ≥20% in 79% (23/29) and resulted in mean GH <1 μg/L and IGF-1 normalization in 33.3% (10/33) of the patients. Metabolic profile including fasting blood glucose, HbA1c, lipids, and blood pressure did not change significantly, but amelioration of arterial stiffness and endothelial function were documented. Notably, the apnea/hypopnea index improved in 61%, remained unchanged in 8.7%, and deteriorated significantly in 30.4% of the patients18.\n\nFougner et al. evaluated the impact of pre-operative octreotide treatment on long-term remission. When both remission criteria of IGF-1 levels ≤ upper limit of normal (ULN) and nadir GH ≤2 mU/L on the oral glucose tolerance test were applied, no beneficial effect was confirmed 1 and 5 years post-operatively for both microadenoma and macroadenoma subgroups19.\n\nSomatostatin analogues as primary therapy. The 2014 Endocrine Society Clinical Practice Guideline recommends that primary therapy with SSAs is principally used for a subgroup of patients with larger tumors when surgical cure is unlikely and, additionally, if surgery is refused or contraindicated4.\n\nPrimary therapy with lanreotide autogel was evaluated in a prospective 48-week multicenter study which recruited 90 naive acromegalics with macroadenomas. Tumor shrinkage ≥20% was observed in 54.1%, 56.3%, and 62.9% of patients at 12, 24, and 48 weeks, respectively, and mean GH ≤1.0 μg/L and IGF-1 normalization was reported in 21.4%, 23.4%, and 30.6% of patients at the same time intervals20.\n\nFurthermore, a meta-analysis of 35 studies on treatment-naive acromegalics showed that in comparison with medical treatment, surgery was associated with higher remission rates at longer follow-up periods (≥24 months) but not at shorter follow-up intervals (≤6 months)21.\n\nThe effect of different treatment modalities on mortality rates was evaluated in 438 acromegalic patients for the period 1966–2009 by Bogazzi et al.22. Interestingly, after correction for several covariates, the risk of death in patients receiving primary SSA therapy was five times higher than the risk in all patients submitted to adenomectomy (hazard ratio [HR]: 5.52, 95% CI 1.06–28.77, p=0.043). Stratification of patients by the presence of diabetes mellitus revealed that diabetic patients receiving primary SSA therapy had a significantly elevated HR of 21.94 (95% CI 1.56–309.04, p=0.022), whereas in non-diabetic ones the HR was only 1.30 (95% CI 0.04–38.09, p=0.881)22.\n\nSomatostatin analogues as adjuvant medical therapy. SSAs are a treatment option for patients with significant persistent disease after surgery4. Adenoma GH granularity and SSTR2A-positive immunohistochemistry may predict the response with the densely granulated and those expressing SSTR2A being more responsive23–27. Sparsely granulated adenomas exhibit lower SSTR2 expression and respond less to somatostatin receptor ligands (SRLs)25,27; it is presumed that, if SSTR5 expression is present, response to novel SSAs may be the case28. It has also been reported that Ki-67 is higher in non-SSA responders29,30.\n\nT2 intensity on magnetic resonance imaging (MRI) has also been proposed as a marker of responsiveness. Hypointense T2-weighted MRI signal is associated with a better responsiveness to octreotide than an isointense or hyperintense one in patients not cured after surgery31. Notably, it has been suggested that hyperintense tumors have lower baseline GH and IGF-1 levels and tend to be larger than the hypointense ones. T2 intensity and granulation pattern correlate and sparsely granulated adenomas tend to be hyperintense32,33.\n\nEffectiveness of somatostatin analogues. A recent meta-analysis on the biochemical response rates to SSAs as primary or adjuvant therapy – including retrospective and prospective studies with both short- and long-acting octreotide and lanreotide formulations (sustained-release and depot/autogel) – between 1987 and 2012 (total of 3787 patients) showed that the average GH control rate was 56% and the IGF-1 normalization rate was 55% with no significant difference in the effectiveness between the different SSA agents34.\n\nFurthermore, a meta-analysis of 41 studies on the effects of octreotide (as first-line or adjuvant therapy) on tumor shrinkage revealed that overall 57% of patients achieved >20% volume decrease. Tumor shrinkage was greater in patients treated with octreotide long-acting release (LAR) rather than subcutaneous octreotide and in those achieving mean GH levels below 2.0–2.5 ng/ml or normal IGF-135.\n\nThe option of discontinuing octreotide in patients well-controlled on low doses offered at long intervals was studied in 12 responders fulfilling the following criteria: single basal GH <1.5 ng/ml and IGF-1 <1.2*ULN, no history of irradiation or recent dopamine agonist use, and tumor remnant <8 mm after discontinuation of octreotide. Five patients (41.7%) remained in remission after 12 months of follow-up and seven (58.3%) relapsed within 1 year of discontinuation. Interestingly, three out of five subjects who remained in remission were on octreotide LAR injections every 12 weeks but none of the seven who recurred36.\n\nFinally, a number of studies have shown that surgical debulking of the adenoma improves the subsequent response to SSAs37–39.\n\nOral octreotide\n\nRecently, oral octreotide capsules (OOCs) have become available. The capsule contains 20 mg non-modified octreotide acetate formulated with a novel transient permeability enhancer, which enables transient and reversible paracellular tight junction passage of molecules <70 kDa. This new formulation facilitates intestinal octreotide absorption.\n\nOOC effectiveness in maintaining biochemical response to injectable SSAs has been assessed in a phase III multicenter, open-label, dose-titration, baseline-controlled study. A total of 155 complete or partially controlled patients (IGF-1 <1.3*ULN, and 2-h integrated GH <2.5 ng/mL) receiving injectable SSAs for ≥3 months were switched to 40 mg daily OOCs; the dose was escalated to 60 mg and then up to 80 mg daily. Subsequent fixed doses were maintained for a 7-month core treatment, followed by a voluntary 6-month extension. A total of 65% of the participants maintained response and achieved the primary endpoint of IGF-1 <1.3*ULN and mean GH <2.5 ng/mL at the end of the core treatment period; this rate was 62% after 13 months’ treatment40. Further studies on the efficacy, safety, and convenience of this formulation are needed.\n\nPasireotide\n\nPasireotide is a novel multi-ligand SRL with high affinity for SSTR1, SSTR2, SSTR3, and particularly SSTR5. In a head-to-head comparison prospective study of pasireotide vs. octreotide in 358 medical treatment-naive patients, biochemical control (defined as mean GH <2.5 μg/L and normal for age IGF-1) at 12 months was achieved in a higher percentage of patients on pasireotide in both the de novo and the post-surgical groups (31.3% vs. 19.2%; p=0.007). Interestingly, the two drugs had the same effect on GH inhibition, but pasireotide was more effective in reducing IGF-141.\n\nThe PAOLA study randomly assigned 198 inadequately controlled patients (previously treated with 30 mg octreotide long-acting or 120 mg lanreotide autogel as monotherapy for 6 months or longer) to pasireotide 40 mg, pasireotide 60 mg, or continued treatment with octreotide or lanreotide. At 24 weeks, 15% of the patients of the pasireotide 40 mg group and 20% of the pasireotide 60 mg group achieved biochemical control; no patient in the sustained octreotide or lanreotide group achieved this goal (absolute difference from control group 15.4%, 95% CI 7.6–26.5, p=0.0006 for pasireotide 40 mg group, 20.0%, 95% CI 11.1–31.8, p<0.0001 for pasireotide 60 mg group)42.\n\nIt should be noted that the impact of pasireotide on blood glucose control remains a concern41,42 and careful monitoring of glycemic status is required. Pasireotide-associated hyperglycemia is related to reduced insulin secretion and incretin hormone responses, without changes in hepatic/peripheral insulin sensitivity43. Pasireotide has the highest affinity for SSTR5, which is known to play an important role in mediating insulin secretion. Dipeptidyl peptidase (DPP)-4 inhibitors (e.g. sitagliptin, vildagliptin, saxagliptin, and linagliptin) and glucagon-like peptide-1 (GLP-1) receptor agonists (e.g. liraglutide and exenatide) may be the most effective agents for controlling pasireotide-associated hyperglycemia44.\n\nPegvisomant is a human GH receptor antagonist which blocks the production of IGF-1. The latest analysis from the ACROSTUDY included 1288 patients and reported IGF-1 control in 63% of them after 5 years on pegvisomant45. Increase in adenoma size was found in 3.2% of the patients and increase in the hepatic enzymes >3 times the ULN in 2.5%; no case of liver failure was described. The current recommendations suggest monitoring liver function tests monthly for the first 6 months and 6-monthly thereafter with consideration of discontinuation of pegvisomant in case of >3-fold elevation of transaminases. As pegvisomant does not have a tumor-suppressive effect, it is proposed that imaging is performed at 6 and 12 months initially and if there is no size change at 1 year, then yearly imaging4.\n\nDopamine agonists are useful in patients with modest elevations of GH and IGF-1 levels with or without concomitant hyperprolactinemia. A meta-analysis (including 160 patients) estimated that cabergoline as single-agent therapy normalizes IGF-1 levels in 34% of acromegalics and as an add-on therapy to SSAs in 52% of them. This effect may occur even in patients with normoprolactinemia and it is dose independent46.\n\nClomiphene citrate – a selective estrogen receptor modulator with organ-specific estrogenic and antiestrogenic properties – has been tried as an “add on” therapy together with octreotide LAR and/or cabergoline in a group of 16 male acromegalics with IGF-1 above the ULN for at least 1 year despite the use of available medical therapies. At the end of the study, serum IGF-1 decreased by 41%, leading to normal values in 44% (7/16) of them47.\n\nThe combination of pegvisomant with cabergoline or SSAs has been reported in two small studies as a potential effective treatment option in patients with acromegaly not responsive to monotherapy. The addition of pegvisomant (10 mg daily for 12 weeks) to cabergoline (offered in a maximum dose of 0.5 mg once daily) reduced significantly the IGF-1 levels in comparison with either cabergoline alone (on a maximum dose of 0.5 mg once daily) or low-dose (10 mg daily) pegvisomant monotherapy; 68% of the patients achieved normal IGF-148. Furthermore, the addition of pegvisomant (median dose 60 mg weekly) in partially controlled acromegalics on high-dose SSAs normalized IGF-1 in 57.9% of them49.\n\n\nRadiotherapy\n\nBased on the recently published Endocrine Society Clinical Practice Guideline on acromegaly, radiotherapy is a third-line option offered if there is residual tumor mass following surgery and if medical therapy is unavailable, unsuccessful, or not tolerated4.\n\nModern stereotactic techniques (stereotactic radiosurgery [SRS] or fractionated stereotactic radiotherapy [FSRT]) deliver more localized irradiation and minimize the long-term toxicity. The volume of the target lesion and its proximity to sensitive structures dictate the choice of the radiotherapy technique. Particle radiation delivered as SRS or FSRT has been also applied successfully in the treatment of pituitary adenomas. The physical properties of proton irradiation can offer superior conformality in dose distribution when compared to photons, and the advantage becomes more apparent for large volumes50.\n\nMinniti et al. reviewed the results of five studies which used FSRT in 115 patients with acromegaly and showed that at a median follow-up of 54 months (range 28–80), the tumor control rate was 97%50. The 5-year biochemical remission of the disease was 42%. Results of 29 studies including 1215 patients treated with SRS showed that at a median follow-up of 50.6 months (range 6–114 months) tumor control was achieved in 98%. When the 2010 criteria were applied, the 5-year hormonal normalization rate assessed in nine studies which included 528 patients was 43%50.\n\nIn a study of 136 acromegalics who received gamma knife (GK) SRS and had a median follow-up 61.5 months, the actuarial remission rates (defined as a normal age- and gender-matched serum IGF-1 level and, in some patients, nadir GH level <1 ng/mL on oral glucose tolerance test, off any medication) at 2, 4, 6, and 8 years post-radiosurgery were 32%, 65%, 73%, and 83%, respectively. New pituitary hormone deficits occurred in 43 patients (32%) and risk factors were a margin dose >25 Gy and tumor volume >2.5 mL51,52.\n\nWattson et al. assessed the value of proton therapy in 50 patients with acromegaly and reported that the actuarial 3-year remission rate was 26% and the median time until remission was 62 months53.\n\nCerebrovascular mortality has been found to be increased in patients with pituitary adenoma treated with radiotherapy compared with the general population, and it is related to atherogenesis in the vascular lining from the radiotoxicity54. Risk factors include older age, previous aggressive intracranial surgery, and total dose >45 Gy. The risk for second brain tumors (most commonly meningiomas, gliomas, and chondrosarcomas) in a series of patients irradiated for pituitary adenoma was 2% at 20 years, but another series comparing patients with pituitary adenoma and treated with surgery alone or post-operative radiotherapy did not confirm increased risk55,56. Furthermore, recent data from a study including 806 patients with a non-functioning pituitary adenoma from the Dutch National Registry of Growth Hormone Treatment in Adults reported that the frequency of secondary intracranial tumors and mortality did not differ between irradiated and non-irradiated subjects57.\n\n\nConclusions\n\nThe tools for the management of acromegaly have shown advances in the last few years, offering further perspectives in the treatment of patients with this condition and particularly of those with challenging tumors. A significant amount of literature has been published in recent years, especially on the area of medical treatment, enlightening the pros and cons of the available agents and facilitating our therapeutic decisions. The impact of the currently accepted treatment algorithms on long-term morbidity and mortality remains to be assessed.\n\n\nAbbreviations\n\nCI, confidence interval; FSRT, fractionated stereotactic radiotherapy; GH, growth hormone; HR, hazard ratio; IGF-1, insulin growth factor-1; LAR, long-acting release; MRI, magnetic resonance imaging; OOCs, oral octreotide capsules; OR, odds ratio; SMR, standardized mortality ratio; SRL, somatostatin receptor ligand; SRS, stereotactic radiosurgery; SSA, somatostatin analogue; SSTR, somatostatin receptor subtype; ULN, upper limit of normal.",
"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\nReferences\n\nArosio M, Reimondo G, Malchiodi E, et al.: Predictors of morbidity and mortality in acromegaly: an Italian survey. Eur J Endocrinol. 2012; 167(2): 189–198. PubMed Abstract | Publisher Full Text\n\nHoldaway IM, Bolland MJ, Gamble GD: A meta-analysis of the effect of lowering serum levels of GH and IGF-I on mortality in acromegaly. Eur J Endocrinol. 2008; 159(2): 89–95. PubMed Abstract | Publisher Full Text\n\nMercado M, Gonzalez B, Vargas G, et al.: Successful mortality reduction and control of comorbidities in patients with acromegaly followed at a highly specialized multidisciplinary clinic. J Clin Endocrinol Metab. 2014; 99(12): 4438–4446. PubMed Abstract | Publisher Full Text\n\nKatznelson L, Laws ER Jr, Melmed S, et al.: Acromegaly: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2014; 99(11): 3933–3951. PubMed Abstract | Publisher Full Text\n\nWang YY, Higham C, Kearney T, et al.: Acromegaly surgery in Manchester revisited--the impact of reducing surgeon numbers and the 2010 consensus guidelines for disease remission. Clin Endocrinol (Oxf). 2012; 76(3): 399–406. PubMed Abstract | Publisher Full Text\n\nJane JA Jr, Starke RM, Elzoghby MA, et al.: Endoscopic transsphenoidal surgery for acromegaly: remission using modern criteria, complications, and predictors of outcome. J Clin Endocrinol Metab. 2011; 96(9): 2732–2740. PubMed Abstract | Publisher Full Text\n\nStarke RM, Raper DM, Payne SC, et al.: Endoscopic vs microsurgical transsphenoidal surgery for acromegaly: outcomes in a concurrent series of patients using modern criteria for remission. J Clin Endocrinol Metab. 2013; 98(8): 3190–3198. PubMed Abstract | Publisher Full Text\n\nSun H, Brzana J, Yedinak CG, et al.: Factors associated with biochemical remission after microscopic transsphenoidal surgery for acromegaly. J Neurol Surg B Skull Base. 2014; 75(1): 47–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHazer DB, Işık S, Berker D, et al.: Treatment of acromegaly by endoscopic transsphenoidal surgery: surgical experience in 214 cases and cure rates according to current consensus criteria. J Neurosurg. 2013; 119(6): 1467–1477. PubMed Abstract | Publisher Full Text\n\nGondim JA, Almeida JP, de Albuquerque LA, et al.: Pure endoscopic transsphenoidal surgery for treatment of acromegaly: results of 67 cases treated in a pituitary center. Neurosurg Focus. 2010; 29(4): E7. PubMed Abstract | Publisher Full Text\n\nSarkar S, Rajaratnam S, Chacko G, et al.: Endocrinological outcomes following endoscopic and microscopic transsphenoidal surgery in 113 patients with acromegaly. Clin Neurol Neurosurg. 2014; 126: 190–195. PubMed Abstract | Publisher Full Text\n\nKhan ZH, Rasouli MR: Intubation in patients with acromegaly: experience in more than 800 patients. Eur J Anaesthesiol. 2009; 26(4): 354–355. PubMed Abstract | Publisher Full Text\n\nFriedel ME, Johnston DR, Singhal S, et al.: Airway management and perioperative concerns in acromegaly patients undergoing endoscopic transsphenoidal surgery for pituitary tumors. Otolaryngol Head Neck Surg. 2013; 149(6): 840–844. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHradec J, Kral J, Janota T, et al.: Regression of acromegalic left ventricular hypertrophy after lanreotide (a slow-release somatostatin analog). Am J Cardiol. 1999; 83(10): 1506–1509, A8. PubMed Abstract | Publisher Full Text\n\nFatti LM, Scacchi M, Lavezzi E, et al.: Effects of treatment with somatostatin analogues on QT interval duration in acromegalic patients. Clin Endocrinol (Oxf). 2006; 65(5): 626–630. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBogazzi F, Di Bello V, Palagi C, et al.: Improvement of intrinsic myocardial contractility and cardiac fibrosis degree in acromegalic patients treated with somatostatin analogues: a prospective study. Clin Endocrinol (Oxf). 2005; 62(5): 590–596. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPita-Gutierrez F, Pertega-Diaz S, Pita-Fernandez S, et al.: Place of preoperative treatment of acromegaly with somatostatin analog on surgical outcome: a systematic review and meta-analysis. PLoS One. 2013; 8(4): e61523. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nAnnamalai AK, Webb A, Kandasamy N, et al.: A comprehensive study of clinical, biochemical, radiological, vascular, cardiac, and sleep parameters in an unselected cohort of patients with acromegaly undergoing presurgical somatostatin receptor ligand therapy. J Clin Endocrinol Metab. 2013; 98(3): 1040–1050. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFougner SL, Bollerslev J, Svartberg J, et al.: Preoperative octreotide treatment of acromegaly: long-term results of a randomised controlled trial. Eur J Endocrinol. 2014; 171(2): 229–235. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCaron PJ, Bevan JS, Petersenn S, et al.: Tumor shrinkage with lanreotide autogel 120 mg as primary therapy in acromegaly: results of a prospective multicenter clinical trial. J Clin Endocrinol Metab. 2014; 99(4): 1282–1290. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nAbu Dabrh AM, Mohammed K, Asi N, et al.: Surgical interventions and medical treatments in treatment-naïve patients with acromegaly: systematic review and meta-analysis. J Clin Endocrinol Metab. 2014; 99(11): 4003–4014. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBogazzi F, Colao A, Rossi G, et al.: Comparison of the effects of primary somatostatin analogue therapy and pituitary adenomectomy on survival in patients with acromegaly: a retrospective cohort study. Eur J Endocrinol. 2013; 169(3): 367–376. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBhayana S, Booth GL, Asa SL, et al.: The implication of somatotroph adenoma phenotype to somatostatin analog responsiveness in acromegaly. J Clin Endocrinol Metab. 2005; 90(11): 6290–6295. PubMed Abstract | Publisher Full Text\n\nFougner SL, Casar-Borota O, Heck A, et al.: Adenoma granulation pattern correlates with clinical variables and effect of somatostatin analogue treatment in a large series of patients with acromegaly. Clin Endocrinol (Oxf). 2012; 76(1): 96–102. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBrzana J, Yedinak CG, Gultekin SH, et al.: Growth hormone granulation pattern and somatostatin receptor subtype 2A correlate with postoperative somatostatin receptor ligand response in acromegaly: a large single center experience. Pituitary. 2013; 16(4): 490–498. PubMed Abstract | Publisher Full Text\n\nCasar-Borota O, Heck A, Schulz S, et al.: Expression of SSTR2a, but not of SSTRs 1, 3, or 5 in somatotroph adenomas assessed by monoclonal antibodies was reduced by octreotide and correlated with the acute and long-term effects of octreotide. J Clin Endocrinol Metab. 2013; 98(11): E1730–1739. PubMed Abstract | Publisher Full Text\n\nLarkin S, Reddy R, Karavitaki N, et al.: Granulation pattern, but not GSP or GHR mutation, is associated with clinical characteristics in somatostatin-naive patients with somatotroph adenomas. Eur J Endocrinol. 2013; 168(4): 491–499. PubMed Abstract | Publisher Full Text\n\nMayr B, Buslei R, Theodoropoulou M, et al.: Molecular and functional properties of densely and sparsely granulated GH-producing pituitary adenomas. Eur J Endocrinol. 2013; 169(4): 391–400. PubMed Abstract | Publisher Full Text\n\nFusco A, Zatelli MC, Bianchi A, et al.: Prognostic significance of the Ki-67 labeling index in growth hormone-secreting pituitary adenomas. J Clin Endocrinol Metab. 2008; 93(7): 2746–2750. PubMed Abstract | Publisher Full Text\n\nKasuki L, Wildemberg LE, Neto LV, et al.: Ki-67 is a predictor of acromegaly control with octreotide LAR independent of SSTR2 status and relates to cytokeratin pattern. Eur J Endocrinol. 2013; 169(2): 217–223. PubMed Abstract | Publisher Full Text\n\nPuig-Domingo M, Resmini E, Gomez-Anson B, et al.: Magnetic resonance imaging as a predictor of response to somatostatin analogs in acromegaly after surgical failure. J Clin Endocrinol Metab. 2010; 95(11): 4973–4978. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHeck A, Ringstad G, Fougner SL, et al.: Intensity of pituitary adenoma on T2-weighted magnetic resonance imaging predicts the response to octreotide treatment in newly diagnosed acromegaly. Clin Endocrinol (Oxf). 2012; 77(1): 72–78. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPotorac I, Petrossians P, Daly AF, et al.: Pituitary MRI characteristics in 297 acromegaly patients based on T2-weighted sequences. Endocr Relat Cancer. 2015; 22(2): 169–177. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCarmichael JD, Bonert VS, Nuño M, et al.: Acromegaly clinical trial methodology impact on reported biochemical efficacy rates of somatostatin receptor ligand treatments: a meta-analysis. J Clin Endocrinol Metab. 2014; 99(5): 1825–1833. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGiustina A, Mazziotti G, Torri V, et al.: Meta-analysis on the effects of octreotide on tumor mass in acromegaly. PLoS One. 2012; 7(5): e36411. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRamírez C, Vargas G, González B, et al.: Discontinuation of octreotide LAR after long term, successful treatment of patients with acromegaly: is it worth trying? Eur J Endocrinol. 2012; 166(1): 21–26. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nColao A, Attanasio R, Pivonello R, et al.: Partial surgical removal of growth hormone-secreting pituitary tumors enhances the response to somatostatin analogs in acromegaly. J Clin Endocrinol Metab. 2006; 91(1): 85–92. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKaravitaki N, Turner HE, Adams CB, et al.: Surgical debulking of pituitary macroadenomas causing acromegaly improves control by lanreotide. Clin Endocrinol (Oxf). 2008; 68(6): 970–975. PubMed Abstract | Publisher Full Text\n\nPetersenn S, Buchfelder M, Reincke M, et al.: Results of surgical and somatostatin analog therapies and their combination in acromegaly: a retrospective analysis of the German Acromegaly Register. Eur J Endocrinol. 2008; 159(5): 525–532. PubMed Abstract | Publisher Full Text\n\nMelmed S, Popovic V, Bidlingmaier M, et al.: Safety and efficacy of oral octreotide in acromegaly: results of a multicenter phase III trial. J Clin Endocrinol Metab. 2015; 100(4): 1699–1708. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nColao A, Bronstein MD, Freda P, et al.: Pasireotide versus octreotide in acromegaly: a head-to-head superiority study. J Clin Endocrinol Metab. 2014; 99(3): 791–799. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGadelha MR, Bronstein MD, Brue T, et al.: Pasireotide versus continued treatment with octreotide or lanreotide in patients with inadequately controlled acromegaly (PAOLA): a randomised, phase 3 trial. Lancet Diabetes Endocrinol. 2014; 2(11): 875–884. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHenry RR, Ciaraldi TP, Armstrong D, et al.: Hyperglycemia associated with pasireotide: results from a mechanistic study in healthy volunteers. J Clin Endocrinol Metab. 2013; 98(8): 3446–53. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBreitschaft A, Hu K, Hermosillo Reséndiz K, et al.: Management of hyperglycemia associated with pasireotide (SOM230): healthy volunteer study. Diabetes Res Clin Pract. 2014; 103(3): 458–65. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nvan der Lely AJ, Biller BM, Brue T, et al.: Long-term safety of pegvisomant in patients with acromegaly: comprehensive review of 1288 subjects in ACROSTUDY. J Clin Endocrinol Metab. 2012; 97(5): 1589–1597. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSandret L, Maison P, Chanson P: Place of cabergoline in acromegaly: a meta-analysis. J Clin Endocrinol Metab. 2011; 96(5): 1327–1335. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDuarte FH, Jallad RS, Bronstein MD: Clomiphene citrate for treatment of acromegaly not controlled by conventional therapies. J Clin Endocrinol Metab. 2015; 100(5): 1863–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHigham CE, Atkinson AB, Aylwin S, et al.: Effective combination treatment with cabergoline and low-dose pegvisomant in active acromegaly: a prospective clinical trial. J Clin Endocrinol Metab. 2012; 97(4): 1187–1193. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nvan der Lely AJ, Bernabeu I, Cap J, et al.: Coadministration of lanreotide Autogel and pegvisomant normalizes IGF1 levels and is well tolerated in patients with acromegaly partially controlled by somatostatin analogs alone. Eur J Endocrinol. 2011; 164(3): 325–333. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMinniti G, Scaringi C, Amelio D, et al.: Stereotactic Irradiation of GH-Secreting Pituitary Adenomas. Int J Endocrinol. 2012; 2012: 482861. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLee CC, Vance ML, Xu Z, et al.: Stereotactic radiosurgery for acromegaly. J Clin Endocrinol Metab. 2014; 99(4): 1273–1281. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSheehan JP, Lee Vance M, Xu Z, et al.: 142 Stereotactic Radiosurgery for Medically and Surgically Refractory Acromegaly: Long-term Rates of Remission and Hypopituitarism. Neurosurgery. 2015; 62(Suppl 1): 1211–2. Publisher Full Text\n\nWattson DA, Tanguturi SK, Spiegel DY, et al.: Outcomes of proton therapy for patients with functional pituitary adenomas. Int J Radiat Oncol Biol Phys. 2014; 90(3): 532–539. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBrada M, Ashley S, Ford D, et al.: Cerebrovascular mortality in patients with pituitary adenoma. Clin Endocrinol (Oxf). 2002; 57(6): 713–7. PubMed Abstract | Publisher Full Text\n\nBrada M, Ford D, Ashley S, et al.: Risk of second brain tumour after conservative surgery and radiotherapy for pituitary adenoma. BMJ. 1992; 304(6838): 1343–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSattler MG, van Beek AP, Wolffenbuttel BH, et al.: The incidence of second tumours and mortality in pituitary adenoma patients treated with postoperative radiotherapy versus surgery alone. Radiother Oncol. 2012; 104(1): 125–30. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nvan Varsseveld NC, van Bunderen CC, Ubachs DH, et al.: Cerebrovascular events, secondary intracranial tumors, and mortality after radiotherapy for nonfunctioning pituitary adenomas: a subanalysis from the Dutch National Registry of Growth Hormone Treatment in Adults. J Clin Endocrinol Metab. 2015; 100(3): 1104–12. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11517",
"date": "11 Dec 2015",
"name": "Maria Fleseriu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11518",
"date": "11 Dec 2015",
"name": "Maria Zatelli",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1426
|
https://f1000research.com/articles/4-1424/v1
|
10 Dec 15
|
{
"type": "Review",
"title": "Progressive Multifocal Leukoencephalopathy",
"authors": [
"Laura Adang",
"Joseph Berger",
"Joseph Berger"
],
"abstract": "Progressive multifocal leukoencephalopathy (PML) is a devastating demyelinating disease with significant morbidity and mortality and no effective, targeted therapies. It is most often observed in association with abnormalities of cell-mediated immunity, in particular human immunodeficiency virus (HIV) infection, but also occurs in association with lymphoproliferative diseases, certain immunosuppressive and immunomodulatory regimens, and other conditions. The etiologic agent of PML is a small, ubiquitous polyomavirus, the JC virus (JCV, also known as JCPyV), for which at least 50% of the adult general population is seropositive. PML results when JCV replicates within cerebral oligodendrocytes and astrocytes, leading to oligodendrocyte death and demyelination. Unfortunately, no treatments have been convincingly demonstrated to be effective, though some have been employed in desperation; treatment otherwise includes attempts to restore any immune system defect, such as the withdrawal of the causative agent if possible, and general supportive care.",
"keywords": [
"Progressive multifocal leukoencephalopathy",
"demyelination",
"JC virus",
"immunocompromised",
"highly active antiretroviral therapy"
],
"content": "Introduction\n\nProgressive multifocal leukoencephalopathy (PML), a typically rapidly progressive, potentially fatal neurologic syndrome, was first described in 1958 as a complication of chronic lymphocytic leukemia and Hodgkin’s disease1. Within two decades of its initial description, in the 1970s, the JC virus (JCV, also known as JCPyV) was discovered as the etiologic agent2. It is named for a subject with PML − patient John Cunningham3. Due to its rarity (1.3 cases per 1000 person-years at risk in human immunodeficiency virus [HIV]+ patients), the disorder is regarded as an orphan disease4. However, following the acquired immunodeficiency syndrome (AIDS) pandemic and with newer immunomodulatory therapies, such as natalizumab, that predispose to the development of PML, the incidence of the disease has increased substantially.\n\n\nThe JC Virus\n\nA ubiquitous infection, the JCV infects over half of the adult population globally3,5,6. Typically the initial exposure occurs during childhood. As no identified clinical illness accompanies acute infection, it is believed that initial infection results in a transient asymptomatic viremia following which the virus establishes as a latent or persistent infection in the kidney and perhaps elsewhere in the body3. The JCPyV found in the urine of approximately one-third of all adults is referred to as the archetype virus and is incapable of replicating effectively in glial tissue. It is theorized that the archetypal form of the virus is responsible for the primary infection. The mode of transmission is also unclear, although it is suspected to be through the tonsils or gastrointestinal tract3. Interestingly, even among the immunocompromised, only a small subset of infected patients develop PML, as the development of this syndrome requires a complex series of events: the virus must be transformed to the prototype (neurotropic) virus, seed the brain, and avoid neuro-immunosurveillance and clearance. Generally, this occurs in the setting of an impairment in cell-mediated immunity, as with HIV/AIDS, or with the use of immunomodulatory agents, such as natalizumab. The transformation of JCV from the archetype to the prototype virus requires genetic modifications in the non-coding control region of the viral DNA. This transformation of the small, circular JCPyV DNA genome impacts the replicative ability, gene transcription patterns, and homing within the body, and ultimately disease pathology3.\n\n\nPML and the JC virus\n\nIn immunocompetent individuals, the JCV is rarely pathogenic, but in immunocompromised patients, it may cause PML, an aggressive, progressive neurologic syndrome that is potentially devastating. Prior to the availability of highly active antiretroviral therapy (HAART), PML was observed in 5–10% of all persons with AIDS, and HIV/AIDS has been an underlying predisposing cause of PML in more than one-half of individuals3,7. Following the advent of HAART, the incidence of PML in this population has declined6. Another cluster of PML cases is observed in patients receiving immunomodulatory therapies. Two therapies in particular appear to predispose to PML, namely natalizumab (trade name Tysabri) and efalizumab (now off the market)8. However, PML has been reported with the use of rituximab, belatacept, fingolimod, infliximab, alemtuzumab, mycophenolate mofetil, fludarabine, leflunomide, and fumaric acid esters as well9. The increased risk of PML from natalizumab is thought to be due to the known mechanism of the drug, namely α4β1 integrin binding. In so doing, this monoclonal antibody prevents lymphocytes from binding to vascular cell adhesion molecule 1 (VCAM) on the central nervous system (CNS) endothelium, decreasing CNS immune surveillance10. While an immune-modulated state is relatively common, PML remains a rare disorder even within these subpopulations. This suggests that immunosuppression alone is insufficient to reactivate the JCV and cause disease.\n\nWhile the JCV predominantly results in the clinical syndrome of PML, it can also cause granule cell neuronopathy (GCN), JCV encephalopathy, and even isolated JCV meningitis11. In GCN, the granule cell neurons of the cerebellum are affected, resulting in symptoms including ataxia, tremor, and nystagmus12. JCV encephalopathy is pathologically characterized by the infection and lysis of the cortical gray matter13.\n\n\nDiagnosis of PML\n\nThe diagnosis of PML requires clinical, radiographic, and virologic evidence5. Clinically, PML can present with a wide constellation of neurologic signs and symptoms due to its ability to affect virtually any area of the brain and the frequently multifocal nature of the lesions. In patients with multiple sclerosis (MS) on natalizumab or other disease-modifying drugs that seem to predispose to PML, distinguishing PML from an acute MS attack can be difficult, as the general symptoms can be similar to the symptoms of an MS flare. The most commonly reported symptoms include gait changes, weakness, cognitive impairment, sensory symptoms, headache, and visual changes14. Visual symptoms are reported in one-quarter to one-half of all PML patients, typically presenting as a field deficit, and can be the initial symptom as well15. Visual system involvement is secondary to involvement of the visual pathways and not as a direct optic neuritis, as seen in other inflammatory, demyelinating diseases15.\n\nSeizures can occur in up to one-third of the general PML population and are more frequent with juxtacortical and T1-hyperintense lesions by magnetic resonance imaging (MRI)13. There is subtle variation in the presentation of PML as determined by the underlying cause: HIV-associated PML vs. immunosuppressant-associated PML. Unlike PML associated with HIV infection where 50% have predominant motor system findings7, natalizumab-associated PML appears to be more commonly associated with cognitive and language changes; motor symptoms are reported in only one-third of natalizumab PML patients14.\n\nPathologically, PML is characterized by multifocal demyelination, with smaller lesions coalescing into larger foci. Overall lesion burden can be extensive, involving entire hemispheres, and can occur throughout the white matter. By histopathology, PML is characterized by multifocal demyelination, enlarged astrocytes that contain lobulated hyperchromatic nuclei and oligodendrocytic hyperpigmented enlarged nuclei16. JC virions can be found by electron microscopy, particularly within reactive astrocytes.\n\nThe appearance of PML by cerebral imaging can be heterogeneous, although it is typically multifocal with frontal or parieto-occipital locations predominating. There are rare reports of PML involvement isolated to the deep gray matter or the brainstem, or even resulting in spinal cord parenchyma abnormalities, but, to date, clinical features of a myelopathy have not been reported with PML17. On computed tomography (CT) imaging, PML lesions are hypointense within the white matter. When the subcortical arcuate fibers are involved, the lesions can have a ‘scalloped’ appearance. By MRI, a far more sensitive measure to detect evidence of PML, lesions are hyperintense by T2 and fluid-attenuated inversion recovery (FLAIR) imaging and hypointense by T1. Gadolinium enhancement is more common in natalizumab cases, one-third vs. 15% of HIV-associated cases14. Of note, similar radiographic patterns can also be found in cytomegalovirus infections, acute disseminated encephalomyelitis, varicella-zoster leukoencephalopathy, and brain tumors, although the diagnosis is often evident within the context of a full history and physical examination14. Patients prescribed medications such as natalizumab should be annually screened by MRI for the imaging characteristics of PML, as it is possible to detect the radiographic findings before the clinical onset.\n\nTo develop PML, a patient must have a latent or persistent infection with the JCV. This is believed to be in an extraneural reservoir, chiefly the kidney. The virus then undergoes genetic rearrangement into the neurotropic form, infects oligodendrocytes and astrocytes within the CNS, and leads to demyelination from oligodendrocyte lysis when the host cannot mount an appropriate immune response to contain the virus. The value of cerebrospinal fluid (CSF) polymerase chain reaction (PCR) for JCPyV is highly dependent on the nature of the test; ultrasensitive PCR techniques for JCPyV have a sensitivity of >95%. Patients on natalizumab at risk for the development of PML are typically screened for the presence of JCV-specific antibodies. A semi-quantitative antibody index is used to indirectly track JC infection and prognosticate PML risk. JCV antibody index exceeding 1.5 has been associated with a higher risk of PML18. Other factors significantly increasing the risk for natalizumab-associated PML is prior immunosuppressive therapy use and duration of natalizumab (>24 months) therapy19.\n\n\nPML survival and treatment\n\nPML is an aggressive, potentially fatal disease. In individuals in whom the immune dysfunction can be restored, such as patients with AIDS in whom immune function is restored by antiretroviral therapy, survival is improved20. Presumably, the same is true of patients on immunosuppressive agents, such as natalizumab, who are diagnosed early and treated with drug cessation, plasma exchange to more quickly eliminate the natalizumab, and supportive care4,14,21. Mortality in natalizumab-related cases is approximately 21%22,23. Survival in the natalizumab cases was associated with younger age, lower pre-PML functional disability, lower viral loads, and more focal brain involvement23. Survival in HIV-associated PML cases is dependent on CD4 count and ranges from 50% to 80%21. PML may be associated with the immune reconstitution syndrome (IRIS) with recovery of immune function. IRIS is defined as a worsening of radiographic and clinical findings of an infection in the context of immune system recovery. Accordingly, patients are often treated with corticosteroids concurrent with supportive care and plasma exchange, although the benefit of this approach still remains unproven9. Among natalizumab-associated PML survivors, one-third of the patients have mild neurologic deficits, one-third have moderate impairment, and one-third are severely affected22,24. Despite the efficacy of certain therapeutic agents in decreasing JCPyV replication in vitro, such as mefloquine and cidofovir, none have demonstrated efficacy in randomized controlled trials.\n\nIn conclusion, PML is an aggressive brain infection caused by the JCV, almost exclusively found in immunosuppressed patients. Although consensus is that high-risk patients on immunosuppressant medications such as natalizumab should be monitored by serial imaging and anti-JCV antibody screening, the frequency of testing and the threshold for concern is a rapidly moving target. The mainstays of treatment include stopping the inciting agent and plasma exchange, although directed anti-viral therapeutics is an active area of investigation. While the duration of therapy with natalizumab influences the risk, many other factors are involved in the development of PML, suggesting a complexity to the predisposition to PML development.\n\n\nAbbreviations\n\nAIDS - Acquired immunodeficiency syndrome\n\nPML - Progressive multifocal leukoencephalopathy\n\nHIV - Human immunodeficiency virus\n\nCNS - Central nervous system\n\nCT - Computed tomography\n\nIRIS - Immune reconstitution syndrome\n\nJC - John Cunningham\n\nJCV - John Cunningham virus\n\nJCPyV - John Cunningham polyomavirus\n\nMRI - Magnetic resonance imaging\n\nHAART - Highly active antiretroviral therapy\n\nVCAM - Vascular cell adhesion molecule 1\n\nGCN - Granule cell neuronopathy\n\nMS - Multiple sclerosis\n\nCSF - Cerebrospinal fluid\n\nPCR - Polymerase chain reaction",
"appendix": "Competing interests\n\n\n\nLaura Adang declares that she has no competing interests.\n\nJoseph R. Berger is or has been a consultant to the following companies: Genentech, Genzyme, Incyte, Inhibikase, Johnson & Johnson, and Novartis. He serves or has served on the PML Adjudication Committees of the following companies: Amgen, AstraZeneca, Bristol-Myers Squibb, Eisai, Janssen, Millennium, PAREXEL, Pfizer, Roche, and Takeda.\n\n\nGrant information\n\nJoseph R. Berger has received grants from the PML Consortium and Biogen Idec.\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\nAstrom KE, Mancall EL, Richardson EP Jr: Progressive multifocal leuko-encephalopathy; a hitherto unrecognized complication of chronic lymphatic leukaemia and Hodgkin's disease. Brain. 1958; 81(1): 93–111. PubMed Abstract | Publisher Full Text\n\nPadgett BL, Walker DL, ZuRhein GM, et al.: Cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy. Lancet. 1971; 1(7712): 1257–60. PubMed Abstract | Publisher Full Text\n\nWhite MK, Khalili K: Pathogenesis of progressive multifocal leukoencephalopathy--revisited. J Infect Dis. 2011; 203(5): 578–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngsig FN, Hansen AB, Omland LH, et al.: Incidence, clinical presentation, and outcome of progressive multifocal leukoencephalopathy in HIV-infected patients during the highly active antiretroviral therapy era: a nationwide cohort study. J Infect Dis. 2009; 199(1): 77–83. PubMed Abstract | Publisher Full Text\n\nBerger JR, Aksamit AJ, Clifford DB, et al.: PML diagnostic criteria: consensus statement from the AAN Neuroinfectious Disease Section. Neurology. 2013; 80(15): 1430–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBozic C, Subramanyam M, Richman S, et al.: Anti-JC virus (JCV) antibody prevalence in the JCV Epidemiology in MS (JEMS) trial. Eur J Neurol. 2014; 21(2): 299–304. PubMed Abstract | Publisher Full Text\n\nBerger JR, Pall L, Lanska D, et al.: Progressive multifocal leukoencephalopathy in patients with HIV infection. J Neurovirol. 1998; 4(1): 59–68. PubMed Abstract | Publisher Full Text\n\nZaheer F, Berger JR: Treatment-related progressive multifocal leukoencephalopathy: current understanding and future steps. Ther Adv Drug Saf. 2012; 3(5): 227–39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliamson EM, Berger JR: Infection risk in patients on multiple sclerosis therapeutics. CNS Drugs. 2015; 29(3): 229–44. PubMed Abstract | Publisher Full Text\n\nO'Connor P, Miller D, Riester K, et al.: Relapse rates and enhancing lesions in a phase II trial of natalizumab in multiple sclerosis. Mult Scler. 2005; 11(5): 568–72. PubMed Abstract | Publisher Full Text\n\nGheuens S, Wüthrich C, Koralnik IJ: Progressive multifocal leukoencephalopathy: why gray and white matter. Annu Rev Pathol. 2013; 8: 189–215. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHenry C, Jouan F, De Broucker T: JC virus granule cell neuronopathy: A cause of infectious cerebellar degeneration. J Neurol Sci. 2015; 354(1–2): 86–90. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKhoury MN, Alsop DC, Agnihotri SP, et al.: Hyperintense cortical signal on magnetic resonance imaging reflects focal leukocortical encephalitis and seizure risk in progressive multifocal leukoencephalopathy. Ann Neurol. 2014; 75(5): 659–69. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBerger JR: Progressive multifocal leukoencephalopathy. Handb Clin Neurol. 2014; 123: 357–76. PubMed Abstract | Publisher Full Text\n\nSudhakar P, Bachman DM, Mark AS, et al.: Progressive Multifocal Leukoencephalopathy: Recent Advances and a Neuro-Ophthalmological Review. J Neuroophthalmol. 2015; 35(3): 296–305. PubMed Abstract | Publisher Full Text\n\nWoodhouse MA, Dayan AD, Burston J, et al.: Progressive multifocal leukoencephalopathy: electron microscope study of four cases. Brain. 1967; 90(4): 863–70. PubMed Abstract | Publisher Full Text\n\nMurayi R, Schmitt J, Woo JH, et al.: Spinal cord progressive multifocal leukoencephalopathy detected premortem by MRI. J Neurovirol. 2015; 21(6): 688–90. 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): 802–12. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBloomgren G, Richman S, Hotermans C, et al.: Risk of natalizumab-associated progressive multifocal leukoencephalopathy. N Engl J Med. 2012; 366(20): 1870–80. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAntinori A, Ammassari A, Giancola ML, et al.: Epidemiology and prognosis of AIDS-associated progressive multifocal leukoencephalopathy in the HAART era. J Neurovirol. 2001; 7(4): 323–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCasado JL, Corral I, García J, et al.: Continued declining incidence and improved survival of progressive multifocal leukoencephalopathy in HIV/AIDS patients in the current era. Eur J Clin Microbiol Infect Dis. 2014; 33(2): 179–87. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVermersch P, Kappos L, Gold R, et al.: Clinical outcomes of natalizumab-associated progressive multifocal leukoencephalopathy. Neurology. 2011; 76(20): 1697–704. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDong-Si T, Gheuens S, Gangadharan A, et al.: Predictors of survival and functional outcomes in natalizumab-associated progressive multifocal leukoencephalopathy. J Neurovirol. 2015; 21(6): 637–44. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLima MA, Bernal-Cano F, Clifford DB, et al.: Clinical outcome of long-term survivors of progressive multifocal leukoencephalopathy. J Neurol Neurosurg Psychiatr. 2010; 81(11): 1288–91. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation"
}
|
[
{
"id": "11444",
"date": "10 Dec 2015",
"name": "Stephen G Waxman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11443",
"date": "10 Dec 2015",
"name": "Meena Subramanyam",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1424
|
https://f1000research.com/articles/4-1423/v1
|
10 Dec 15
|
{
"type": "Case Report",
"title": "Case Report: Genetic analysis and anesthetic management of a child with Niemann-Pick disease Type A",
"authors": [
"Priti G. Dalal",
"Melissa Coleman",
"Meagan Horst",
"Dorothy Rocourt",
"Roger L. Ladda",
"Piotr K. Janicki",
"Melissa Coleman",
"Meagan Horst",
"Dorothy Rocourt",
"Roger L. Ladda",
"Piotr K. Janicki"
],
"abstract": "A 14-month-old child, recently diagnosed with Niemann-Pick disease type A, presented for a laparoscopic placement of a gastrostomy tube under general anesthesia. The disease was confirmed and further characterized by genetic testing, which revealed evidence of the presence of two known pathogenic mutations in the SMPD1 gene, and enzyme studies showed a corresponding very low level of enzymatic activity of acidic sphingomyelinase. The anesthetic management involved strategies to manage an anticipated difficult intubation and avoid post-operative ventilation.",
"keywords": [
"Anesthesia",
"Niemann-Pick disease",
"genomic and biochemical analysis"
],
"content": "Introduction\n\nNiemann-Pick disease (NPD) is a rare inherited autosomal recessive lysosomal storage disorder (incidence about 1:40,000 in general population) caused by pathogenic mutations in the SMPD1 gene and characterised by enzyme studies showing a corresponding very low level of enzymatic activity of acidic sphingomyelinase (ASM), associated with intracellular accumulation of lipids1–3. People with NPD type A (NPA; generally a very rare presentation of NPD) have little or no ASM production (less than 1% of normal) while those with NPD type B (the most frequent presentation of NPD) have approximately 10% of the normal level of ASM. There is no information on the anesthetic management of a patient with fully genetically characterized and biochemically confirmed NPA, and only one previous report describing a pediatric patient with presumably diagnosed NPA4. In this case report, we describe the genetic background, pathophysiology and anesthetic-related problems in a patient with NPA who presented for surgery.\n\n\nCase report\n\nA 14-month-old Caucasian child (residing in the United States, diagnosed with NPA, presented to our hospital pre-anesthesia assessment clinic for laparoscopic placement of a gastrostomy tube. There was no known family member or relatives diagnosed with NPD; mother reported a distant relative of Jewish origin. On physical exam, he was a well-proportioned child, weight was 8.32 kg (10th percentile) and height was 77cm (50–75th percentile), head circumference was 48.3 cm (90–95th percentile). Craniofacial features included frontal bossing, protuberant tongue and mild bilateral ptosis. Generalized hypotonia with head lag and weakness in upper girdle and lower leg muscles, decreased deep tendon reflexes and hepatosplenomegaly (3 cm below the costal margin) were noted. Liver function tests revealed elevation of alanine transaminase (ALT) levels - 90 uts/L (normal range 13–69), aspartate transaminase (AST) levels - 271 uts/L (normal range 9–80), alkaline phosphatase levels - 252 uts/L (normal 8–240) and creatine phosphokinase (CPK) - <20 uts/L (normal range 55–170).\n\nThe child was scheduled as the first case on the day of surgery. General anesthesia was induced with a 50:50 mixture of nitrous oxide: oxygen and sevoflurane. Following a smooth inhalational induction, intravenous access was established and a single dose of propofol 2 mg/kg was administered intravenously to facilitate tracheal intubation. Tracheal intubation was performed under deep anesthesia with a 4 mm internal diameter cuffed endotracheal tube with a c-MAC videolaryngoscope blade (Cormack and Lehane grade 2 view, Figure 1). Anesthesia was maintained with air: oxygen and sevoflurane mixture (Minimum Alveolar Concentration i.e. MAC=1). A single dose of per rectal acetaminophen 240 mg (30–40 mg/kg) and local anesthetic infiltration with 0.25% bupivacaine were used to facilitate analgesia. Tracheal extubation was uneventful. The child was transported to the post-anesthesia care unit and made an uneventful post-operative recovery.\n\nFollow-up The child had G-tube feeds commenced following surgery and subsequently discharged to home the following day and made an uneventful clinical recovery. Subsequently over the course of next few months, the child had episodes of intractable seizures which were medically managed. The patient was referred to home hospice care and expired at home at the age of 2 years.\n\nThe results of enzymatic evaluation of patient blood and leukocytes by an outside laboratory are presented in Table 1.\n\nP – Plasma, L – Leukocytes – Bold face denotes abnormal results\n\nExtracted DNA was PCR-amplified for analysis of the coding exons 1 to 6 of the SPMD1 gene and their flanking splice sites, using a standard Sanger sequencing approach. Bi-directional sequence was obtained and the DNA sequence was analyzed and compared to the published gene sequence. Reportable variants were confirmed by repeat sequence analysis. Based on the genetic diagnostic laboratory (Proprietary information from GeneDx, Gaithersburg, MD) 99% sensitivity is expected in detecting mutations identifiable by sequencing. Please refer to Table 2.\n\n*The normal sequence with the base that is deleted in braces is: ACCCCC(T)AGCC. **The normal sequence with the bases that are deleted in braces is: TACT(CT)TTGT\n\n\nDiscussion\n\nOur anesthesia management strategy focused on avoiding muscle relaxants and narcotic analgesics. Hence, endotracheal intubation under deep anesthesia without muscle paralysis and non-opioid analgesics, i.e., acetaminophen, and infiltration of the surgical wound with local anesthetic were contemplated. Adequate pain control was possible with this technique since the procedure was performed laparoscopically. Although the child had mild elevation of liver enzymes, acetaminophen was preferred as a short-term analgesic. Use of atracurium, midazolam, sevoflurane and fentanyl has been described in the management of anesthesia in a 2-year-old patient with presumable NPA presenting for emergency splenectomy4. The child had severe hepatosplenomegaly, ventilation improved after splenectomy and the child required a post-operative ICU stay for 9 days.\n\nThe observed c.573delT mutation in the SMPD1 gene in our case has been reported previously in association with NPD type A5. The deletion causes a frame shift starting with codon Serine 192, changes this amino acid to an Alanine residue and creates a premature Stop codon at position 65 in the new reading frame denoted p.Ser192AlafsX65. This mutation is predicted to cause abnormal protein function either through protein truncation or nonsense-mediated mRNA decay. The c1783_1784delCT deletion causes a frame shift starting with codon Alanine 597, changes this amino acid to a Proline residue, and creates a premature stop codon at position 7 of the new reading frame denoted p.Ala597ProfsX7. This mutation is predicted to cause loss of normal protein function through protein truncation. Specifically, the last 35 correct residues are replaced by six incorrect residues. Therefore, the presence of these mutations is consistent with the diagnosis of an SMPD1-related disorder, if the mutations were inherited on different alleles (in trans), i.e., each mutation coming from a different parent6. This result therefore permits mutation-specific carrier testing for family members and prenatal diagnosis for the parents of this child, if desired. However, targeted carrier testing of both parents is necessary prior to or concurrently with any carrier testing or predictive testing in this family.\n\nPatients with NPA and NPB have significantly different clinical course. While NPA is associated with severe neurological deficits leading to early death, patients with NPB have minimal neurological involvement and may survive to adulthood, although hepatosplenomegaly and cardiorespiratory problems may occur. We believe therefore that it is important to distinguish these form of NPD preoperatively, because NPA requires much more strict anesthetic management (avoidance of muscle relaxants and opioids, if at all possible) and it could associated more frequently with step-up level of postoperative care.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and/or images was obtained from the parent of the patient.",
"appendix": "Author contributions\n\n\n\nPD and PJ prepared the initial draft of the manuscript. MC, MH, RL and DR did further literature review and modifications of the manuscript. RL gave considerable input on the genetic concepts in the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nVanier MT: Niemann-Pick diseases. Handb Clin Neurol. 2013; 113: 1717–21. PubMed Abstract | Publisher Full Text\n\nVanier MT: Lysosomal diseases: biochemical pathways and investigations. Handb Clin Neurol. 2013; 113: 1695–9. PubMed Abstract | Publisher Full Text\n\nMiao N, Lu X, O'Grady NP, et al.: Niemann-pick disease type C: implications for sedation and anesthesia for diagnostic procedures. J Child Neurol. 2012; 27(12): 1541–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBujok LS, Bujok G, Knapik P: Niemann-Pick disease: a rare problem in anaesthesiological practice. Paediatr Anaesth. 2002; 12(9): 806–8. PubMed Abstract | Publisher Full Text\n\nGluck I, Zeigler M, Bargal R, et al.: Niemann Pick Disease type A in Israeli Arabs: 677delT, a common novel single mutation. Mutations in brief no. 161. Online. Hum Mutat. 1998; 12(2): 136. PubMed Abstract | Publisher Full Text\n\nSimonaro CM, Desnick RJ, McGovern MM, et al.: The demographics and distribution of type B Niemann-Pick disease: novel mutations lead to new genotype/phenotype correlations. Am J Hum Genet. 2002; 71(6): 1413–9. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11964",
"date": "01 Feb 2016",
"name": "James J. Fehr",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 novel case report of a 14 month old with NPA who is undergoing an anesthetic. The manuscript is generally written, a few missing articles were noted, but the substance of the article is sound. The report is supported by appropriate references from the literature and the case description is succinct but complete. A modest improvement might be made by including the clinical presentation of NPA (what to be concerned about) in the introduction which would serve to focus the reader on the subsequent anesthetic management.",
"responses": []
},
{
"id": "12222",
"date": "01 Feb 2016",
"name": "Rakesh Garg",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlease provide more details of airway management i.e. difficulty level. Was it predicted and preparation done before inducing anaesthesia?Please provide detail (positive or negative ) for any other additional comorbidities - structural or functional.Please mention duration of surgery, need of any NMB drugs, other analgesia, hemodynamic status intraoperatively.Since the title says anaesthetic management as well, It would be good if authors could add an table for various perioperative concerns in such child and their suggestions for the management.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1423
|
https://f1000research.com/articles/4-1410/v1
|
08 Dec 15
|
{
"type": "Review",
"title": "Advances in Laryngoscopy",
"authors": [
"Michael Aziz"
],
"abstract": "Recent technological advances have made airway management safer. Because difficult intubation remains challenging to predict, having tools readily available that can be used to manage a difficult airway in any setting is critical. Fortunately, video technology has resulted in improvements for intubation performance while using laryngoscopy by various means. These technologies have been applied to rigid optical stylets, flexible intubation scopes, and, most notably, rigid laryngoscopes. These tools have proven effective for the anticipated difficult airway as well as the unanticipated difficult airway.",
"keywords": [
"Laryngoscopy",
"Optical Stylets",
"Flexible bronchoscopy",
"Video laryngoscopy",
"Airway management"
],
"content": "Introduction\n\nAirway management remains a difficult skill to master. Competency requires didactic instruction as well as hands-on training with simulators and human patients. To augment challenges with training, airway management is burdened with poor prediction models for difficulty or failure. Indeed, most studies of bedside tests may only marginally predict that direct laryngoscopy fails to achieve an adequate laryngeal view. These tests have very limited application to actual intubation success rates and tools other than direct laryngoscopy with a Macintosh blade. Fortunately, recent advances in airway management have facilitated easier intubation by augmenting the laryngeal view with video technologies. Application of light-emitting diode (LED) light, liquid crystal display (LCD) screens, and complementary metal–oxide–semiconductor (CMOS) video chip technology has made video augmentation more portable, easier to use, and feasible in today’s economic climate. This review will briefly discuss how video technology has changed optical stylets and flexible intubation scopes. Further emphasis will be placed on newer-generation rigid video laryngoscopes.\n\n\nOptical stylets\n\nOlder optical stylets used fiberoptic bundles and thus required an eyepiece that the provider would have to place their eye on. Today’s optical stylets use video chips and thus can incorporate an easy-to-view video screen on the handle itself. This simplicity and ease of use result in improvements in intubation performance, especially when the neck is mobilized1. Other newer optical stylets have also incorporated technologies with flexible tips2. These attached video screens have overcome a classic barrier to optical stylets—that the head and eye of the provider need to follow a moving target as the scope is advanced through the pharynx and redirected toward the trachea. Instead, a screen that swivels allows the provider to remain in a neutral position to facilitate laryngoscopy. Furthermore, the use of a video camera provides a much wider field of view than the narrow field of view provided by fiberoptic devices.\n\n\nFlexible intubation scopes\n\nFlexible intubation scopes have done away with an old name of “flexible fiberoptic” as the optical component no longer utilizes fiberoptic bundles. Today’s technologies also use a video chip carried on the distal end of the bronchoscope. As flexible intubation remains the standard tool for the anticipated difficult airway, better optics and fewer degradation problems related to fiberoptic bundle fracture stand to improve the reliability of awake flexible bronchoscopic airway management. The literature has yet to offer comparisons of video bronchoscopic techniques with older fiberoptic techniques. However, these technologies have allowed them to become more portable. They do not require a large cart with a light source and video processor.\n\nIn recent years, bronchoscopes have become more portable and have even come in disposable forms. A disposable approach permits availability of this gold-standard airway management tool in areas where difficult airways are managed less frequently, such as an ambulatory surgery center. Although the technology is not quite as robust as a traditional platform bronchoscopic system, it can be effectively used for difficult airway management3,4.\n\n\nVideo laryngoscopy\n\nVideo laryngoscopy made its introduction to airway management many years ago with the introduction of the GlideScope video laryngoscope (Verathon, Seattle, WA, USA). Since that time, we have learned much about the utility of the devices and have generated questions to guide future investigation. These tools have been shown to consistently provide an improved view of the larynx compared with direct laryngoscopy. Research has attempted to identify whether these benefits translate to an improvement in actual intubation difficulty or success rate, or both. As success rates for tracheal intubation using direct laryngoscopy in experienced hands are very high, there does not seem to be added benefit beyond improvement of laryngeal view for the undifferentiated airway across age spectrums5,6. However, evidence has made it clear that video laryngoscopy eases intubation difficulty and increases first-attempt success rates in the airway predicted to be difficult to intubate by direct laryngoscopy7–10. These benefits are seen for patients who are obese, have a raised Mallampati score, or have reduced cervical motion from pathology or cervical spine precautions.\n\n\nVideo laryngoscopy: the novice provider\n\nVideo laryngoscopy offers significant benefits for the provider who is less experienced with airway management. Compared with direct laryngoscopy, intubation success rates are higher with video laryngoscopy in the hands of novice providers11,12. These benefits come both with devices that are video-assisted direct laryngoscopes and with those with acutely curved indirect laryngoscopes. An instructor-guided laryngoscopy with a video device appears to accelerate even direct laryngoscopy skills13.\n\n\nVideo laryngoscopy: awake airway management\n\nCompared with awake fiberoptic intubation, awake video laryngoscopy has been evaluated in the potential difficult airway14. Findings demonstrated similar performance with both techniques. However, validity of the study findings is limited by sedation of study patients, exclusion of those with neck pathologies, and inappropriate post-randomization exclusions15. In another study of awake nasal intubation with flexible bronchoscopy versus video laryngoscopy, techniques performed similarly16. In this study, patients in both groups were sufficiently sedated to avoid recall of the procedure. As such, findings may not apply to the true “awake” intubation. Despite these limitations, awake video laryngoscopy may be a tool that is easier to learn and master than flexible bronchoscopic intubation and likely has a future role in awake airway management.\n\n\nVideo laryngoscopy: recording and archiving\n\nOne of the potential benefits of video technology is the capacity to record and archive still images or video clips from the laryngoscopy. These recordings can be used to confirm tracheal tube placement, document laryngeal view, document absence of trauma, and teach future laryngoscopists. Today’s anesthesia record contains a narrative describing the device, laryngeal view on the Cormack and Lehane scale, absence of trauma, and confirmation of tube placement with various tools. The future electronic record could feasibly store a single picture or video clip that tells the airway management story. To date, anesthesia information management systems have not incorporated archiving of digital airway images as part of the anesthesia record, but this future seems feasible. This advancement is poised to change how we define difficult airway management. Instead of describing a laryngeal view by Cormack-Lehane grading, a video recording may better tell the story and also better describe challenges related to tube passage that may be difficult to describe in a narrative.\n\n\nVideo laryngoscopy: outside of the operating room\n\nEarly investigations in various clinical environments outside of the operating room suggest potential benefit. These environments contain both difficult airway scenarios and providers with less airway management experience than is often found in the operating room. In critical care and emergency medicine environments, video laryngoscopy was associated with a higher intubation success rate in patients with predictors of difficult direct laryngoscopy10,17–19. There is one randomized trial to confirm benefit of video laryngoscopy in the critical care environment20. The findings are impressive, but interpretation of the results is limited by a select patient and provider population. In the setting of trauma, the use of video laryngoscopy was associated merely with longer intubation times21. In the obstetric environment, video laryngoscopy has been used for emergency airway management, potential difficult intubations, and rescue of failure of direct laryngoscopy22. Finally, in prehospital emergency medicine, video laryngoscopy is associated with a reduction in the number of intubation attempts and shorter laryngoscopy time than direct laryngoscopy23,24. However, these studies are observational in nature and interpretation of the results deserves some caution because of the possibility of confounding factors. Prospective randomized studies are much more difficult to perform in these dynamic and emergent environments. Clinical care appears to be transitioning to primary use of video laryngoscopy in these environments, but the literature has not yet confirmed that this practice should be standard in these environments. Prospective randomized controlled trials are still needed to confirm the potential benefit for all airway management within each of these environments.\n\n\nVideo laryngoscopy: as a rescue device\n\nWhen direct laryngoscopy fails, it is unclear how to proceed with airway management. The American Society of Anesthesiologists’ difficult airway algorithm calls for transitioning to alternate airway management techniques, but it is not clear which techniques are optimal25. Current intubation rescue techniques include video laryngoscopy, flexible bronchoscopic intubation, use of a lighted stylet, or insertion of a supraglottic airway as a conduit to tracheal intubation. One study across two centers demonstrated that rescue using the GlideScope video laryngoscope was successful in 94% of cases (224/239) after failed direct laryngoscopy26. The Pentax AWS (Ambu, Ballerup, Denmark) was found to achieve successful intubation in 99% of cases (268/270) in which direct laryngoscopy failed to achieve an adequate laryngeal view for intubation27. In a large observational study of a new algorithm involving a small group of providers and select group of patients, failed direct laryngoscopy was rescued by using the Airtraq system (Prodol Meditec SA, Getxo, Spain) in 27 out of 28 cases28. Others reported that when mask ventilation and intubation are both difficult, supraglottic airways restore ventilation in 94% of cases (16/17)29. In summary, video laryngoscopy appears to have a promising role in the management of failed direct laryngoscopy as a rescue technique with a high success rate.\n\n\nVideo laryngoscopy: difficulty or failure\n\nDespite these many benefits for the difficult airway, video laryngoscopy can fail. One source of failure is an inability to achieve a laryngeal view. However, a frequent and more perplexing scenario is that of an adequate laryngeal view but inability to pass the tube into the trachea. This difficulty may occur with acutely curved indirect laryngoscopes, channeled video laryngoscopes, or even video-assisted direct laryngoscopes. A recent study identified predictors of failure with the GlideScope video laryngoscope26. The strongest predictor for failure is neck pathology from tumor, radiation, or surgical scar. This evidence suggests that flexible techniques remain an important tool to master and maintain competency with. Indeed, awake airway management with flexible techniques remains the gold standard for difficult airway management in the cooperative patient for experienced providers. The key challenge is to maintain this competency.\n\nOne potential cause of difficult tube passage results when an acutely curved video laryngoscope is inserted too deep. Figure 1–Figure 3 display this phenomenon in which an adequate laryngeal view is achieved, but the glottis is lifted too anteriorly to permit tube passage. When the laryngoscope is withdrawn so that the tip of the blade is in the vallecula, the glottis falls into a more posterior position and is better aligned for tube passage. While intubating nasally, the use of adjunct techniques such as Magill forceps may be very useful to overcome the acute curvature of devices such as the GlideScope of C-MAC with D-blade.\n\nA good laryngeal view is achieved.\n\n\nVideo laryngoscopy: stress\n\nMany authors believe that video laryngoscopy causes less patient stress than direct laryngoscopy. Indeed, less suspension pressure and extension of the cervical spine may be necessary to achieve an adequate laryngeal view. However, well-designed clinical trials have been inconsistent in demonstrating that video laryngoscopy is associated with less cervical traction than direct laryngoscopy when manual in-line stabilization is applied30.\n\n\nVideo laryngoscopy: trauma\n\nVideo laryngoscopy appears to expose the patient to different kinds of iatrogenic trauma compared with direct laryngoscopy. Reports continue to warn that pharyngeal injury is a problem with video laryngoscopy, although the incidence appears to be low. In particular, there are reports of tracheal tubes passing through the pharynx during video laryngoscopy31. In fact, the operator, not the device, is responsible for this injury. However, despite more widespread knowledge of this potential problem, this complication occurs even in experienced clinical hands. Caution is warranted when advancing a tracheal tube through the pharynx during video laryngoscopy, especially when visual attention may be distracted from the patient. Specifically, the provider needs to watch the tube pass into the mouth and make the turn toward the larynx prior to focusing further attention on the video screen.\n\n\nVideo laryngoscopy: future directions\n\nFuture research is poised to address some new questions. In particular, very few studies have compared video laryngoscope types and designs to determine the ideal device characteristics. Some limited data suggest that devices with channeled components (i.e. with a preloaded tube) result in faster and easier intubation than non-channeled devices32. Furthermore, questions surrounding blade design continue to arise. For example, video-assisted direct laryngoscopes (i.e. Macintosh blade design) have the potential benefit of familiarity, simple tube passage, and narrow blade profile. On the other hand, acutely curved blade designs may further augment laryngeal view for the anterior airway beyond what may be achieved with a video-assisted direct laryngoscope. Thus far, studies have not shown a clear difference in success rate between various blade design types33,34.\n\n\nConclusions\n\nIn summary, video laryngoscopy has established a permanent role in difficult airway management. Future investigations will help guide the clinical scenarios for use, algorithm approaches, and device designs. It is less clear whether video laryngoscopy will one day replace direct laryngoscopy. Currently, cost remains a barrier to such transitions for routine clinical care in the operating room. In other environments, that transition has occurred already on the basis of observational data, suggesting a reduction in intubation attempts.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nI have received research funding from Karl Storz endoscopy (Tuttlingen, Germany), the vendor of various airway management tools.\n\n\nReferences\n\nKim JK, Kim JA, Kim CS, et al.: Comparison of tracheal intubation with the Airway Scope or Clarus Video System in patients with cervical collars. Anaesthesia. 2011; 66(8): 694–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSetty H, Rawlings JL, Dubin S: Video RIFL: a rigid flexible laryngoscope to facilitate airway management. J Clin Anesth. 2010; 22(8): 642–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPujol E, López AM, Valero R: Use of the Ambu(®) aScope™ in 10 patients with predicted difficult intubation. Anaesthesia. 2010; 65(10): 1037–40. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKristensen MS, Fredensborg BB: The disposable Ambu aScope vs. a conventional flexible videoscope for awake intubation -- a randomised study. Acta Anaesthesiol Scand. 2013; 57(7): 888–95. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFiadjoe JE, Gurnaney H, Dalesio N, et al.: A prospective randomized equivalence trial of the GlideScope Cobalt® video laryngoscope to traditional direct laryngoscopy in neonates and infants. Anesthesiology. 2012; 116(3): 622–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSun DA, Warriner CB, Parsons DG, et al.: The GlideScope Video Laryngoscope: randomized clinical trial in 200 patients. Br J Anaesth. 2005; 94(3): 381–4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMalik MA, Subramaniam R, Maharaj CH, et al.: Randomized controlled trial of the Pentax AWS, Glidescope, and Macintosh laryngoscopes in predicted difficult intubation. Br J Anaesth. 2009; 103(5): 761–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nJungbauer A, Schumann M, Brunkhorst V, et al.: Expected difficult tracheal intubation: a prospective comparison of direct laryngoscopy and video laryngoscopy in 200 patients. Br J Anaesth. 2009; 102(4): 546–50. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAziz MF, Dillman D, Fu R, et al.: Comparative effectiveness of the C-MAC video laryngoscope versus direct laryngoscopy in the setting of the predicted difficult airway. Anesthesiology. 2012; 116(3): 629–36. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSakles JC, Patanwala AE, Mosier JM, et al.: Comparison of video laryngoscopy to direct laryngoscopy for intubation of patients with difficult airway characteristics in the emergency department. Intern Emerg Med. 2014; 9(1): 93–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHoward-Quijano KJ, Huang YM, Matevosian R, et al.: Video-assisted instruction improves the success rate for tracheal intubation by novices. Br J Anaesth. 2008; 101(4): 568–72. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNouruzi-Sedeh P, Schumann M, Groeben H: Laryngoscopy via Macintosh blade versus GlideScope: success rate and time for endotracheal intubation in untrained medical personnel. Anesthesiology. 2009; 110(1): 32–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLow D, Healy D, Rasburn N: The use of the BERCI DCI Video Laryngoscope for teaching novices direct laryngoscopy and tracheal intubation. Anaesthesia. 2008; 63(2): 195–201. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRosenstock CV, Thøgersen B, Afshari A, et al.: Awake fiberoptic or awake video laryngoscopic tracheal intubation in patients with anticipated difficult airway management: a randomized clinical trial. Anesthesiology. 2012; 116(6): 1210–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTodd MM, Bayman EO: Fiberoptic versus videolaryngoscopic management of the difficult airway: problems with postrandomization patient exclusion. Anesthesiology. 2013; 118(2): 460. PubMed Abstract | Publisher Full Text\n\nKramer A, Müller D, Pförtner R, et al.: Fibreoptic vs videolaryngoscopic (C-MAC(®) D-BLADE) nasal awake intubation under local anaesthesia. Anaesthesia. 2015; 70(4): 400–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNoppens RR, Geimer S, Eisel N, et al.: Endotracheal intubation using the C-MAC® video laryngoscope or the Macintosh laryngoscope: a prospective, comparative study in the ICU. Crit Care. 2012; 16(3): R103. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSakles JC, Mosier J, Chiu S, et al.: A comparison of the C-MAC video laryngoscope to the Macintosh direct laryngoscope for intubation in the emergency department. Ann Emerg Med. 2012; 60(6): 739–48. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSakles JC, Mosier JM, Chiu S, et al.: Tracheal intubation in the emergency department: a comparison of GlideScope® video laryngoscopy to direct laryngoscopy in 822 intubations. J Emerg Med. 2012; 42(4): 400–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSilverberg MJ, Li N, Acquah SO, et al.: Comparison of video laryngoscopy versus direct laryngoscopy during urgent endotracheal intubation: a randomized controlled trial. Crit Care Med. 2015; 43(3): 636–41. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nYeatts DJ, Dutton RP, Hu PF, et al.: Effect of video laryngoscopy on trauma patient survival: a randomized controlled trial. J Trauma Acute Care Surg. 2013; 75(2): 212–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAziz MF, Kim D, Mako J, et al.: A retrospective study of the performance of video laryngoscopy in an obstetric unit. Anesth Analg. 2012; 115(4): 904–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWayne MA, McDonnell M: Comparison of traditional versus video laryngoscopy in out-of-hospital tracheal intubation. Prehosp Emerg Care. 2010; 14(2): 278–82. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nJarvis JL, McClure SF, Johns D: EMS Intubation Improves with King Vision Video Laryngoscopy. Prehosp Emerg Care. 2015; 19(4): 482–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nApfelbaum JL, Hagberg CA, Caplan RA, et al.: Practice guidelines for management of the difficult airway: an updated report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway. Anesthesiology. 2013; 118(2): 251–70. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAziz MF, Healy D, Kheterpal S, et al.: Routine clinical practice effectiveness of the Glidescope in difficult airway management: an analysis of 2,004 Glidescope intubations, complications, and failures from two institutions. Anesthesiology. 2011; 114(1): 34–41. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAsai T, Liu EH, Matsumoto S, et al.: Use of the Pentax-AWS® in 293 patients with difficult airways. Anesthesiology. 2009; 110(4): 898–904. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAmathieu R, Combes X, Abdi W, et al.: An algorithm for difficult airway management, modified for modern optical devices (Airtraq laryngoscope; LMA CTrach™): a 2-year prospective validation in patients for elective abdominal, gynecologic, and thyroid surgery. Anesthesiology. 2011; 114(1): 25–33. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nParmet JL, Colonna-Romano P, Horrow JC, et al.: The laryngeal mask airway reliably provides rescue ventilation in cases of unanticipated difficult tracheal intubation along with difficult mask ventilation. Anesth Analg. 1998; 87(3): 661–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAziz M: Airway management in neuroanesthesiology. Anesthesiol Clin. 2012; 30(2): 229–40. PubMed Abstract | Publisher Full Text\n\nNestler C, Reske AP, Reske AW, et al.: Pharyngeal wall injury during videolaryngoscopy-assisted intubation. Anesthesiology. 2013; 118(3): 709. PubMed Abstract | Publisher Full Text\n\nTeoh WH, Saxena S, Shah MK, et al.: Comparison of three videolaryngoscopes: Pentax Airway Scope, C-MAC, Glidescope vs the Macintosh laryngoscope for tracheal intubation. Anaesthesia. 2010; 65(11): 1126–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMosier J, Chiu S, Patanwala AE, et al.: A comparison of the GlideScope video laryngoscope to the C-MAC video laryngoscope for intubation in the emergency department. Ann Emerg Med. 2013; 61(4): 414–420.e1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg I, Hill AL, Williams DL, et al.: Randomized controlled trial comparing the McGrath videolaryngoscope with the C-MAC videolaryngoscope in intubating adult patients with potential difficult airways. Br J Anaesth. 2012; 109(3): 439–43. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11446",
"date": "08 Dec 2015",
"name": "Harald Groeben",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11445",
"date": "08 Dec 2015",
"name": "John Sakles",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1410
|
https://f1000research.com/articles/4-1408/v1
|
07 Dec 15
|
{
"type": "Review",
"title": "Capturing relevant extracellular matrices for investigating cell migration",
"authors": [
"Patricia Keely",
"Amrinder Nain",
"Amrinder Nain"
],
"abstract": "Much progress in understanding cell migration has been determined by using classic two-dimensional (2D) tissue culture platforms. However, increasingly, it is appreciated that certain properties of cell migration in vivo are not represented by strictly 2D assays. There is much interest in creating relevant three-dimensional (3D) culture environments and engineered platforms to better represent features of the extracellular matrix and stromal microenvironment that are not captured in 2D platforms. Important to this goal is a solid understanding of the features of the extracellular matrix—composition, stiffness, topography, and alignment—in different tissues and disease states and the development of means to capture these features",
"keywords": [
"cell migration",
"extracellular matrix",
"ECM",
"stromal microenvironment",
"Matrix remodeling"
],
"content": "Introduction\n\nCell migration is a fundamental process necessary for the creation of tissues during embryogenesis, immune surveillance, wound repair, inflammation, and the invasion and metastasis of cancer cells. All of these processes occur in the context of the extracellular matrix (ECM). For decades, a concerted effort to understand the basic mechanisms of cell migration and other cellular behaviors has focused largely on cells cultured on two-dimensional (2D) surfaces. This body of work has created important knowledge regarding cell migration. Moreover, certain aspects of in vivo cell migration are well represented by 2D assays; for example, recapitulating the movement of keratinocytes closing a wound (for example, 1) or capturing melanoma cell migration on deep dermal tissue layers2.\n\nStudies in vivo demonstrate that cells also use migratory approaches different from those observed on 2D surfaces in vitro, and even 2D migration in vivo occurs in the context of 3D tissue. The variety of possible migratory approaches has created controversy about the mechanism of cell migration, the localization of cell signals, and the necessity of cell-matrix adhesions3,4. Thus, there is a disconnect between what we know about 2D migration and what we might suppose about migration in vivo.\n\nRecently, there has been a move to apply the wealth of knowledge regarding what we know about 2D cell migration to understand cell migration within the context of 3D matrices and in vivo. Leaders in the field have identified several challenges to this task, which include the fact that migration in 3D matrices in vivo is quite different from that in 2D matrices, the difficulty creating relevant in vitro matrices that capture matrix composition and topography of in vivo microenvironments, and the challenges of manipulating the environment in vivo5.\n\nHere, we will investigate features of the ECM that are emerging as key to consider when creating appropriate experimental platforms that can be used to understand the role of the ECM in determining cellular migration, differentiation, development, and pathological processes, and we will discuss ways that these features can be captured by various in vitro approaches. Although no single approach can capture all relevant features, by understanding the strengths and limits of different platforms, one can design the most appropriate approach to address specific questions related to cell migration in vivo.\n\n\nHow do we define a relevant matrix?\n\nThe ECM can be oversimplified into two major types: interstitial ECM (loose or dense connective tissue) and the basement membrane. The 50–200 nm-thick basement membrane is composed predominantly of laminins, proteoglycans (perlecan and others), nidogen/entactin, and collagen IV. Basement membrane surrounds most epithelial units (ascini and alveoli) and vasculature, providing a defining barrier that provides architectural context and a surface on which epithelial and endothelial cells attach and create basal-to-apical polarity6. Specific differences in basement membranes across different tissues are not well understood.\n\nThe interstitial matrix is a complex mixture of proteins that includes predominantly fibrillar collagens supplemented by various proteoglycans and glycoproteins such as fibronectin, laminin, and tenascin. Interstitial matrices vary significantly across different tissues, across developmental time frames, and across disease processes. Interstitial matrices are constructed in an active manner by fibroblasts, which themselves vary across tissues and are altered in pathological conditions. Bone and cartilage are constructed by specialized mesenchymal cells related to fibroblasts: chondrocytes and osteocytes. Other cells such as macrophages, epithelial cells, tumor cells, and adipocytes, are some of the cells that can contribute to ECM production either directly by making ECM proteins or by stimulating fibroblasts to secrete ECM. A complete consideration of how ECM production is regulated has recently been reviewed7.\n\nThus, depending on the question at hand, the investigator is tasked with determining what is an appropriate ECM in terms of composition and architecture to recapitulate a particular extracellular environment. Mass spectrometry analysis of the ECM had been difficult because of its insolubility, which was overcome recently by important technical advances8. Additional informatics applied to analyzing the proteome of the ECM, termed the “matrisome”, have uncovered important knowledge about the composition of the ECM9. From proteomics, it is clear that carcinoma tissue differs from normal tissue in the composition of the ECM10,11. From these studies, tenascin C emerges as an important player in mammary carcinogenesis12,13. Moreover, analysis of metastatic tissue identifies several candidates as possible metastasis promoters10,11. A complete readout of the matrisome of tissues, such as the lung, allows better tissue engineering approaches14.\n\n\nStiffness and cross-linking\n\nMatrix stiffness has emerged as a key consideration in understanding cellular response to the ECM. Matrix stiffness varies considerably across tissues, and elastic modulus values are around 0.1–1 Pa for neuronal tissue, approximately 8–17 kPa for muscle, and 25–40 kPa for bone15–17. Moreover, stiffness changes with pathological conditions. For example, whereas normal breast tissue has an elastic modulus on the order of 1.2 kPa, breast tumors are significantly stiffer (moduli of 2.4–4.8 kPa)18. These moduli can be represented by increasing the concentration of collagen gels; gels around 1 mg/mL are similar to normal breast tissue, whereas collagen gels around 4–6 mg/mL have a modulus similar to that of tumors18–20. The fate of mesenchymal stem cells can be manipulated by matrix stiffness such that a neuronal fate is promoted by culture on a compliant/soft matrix, whereas an osteogenic fate is promoted by a stiff matrix21.\n\nMatrix stiffness increases with the progression of carcinomas and this is due in part to increased deposition of collagen and matrix remodeling19,22. Moreover, in cancer progression, there is a dramatic upregulation of matrix cross-linkers, including lysyl oxidases (LOXs) and tissue transglutaminases, that correlates to increased matrix stiffness23–25. The normal physiological role of these cross-linkers is increased stiffness (for example, in dermal wound healing)26,27. In breast cancer, upregulation of LOX contributes to the increased stiffness surrounding breast tumors23. The importance of accounting for stiffness is demonstrated by findings that altering stiffness dramatically changes the expression of genes and drives a proliferative and metastatic phenotype in breast carcinoma19,22. Moreover, matrix stiffness alters the response of cells to hormones and growth factors21,23–28.\n\n\nImaging the extracellular matrix\n\nIt is worth spending a moment discussing how one sees the structure of the ECM, as this has led to experimental questions we might otherwise have failed to ask. The ability to see collagen fibers in live samples without adding exogenous fluorophores is feasible through the techniques of confocal reflectance microscopy, second harmonic generation (SHG), or third harmonic generation (THG). These approaches discern most, if not all, types of fibrillar collagen but cannot discern subtypes of collagen composition. As such fibers represent the major structural feature of the ECM, capturing their structure and organization is often a good place to begin an understanding of what comprises appropriate ECM architecture.\n\nConfocal reflectance microscopy relies on the backward scatter of light and is easily obtained on a standard confocal microscope29,30, making it readily available to a majority of biologists. Of potential harmonophores, collagen fibrils have a particularly strong SHG signal and are readily imaged31–34, even in the context of multiple fluorophores (for example35). SHG is the preferred approach for imaging thicker 3D and in vivo samples, as the longer wavelengths used can penetrate deeper into tissue, and the use of two photons eliminates out-of-plane focus. SHG misses some fibers, as it depends on the collagen structure to generate the signal. The third harmonic, THG, is useful in combination with SHG, as it captures some structures that are invisible to SHG, including elastin fibers2,33,36–38. Other approaches are being advanced to visualize collagen by using its unique structural properties. Coherent anti-Stokes Raman scattering (CARS) imaging makes use of molecular vibrations to visualize collagen and elastin fibers and discern them from cellular structures39,40. Optical coherence tomography (OCT) can make use of polarization to discern highly ordered collagen structures such as those in tendon41, and has recently been combined with multiphoton imaging42. There is also interest in exploiting collagen, and the structural information it conveys for meso- and macro-scopic imaging approaches using OCT43, which is being exploited for intra-operative imaging of collagen structures44.\n\n\nTopography and alignment\n\nUsing SHG of tissues, the Keely lab has characterized a set of collagen changes, termed Tumor-Associated Collagen Signatures (TACS), that accompany tumor progression (Figure 1). Notably, these changes manifest in predictable ways and are characterized by the deposition of bundled, straight collagen (TACS-2) that becomes oriented perpendicularly to the tumor-stromal boundary (TACS-3)45. Importantly, these changes are observed in human breast cancer, and the presence of TACS-3 collagen is an independent predictor of poor outcome46. Collagen alignment is also observed in the ECM of the involuting mammary gland during a window in which the ECM demonstrates increased ability to promote mammary carcinogenesis47. Recent findings demonstrate that haplo-insufficiency for collagen III, which can form mixed fibrils with collagen I, leads to an increase in aligned collagen and tumor progression in murine models48. Several additional aspects likely contribute to collagen alignment, as discussed in a recent review7. It is becoming appreciated that the structure of collagen around tumors of various origins in addition to breast carcinoma, including ovarian49, colon50, and prostate51 cancers, changes during cancer progression. It is of interest that the collagen structures of these carcinomas are not identical to each other or to that of breast carcinoma, yet each has a structure that is distinguishable from the normal tissue. A common feature is the increased organization of the collagen to be more aligned, but the actual structure of the collagen (wavy or straight, thick or thin) varies by tissue. Thus, during attempts to capture topography in vitro, it will be important to consider tissue-specific structures.\n\n(A) Second harmonic generation (SHG) image of a normal mouse mammary gland. Collagen appears white. (B) SHG of a mouse mammary PyMT tumor. Note the straightened and aligned collagen perpendicular to the tumor boundary. Both images are reprinted with permission from Provenzano et al.34 (2008). (C) Intravital image of human MDA-MB-231 breast carcinoma cells in mouse mammary gland. Cells are transfected with green fluorescent protein. Collagen appears white. Note that cells polarize along collagen. (D) Zoom of boxed region in (C). The arrow points to a single collagen fiber in the field of view. Scale bars = 50 μm.\n\nAlthough the definition of TACS is a useful means to quantify topography in breast cancer, it is becoming clear that a broader means to quantify and describe collagen structure is needed, as there are tissue-specific and disease-specific differences in collagen structure. To address this, a concerted effort has gone into developing image analysis tools to capture and quantify collagen features such as fibril width, alignment, spacing, density, and curliness (www.loci.wisc.edu/software)52. It is our vision that these additional collagen features will allow the field to provide quantifiers to the structure of the ECM, and to parse out which features may track with pathological changes.\n\nAligned collagen facilitates the migration of carcinoma cells away from the primary tumor and toward the vasculature35,53–55. Cells overwhelmingly track along fibers and are not efficient at migration across the same density of collagen fibers when they are oriented parallel to the cell-matrix boundary53. This observation may reflect the fact that collagen is stiffer along the axis of alignment compared with across the axis of alignment56. Moreover, cells migrating on aligned fibers demonstrate more persistent migration and adopt a bipolar phenotype with fewer lateral protrusions and increased membrane blebbing56, much like the lobopodial migration described by Petrie et al.3. As cell migration can be limited by nuclear deformation, especially when proteases are not available, alignment may provide open tracks through which cells move their nuclei57. Although it is intuitively obvious that cells would track along a collagen fiber, the molecular mechanism by which aligned collagen facilitates invasion is unknown. Below, we discuss approaches that should facilitate investigation of migration on aligned fibers.\n\nMatrix remodeling is likely concomitant with cell migration, and the cleavage and straightening of fibers have been observed with careful microscopy58. Moreover, as cancer cells migrate through the ECM, their ability to cleave the matrix can be a distinct advantage59. When protease activity is broadly blocked, cells must change their shape to adapt to the available space because they are more limited by the confinement of the matrix57. One choice cells make to navigate confined spaces is the use of amoeboid migration57. Another choice may be lobopodial migration, which is observed within dense dermal matrices that may be quite confined and is lost when cells are unconfined on top of these same matrices3. Thus, migrating along aligned fibers provides cells with multiple advantages: a stiffer environment on which to move, cues to achieve cellular polarization and limit “distracting” lateral protrusions, and the creation of open areas that allow the nucleus to be readily moved with the cell. The result is efficient and persistent migration on collagen fibers.\n\n\nModeling the complex features of the extracellular matrix in vitro\n\nAlthough animal studies and investigation of cell migration by intravital imaging trump any in vitro approach when considering the perfect representation of the relevant ECM, they are limited by the difficulty of the approach. More importantly, it is often difficult to precisely control ECM features in a manner that allows mechanistic understanding of cell response to a particular feature, and this approach is not amenable to large-scale screening or multi-factorial manipulations. Thus, relevant in vitro systems are needed to inform and complement the in vivo studies.\n\n\nCapturing extracellular matrix stiffness\n\nOne of the most often overlooked aspects of in vitro systems by those not studying mechano- signal transduction is the need to consider ECM stiffness, which profoundly regulates gene expression, stem cell fate, differentiation, and cell phenotype16,19,21,22,28,60. Although investigators may not realize it, the majority of in vitro experimental approaches set the cellular microenvironment orders of magnitude stiffer than the relevant tissue by coating ECM components on plastic or glass surfaces. In fact, many ignore the ECM altogether by performing experiments on cells cultured on uncoated surfaces, not appreciating that in fact they are culturing on an ill-defined combination of fibronectin, vitronectin, and several other proteins, adsorbing from the bovine serum in the medium onto the plastic. The fact that cell behavior on these surfaces is not the same as in vivo cautions us to consider the ways such studies may be limited. For example, in contrast to the more uniformly spread cells that are accompanied by stress fibers and large focal adhesions that are observed on such stiff 2D surfaces, cells in 3D matrices tend to have more elongated shapes, minimal stress fiber formation, and smaller focal adhesions3,61–64.\n\nECM stiffness is most readily captured by the use of polyacrylamide substrata (PAS), in which the amount of bisacrylamide can be varied to tune stiffness in a near-linear manner65–67. Similar approaches make use of alginate gels or mixed alginate-polyacrylamide68. These surfaces are functionalized by the addition of a cross-linker and then coated with the desired ECM component. The result is the ability to tune stiffness in a precise way and test specific questions about stiffness and cell response. Moreover, nanopatterning allows precise manipulation of the spatial organization and topography of varied stiffness66, and the use of hydrogel columns allows simultaneous measurement of cellular force on the substratum69.\n\nPolyethylene glycol (PEG) hydrogels with controlled stiffness allow incorporation of cells into 3D environments and measurement of forces within70. Hyaluronic acid-based 3D gels demonstrate the importance to stem cell differentiation of adding the third dimension71. Cross-linking of alginate gels with carbonate allows tuning of stiffness72. However, often a limit of hydrogels is that they are dense with minimal pores compared with a natural ECM, and thus cells either confront them as a 2D surface or invade them by uncertain mechanisms. For example, the addition of hyaluronic acid to otherwise soft substrata results in cellular behavior appropriate for stiff surfaces73. In some cases, this property is exploited to create barriers, define geometries, and confinement conditions74.\n\nAn additional feature of hyaluronic acid and proteoglycans is their ability to sequester water in the interstitial matrix, which adds to their effect on matrix stiffness. When combined with the effects of pathological conditions such as diabetes, inflammation, cancer, or surgery, all of which can damage or limit lymphatics, the result can be increased interstitial pressure and altered fluid flow. In pancreatic cancer, hyaluronic acid limits adequate perfusion of the tissue with anti-cancer chemotherapeutics, which can be reversed with hyaluronidase75,76. Interstitial pressure and fluid dynamics are emerging as key regulators of cell behavior, and can enhance cell migration77.\n\nPerhaps the simplest manner in which to create a relevant 3D matrix is the use of collagen gels made from neutralized rat tail or bovine skin collagen. Depending on the extraction method, rat tail collagen typically retains the non-collagenous N- and C-terminal telopeptide domains that allow cross-links of lysine residues. In contrast, collagen obtained from dermis is usually extracted with trypsin, removing the bulk of these regions. These two sources have been directly compared and demonstrate differences in the phenotype and migration of embedded cells57. Stiffness is easily manipulated by contrasting gels that are attached to the culture dish (“restrained” or stiff) compared with those that are released from the dish to float in the medium (“contractible” or compliant; reviewed in 78). Varying the concentration of collagen results in exponential stiffening of the gels over a concentration range of 1–5 mg/mL collagen22,79.\n\nVarious additional ECM components, such as collagen V, elastin, fibronectin, and other proteins, can be added to these gels80. The cross-linking and stiffness of collagen gels can be further modified by the addition of glutaraldehyde, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS)81–83, hydroxyapatite84,85, or sugars such as ribose or glucose to cause glycation86,87. Gels composed of fibrin are also used for several applications; for example, fibrin gels allow endothelial cells to undergo vasculogenesis88. Although gels composed of recombinant basement membrane (matrigel) mimic the composition of the basement membrane, they are not readily manipulated and are too soft to present cells with a stiff environment.\n\n\nCapturing extracellular matrix topography and porosity\n\nAs described above, the architecture and topography of the ECM have profound ability to alter cellular response. Intravital imaging of carcinoma cell migration on thick collagen fibers (approximately 1–3 μm in thickness) suggests that for some studies it is important to capture this feature4,89. Recent approaches to recapitulate this sort of migration in simple culture systems have led to the approach of painting thin isolated strips of collagen on a surface to create “1D” cell migration studies90. With this approach, it was recently noted that the spatial organization of the well-known favorite regulators of cell migration, Cdc42, Rac, and Rho, is completely different from when cells are migrating on a 2D surface or within a disorganized 3D collagen matrix3,91.\n\nInherently, 1D patterning lacks control of two features that are likely to be important: the diameter of ECM fibers and their stiffness. Cell interaction on fibrous ECM can be categorized at two scales: (a) cells stretching across and interacting with the whole collagen fiber network, and thus responding to bulk material properties or (b) cells interacting with a small number of fibrils or bundles of fibers. Therefore, in vitro models mimicking the ECM need to account for both the elastic modulus of the whole mesh, as well as the bending stiffness of individual ECM fibrils of varying diameters. In vivo, the tissue architecture varies considerably on the basis of tissue and disease state, and optimal fiber diameter and network pore size result in efficient migration (speed, distance travelled, and persistence). Either a more or a less dense network can lead to less efficient migration: in a dense network, a large number of contacts cause cells to encounter confinement, whereas less dense matrices with larger pores can lead to insufficient contact points for efficient migration91–93.\n\nOne means to precisely control fiber properties is the use of fibers of polycarbonate, or polycaprolactone (polyurethane), which can be combined with collagen94,95. Arguably, electrospinning is the most widely known and thoroughly studied method of forming polymeric nanofibers. In this process, the polymer solution is pumped through a syringe to a needle where an electrical charge extrudes polymer fibers onto a collecting target96,97. The principles underlying the process were first observed over 100 years ago, and modern refinements allow electrospinning to generate micro/nanoscale fibers96,98. With the realization that electrospinning could produce fibers with diameters on the order of those in native tissue, there has been rapid growth in the use and improvement of electrospinning techniques to achieve greater control of alignment and spatial organization. However, the manufacturing challenges in controlling diameter, spacing, and alignment restrict the questions that can be investigated by using electrospinning methods96,98. Non-electrospinning spinneret-based tunable engineered parameter (STEP) technique is a pseudo-dry spinning nanofiber fabrication technique that does not rely on an electric field to stretch the solution filament, thus allowing arrays of highly aligned fibers to be created. The STEP fiber manufacturing platform allows suspended fibers of a variety of polymers to be deposited with control of fiber dimensions (diameter of less than 50 nm to microns and length in centimeters) and orientation (0–90° and sub-micrometer fiber-spacing in single and multiple layers). With this approach, a network of suspended fibers can be generated that allows the manipulation of fiber stiffness, diameter, and topographical features such as fiber spacing, orientation relative to one another, and the ability to juxtapose fibers of different dimensions, such as micro- and nanofibers (Figure 2)99–102. Cells on suspended fibers adapt to the underlying fibrous arrangement, acquiring a spindle morphology on single or parallel fibers, and a polygonal morphology on intersecting fibers.\n\nThe platform was created by using the spinneret-based tunable engineered parameter (STEP) technique as described by Nain et al.100 (2009). (A) STEP manufacturing platform and scanning electron microscope images of fibers fabricated (i) same diameter crosshatch, (ii) mix of diameters in three layers, and (iii) varying orientations. (B) Immunofluorescence images of single cells in (i) spindle, (ii) parallel with star showing leading edge, and (iii) polygonal shapes (red: f-actin stress fibers, blue: nucleus, and green: paxillin focal adhesion clusters). Scale bars: 10 µm (Ai and Aii), 20 µm (Aiii), 50 µm (B).\n\nFiber arrangements can thus be created to provide cells with simultaneous 1, 2, and 3D cues, by which cells can align along the fiber axis (1D), spread and stretch between fibers (2D), and wrap around fibers, thus sensing the curvature (3D). Indeed, considering the diameter or curvature of the fibers is important, as resulting cell phenotypes are profoundly affected by the diameter of nano- and micro-fibers95,103–105. For example, Meehan and Nain show that spindle cell migration speed increases with larger-diameter fibers of similar structural stiffness, but that focal adhesion length decreases105. Cells attached to small-diameter fibers (less than approximately 250 nm) exhibit rounded morphology with active protrusive rates (Supplementary movie 1), and single cells attached to multiple small-diameter fibers are able to spread (Supplementary movie 2). This suggests the role of curvature and attachment sites in cell behavior, probably reflecting the need to achieve a minimum threshold area required to establish mature focal adhesions for spreading. Furthermore, through the ability to juxtapose fibers of different dimensions, such as micro- and nanofibers, deposited at different spacing, different migratory modes and associated cell forces can be determined (Supplementary movie 3 and Supplementary movie 4).\n\nA key feature of migration on a collagen fiber or a 1D strip is that the cell is confined by adhesive choices. Physical confinement due to limited spacing within the ECM is another important feature of the microenvironment that cells must navigate. Various approaches have been used to model this question. The use of microfluidic chambers with widths that vary from 3–50 μm demonstrates that cells are significantly confined and slowed in their migration speed below their nuclear diameter (approximately 10 μm in this study)106,107. By varying the porosity of collagen gels, Wolf et al. determined that cells deform their nucleus during migration but that this is limited; cells cannot traverse through pores smaller than 10% of their nuclear diameter57. The use of proteases to cleave the ECM allows cells to overcome this confinement. Similar results are obtained when a laser is used to etch tracks into 3D collagen matrices, in which there is a lower size limit to tracks through which cells can traverse. Moreover, the tracks guide cells as the path of least resistance108. 3D microchannels have also been used to create physical matrix confinement at defined 2D interfaces. With polydimethylsiloxane (PDMS) pillars to hold up a coverslip under pressure, cells can be forced to migrate through confined spaces, demonstrating the effect on nuclear architecture and profound changes in gene expression109. PDMS microchannels have also been used to create confined spaces through which cells migrate in response to chemotactic gradients107.\n\nAn alternative way to engineer topography is through light-based nanofabrication. Here, photoactivation of a hydrogel environment is controlled at the nanoscale to allow incorporation of desired ECM components into a 3D environment110, and has recently been adapted to take advantage of multiphoton microscopy111. Gradients of ECM molecules, including fibronectin, can be created with precise spatial control112. Moreover, the composition can be matched to in vivo analysis with spatial control based on imaging; for example, the dense, wavy, and aligned nature of the ovarian carcinoma microenvironment can be recapitulated in terms of nanotopography113,114. Photochemistry production of defined matrices has been used to control stem cell differentiation and mimic developing heart tissue114,115. A converse approach is to create a collagen gel scaffold and use multiphoton microscopy to generate microtracks within the scaffold. This approach was used to demonstrate the role of confinement and nuclear deformability as a limiting factor in cell migration57,108.\n\n\nCapturing collagen alignment\n\nA limit to many of the above approaches is that, although they can incorporate collagen, they do not fully capture the collagen fibril or fiber structure that is observed in vivo. Collagen assembles into a wide variety of fiber structures on the basis of collagen packing, the incorporation of different collagen types, cross-linking, and the addition of other molecules such as fibronectin or proteoglycans. Although in vitro collagen can self-assembly, fibronectin and minor collagens in particular are crucial for collagen fibril assembly in vivo (reviewed in 116). Moreover, as detailed above, cells track along collagen fibers by using mechanisms that differ from migration within disorganized matrices3,56. Thus, there is a great need to create matrices that capture both collagen fiber structure, as well as topography and alignment.\n\nMany approaches take advantage of strain-induced alignment. We have been able to align large gels by strain and then cut out smaller regions to test alignment in the parallel and perpendicular orientations56. Cells themselves can exert strain on the collagen, and a highly aligned region can be created between two plugs of dense cells within a 3D collagen gel53. Force based on shear and flow has also been used to align collagen. Collagen extruded from a syringe into a narrow tube creates a high level of alignment and can be further stiffened with cross-linkers to mimic the stiffness of tissue, such as tendon117,118.\n\nWe have exploited micro-chambers to generate aligned collagen by using vacuum-induced flow (Figure 3, 56). If the diameter of the microchannel is narrow (1 mm), an aligned matrix is formed68. These channels can be compared to wider channels (3 mm) in which collagen is randomly organized56. A consideration when working with collagen is that the fibril and fiber diameter can be manipulated by changing the temperature of nucleation and polymerization or by changing the pH of the medium57. Nucleation conditions, polymerization conditions, and flow conditions can be combined to further tune the effects.\n\n(A) Design of microchannels. Collagen is neutralized and pre-incubated for several minutes prior to pulling it across a narrow (1 mm wide × 250 μm tall) or wide (3 mm wide × 250 μm tall) microfluidic chamber. The chambers are incubated for several hours at 37°C, and then the mask for the central port is removed, allowing cells to be seeded into the central port. Cells are incubated and imaged by time-lapse microscopy for 3–4 days. (B) Second harmonic generation image of collagen in a narrow and wide chamber demonstrates aligned fibers in the narrow chamber. (C) Cells in aligned matrices (narrow channel) exhibit elongated morphology and minimal protrusions, whereas cells in random matrices (wide channel) have more abundant protrusions. MDA-MB-231 breast carcinoma cells were transfected with LifeAct-GFP. All images are reprinted with permission from Riching et al.56.\n\nFor many investigations, a single ECM component is used as a substratum for investigation, which is an advantage when the biological question is to compare isolated ECM molecules. However, often the desire is to create a relevant context in which to ask other questions. Recent advances in proteomics demonstrate the molecular complexity of the endogenous ECM9. Even once we know all the components that make up a particular tissue-specific ECM, it may be impossible to re-create the ECM environment in vitro. One approach is to culture tissue-specific fibroblasts and allow the fibroblasts to assemble a complex cell-derived matrix (CDM). After several days, the fibroblasts are removed by a gentle alkaline lysis, resulting in a matrix that is 3D at the cellular scale. A nuanced approach is to use tissue-specific mesenchymal stem cells derived from periods of tissue development rather than from the adult. For example, the use of fetal synovium-derived stem cells (SDSCs) creates an ECM that is superior to that of adult SDSCs in promoting chondrocyte differentiation119.\n\nThe use of CDMs has allowed an increased understanding of cell adhesions and behavior. By combining this approach with genetically engineered fibroblasts, one can further manipulate the CDM and in many cases achieve a complex matrix that is also aligned7. Overexpression of integrin-linked kinase (ILK) in cardiac fibroblasts leads to increased collagen deposition and fibrosis120, whereas overexpression of fibroblast activation protein (FAP) in fibroblasts leads to deposition and organization of aligned collagen and fibronectin in pancreatic carcinoma121. Deposition of an aligned matrix also requires syndecan-1 expression in fibroblasts122. Conversely, knockdown of caveolin-1 (Cav-1) in fibroblasts results in deposition of a less organized matrix123. By combining fibroblast CDMs with substrata of various stiffness, one can additionally control the microenvironment. In addition, stiffness of the ECM can be achieved by transfection of fibroblasts with LOX23,122,123.\n\nA limit of CDM is that generic fibroblast cell lines such as 3T3 cells may not accurately represent the specific ECM of the tissue under investigation and this can be mitigated by using tissue-specific primary fibroblasts or cell lines derived from them when possible. An additional complication is that fibroblasts in healthy adult tissue are not functionally the same as those found in fetal tissues or wound healing, or those associated with carcinomas124. Recent developments using patient-specific fibroblasts to create CDMs show great promise in capturing ECM features of the tumor microenvironment125.\n\nAn alternative approach has been to decellularize isolated tissue and use the remaining ECM as a scaffold for cell studies and tissue engineering. The reader is referred to a recent review that covers several examples of decellularized dermis and other tissues126. Cardiac progenitor cells will migrate into decellularized pericardium and differentiate127,128. Human decellularized adipose tissue not only promotes the culture of adipocytes but works as an appropriate matrix for breast cells, which exist in the mammary fat pad129. Neuronal matrix scaffolds promote the migration of neural crest-derived cells130, suggesting that there may be usefulness for nerve regeneration. Dermal matrix promotes the migration of keratinocytes to aid healing of wounds and burns131,132, and tendon matrix promotes stem cell migration and differentiation to repair tendon133. One issue has been the rapid degradation of these matrices in vivo, and the addition of PEG into the ECM scaffold adds stability134.\n\n\nAdding complexity\n\nAs our understanding of cellular behavior deepens, there is an increased need to create complex mimetics of tissue structure. For example, hydrogel-based microfluidic molds can be patterned in a manner that a lumen-based structure is created and surrounded by collagen and stromal cells. In this manner, it is possible to capture features of endothelial-lined blood vessels or epithelial-lined ducts. By the addition of stromal cells, the complex interaction between the endothelium and the surrounding stroma is captured135,136. Moreover, it is possible to place a breast ductal structure near a blood vessel structure with stroma in between to probe the processes of invasion, intravasation, and extravasation137. Layered microchannel scaffolds of collagen and alginate have been created to mimic the layers of smooth muscle cells that surround blood vessels138. From these approaches, it is now possible to build complexity by including multiple cell types such as macrophages, endothelium, epithelium, smooth muscle, and fibroblasts in the same culture system.\n\n\nSummary\n\nWith increased understanding of the complex composition and structure of the ECM and how that varies in different developmental stages and during normal and disease processes, it will be possible to create in vitro microenvironments that better capture the complex in vivo ECM. Such relevant ECMs will allow a more complete understanding of basic questions related to cell migration, wound healing, and a variety of other cellular behaviors. By adding tissue-specific cells, fibroblasts, vascular components, and immune cells, the bi-directional signaling between these compartments will be more readily investigated. In addition to advancing our understanding, these approaches should lead to the development of disease-specific and personalized tissue mimetics for testing drug efficacy across a variety of patients and conditions.",
"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\nSupplementary materials\n\nMovie 1. Cells attached to small-diameter fibers. STEP (spinneret-based tunable engineered parameter)-based fiber assembly of two small-diameter fibers (less than approximately 250 nm) suspended between two large-diameter fibers (north-south direction, diameter of approximately 2 µm). Cells attached to small-diameter fibers (approximate location shown by two arrows) exhibit rounded morphology, and active protrusive rates suggest insufficient surface area to mature focal adhesions. Cells attached to the large-diameter fibers are stretched in spindle morphologies. Time is presented in hours:minutes:seconds https://f1000researchdata.s3.amazonaws.com/supplementary/6623/ef7d9031-4085-4a6f-8ae2-ed78248caebd.wmv\n\nMovie 2. Cell attached to multiple small-diameter fibers. STEP (spinneret-based tunable engineered parameter)-based fiber assembly of multiple small-diameter fibers (less than approximately 250 nm) suspended between three large-diameter fibers (north-south direction, diameter of approximately 2 µm). Single cell attached to multiple small-diameter fibers is able to stretch and migrate while simultaneously putting multiple protrusions on individual fibers (shown by arrows). Time is presented in hours:minutes:seconds https://f1000researchdata.s3.amazonaws.com/supplementary/6623/57923fd9-0179-4d99-bed1-5b393e6a1b67.wmv\n\nMovie 3. Cells on single and parallel fibers. STEP (spinneret-based tunable engineered parameter)-based fiber assembly of aligned fibers with varied spacing causing cells to form spindle and parallel shapes of varying widths, each having different migratory response. Time is presented in hours:minutes https://f1000researchdata.s3.amazonaws.com/supplementary/6623/7e378b7d-9112-4581-9dad-822e4ab95957.wmv\n\nMovie 4. Cells on single and intersecting fibers. STEP (spinneret-based tunable engineered parameter)-based fiber assembly of aligned parallel and intersecting fibers. Cells attached to single fibers form spindle morphologies, while a dividing cell at the intersecting fiber forms polygonal shape (kite) and then emerges on the single fiber as spindle. Time is presented in hours:minutes https://f1000researchdata.s3.amazonaws.com/supplementary/6623/ab307a40-d2e0-4845-8962-c2bae2091979.wmv\n\n\nReferences\n\nTscharntke M, Pofahl R, Chrostek-Grashoff A, et al.: Impaired epidermal wound healing in vivo upon inhibition or deletion of Rac1. J Cell Sci. 2007; 120(Pt 8): 1480–90. 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Macromol Rapid Commun. 2009; 30(16): 1406–12. PubMed Abstract | Publisher Full Text\n\nNain AS, Phillippi JA, Sitti M, et al.: Control of cell behavior by aligned micro/nanofibrous biomaterial scaffolds fabricated by spinneret-based tunable engineered parameters (STEP) technique. Small. 2008; 4(8): 1153–9. PubMed Abstract | Publisher Full Text\n\nWang J, Nain AS: Suspended micro/nanofiber hierarchical biological scaffolds fabricated using non-electrospinning STEP technique. Langmuir. 2014; 30(45): 13641–9. PubMed Abstract | Publisher Full Text\n\nKhandalavala K, Jiang J, Shuler FD, et al.: Electrospun nanofiber scaffolds with gradations in fiber organization. J Vis Exp. 2015; (98). PubMed Abstract | Publisher Full Text\n\nSheets K, Wunsch S, Ng C, et al.: Shape-dependent cell migration and focal adhesion organization on suspended and aligned nanofiber scaffolds. Acta Biomater. 2013; 9(7): 7169–77. PubMed Abstract | Publisher Full Text\n\nSharma P, Sheets K, Elankumaran S, et al.: The mechanistic influence of aligned nanofibers on cell shape, migration and blebbing dynamics of glioma cells. Integr Biol (Camb). 2013; 5(8): 1036–44. PubMed Abstract | Publisher Full Text\n\nMeehan S, Nain AS: Role of suspended fiber structural stiffness and curvature on single-cell migration, nucleus shape, and focal-adhesion-cluster length. Biophys J. 2014; 107(11): 2604–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalzer EM, Tong Z, Paul CD, et al.: Physical confinement alters tumor cell adhesion and migration phenotypes. FASEB J. 2012; 26(10): 4045–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTong Z, Balzer EM, Dallas MR, et al.: Chemotaxis of cell populations through confined spaces at single-cell resolution. PLoS One. 2012; 7(1): e29211. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBischel LL, Casavant BP, Young PA, et al.: A microfluidic coculture and multiphoton FAD analysis assay provides insight into the influence of the bone microenvironment on prostate cancer cells. Integr Biol (Camb). 2014; 6(6): 627–35. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBischel LL, Beebe DJ, Sung KE: Microfluidic model of ductal carcinoma in situ with 3D, organotypic structure. BMC Cancer. 2015; 15: 12. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nBischel LL, Sung KE, Jiménez-Torres JA, et al.: The importance of being a lumen. FASEB J. 2014; 28(11): 4583–90. 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}
|
[
{
"id": "11461",
"date": "08 Dec 2015",
"name": "Edna Cukieman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11462",
"date": "08 Dec 2015",
"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": []
}
] | 1
|
https://f1000research.com/articles/4-1408
|
https://f1000research.com/articles/4-1407/v1
|
07 Dec 15
|
{
"type": "Software Tool Article",
"title": "Crambled: A Shiny application to enable intuitive resolution of conflicting cellularity estimates",
"authors": [
"Andy G. Lynch"
],
"abstract": "It is now commonplace to investigate tumour samples using whole-genome sequencing, and some commonly performed tasks are the estimation of cellularity (or sample purity), the genome-wide profiling of copy numbers, and the assessment of sub-clonal behaviours. Several tools are available to undertake these tasks, but often give conflicting results – not least because there is often genuine uncertainty due to a lack of model identifiability. Presented here is a tool, \"Crambled\", that allows for an intuitive visual comparison of the conflicting solutions. Crambled is implemented as a Shiny application within R, and is accompanied by example images from two use cases (one tumour sample with matched normal sequencing, and one standalone cell line example) as well as functions to generate the necessary images from any sequencing data set. Through the use of Crambled, a user may gain insight into why each tool has offered its given solution and combined with a knowledge of the disease being studied can choose between the competing solutions in an informed manner.",
"keywords": [
"Cancer",
"Whole-genome sequencing",
"Cellularity",
"Copy number",
"Sub-clonality",
"Cell lines",
"Shiny",
"R",
"Bioconductor"
],
"content": "Introduction\n\nThe generation of whole-genome sequencing data to investigate tumour samples has become commonplace, thanks in particular to initiatives such as the International Cancer Genome Consortium (ICGC)1. Among the analyses being applied to the data generated are investigations of copy number changes2, structural variants3, and sub-clonality4. These analyses typically require the establishment of segmented copy number profiles for the sample, which in turn rely on estimating the degree of contamination of normal tissue in a sample and the mean ploidy (equivalently the depth of coverage associated with a particular copy number state or the increase in depth associated with an increment in copy number).\n\nMany tools have been developed that allow for the estimation of one or more of sample purity, copy number profile and clonality. These include e.g. ABSOLUTE (www.broadinstitute.org/cancer/cga/absolute)5, ASCAT (heim.ifi.uio.no/bioinf/Projects/ASCAT)6, CloneHD (github.com/andrej-fischer/cloneHD)7, OncoSNP-SEQ (sites.google.com/site/oncosnpseq/)8 and QPure (sourceforge.net/projects/qpure/)9 (for a more complete review see Yadav and De.10). There are real problems of model identifiability in performing this task, particularly when sub-clonal solutions are allowed, as any errors or discrepancies in the purity, segmentation or identification of mean ploidy can often be explained as sub-clonal behaviour (many of the issues are discussed in Lonnstedt et al.11). Indeed, many tools acknowledge the inherent uncertainty in the calculations. As a consequence, applying two tools to a data set may return two conflicting solutions.\n\nContrasting this problem with another common analysis applied to whole-genome cancer sequencing data, that of identifying somatic single nucleotide variants and indels then there is one striking difference: SNV calls can be easily validated. This can be via a targeted experimental approach, or increasingly commonly through visual assessment using a tool such as the Integrative Genomics Viewer12.\n\nHerein is presented the Crambled tool to enable the visual assessment of the alternative purity/depth-of-sequencing solutions that can arise for a tumour sample. Through exploration of the solutions using Crambled one may achieve an appreciation of the reasons for each to have been offered. Coupled with knowledge of the tumour type, and the case history and pathology of the particular patient under examination, it may be possible to then select a preferred solution.\n\n\nMethods\n\nOverview. The Crambled tool is implemented as a Shiny13 application in R14. In essence it takes in a figure depicting the sequencing data (akin to a patchwork plot15 or grid plot11), constructs a second figure based on a solution for the cellularity/depth conditions, and displays them in a superimposed manner. By allowing for a dynamic adjustment of the cellularity/depth solution being considered, Crambled enables an intuitive comparison of competing solutions and a tactile exploration of the solution space that provides insight into why different tools offer different solutions and informs the user in choosing between them.\n\nThe Crambled tool is divided into two sections as depicted in Figure 1, nominally a ‘client’ side and a ‘server’ side, to avoid having to upload across a network the large BAM files that contain the genome sequencing data.\n\nTasks are divided between those on the ‘client’ side (that aim to reduce the size of the data that need to be transferred) and the main application on the ‘server’ side (where the cellularity/depth solutions can be investigated).\n\nEven when both ‘client’ and ‘server’ sides are run on the same machine, as is envisaged for a typical user, there are clear benefits to the division of labours. The initial data processing is an expensive operation and should only be performed once, with the output stored for potential repeated and spontaneous investigations in the future.\n\n‘Server’-side tools. The Crambled application consists primarily of a dialogue box for selecting an image representing a sequencing experiment, two sliders for specifying cellularity and the depth of a single copy (that is the depth associated with a single copy present in all cells in a sample or, equivalently, half of the depth of coverage in diploid regions), and a display that dynamically updates the selected image with superimposed predictions based on the chosen values.\n\nThe display figure is generated by first writing out the predictions to a temporary image before the two image files are combined. For two values, cellularity (C) and single-copy depth (D), the initial predictions for a region with n1 copies of one allele and n2 copies of the second are:\n\n\n\nand\n\n\n\nThese values are depicted in Figure 2.\n\nTop left: The basic predicted depths of sequencing and allele fractions that are expected for a number of copy number states. Top right: For six cases the uncertainty in the allele fraction that will be observed is illustrated. Bottom left: When the distribution folds at the 0.5 level, the expected value of the allele fraction is reduced. Bottom right: The predicted depths of sequencing and allele fractions that are used in the Crambled tool.\n\nFor a locus that is heterozygous in the germline sample, the observed minor allele fraction is recorded. If the SNPs are phased, and one is considering a region in allelic balance, then it is possible to take all the allele fractions from one allele and gain a mean fraction of 0.5. Without the phasing information, the minor allele will be taken at each locus and so the mean allele fraction recorded in the region will be below 0.5. Even in regions of strong allelic imbalance, it is possible that the observed allele fraction can exceed 0.5 when the true value does not, and so the mean allele fraction in those regions will also be biased.\n\nGiven the true minor allele fraction and depth, the observed fraction of the true minor allele has an estimable probability distribution. The mass of probability for allele fractions greater than 0.5 then ‘folds’ below 0.5 to reflect the distribution of the observed fraction of the observed minor allele, which is the value recorded. In this manner, the mean of the distribution is reduced. This effect is greater for small depths of sequencing due to the greater variance in observed allele fraction that comes from having a smaller denominator. The effect is greater also for allele fractions that are close to 0.5 (Figure 2).\n\nThe final options available in the server interface allow the user to edit some of the metadata concerning the image loaded into Crambled. Specifically, the user can depict a different range of depths as appropriate for their experimental data, and can specify the size of the image if this is different to the default.\n\n‘Client’-side tools. It may be that the user will already have details of germline variants, and their depths and allelic fractions in the tumour sample, from which to create an image to load into Crambled. Such data may be purposefully sought from tools such as GATK (www.broadinstitute.org/gatk/)16, SAMtools (www.htslib.org/)17, or parabam (parabam.readthedocs.org/). Alternatively, the information may be available as the side effect of running a somatic mutation caller. In the event that such data are not available, functions are supplied with Crambled to enable suitable data to be generated from BAM files.\n\nThe function CrambledScan() is accompanied by a file that lists (for Human Genome Issue 19) 177,299 sites that are highly likely to be heterozygous in a sample. This was generated from the “snp138Common” table for hg19 from the UCSC Table Browser18. Rsamtools19 is used to interrogate the BAM files at those locations. At a depth of coverage of about 40×, this typically returns approximately 80,000 heterozygous sites once quality filters have been put in place.\n\nA running median is then applied to the depths and allele fractions at these sites to reduce noise. Note that this is not an attempt to characterize the entire genome, but merely to capture the cellularity information. Thus it does not matter if fine-grain copy number changes are lost in this smoothing. Ideally one would have as many germline heterozygous loci as possible (typically 2,000,000 such loci for an individual), but extracting them comes at a computational cost. The approximately 80, 000 loci used here will usually suffice for this limited task, and fewer may be feasible as seen in Figure 3.\n\nTop left: A smoothed density plot of depth and B-allele fraction for sample SS6003302 (see Use Case 1) using 84,252 loci and no running average (equivalently a running average with a window length of 1). Top right: The same density plot with a running average (window size: 51 loci) first applied to the depth and minor allele fractions. Middle left, Middle right, Bottom left: The same density plots produced from samples of 50,000, 10,000 and 5,000 loci respectively. Bottom right: The same density plot produced from a sample of 1,000 loci and using a window of 11 loci for the running average.\n\nThe need for using a running average is also demonstrated in Figure 3. Note that as the number of heterozygous loci being used decreases, then a window of, e.g., 50 loci represents a much larger genomic region, making it more likely that the values being averaged represent several distinct states and that the average will not represent a true state. Reducing that window will increase the noise in the picture and so a balance must be sought that reflects the complexity of the genome being studied.\n\nThe plotting function (CrambledPlot) takes the output of this approach and produces a standard R plot, but with parameters set to values that the Crambled application will anticipate. Should it be necessary to change these, e.g. to increase the limits on the plotting area because a sample has been sequenced to 500× depth of coverage, then the Crambled app needs to be informed via the metadata input options.\n\nThe tool and code presented here are built on top of R14 and Shiny13, and thus will run on a large number of operating systems. The dependencies within R are on the ‘Shiny’ package (obtainable from The Comprehensive R Archive Network [cran.r-project.org]) to run the application, and the ‘Rsamtools’ packages from Bioconductor20 required for the code to prepare images for Crambled. Naturally, if the BAM files have been aligned against a genome other than human genome issue HG-19, then the list of suggested loci for investigation that accompanies the Crambled tool will not be relevant and an alternative list must be generated. The user must create an image from the sequencing data of interest (either using the code provided, or independently in the style of the example images) and then load this into the Crambled application. The Crambled application can be run on a local machine that has R and the R shiny package installed, or it can be run on a Linux machine running the Shiny Server software (www.rstudio.com/products/shiny/shiny-server/). It has been extensively tested on a Ubuntu 12.04.5 machine running R version 3.2.1, Shiny 0.12.2 and Rsamtools 1.20.5. and an Apple computer running OS X version 10.9.5, R version 3.1.2, Shiny 0.11.1 and Rsamtools 1.18.3.\n\nThe code provided with Crambled for creating the images takes approximately 20 minutes to run (using a single processor on a reasonable desktop machine) on a single 100GB cell line BAM file, or approximately an hour to run on two 150GB BAM files representing a tumour/normal pair.\n\n\nUse cases\n\nThe first illustration of Crambled is an application to a previously published tumour/normal pair from an oesophageal adenocarcinoma (OAC) patient21. Two estimates of cellularity have been suggested in this case: 81% and 68%. While a small variation in estimates is natural, in this case the difference is too great to put down to the (in)stability of the estimate and it warrants investigation with the Crambled tool.\n\nThe first step is to generate the image to load into the Crambled Shiny application. Assuming that one has obtained the two BAM files (here called “SS6003301.bam” and “SS6003302.bam”), within the R environment one types:\n\n\n\nThis produces the file ‘SS6003022-shiny.png’ (available in the ExamplePlots folder at https://github.com/dralynch/crambled.git), that can be loaded into the Crambled application. The application may be operating on a server or, after installing Shiny, within R one can type:\n\n\n\nto begin the tool. Uploading the figure, one can then test the two solutions as seen in Figure 4.\n\nLeft: The solution at 81% cellularity fits the extremes of the data (the red contour) well, suggests a (hypo)diploid solution and leaves two states (blue regions in the density plot) unexplained by the clonal model (presumably representing sub-clonal behaviour). Right: The solution at 68% cellularity fits the extremes of the data equally well, and explains all of the main observed states, but suggests that the tumour is largely tetraploid.\n\nOne can see that the solution with 81% tumour assumes that the sample is primarily diploid, with some regions of copy number loss and, crucially, some regions of data that lie off of the predicted grid and so must represent ‘sub-clonal’ states. By contrast, the 68% cellularity solution shows that all of the data lie on the predicted grid (i.e. there is little-to-no evidence of sub-clonality). However this solution suggests that the sample is broadly tetraploid.\n\nSince OAC cases are often tetraploid, and this solution explains away all of the sub-clonality, the 68% cellularity solution seems to be the favourable one. While sub-clonal behaviour is also seen frequently in OAC, and the 81% solution is entirely valid, that the suggested sub-clonality conveniently only occurs at frequencies that can be explained by the tetraploid solution leads to a favouring of 68%.\n\nCell lines present a different problem. Typically one would not be trying to estimate the cellularity of a cell line (which should be pure), but resolving the depth-of-sequencing/copy number can still be an issue, as can confirming that only a single population of cells is present.\n\nCommonly, there will be no germline reference sample for a tumour cell line. Coupled with this, due to the (near-)perfect purity of a cell line, it is not possible to distinguish a germline homozygous site from a heterozygous site that has undergone loss-of-heterozygosity (LOH). Nor is it possible to distinguish a clonal somatic mutation from a germline-heterozygous site.\n\nFor the purposes of inferring cellularity, neither of these confusions matters except that in producing a patchwork- or grid- plot the signal from sites with no heterozygosity will drown out any observations from the (more informative) loci that have gained or retained heterozygosity. The purity of the cell line, and the consequent separating of LOH-representing regions from other regions on the plot allows for separation of the two groups (with a simple threshold on allele fraction) and subsequent down-sampling of the LOH-like regions. Since this case requires different preparation, a separate command is provided for the creation of the plot.\n\nThis usage is illustrated using whole genome sequencing from the Genome Modelling System (github.com/genome/gms)22. Three lanes of whole genome sequencing data for the HCC1395 breast cancer cell line (gerald_D1VCPACXX_1.bam, gerald_D1VCPACXX_2.bam, and gerald_D1VCPACXX_3.bam) were downloaded (via github.com/genome/gms/wiki/HCC1395-WGS-Exome-RNA-Seq-Data) and aligned to the EnsEMBL release 71 assembly of the GRCh37 (hg19) human genome (apr2013.archive.ensembl.org/index.html).\n\n\n\nThis produces the file ‘HCC1395-shiny.png’ (available in the ExamplePlots folder at https://github.com/dralynch/crambled.git). This image can be loaded into the Crambled application as per the previous use case. Therefore, within R one would type:\n\n\n\nThe results can be seen in Figure 5. The cell line appears to be a single population of cells, with copy numbers mainly in the 2 to 4 range (with some regions at a copy number of 1, and others at a copy number of 5 but with a four-to-one allele balance). Note that the thresholding of allele fractions may, at low depths, cause an artefactual data cloud to appear in the plot close to the threshold and suggestive of sub-clonality, but that this should be ignored.\n\n\nDiscussion\n\nThe code provided to produce the images to load into the Crambled application suffices for the intended purpose, but has shortcomings with regard to other tasks. Firstly, it is prohibitively slow if one wishes to search for the complete set of informative loci in the genome, and the resolution that such a set brings to the problem is useful. One of the other tools mentioned in the introduction can be used if this is desired.\n\nSecondly, there is no correction either for biases such as GC-related biases in the tumour sample, nor for regions of the normal sample that are polyploid, nor for artefactual mapping. Any of these would reduce the noise in the figures (particularly that about the depth estimate) and could potentially allow fewer loci, or a smaller window for the running average, to be used.\n\nFinally, it should be noted that little effort is required to annotate the uploaded image more than is currently the case. Individual chromosomes could be picked out and highlighted using different colours. Alternatively, if one wanted to know whether e.g. TP53 had undergone LOH, it would be straightforward to mark the location of nearby SNPs and see where they lie in the plot.\n\n\nSummary\n\nCrambled is a tool that allows for interactive investigation of the multiple solutions that are possible when inferring the cellularity (and related attributes) of a tumour sample that has undergone whole genome sequencing. Implemented as a Shiny application it can be used on most platforms and comes with example files and code to prepare such files from one’s own sequencing data.\n\n\nSoftware availability\n\n1. Software available from: https://dralynch.shinyapps.io/crambled_app (limited usage server - the preference is for the user to download the Crambled application and run it locally)\n\n2. Latest source code: https://github.com/dralynch/crambled.git\n\n3. Link to archived source code as at time of publication: http://www.dx.doi.org/10.5281/zenodo.3414723\n\n4. License: Lesser GNU Public License 2.0: https://www.gnu.org/licenses/old-licenses/lgpl-2.0.html",
"appendix": "Author contributions\n\n\n\nAGL conceived and conducted this research.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAGL is supported by a Cancer Research UK programme grant (C14303/A20406) to Simon Tavaré and additionally acknowledges funding from the European Commission through the Horizon 2020 project SOUND (Grant Agreement no. 633974).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe author acknowledges the access to test data provided by the Oesophageal cancer clinical and molecular stratification (OCCAMS) consortium.\n\n\nReferences\n\nInternational Cancer Genome Consortium, Hudson TJ, Anderson W, et al.: International network of cancer genome projects. Nature. 2010; 464(7291): 993–998. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZack TI, Schumacher SE, Carter SL, et al.: Pan-cancer patterns of somatic copy number alteration. Nat Genet. 2013; 45(10): 1134–1140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPleasance ED, Cheetham RK, Stephens PJ, et al.: A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 2010; 463(7278): 191–196. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnight SJ, Yau C, Clifford R, et al.: Quantification of subclonal distributions of recurrent genomic aberrations in paired pre-treatment and relapse samples from patients with B-cell chronic lymphocytic leukemia. Leukemia. 2012; 26(7): 1564–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarter SL, Cibulskis K, Helman E, et al.: Absolute quantification of somatic DNA alterations in human cancer. Nat Biotechnol. 2012; 30(5): 413–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Loo P, Nordgard SH, Lingjaerde OC, et al.: Allele-specific copy number analysis of tumors. Proc Natl Acad Sci U S A. 2010; 107(39): 16910–16915. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFischer A, Vázquez-García I, Illingworth CJ, et al.: High-definition reconstruction of clonal composition in cancer. Cell Rep. 2014; 7(5): 1740–1752. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYau C: OncoSNP-SEQ: a statistical approach for the identification of somatic copy number alterations from next-generation sequencing of cancer genomes. Bioinformatics. 2013; 29(19): 2482–2484. PubMed Abstract | Publisher Full Text\n\nSong S, Nones K, Miller D, et al.: qpure: A tool to estimate tumor cellularity from genome-wide single-nucleotide polymorphism profiles. PLoS One. 2012; 7(9): e45835. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYadav VK, De S: An assessment of computational methods for estimating purity and clonality using genomic data derived from heterogeneous tumor tissue samples. Brief Bioinform. 2015; 16(2): 232–241. PubMed Abstract | Publisher Full Text\n\nLönnstedt IM, Caramia F, Li J, et al.: Deciphering clonality in aneuploid breast tumors using SNP array and sequencing data. Genome Biol. 2014; 15(9): 470. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson JT, Thorvaldsdóttir H, Winckler W, et al.: Integrative genomics viewer. Nat Biotechnol. 2011; 29(1): 24–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. R package version 0.11.1. 2015.\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. 2014. Reference Source\n\nMayrhofer M, DiLorenzo S, Isaksson A: Patchwork: allele-specific copy number analysis of whole-genome sequenced tumor tissue. Genome Biol. 2013; 14(3): R24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcKenna A, Hanna M, Banks E, et al.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–303. PubMed Abstract | Publisher Full Text | Free Full Text\n\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\nKarolchik D, Hinrichs AS, Furey TS, et al.: The UCSC Table Browser data retrieval tool. Nucleic Acids Res. 2004; 32(Database issue): D493–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan M, Pagès H, Obenchain V, et al.: Rsamtools: Binary alignment (BAM), FASTA, variant call (BCF), and tabix file import. R package version 1.18.3. 2015. Reference Source\n\nGentleman RC, Carey VJ, Bates DM, et al.: Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol. 2004; 5(10): R80. PubMed Abstract | Publisher Full Text | Free Full Text\n\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\nGriffith M, Griffith OL, Smith SM, et al.: Genome Modeling System: A Knowledge Management Platform for Genomics. PLoS Comput Biol. 2015; 11(7): e1004274. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLynch AG: Crambled: Release v1.0 to accompany manuscript. Zenodo. 2015. Data Source"
}
|
[
{
"id": "11450",
"date": "05 Jan 2016",
"name": "Jan Oosting",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Lynch presents an R app ‘Crambled’ to ease the determination, in whole genome DNA sequencing experiments, of cellularity in tumour samples or to show the presence of sub clones in cellline samples.A shiny app displays an image of the behavior of heterozygous SNPs according to read-depth and minor allele frequency, and overlays that with the theoretical positions of chromosomal aberrations at user- defined cellularity and single copy coverage levels.The image can be produced, starting from BAM-files, by the accompanying functions of the app.The manuscript is written clearly, and describes the functionality of the package well. I have not tested all functions of the package, but the R code is understandable and straightforward. The functions are commented throughout. As described in the paper, during the use of the shiny app, it is important to have an idea of the cellularity of the sample. Just playing around with the sliders does not converge to logical solutions easily. The example case 1 illustrates the usefulness of the app because it shows clearly that one of the cellularity solutions is to be preferred over the other. I would like more guidance on how to get the cellularity estimates. The references 5-9 seem to be geared mostly towards array type of data. It would be a good addition to provide a preferred solution in the workflow description. It would be good to mention the values for cellularity and single copy depth that were chosen for figure 2.In conclusion, the manuscript presents a tool that is useful in the assessment of copy-number aberrations. There are a few minor details that could be improved.",
"responses": []
},
{
"id": "12383",
"date": "19 Feb 2016",
"name": "Ingrid Lönnstedt",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article describes \"Crambled\", an online application to investigate cellularity/ploidy solutions of genome sequencing copy number data. The easily accessible and soundly developed tool, enabling informed choices between potential solutions, is the main contribution of this study. It also comprises additional advances to existing methods, as (i) the naturally occurring bias in minor allele fractions of heterozygous loci is assessed and adjusted for, and (ii) crambled suggests potentially heterozygous sites which can add substantially to the information a solution choice is based upon.These are minor suggestions to improve the article:I would have enjoyed some performance statistics on Crambled, for example showing the fraction of samples tried that were successfully resolved, or the fractions of samples tried that had in practice a good fit to expected cluster centers for at least say 70% of the genome. At the moment I can only see the overall performance of a couple of of samples, and Figure 2 seems to be based on simulated data so I cannot assess whether this strategy works out well with real data. I have only tried the online Crambled application, not the full from bam file setup which requires source download. Based on the current version of the article, it is not obvious to me whether the full downloadable version of Crambled returns a list with the copy numbers of a chosen cellularity solution are assigned to genomic locations, or whether this solutions (which will commonly be a central desired outcome of the analysis) have to be extracted with a different software. In the last section before Discussion, I do not understand where the \"cloud close to the threshold\" is, which is falsely suggesting subclonality. Could that be explained better? The png figures to be supplied to Crambled should show \"minor allele proportion\" versus \"depth of sequencing\". Preferably explain that \"depth of sequencing\" is different from the definition of \"Depth\" in equation 1 (assuming you mean the total depth, not just the depth of one copy). I think many readers would have enjoyed some more instructions on how to play with the cellularity and depth in the application. What should I try to achieve, what should I try to avoid?",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1407
|
https://f1000research.com/articles/4-1406/v1
|
07 Dec 15
|
{
"type": "Review",
"title": "Overactive bladder",
"authors": [
"Karen M. Wallace",
"Marcus J. Drake",
"Karen M. Wallace"
],
"abstract": "Overactive bladder syndrome is highly prevalent, and increasingly so with aging. It is characterized by the presence of urinary urgency, and can be associated with incontinence, increased voiding frequency, and nocturia. Assessment needs to exclude serious medical disorders that might present with similar symptoms, and a bladder diary is an invaluable part of understanding the presentation. Initial management is conservative, comprising education, bladder training, and advice on fluid intake. Drug therapy options include antimuscarinic medications and beta-3 adrenergic receptor agonists. Persistent overactive bladder syndrome, despite initial therapy, requires a review of the patient’s understanding of conservative management and compliance, and adjustment of medications. For refractory cases, specialist review and urodynamic testing should be considered; this may identify detrusor overactivity or increased filling sensation, and needs to exclude additional factors, such as stress incontinence and voiding dysfunction. Botulinum neurotoxin-A bladder injections can be used in severe overactivity, provided the patient is able and willing to do intermittent self-catheterisation, which is necessary in about 5% of treated patients. Sacral nerve stimulation and tibial nerve stimulation are other approaches. Major reconstructive surgery, such as augmentation cystoplasty, is rarely undertaken in modern practice but remains a possibility in extreme cases.",
"keywords": [
"Overactive bladder syndrome",
"urinary urgency",
"nocturia",
"detrusor overactivity",
"mirabegron"
],
"content": "Introduction\n\nThe International Continence Society1, with slight modification by the International Consultation on Incontinence Research Society2, states that overactive bladder syndrome is urinary urgency, with or without urgency incontinence, usually with increased daytime frequency and nocturia, if there is no proven infection or obvious pathology.\n\nThe components of the symptom syndrome of overactive bladder syndrome are as follows. The key symptom is urinary urgency, which is the complaint of a sudden compelling desire to void that is difficult to defer1. Urgency should not be confused with a strong desire to void, which is effectively the normal sensation when functional bladder capacity is reached. Patients often express anxiety around the “fear of leakage” due to urgency. It is the “fear of leakage” and the “fear of pain” that differentiates patients suffering from urgency in overactive bladder syndrome from those with bladder pain syndrome3. In overactive bladder syndrome, urgency is typically felt at the perineum or the base of penis or vagina/urethra. In contrast, in bladder pain syndrome suprapubic pain is characteristic, though additional perineal (urethral/vaginal/penile) discomfort can occur4.\n\nUrgency urinary incontinence is defined as involuntary leakage of urine, accompanied or immediately preceded by urgency1. Urgency urinary incontinence can cause concern to the patient, such as social or hygiene effects, but the extent to which patients complain of such problems is very varied.\n\nIncreased daytime frequency is the complaint made by the patient who considers that he or she voids too often by day1. This standardised definition does not include any minimum number of voids, since there is considerable overlap between normal and overactive bladder syndrome in terms of objective voiding frequency. Currently, evidence to set a threshold for defining increased daytime frequency is lacking, so a minimum number of voids is not included in the definition.\n\nNocturia is the complaint made by the individual who has to wake at night one or more times to void1,5.\n\nMixed urinary incontinence encompasses overactive bladder syndrome and stress urinary incontinence. The complaint is of involuntary leakage associated with urgency and with exertion, effort, sneezing, or coughing1.\n\nThe current definition of overactive bladder syndrome is a symptomatic diagnosis. In contrast, detrusor overactivity (DO) is a urodynamic observation, characterised by involuntary detrusor contractions during the filling phase, which may be spontaneous or provoked1. These are not terms that can be interchanged, as overactive bladder syndrome sufferers may not have detrusor activity on urodynamic testing.\n\nThe overall prevalence of overactive bladder syndrome is approximately 12%6. Men have a higher prevalence of “overactive bladder syndrome dry”, meaning urgency without urgency urinary incontinence, and women had a higher prevalence of “overactive bladder syndrome wet”7. The differing prevalence of incontinence is presumed to be due to the relative weakness of the bladder neck and urethral sphincter mechanism in women. The prevalence of overactive bladder syndrome wet in women was also noted to increase significantly with age, from 2.0% in those aged between 18 and 24 to 19.1% in those 65 to 74 years of age.\n\nOveractive bladder syndrome affects many patients and their quality of life; the symptom of urgency having the biggest impact. A large study carried out in the US, UK and Sweden reported that storage symptoms held greater impact than other lower urinary tract symptoms8. Substantial cost is associated with overactive bladder syndrome. The population cost estimates are dependent on the accuracy of prevalence data. In estimate, the average annual per capita cost of overactive bladder syndrome in the US was $1925 in 2000, bringing a national cost of tens of billions of dollars9. This can be inferred to include indirect impact, such as falls when having to void urgently10, and impaired social or occupation functioning11.\n\n\nPathophysiology and aetiology\n\nAs overactive bladder syndrome is a condition based on symptoms, there is no equivalent animal model12. Instead, surrogates are used, and assumptions have to be made, which should not be over-interpreted. The research focus has narrowed in on three key aspects.\n\nThe first of these is lower urinary tract sensory activity, which is due to the sensations driving the symptom syndrome. Sensory activity involves the understanding of afferent signalling, including signal transduction and afferent traffic, gating, sensitization, and conscious perception. The lower urinary tract nerve endings are densely concentrated under the urothelium. In this location, they may be exposed to release of mediators by the urothelium13,14, cellular influences, and cytokines15. Thus, many patients may experience urinary urgency as a consequence of altered sensory input.\n\nThe second aspect is motor control, which is due to the presence of detrusor overactivity in many people with overactive bladder syndrome. Motor function involves the processes giving rise to the contractility in the detrusor muscle of the bladder. Many changes in properties of bladder smooth muscle have been described in overactive bladder syndrome and detrusor activity, leading to the “myogenic hypothesis of detrusor activity”. This suggests that overactive detrusor contractions result from increased excitability and spread of contraction within the muscle16. This may be further altered via the efferent nerves, interstitial cells and local mediators17. Such excitation is normal during voiding, but should not occur during storage, since there are CNS inhibitory influences to suppress them.\n\nThe third aspect of research focus is on reflexes of the lower urinary tract, since bladder urine storage requires inhibition of the muscle18, such that disinhibition of bladder motility or voiding can be presumed to cause detrusor overactivity or urgency urinary incontinence. The motor behaviour of lower urinary tract reflexes is underpinned by the ascending afferents of sensory information that are integrated at various CNS levels (notably the brainstem and the sacral spinal cord)19,20. The potential importance of the reflexes is encapsulated in the neurogenic hypothesis of detrusor overactivity, which states that detrusor overactivity arises from generalized, nerve-mediated, excitation of the detrusor muscle21.\n\nOveractive bladder syndrome prevalence has some gender disparity, and there is a higher prevalence in women, particularly in younger people. A small influence of racial factors is also present, with slightly higher prevalence among African Americans than among Hispanics and whites, for both men and women22.\n\nNeurological disease is highly associated with lower urinary tract dysfunction, due to impaired regulatory influence of the innervation on the lower urinary tract. The CNS should allow people to store urine asymptomatically; consequently, neurological disease commonly impairs urine storage, leading to detrusor overactivity.\n\nBladder outlet obstruction has also been considered to play a part in detrusor overactivity and overactive bladder syndrome, although the relationship is not clear. Detrusor overactivity and overactive bladder syndrome increase with aging, and co-existing bladder outlet obstruction may enhance this effect.\n\nOveractive bladder syndrome can often be described by patients also reporting functional problems, including altered bowel function23 and fibromyalgia24.\n\n\nClinical assessment\n\nGiven that potentially serious mechanisms could present with similar features, assessment needs to exclude disorders such as neurological, malignant, or systemic disease. Careful history taking, physical exam and urinalysis are mandatory25,26. The history should include symptoms such as urgency, urgency incontinence, nocturia, increased frequency, dysuria, haematuria, and lower urinary tract pain. These symptoms are often best explored with a symptom-based questionnaire. The clinician should also enquire about fluid intake to assess for polydipsia and stimulants that could worsen lower urinary tract symptoms27. Other symptoms considered may include new-onset tremor or erectile dysfunction, which may hint at neurological disease. Other existing medical conditions, such as closed angle glaucoma, history of urinary retention, and cognitive impairment, should be ascertained, as these are relative contraindications to anti-muscarinic therapy. Likewise, hypertension may be a contraindication to beta-3 adrenergic agonist therapy.\n\nA general examination, abdominal and pelvic examination, and a basic neurological examination should be carried out. By palpating the abdomen after recent voiding it is possible to identify significant post void bladder residual, noting that such a situation may impair response to bladder storage medications. Urinalysis is crucial in all assessments to exclude urinary tract infections, leukocyturia, and haematuria.\n\nEvaluating frequency of voiding and nocturia is carried out by getting the patient to complete a frequency volume chart or bladder diary. In overactive bladder syndrome, the pattern of voided volumes is often characteristically erratic. On a frequency volume chart, frequency is the number of voids recorded during waking hours, including the last void before sleep and the first void on waking and rising in the morning1. Completion of such a record can identify hindering factors that affect management, such as nocturnal polyuria, or potential factors leading to sleep disturbance28. The maximum voided volume recorded can indicate the severity of overactive bladder syndrome and detrusor overactivity. A bladder diary is used to capture information beyond that seen on a frequency volume chart, such as symptom scores. Good examples of scores used in a diary include the ICIQ Bladder diary score29 or the Patient Perception of Intensity of Urgency Scale (PPIUS)30. Scales allow patients to rank a score for each time they void, such as 0 (no urgency) through to 4 (urgency incontinence: leaking before arriving at the toilet). A quantitative method of assessing the bladder diary is the Total Urgency and Frequency Score (TUFS), where the daily sum of the PPIUS scores is calculated31.\n\nUrodynamics, cystoscopy, and diagnostic urinary tract ultrasound should not generally be used in initial workup of the uncomplicated patient25,26. They may have a role where symptoms persist despite compliance with appropriate initial therapy.\n\n\nTreatment\n\nThe nature of the overactive bladder syndrome definition enables an empirical diagnosis, allowing clinicians to initiate preliminary treatment. Thus, it is common practice to employ conservative management and oral pharmacotherapy without a urodynamic diagnosis. In overactive bladder syndrome, management of expectations necessitates taking an honest approach explaining the nature of the symptom syndrome and that “cure” is not a realistic goal. Lifestyle interventions play a large part in managing overactive bladder syndrome: educating the patient about their condition and certain aspects that influence it, such as volume of fluid intake, smoking cessation, and making certain dietary alterations. Bladder training and pelvic floor muscle training are valuable components32 and may re-establish some inhibitory control over bladder storage.\n\nDrug treatments should be initiated after conservative methods have been tried. Anti-muscarinic medication is a main drug class used for treatment33. The efficacy of the drugs is counterbalanced by potential side effects that the patient should be warned about, such as dry mouth, constipation, cognitive effects, and visual impairment, amongst others. The cognitive effects may prevent the use of antimuscarinics in older patients34. Accordingly, a balance of beneficial and adverse effects has to be considered when prescribing therapy and reviewing outcomes35. As a drug class, persistence rates are low (12–39% at 12 months and 6–12% at 24 months)36. Part of this may result from the well-recognised placebo effect in overactive bladder syndrome drug therapy37, which may bring an initial but poorly sustained response, and may reflect the potentially heterogeneous populations included within the clinical trials.\n\nA beta-3 adrenergic agonist has also been introduced as a means of medical management of overactive bladder syndrome, which works by detrusor relaxation through binding to the subtype sympathetic receptor. Large phase 3 clinical trials have found therapeutic efficacy38,39 and a differing adverse effect profile from antimuscarinics. Accordingly, mirabegron can be considered for older patients40. Mirabegron can bring symptom improvement to people who have not had adequate response to antimuscarinics41. Combination therapy, using both antimuscarinic and mirabegron, is under development42,43.\n\nMen with overactive bladder syndrome can legitimately be prescribed antimuscarinic drugs44. Men with concurrent voiding symptoms should be given an additional alpha-1 adrenergic blocker as first line45. While caution is advised in prescribing antimuscarinics to men45, long-term (up to a year) combination therapy of alpha blocker and antimuscarinic in men with moderate-to-severe lower urinary tract symptoms is associated with a very low rate of acute urinary retention46.\n\nThe treatment will need to be followed up and tailored according to the efficacy and adverse effects of the drugs45. Once medication has begun, repeating a symptom assessment questionnaire can be used to evaluate response.\n\nIf the initial treatment seems inadequate, then a specialist referral is appropriate. In this situation it is necessary to review the diagnosis, look for complicating factors, ensure that suitable initial therapy has been given and complied with, and that response has been appropriately evaluated. Adverse effects can influence willingness to continue medication47.\n\nUrodynamic evaluation is potentially part of specialist assessment. It applies when conservative and medical management is suboptimal and if overactive bladder syndrome is having a significant impact on a patient’s quality of life. The patient should be sufficiently healthy and in a position to consider more invasive therapeutic intervention if appropriate. The aim of urodynamics is to recreate the patient’s symptoms and identify what factors can influence the treatment. The diagnoses associated with overactive bladder syndrome in this investigation are detrusor overactivity and increased filling sensation. Detrusor overactivity can be phasic (characterised by increasing amplitude in contractions when the bladder volume increases) or terminal (a single involuntary detrusor contraction at cystometric capacity, causing incontinence often leading to bladder emptying). It is important to realise that some patients with detrusor overactivity are asymptomatic and do not, strictly-speaking, have overactive bladder syndrome. In women, detrusor overactivity may not be present in some patients with overactive bladder syndrome48,49. If detrusor overactivity is absent, the patient may report increased bladder sensation during urodynamics, that is, an early and persistent desire to void1. If detrusor overactivity isn’t observed during bladder filling, then a provocation test can be initiated, such as generating the sound of running water. If urodynamic stress incontinence is seen, it should be ascertained whether the urodynamic stress incontinence leads to a sensation of urgency, which can occur due to urethral stimulation, and should not be confused with detrusor overactivity. The voiding phase in a urodynamic test may reveal bladder outlet obstruction or incomplete bladder emptying.\n\nConservative methods are revisited, including bladder training, pelvic floor muscle training, and fluid advice, as this important aspect is often overlooked in initial treatment, and specialist centres may be better set up for more effective delivery. Likewise, pharmacotherapy is fully reviewed, based on antimuscarinics and/or beta-3 adrenergic agonist, to ensure a suitable agent, dose level, and timing.\n\nInterventional therapy may be offered, potentially including the following options.\n\nIntravesical botulinum neurotoxin-A injection has become established as a mainstream method of management of refractory overactive bladder syndrome26. The technique involves injection of 100 units of onabotulinum-A throughout the bladder wall50–52. Prior to injection, patients need to be taught intermittent self-catheterisation, as there is a small risk of significant urinary retention after injections. The response is temporary, but the majority of patients will respond to repeat injections, which typically are needed between six months and a year later. The major adverse effect is urinary tract infection. Furthermore, the extent of symptom control fluctuates, peaking at about a month, and declining thereafter until retreatment.\n\nSacral nerve stimulation, can be used as an alternative to botulinum injections, and patients who are dissatisfied with botulinum toxin-A treatment, or in whom such treatment fails, can respond successfully to sacral neuromodulation53. Sacral nerve stimulation follows a test phase, in which temporary electrodes are placed adjacent to the S3 sacral nerve root and connected to an external battery pack. If sufficient symptomatic improvement is seen, the definitive electrode can be placed, with a subcutaneous stimulator pack. The implant is expensive, and the battery pack has to be replaced once it has discharged. However, if viewed in the longer term, the cost may be comparable to that of other treatments54. Incontinence episodes and voiding frequency are both reduced while receiving sacral nerve stimulation, and this is associated with improved quality of life55,56. Device-related adverse events were reported in one study for 16% of subjects during test stimulation and 30% of subjects post-implant55.\n\nTibial nerve stimulation is achieved with a fine needle electrode temporarily placed at the level of the medial malleolus of the ankle, observing for intrinsic foot muscle contraction as an indicator of proximity to the nerve57. The stimulus is applied at close to tolerance threshold for half an hour. Treatment is repeated at weekly intervals for an induction phase, and then the interval is increased to maintain efficacy. This approach is labour-intensive, though some patients can learn to self-administer. Efficacy is comparable to that seen with antimuscarinics, but with fewer adverse effects58. Due to the intensive nature of treatment delivery and uncertain long-term efficacy, tibial nerve stimulation is not widely used in mainstream healthcare.\n\nThe exact pathway for interventional therapy options varies between centres. In addition, patient preferences are an important factor when considering next steps for managing overactive bladder syndrome persisting despite drug therapy59.\n\nAugmentation cystoplasty is a rare procedure in modern management of overactive bladder syndrome60,61. It requires splitting the bladder in half, and incorporating an isolated section of small intestine that has been detubularised. The operation is a major undertaking, and there is a risk of major complications, such as rupture of the reconstructed bladder, metabolic effects (due to reabsorption of toxins from the urine by the bowel segment), new bowel symptoms, and sepsis. Accordingly, this option is only considered once all alternatives have been fully explored.\n\n\nConclusions\n\nOveractive bladder syndrome is a common problem, with potentially substantial impact on quality of life. Assessment requires exclusion of serious diagnoses and other influential factors. Management is initially conservative, followed by a tailored use of antimuscarinics or mirabegron, with follow up review. Refractory cases need specialist reassessment, typically with urodynamic testing. Management may then require botulinum neurotoxin injections, sacral nerve stimulation, tibial nerve stimulation, or even augmentation cystoplasty. Each option has significant considerations, which must be fully evaluated with the patient to avoid major problems.",
"appendix": "Competing interests\n\n\n\nMarcus J. Drake is on the Research and Advisory Boards of, and is a Speaker for, Allergan, Astellas and Ferring.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbrams P, Cardozo L, Fall M, et al.: The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Neurourol Urodyn. 2002; 21(2): 167–78. PubMed Abstract | Publisher Full Text\n\nDrake MJ: Do we need a new definition of the overactive bladder syndrome? ICI-RS 2013. Neurourol Urodyn. 2014; 33(5): 622–4. PubMed Abstract | Publisher Full Text\n\nHanno P, Nordling J, Fall M: Bladder pain syndrome. Med Clin North Am. 2011; 95(1): 55–73. PubMed Abstract | Publisher Full Text\n\nFitzGerald MP, Kenton KS, Brubaker L: Localization of the urge to void in patients with painful bladder syndrome. Neurourol Urodyn. 2005; 24(7): 633–7. PubMed Abstract | Publisher Full Text\n\nvan Kerrebroeck P, Abrams P, Chaikin D, et al.: The standardization of terminology in nocturia: report from the standardization subcommittee of the International Continence Society. BJU Int. 2002; 90(Suppl 3): 11–5. PubMed Abstract | Publisher Full Text\n\nIrwin DE, Milsom I, Hunskaar S, et al.: Population-based survey of urinary incontinence, overactive bladder, and other lower urinary tract symptoms in five countries: results of the EPIC study. Eur Urol. 2006; 50(6): 1306–14; discussion 1314–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nStewart WF, Van Rooyen JB, Cundiff GW, et al.: Prevalence and burden of overactive bladder in the United States. World J Urol. 2003; 20(6): 327–36. PubMed Abstract\n\nSexton CC, Coyne KS, Kopp ZS, et al.: The overlap of storage, voiding and postmicturition symptoms and implications for treatment seeking in the USA, UK and Sweden: EpiLUTS. BJU Int. 2009; 103(Suppl 3): 12–23. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGanz ML, Smalarz AM, Krupski TL, et al.: Economic costs of overactive bladder in the United States. Urology. 2010; 75(3): 526–32, 532.e1–18. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKurita N, Yamazaki S, Fukumori N, et al.: Overactive bladder symptom severity is associated with falls in community-dwelling adults: LOHAS study. BMJ Open. 2013; 3(5): pii: e002413. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGoren A, Zou KH, Gupta S, et al.: Direct and indirect cost of urge urinary incontinence with and without pharmacotherapy. Int J Clin Pract. 2014; 68(3): 336–48. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nParsons BA, Drake MJ: Animal models in overactive bladder research. Handb Exp Pharmacol. 2011; (202): 15–43. PubMed Abstract | Publisher Full Text\n\nBirder L, Andersson KE: Urothelial signaling. Physiol Rev. 2013; 93(2): 653–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBirder LMD, de Groat W, Fowler C, et al.: Neural Control. In: Incontinence. edn. Edited by Abrams P, Cardozo L, Khoury S, Wein A. Paris: Health Publication Ltd. 2009; 167–254.\n\nAndersson KE, McCloskey KD: Lamina propria: the functional center of the bladder? Neurourol Urodyn. 2014; 33(1): 9–16. PubMed Abstract | Publisher Full Text\n\nBrading AF: A myogenic basis for the overactive bladder. Urology. 1997; 50(6A Suppl): 57–67; discussion 68–73. PubMed Abstract | Publisher Full Text\n\nDrake MJ, Mills IW, Gillespie JI: Model of peripheral autonomous modules and a myovesical plexus in normal and overactive bladder function. Lancet. 2001; 358(9279): 401–3. PubMed Abstract | Publisher Full Text\n\nSadananda P, Drake MJ, Paton JF, et al.: A functional analysis of the influence of β3-adrenoceptors on the rat micturition cycle. J Pharmacol Exp Ther. 2013; 347(2): 506–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBirder L, de Groat W, Mills I, et al.: Neural control of the lower urinary tract: peripheral and spinal mechanisms. Neurourol Urodyn. 2010; 29(1): 128–39. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDrake MJ, Fowler CJ, Griffiths D, et al.: Neural control of the lower urinary and gastrointestinal tracts: supraspinal CNS mechanisms. Neurourol Urodyn. 2010; 29(1): 119–27. PubMed Abstract | Publisher Full Text\n\nde Groat WC: A neurologic basis for the overactive bladder. Urology. 1997; 50(6A Suppl): 36–52; discussion 53–6. PubMed Abstract | Publisher Full Text\n\nCoyne KS, Sexton CC, Bell JA, et al.: The prevalence of lower urinary tract symptoms (LUTS) and overactive bladder (OAB) by racial/ethnic group and age: results from OAB-POLL. Neurourol Urodyn. 2013; 32(3): 230–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDaly D, Chapple C: Relationship between overactive bladder (OAB) and irritable bowel syndrome (IBS): concurrent disorders with a common pathophysiology? BJU Int. 2013; 111(4): 530–1. PubMed Abstract | Publisher Full Text\n\nChung JH, Kim SA, Choi BY, et al.: The association between overactive bladder and fibromyalgia syndrome: a community survey. Neurourol Urodyn. 2013; 32(1): 66–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGormley EA, Lightner DJ, Burgio KL, et al.: Diagnosis and treatment of overactive bladder (non-neurogenic) in adults: AUA/SUFU guideline. J Urol. 2012; 188(6 Suppl): 2455–63. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLucas MG, Bosch RJ, Burkhard FC, et al.: EAU guidelines on assessment and nonsurgical management of urinary incontinence. Eur Urol. 2012; 62(6): 1130–42. PubMed Abstract | Publisher Full Text\n\nRobinson D, Giarenis I, Cardozo L: You are what you eat: the impact of diet on overactive bladder and lower urinary tract symptoms. Maturitas. 2014; 79(1): 8–13. PubMed Abstract | Publisher Full Text\n\nCornu JN, Abrams P, Chapple CR, et al.: A contemporary assessment of nocturia: definition, epidemiology, pathophysiology, and management--a systematic review and meta-analysis. Eur Urol. 2012; 62(5): 877–90. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBright E, Cotterill N, Drake M, et al.: Developing and validating the International Consultation on Incontinence Questionnaire bladder diary. Eur Urol. 2014; 66(2): 294–300. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCartwright R, Srikrishna S, Cardozo L, et al.: Validity and reliability of the patient's perception of intensity of urgency scale in overactive bladder. BJU Int. 2011; 107(10): 1612–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChapple CR, Drake MJ, van Kerrebroeck P, et al.: Total urgency and frequency score as a measure of urgency and frequency in overactive bladder and storage lower urinary tract symptoms. BJU Int. 2014; 113(5): 696–703. PubMed Abstract | Publisher Full Text\n\nBurgio KL: Update on behavioral and physical therapies for incontinence and overactive bladder: the role of pelvic floor muscle training. Curr Urol Rep. 2013; 14(5): 457–64. PubMed Abstract | Publisher Full Text\n\nNovara G, Galfano A, Secco S, et al.: A systematic review and meta-analysis of randomized controlled trials with antimuscarinic drugs for overactive bladder. Eur Urol. 2008; 54(4): 740–63. PubMed Abstract | Publisher Full Text\n\nMoga DC, Carnahan RM, Lund BC, et al.: Risks and benefits of bladder antimuscarinics among elderly residents of Veterans Affairs Community Living Centers. J Am Med Dir Assoc. 2013; 14(10): 749–60. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBuser N, Ivic S, Kessler TM, et al.: Efficacy and adverse events of antimuscarinics for treating overactive bladder: network meta-analyses. Eur Urol. 2012; 62(6): 1040–60. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVeenboer PW, Bosch JL: Long-term adherence to antimuscarinic therapy in everyday practice: a systematic review. J Urol. 2014; 191(4): 1003–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLee S, Malhotra B, Creanga D, et al.: A meta-analysis of the placebo response in antimuscarinic drug trials for overactive bladder. BMC Med Res Methodol. 2009; 9: 55. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nChapple CR, Kaplan SA, Mitcheson D, et al.: Randomized double-blind, active-controlled phase 3 study to assess 12-month safety and efficacy of mirabegron, a β3-adrenoceptor agonist, in overactive bladder. Eur Urol. 2013; 63(2): 296–305. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nNitti VW, Auerbach S, Martin N, et al.: Results of a randomized phase III trial of mirabegron in patients with overactive bladder. J Urol. 2013; 189(4): 1388–95. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nWagg A, Cardozo L, Nitti VW, et al.: The efficacy and tolerability of the β3-adrenoceptor agonist mirabegron for the treatment of symptoms of overactive bladder in older patients. Age Ageing. 2014; 43(5): 666–75. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKhullar V, Cambronero J, Angulo JC, et al.: Efficacy of mirabegron in patients with and without prior antimuscarinic therapy for overactive bladder: a post hoc analysis of a randomized European-Australian Phase 3 trial. BMC Urol. 2013; 13: 45. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nvan Gelderen M, Tretter R, Meijer J, et al.: Absence of clinically relevant cardiovascular interaction upon add-on of mirabegron or tamsulosin to an established tamsulosin or mirabegron treatment in healthy middle-aged to elderly men. Int J Clin Pharmacol Ther. 2014; 52(8): 693–701. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAbrams P, Kelleher C, Staskin D, et al.: Combination treatment with mirabegron and solifenacin in patients with overactive bladder: efficacy and safety results from a randomised, double-blind, dose-ranging, phase 2 study (Symphony). Eur Urol. 2015; 67(3): 577–88. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKaplan SA, Roehrborn CG, Abrams P, et al.: Antimuscarinics for treatment of storage lower urinary tract symptoms in men: a systematic review. Int J Clin Pract. 2011; 65(4): 487–507. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nOelke M, Bachmann A, Descazeaud A, et al.: EAU guidelines on the treatment and follow-up of non-neurogenic male lower urinary tract symptoms including benign prostatic obstruction. Eur Urol. 2013; 64(1): 118–40. PubMed Abstract | Publisher Full Text\n\nDrake MJ, Chapple C, Sokol R, et al.: Long-term safety and efficacy of single-tablet combinations of solifenacin and tamsulosin oral controlled absorption system in men with storage and voiding lower urinary tract symptoms: results from the NEPTUNE Study and NEPTUNE II open-label extension. Eur Urol. 2015; 67(2): 262–70. PubMed Abstract | Publisher Full Text\n\nWagg A, Compion G, Fahey A, et al.: Persistence with prescribed antimuscarinic therapy for overactive bladder: a UK experience. BJU Int. 2012; 110(11): 1767–74. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nvan Leijsen SA, Hoogstad-van Evert JS, Mol BW, et al.: The correlation between clinical and urodynamic diagnosis in classifying the type of urinary incontinence in women. A systematic review of the literature. Neurourol Urodyn. 2011; 30(4): 495–502. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHashim H, Abrams P: Is the bladder a reliable witness for predicting detrusor overactivity? J Urol. 2006; 175(1): 191–4; discussion 194–5. PubMed Abstract | Publisher Full Text\n\nAnger JT, Weinberg A, Suttorp MJ, et al.: Outcomes of intravesical botulinum toxin for idiopathic overactive bladder symptoms: a systematic review of the literature. J Urol. 2010; 183(6): 2258–64. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDmochowski R, Chapple C, Nitti VW, et al.: Efficacy and safety of onabotulinumtoxinA for idiopathic overactive bladder: a double-blind, placebo controlled, randomized, dose ranging trial. J Urol. 2010; 184(6): 2416–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDuthie JB, Vincent M, Herbison GP, et al.: Botulinum toxin injections for adults with overactive bladder syndrome. Cochrane Database Syst Rev. 2011; (12): CD005493. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSmits MA, Oerlemans D, Marcelissen TA, et al.: Sacral neuromodulation in patients with idiopathic overactive bladder after initial botulinum toxin therapy. J Urol. 2013; 190(6): 2148–52. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHassouna MM, Sadri H: Economic evaluation of sacral neuromodulation in overactive bladder: A Canadian perspective. Can Urol Assoc J. 2015; 9(7–8): 242–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nNoblett K, Siegel S, Mangel J, et al.: Results of a prospective, multicenter study evaluating quality of life, safety, and efficacy of sacral neuromodulation at twelve months in subjects with symptoms of overactive bladder. Neurourol Urodyn. 2014. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHerbison GP, Arnold EP: Sacral neuromodulation with implanted devices for urinary storage and voiding dysfunction in adults. Cochrane Database Syst Rev. 2009; (2): CD004202. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGaziev G, Topazio L, Iacovelli V, et al.: Percutaneous Tibial Nerve Stimulation (PTNS) efficacy in the treatment of lower urinary tract dysfunctions: a systematic review. BMC Urol. 2013; 13: 61. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nPreyer O, Umek W, Laml T, et al.: Percutaneous tibial nerve stimulation versus tolterodine for overactive bladder in women: a randomised controlled trial. Eur J Obstet Gynecol Reprod Biol. 2015; 191: 51–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHashim H, Beusterien K, Bridges JF, et al.: Patient preferences for treating refractory overactive bladder in the UK. Int Urol Nephrol. 2015; 47(10): 1619–27. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVeeratterapillay R, Thorpe AC, Harding C: Augmentation cystoplasty: Contemporary indications, techniques and complications. Indian J Urol. 2013; 29(4): 322–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReyblat P, Ginsberg DA: Augmentation cystoplasty: what are the indications? Curr Urol Rep. 2008; 9(6): 452–8. PubMed Abstract | Publisher Full Text"
}
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[
{
"id": "11457",
"date": "08 Dec 2015",
"name": "Emmanuel Chartier-kastler",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11458",
"date": "08 Dec 2015",
"name": "Karl-Dietrich Sievert",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11460",
"date": "08 Dec 2015",
"name": "George Kasyan",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1406
|
https://f1000research.com/articles/4-1393/v1
|
03 Dec 15
|
{
"type": "Research Article",
"title": "World Health Organization estimates of the global and regional disease burden of four foodborne chemical toxins, 2010: a data synthesis",
"authors": [
"Herman Gibb",
"Brecht Devleesschauwer",
"P. Michael Bolger",
"Felicia Wu",
"Janine Ezendam",
"Julie Cliff",
"Marco Zeilmaker",
"Philippe Verger",
"John Pitt",
"Janis Baines",
"Gabriel Adegoke",
"Reza Afshari",
"Yan Liu",
"Bas Bokkers",
"Henk van Loveren",
"Marcel Mengelers",
"Esther Brandon",
"Arie H. Havelaar",
"David Bellinger",
"Brecht Devleesschauwer",
"P. Michael Bolger",
"Felicia Wu",
"Janine Ezendam",
"Julie Cliff",
"Marco Zeilmaker",
"Philippe Verger",
"John Pitt",
"Janis Baines",
"Gabriel Adegoke",
"Reza Afshari",
"Yan Liu",
"Bas Bokkers",
"Henk van Loveren",
"Marcel Mengelers",
"Esther Brandon",
"Arie H. Havelaar",
"David Bellinger"
],
"abstract": "Background Chemical exposures have been associated with a variety of health effects; however, little is known about the global disease burden from foodborne chemicals. Food can be a major pathway for the general population’s exposure to chemicals, and for some chemicals, it accounts for almost 100% of exposure. Methods and Findings Groups of foodborne chemicals, both natural and anthropogenic, were evaluated for their ability to contribute to the burden of disease. The results of the analyses on four chemicals are presented here - cyanide in cassava, peanut allergen, aflatoxin, and dioxin. Systematic reviews of the literature were conducted to develop age- and sex-specific disease incidence and mortality estimates due to these chemicals. From these estimates, the numbers of cases, deaths and disability adjusted life years (DALYs) were calculated. For these four chemicals combined, the total number of illnesses, deaths, and DALYs in 2010 is estimated to be 339,000 (95% uncertainty interval [UI]: 186,000-1,239,000); 20,000 (95% UI: 8,000-52,000); and 1,012,000 (95% UI: 562,000-2,822,000), respectively. Both cyanide in cassava and aflatoxin are associated with diseases with high case-fatality ratios. Virtually all human exposure to these four chemicals is through the food supply. Conclusion Chemicals in the food supply, as evidenced by the results for only four chemicals, can have a significant impact on the global burden of disease. The case-fatality rates for these four chemicals range from low (e.g., peanut allergen) to extremely high (aflatoxin and liver cancer). The effects associated with these four chemicals are neurologic (cyanide in cassava), cancer (aflatoxin), allergic response (peanut allergen), endocrine (dioxin), and reproductive (dioxin).",
"keywords": [
"public health",
"epidemiology",
"foodborne diseases",
"DALYs",
"aflatoxin",
"cassava",
"cyanide",
"dioxin",
"peanut allergen"
],
"content": "Introduction\n\nChemicals in food are a worldwide health concern1. Foodborne chemicals, both natural and anthropogenic, have been a source of concern with respect to international trade2–8, and various articles in the scientific literature have reported the health risks of chemical food contaminants9–11. The Dutch National Institute for Public Health and the Environment (RIVM) found that chemicals in food contributed as much as infectious agents to the foodborne burden of disease in the Netherlands12.\n\nIn September 2006 the World Health Organization (WHO) organized a consultation to develop a strategy to estimate the global burden of foodborne disease13. The first meeting of the WHO Foodborne Disease Burden Epidemiology Reference Group (FERG), convened in September 200714, was the first of several meetings15–17. The FERG includes three hazard-based task forces: Enteric Disease Task Force, Parasitic Disease Task Force, and the Chemical and Toxins Disease Task Force (CTTF). A Country Studies Task Force, a Source Attribution Task Force, and a Computational Task Force were subsequently added to FERG. In the current study, the CTTF reports the estimates of the burden of disease of four chemicals.\n\n\nMethods\n\nAt its first meeting, the CTTF identified groups of chemicals and toxins that are of highest priority in estimating the burden of foodborne disease. These included:\n\nElemental contaminants (e.g., lead, mercury, cadmium, manganese, arsenic)\n\nMycotoxins (e.g., aflatoxins, ochratoxins, fumonisins, trichothocenes)\n\nFood additives (e.g., sulphites, nitrites/nitrates, benzoic acid)\n\nPesticides/residues (e.g., organophosphates, carbamates, DDT, pyrethrins)\n\nOrganic industrial pollutants (e.g., persistent organic pollutants)\n\nVeterinary drugs/residues (e.g., antibiotics, hormones – but not antimicrobial residues)\n\nSeafood toxins (e.g., tetrodotoxin, ciguatera, shellfish toxins, DSPs, PSPs, histamines)\n\nProcess contaminants (e.g., acrylamide, PAHs, choropropanol)\n\nAllergens (e.g., peanuts)\n\nNatural toxicants (e.g., cyanide in cassava, aminoglycosides)\n\nRadionuclides and depleted uranium\n\nThe hazards were ranked on (1) the severity of potential health effects, (2) the prevalence of exposure, and (3) the availability of data to make burden estimates. After considerable discussion, the final list of chemicals/toxins for which the CTTF believed that burdens could be estimated were aflatoxin, cyanide in cassava, peanut allergen, dioxin and dioxin-like compounds, methylmercury, lead, arsenic, and cadmium. Only the results for aflatoxin, cyanide in cassava, peanut allergen, and dioxin are presented here. The results for the metals will be provided in a subsequent publication.\n\nFor each of the four chemicals, a systematic literature review was conducted. It was concluded that burden estimates could be developed for (1) cyanide in cassava and konzo; (2) peanut allergy; (3) aflatoxin and hepatocellular carcinoma (HCC); and (4) dioxin and hypothyroidism; and (5) dioxin and decrease in sperm count. The methodology employed for each is described below. Additional information may be found in the Supplementary material.\n\nThe metrics used to express burden are those of the WHO19. DALYs are the sum of years lived with disability (YLD) and years of life lost (YLL)18. YLD are estimated from the number of incident cases multiplied by the disability weight (DW) assigned to the disease and the duration of the disease from onset until remission or death18. YLL are estimated from the number of deaths, the distribution of age at death, and life expectancy18. The life expectancy used for the calculations is the projected life expectancy for the year 2050. Estimates of the number of incident cases were produced using United Nations country-level population data for 2010 using the 2012 Revision of World Population Prospects. Uncertainty around input parameters was estimated using Monte Carlo simulations; 10,000 samples from each input parameter were used to calculate 10,000 estimates of cases, deaths or DALYs. The 2.5th and 97.5th percentile of each set of the 10,000 estimates yielded a 95% uncertainty interval (UI) which is presented around the median19. Detailed information on the input parameters used in the DALY calculations for the different hazards is provided in the Supplementary material.\n\nCassava is an important staple for over 800 million people in approximately 80 countries, mostly in sub-Saharan Africa but also in Asia, the Pacific, and South America20. Cassava tubers contain a varying quantity of cyanogenic glucosides which protect the root against attack by animals and insects. Appropriate processing before consumption can reduce cyanogenic glucoside content of cassava. When high cyanogenic cassava is not processed correctly, high dietary cyanide exposure occurs. This often happens during times of famine and war. Cyanide in cassava is associated with acute cyanide poisoning and several diseases including konzo21. Worldwide reports exist of acute poisoning from cyanide in cassava21 exist, but the data are inadequate to make burden estimates. The data are sufficient, however, to make burden estimates of konzo. Konzo is an irreversible spastic paraparesis of sudden onset, associated with the consumption of bitter cassava22,23 and a low protein intake24. It is a disease of extreme poverty. Konzo mostly occurs in epidemics, but sporadic cases are also reported. The case definition includes the following criteria: (1) a visible symmetrically spastic abnormality of gait while walking and/or running; (2) a history of abrupt onset (less than one week), followed by a non-progressive course in a formerly healthy person; (3) bilaterally exaggerated knee and/or ankle jerks without signs of disease in the spine24,25.\n\nBecause konzo mostly affects remote rural areas where health infrastructure is poor or non-existent, many cases remain undiagnosed or unreported, so the true burden of disease remains unknown. No cases have been reported from urban areas. A total of 2376 konzo cases have been reported in 5 countries in Africa (Cameroon, Central African Republic, Democratic Republic of Congo [DRC], Mozambique, and United Republic of Tanzania)21, corresponding to 149 cases per year for 122 million people. Dividing the average annual number of cases for each country by the corresponding country population produces an observed incidence ranging from 0.043 to 0.179 per 100,000. The degree of underestimation is difficult to determine as konzo occurs in rural areas, often under conditions of war, and the disease is not notifiable. The only reported calculation of underestimation was that of Tylleskar25 in the DRC in 1994, when he estimated that at least twice as many cases may have occurred as those reported. The underestimation in the DRC is likely to be much greater more recently, due to war and displacement. It was therefore decided to account for the uncertainty in the underreporting by applying an expansion factor ranging uniformly from 1 to 10 to the observed cases. The mean annual incidence rate was therefore estimated as 0.9/100,000 (0.04 to 1.8/100,000). Our estimate of the burden of konzo is restricted to the 5 African countries described above and Angola. The decision to include Angola is based on a report to the World Congress on Neurology suggesting that cases have occurred in that country26. Although cassava consumption occurs in tropical areas throughout the world, the term konzo has only been used to describe cases in Africa. The incidence of konzo in other countries in Africa and other parts of the world is assumed to be zero.\n\nWe assumed the age of onset and gender distribution of these cases to be that observed by Tylleskar25. The konzo case-fatality ratio is approximately 21% based on four studies25,27–29. The age and gender distribution of fatal cases was assumed to be that of Tshala-Katumbay27.\n\nThe onset of paraparesis in konzo is abrupt, usually within minutes or hours, with occasional progression during the first days of the illness. After that time, the paraparesis is non-progressive and permanent. As a result, duration is defined as lifelong for non-fatal cases. For fatal cases, it was assumed that death occurred one to seven years after onset, with a most likely value of three years after onset, following Banea et al.28 and Tylleskar et al.30.\n\nThere is no DW specifically for konzo. The WHO defined three severity levels for konzo: (1) Mild = able to walk without support; (2) Moderate = uses one or two sticks or crutches to walk; and (3) Severe = not able to walk24. The Global Burden of Disease (GBD) 2010 DWs for mild, moderate, and severe motor impairment are 0.012, 0.076, and 0.377, respectively31. The distribution of konzo severity among 753 patients from nine different studies were mild (63%), moderate (27%) and severe (10%)27,28,30,32–37. This distribution and the disability weights described above were used to assign a disability weight of 0.065 to konzo.\n\nPrevalence data on peanut allergy were used to make estimates of incidence since allergy occurs early in life (< 5years) and is believed to be lifelong38–42. All peanut allergy cases are assumed to be the result of eating peanuts or peanut products. In western countries, the prevalence of clinical peanut allergy in children is 0 to 1.8% of the population38, corresponding to incidence rates of 0 to 22.6 per 100,000. Limited data exist on the mortality rate of peanut-induced anaphylaxis, but the majority of studies found similar rates, ranging from 0 to 0.006 deaths per 100,000 person-years38. Incidence was estimated only for the WHO A level (high income) subregions; too few data exist to make estimates for other subregions38. Several studies have reported that 63–66% of cases are male38, but given the uncertainty in this number, the gender distribution was assumed to be equal for the burden of disease calculations. No DW exists for peanut allergy. Mullins et al.39 reported that 52% of cases referred to a specialist allergy medical practice in Australia suffered from mild symptoms (skin and subcutaneous tissue involvement only), 42% from moderate symptoms (features suggestive of respiratory, cardiovascular or gastrointestinal involvement), and 6% from severe symptoms (cyanosis, hypotension, confusion, collapse, loss of consciousness, incontinence). We propose the DW for peanut allergy be a weighted average accounting for this severity distribution. GBD 2010 DWs31 for the health states defined in the category “Asthma: controlled” (DW=0.009) are considered applicable for mild and moderate cases (94%), and “Generic uncomplicated disease: anxiety about the diagnosis” (DW=0.054) for severe cases (6%), because anxiety is known to impact quality of life in food allergic patients43, leading to a severity-weighted DW of 0.012 for clinically relevant peanut allergy. Unlike other childhood allergies such as cow’s milk and egg allergy, peanut allergy rarely resolves44,45.\n\nAflatoxins are secondary metabolites of the fungi Aspergillus flavus and A. parasiticus, and less frequently other Aspergillus species such as A. nomius46. These species can be found in maize, peanuts (groundnuts), oilseeds, and tree nuts in tropical and subtropical regions46. It is believed that all aflatoxin exposure results from food consumption. We assumed a multiplicative model for the effects of aflatoxin exposure and hepatitis B virus (HBV) infection and estimated excess risk due to aflatoxin exposure as described by Liu and Wu46. To account for differences in background rates between the study population from which the cancer potency factor was derived47 and global populations, we estimated population attributable fractions (PAFs) by country, and applied them to HCC incidence and mortality based on48,49. A Bayesian log-normal random effects model50 was used to extrapolate available PAFs to countries without data. Age-specific incidence estimates were derived from a study in China comparing age-specific incidence of HCC in Qidong, a city in China with high aflatoxin exposure, and Beijing, a city with low aflatoxin exposure51. The YLD and YLL envelopes for HCC that are available from WHO were multiplied by the proportion of the burden due to aflatoxin. Thus no DW was directly involved in the calculation.\n\nDioxins are mainly byproducts of industrial processes, but can also result from natural phenomena such as volcanic eruptions and forest fires. More than 90% of human exposure to dioxins is through the food supply. The foods most often associated with dioxin contamination are meat, dairy products, fish, and shellfish52. Due to the bioaccumulation and lipophilic characteristics of dioxins, daily dietary exposure leads to accumulation of these compounds in human body fat. In adults this accumulation is thought to reach a constant level (i.e., a steady state). Consequently, the dioxin body burden, rather than the daily exposure, is taken as the dose metric for chronic toxicity risk and the assessment of dioxins53–58. In this context the dioxin concentration in breast milk fat directly reflects the concentration in body fat58–61.\n\nMany national authorities have programs in place to monitor dioxin in the food supply and breast milk61–63. Dioxin-induced prenatal and postnatal hypothyroidism and prenatally induced reduced sperm production have been found to be the most sensitive non-cancer toxic endpoints for dioxins. Estimates for dioxin-induced prenatal and postnatal hypothyroidism and reduced fertility due to disturbed sperm formation were based on an exposure assessment, toxicity assessment, and the comparison of both assessments64,65. The exposure assessment is based on breast milk concentrations of dioxin from 50 countries63. The toxicity assessment utilizes the benchmark dose (BMD) approach66–68 in which the dose response of postnatal total thyroxine (TT; decrease of TT4 in adult blood), prenatal thyroid stimulating hormone (TSH; increase in TSH in neonatal blood), and sperm production (reduced concentration of sperm cells) is analyzed. The toxicity and exposure assessments are compared to derive the transgression of a dioxin induced decrease in TT4, decrease in sperm cell count and increase in TSH across a physiological threshold indicating a disease status (i.e., incidence of hypothyroidism or impaired fertility). Additional details of these assessments may be found in Zeilmaker et al.69. The BMD analysis was performed on studies which served as the starting point for the derivation of a tolerable weekly intake (TWI)54–57 or reference dose for dioxin (RfD)58.\n\nIn a study of a mother-child cohort, Baccarelli et al. determined the relationship between maternal plasma dioxin concentration and TSH level70. A BMD analysis of these data resulted in a population distribution of the maternal body burden of dioxin corresponding to an increased TSH level of 5 µU/mL in offspring, a level not to be exceeded in 3% of newborns in iodine-replete populations71.\n\nFollowing administration of an acute oral dose to pregnant Long Evans rats on day 15 of gestation, Gray et al. measured the reduction in cauda epididymis sperm count in male offspring72. The resulting dose response data were used to calculate a BMD lower confidence limit (BMDL) and upper confidence limit (BMDU) dioxin body burden for various levels of reduction in sperm count. A WHO reference cut-off value for impaired fertility of 20 × 106 sperm cells/mL was used to link toxicity (sperm count reduction) to a disease status (impaired fertility) (i.e., the calculation of the probability of a male being born with dioxin-impaired fertility)73.\n\nA BMD analysis of a National Toxicology Program (NTP) two year feeding study in rats was used to make estimates of dioxin-induced thyroid toxicity. The NTP study administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)74 and 2,3,4,7,8-pentachlorodibenzofuran75 for periods of 14, 31, and 53 weeks. The concentrations were converted to toxic equivalent quotients76 to enable a combined analysis of both congeners. BMDL and BMDU body burdens for reduction in TT4 were calculated for each of the exposure periods. A distribution of TT4 in human blood has been reported by Aoki et al.71. The 5th percentile of this distribution (65 nmol/L) was used as the cut-off for overt clinical hypothyroidism in adults.\n\nThe results of the BMD analyses and the breast milk concentrations for 50 countries were compared, taking account of possible differences between experimental animals and humans and among individual humans64,65. This comparison provided country-specific estimates of the incidence of dioxin induced prenatal and postnatal hypothyroidism and impaired fertility. The estimates were extrapolated to other countries for which no breast milk concentrations were available by means of Bayesian random effects modeling50.\n\n\nResults\n\nThe analyses presented here show that four selected chemicals already have a substantial impact on the foodborne burden of disease, particularly in low- and middle-income countries. Just these four agents are estimated to be associated with 339,000 illnesses (95% UI: 186,000–1,239,000); 20,000 deaths (95% UI: 8,000–52,000); and 1,012,000 DALYs (95% UI: 562,000–2,822,000), respectively, in the year 2010. These should be considered the “tip of the iceberg” in terms of foodborne chemicals and their impact on the global burden of disease. For peanut allergens, we were unable to estimate a burden for low- and middle-income countries due to data gaps. We also had to use an approximate disability weight, as there are data only on quality of life of patients with food allergy38 and no specific data are available for peanut allergy.\n\nThe estimated number of incident cases, deaths, and DALYs of each of the diseases associated with chemicals is given in Table 1. The chemical associated with the most number of illnesses is dioxin; however, no deaths have been reported from the presence of dioxin in the food supply. The chemical associated with the greatest number of DALYs is aflatoxin. The DALY estimates for aflatoxin and dioxin have the least uncertainty; more uncertainty is associated with the DALY estimates for peanut allergen and cyanide in cassava. The annual incidence, mortality, and DALY rate of each chemical-associated disease per 100,000 population for each of the WHO regions is reported in Table 2. Peanut allergy is not reported in Table 2 because burden was estimated only for Americas Region A (AMR A) - United States, Canada, and Cuba); Europe A (EUR A) - primarily countries in western Europe; and Western Pacific Region A (WPR A) - Australia, Brunei Darussalam, Japan, and New Zealand. Burden estimates for cyanide in cassava are provided only for the African region (AFR) and assumed to be zero for other regions.\n\n*Only the burden for AMR A, EUR A, and WPR A was assessed.\n\nFigure 1 provides the DALYs per 100,000 inhabitants by global region. The regions with the highest burden per 100,000 inhabitants are the Southeast Asia Region (SEAR), Western Pacific Region (WPR), and the African Region (AFR). The AMR, Eastern Mediterranean Region (EMR), and EUR have the lowest DALYs per 100,000. Aflatoxin is the largest contributor to the burden in AFR and WPR. Dioxin makes the largest contribution in SEAR. Figure 2 contrasts the proportion of DALYs due to YLL and YLD for each of the four chemicals. Virtually all of the DALYs for aflatoxin and most of the DALYs for cyanide in cassava are due to YLL, whereas most of the DALYs for peanut allergen and all of the DALYs for dioxin are due to YLD. Figure 3 shows the uncertainty around the DALY estimates for each of the four chemicals. The chemical with the least uncertainty and the most number of DALYs is aflatoxin.\n\n\nDiscussion\n\nThe assessment of burden of disease from chemicals in the food is a challenge on several levels. There are thousands of chemicals in production and many naturally occurring toxins. How many of these chemicals and toxins make it into the food supply is unknown. The health effects of chemicals may not be observed for years following exposure (e.g., aflatoxin and liver cancer, lead and cardiovascular disease). Longitudinal studies of these effects are expensive and time-consuming. Sufficient information is available, however, to make estimates of the burden for arsenic, cadmium, methyl mercury, and lead and possibly for other chemicals and toxins (e.g., fish toxins, aristolochic acid). Other chemicals (e.g., persistent organic pollutants) may not require elaborate epidemiological studies because the burden can be derived from biomonitoring data in combination with relevant toxicity data. Estimates of the burden for these chemicals will provide a much more comprehensive understanding of the impact that chemicals in the food supply have on the burden of disease.\n\nAs the relevant disease endpoints due to foodborne chemicals may arise from different causes, various approaches are possible for estimating incidence and mortality. A “top-down” approach uses an existing estimate of morbidity or mortality of the disease endpoint by all causes (“envelope”) as a starting point. A population attributable fraction is then calculated for the hazard under consideration, and applied to the envelope to estimate the hazard-specific incidence. This method, which is the standard in global burden of disease estimations, was used for aflatoxin. A “bottom-up” or dose response approach uses dose-response and exposure information. The approach begins with selection of the appropriate dose response relationship between the chemical and the particular disease. This dose response relationship is then combined with the distribution of exposure within a population to derive an estimate of the incidence of the disease that is attributable to the exposure. A probabilistic version of this method, which is applied in chemical risk assessment, was used for dioxin64,65. The two approaches would result in the same results if perfect data were available, and if it can be assumed that the risk of exposure to a chemical is additive to the background risk from other causes. In reality, the available data for both approaches are limited and there is insufficient information to decide conclusively whether risks are additive, multiplicative or otherwise. This may result in considerable discrepancies between results from these methods. In this study, we chose a “top-down” approach for aflatoxin because the cancer potency factor derived by the Joint FAO/WHO Expert Committee on Food Additives (JECFA)47 was based on a multiplicative model, and there is evidence for a high background rate in the study population underlying this estimate and the global population (see Supplementary material). Using the population attributable fraction approach, we estimated there were approximately 22,000 (95% UI 9,000–57,000) cases of aflatoxin-related HCC in 2010. A dose response approach46 estimated that annually, 25,200–155,000 cases of HCC may be attributable to aflatoxin exposure. Even though the uncertainty intervals overlap, there is significant difference between these two approaches. There is evidence for a high background rate in the study population underlying this estimate and the global population (see Supplementary material), which may result in overestimation of mortality by the dose response approach. On the other hand, the global liver cancer envelope may be underestimated, particularly in Africa77,78, leading to underestimation of the aflatoxin attributable incidence.\n\nIt is hoped that the presentation here will raise awareness among countries planning their own foodborne burden of disease assessments to consider natural and anthropogenic chemicals. It is also hoped that this publication will lead to the development of chemical specific biomonitoring data to assess exposure and of epidemiologic data on other diseases associated with chemicals in food.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for Gibb et al. 2015, ‘World Health Organization estimates of the global and regional disease burden of four foodborne chemical toxins, 2010’, 10.5256/f1000research.7340.d10725479",
"appendix": "Author contributions\n\n\n\nConceived and designed the experiments: HG, PMB, FW, JE, JC, MZ, PV, JIP, JB, GA, RA, YL, BB, HL, ML, AH, DB, EB.\n\nPerformed the experiments: FW, JE, JC, MZ, YL, BB, HL, MM, EB.\n\nAnalyzed the data: BD, FW, JE, JC, MZ, YL, BB, HL, MM, EB.\n\nWrote the first draft of the manuscript: HG.\n\nContributed to the writing of the manuscript: HG, AH, BD, DB, PMB, MZ, BB, JP, JB.\n\nICMJE criteria for authorship read and met: HG, BD, PMB, FW, JE, JC, MZ, PV, JIP, JB, GA, RA, YL, BB, HL, ML, AH, DB, EB.\n\nAgree with manuscript results and conclusions: HG, BD, PMB, FW, JE, JC, MZ, PV, JIP, JB, GA, RA, YL, BB, HL, ML, AH, DB, EB.\n\n\nCompeting interests\n\n\n\nHJG, BD, MPB, AHH, JB, PV, JIP, GA, RA, and DCB serve as members of the World Health Organization advisory body—the Foodborne Disease Burden Epidemiology Reference Group - without remuneration. The authors declare no competing interests.\n\n\nGrant information\n\nThis study was commissioned by the World Health Organization (WHO). Funding for the work on aflatoxin and cyanide in cassava was provided by the WHO. The work on peanut allergy and dioxin was done through in-kind support provided by the National Institute for Public Health and the Environment, Bilthoven, the Netherlands and the Ministry of Public Health, Welfare, and Sports, the Hague. Copyright in the original work on which this article is based belongs to WHO. The authors have been given permission to publish this article. The author(s) alone are responsible for the views expressed in this publication and they do not necessarily represent the views, decisions or policies of the World Health Organization.\n\n\nAcknowledgments\n\nWe would like to acknowledge the assistance of the WHO Secretariat over the life of the initiative, particularly Amy Cawthorne, Tim Corrigan, Tanja Kuchenmüller, Yuki Minato, and Claudia Stein. We also would like to acknowledge the assistance of Ms. Alison Chiaramonte of the George Washington University Milken Institute School of Public Health in the preparation of the manuscript.\n\n\nSupplementary material\n\nIncidence, clinical outcomes, duration, disability weights, mortality, age and sex distribution of 4 chemicals/toxins transmitted through food.\n\nClick here to access the data.\n\n\nReferences\n\nWorld health organization: Food Safety: Chemical Risks. 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Reference Source\n\nWHO: Health Statistics and Information Systems. Estimates for 2000–2012: Estimated deaths by cause, sex and WHO Member State, 2010; Estimated YLDs by cause, sex and WHO Member State, 2010; and Estimated YLLs by cause, sex and WHO Member State, 2010. Reference Source\n\nMcDonald SA, Devleesschauwer B, Speybroeck N, et al.: Data-driven methods for imputing national-level incidence in global burden of disease studies. Bull World Health Organ. 2015; 93(4): 228–236. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKensler TW, Qian GS, Chen JG, et al.: Translational strategies for cancer prevention in liver. Nat Rev Cancer. 2003; 3(5): 321–329. PubMed Abstract | Publisher Full Text\n\nWHO: Dioxins and their effects on human health: Fact Sheet No 225. 2014. Reference Source\n\nVan Leeuwen FXR, Younes MM: Consultation on assessment of the health risk of dioxins: re-evaluation of the tolerable daily intake (TDI): Executive summary. Food Addit Contam. 2000; 17(4): 223–240. PubMed Abstract | Publisher Full Text\n\nScientific Commission on Food: Opinion of the Scientific Committee on Food on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food. SCF/CS/CNTM/DIOXIN/8 Final, 2000. Reference Source\n\nScientific Commission on Food: Opinion of the Scientific Committee on Food on the Risk Assessment of Dioxins and Dioxin-like PCBs in Food; Update based on new scientific information available since the adoption of the SCF opinion of the 22nd November 2000. CS/CNTM/Dioxin/20 Final, 2001. Reference Source\n\nJECFA: Joint FAO/WHO Expert Committee on Food Additives (JECFA), Safety evaluation of certain food additives and contaminants. WHO Food Additives Series 48, 2002. Reference Source\n\nJECFA: Joint FAO/WHO Expert Committee on Food Additives (JECFA), Summary and Conclusions of the sixty-fourth meeting. 2005. Reference Source\n\nUS EPA: Reanalysis of Key Issues Related to Dioxin Toxicity and Response to NAS Comments. 2012; 1. (CAS No. 1746-01-6), EPA/600/R-10/038F. Reference Source\n\nPatterson DG Jr, Needham LL, Pirkle JL, et al.: Correlation between serum and adipose tissue levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin in 50 persons from Missouri. Arch Environ Contam Toxicol. 1988; 17(2): 139–143. PubMed Abstract | Publisher Full Text\n\nLaKind JS, Berlin CM Jr, Sjödin A, et al.: Do human milk concentrations of persistent organic chemicals really decline during lactation? Chemical concentrations during lactation and milk/serum partitioning. Environ Health Perspect. 2009; 117(10): 1625–1631. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNakamura T, Nakai K, Matsumura T, et al.: Determination of dioxins and polychlorinated biphenyls in breast milk, maternal blood and cord blood from residents of Tohoku, Japan. Sci Total Environ. 2008; 394(1): 39–51. PubMed Abstract | Publisher Full Text\n\nWittsiepe J, Fürst P, Schrey P, et al.: PCDD/F and dioxin-like PCB in human blood and milk from German mothers. Chemosphere. 2007; 67(9): S286–294. PubMed Abstract | Publisher Full Text\n\nConference of the Parties to the Stockholm Convention on Persistent Organic Pollutants, Sixth meeting. United Nations Environmental Programme (UNEP): Human exposure to POPs across the globe: POPs levels and human health implication: Results of the WHO/UNEP human milk survey. 2013. Geneva, Switzerland. Reference Source\n\nVan der Voet H, Slob W: Integration of probabilistic exposure assessment and probabilistic hazard characterization. Risk Anal. 2007; 27(2): 351–371. PubMed Abstract | Publisher Full Text\n\nSlob W, Bakker MI, Biesebeek JD, et al.: Exploring the uncertainties in cancer risk assessment using the integrated probabilistic risk assessment (IPRA) approach. Risk Anal. 2014; 34(8): 1401–1422. PubMed Abstract | Publisher Full Text\n\nCrump KS: A new method for determining allowable daily intakes. Fundam Appl Toxicol. 1984; 4(5): 854–871. PubMed Abstract | Publisher Full Text\n\nEuropean Food Safety Authority: Use of the benchmark dose approach in risk assessment. Guidance of the Scientific Committee (Question No. EFSA-Q-2005-232). EFSA J. 2009; 1150: 1–72. Publisher Full Text\n\nSlob W: Dose-response modeling of continuous endpoints. Toxicol Sci. 2002; 66(2): 298–312. PubMed Abstract | Publisher Full Text\n\nZeilmaker MJ, DeVleesschauwer B, Mengelers MJB, et al.: The disease burden of dioxins: A global perspective (manuscript in preparation).\n\nBaccarelli A, Giacomini SM, Corbetta C, et al.: Neonatal thyroid function in Seveso 25 years after maternal exposure to dioxin. PLoS Med. 2008; 5(7): e161. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAoki Y, Belin RM, Clickner R, et al.: Serum TSH and Total T4 in the United States population and their association with participant characteristics: National Health and Nutrition Examination Survey (NHANES 1999-2002). Thyroid. 2007; 17(12): 1211–1223. PubMed Abstract | Publisher Full Text\n\nGray LE, Ostby JS, Kelce WR: A dose-response analysis of the reproductive effects of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans Hooded rat offspring. Toxicol Appl Pharmacol. 1997; 146(1): 11–20. PubMed Abstract | Publisher Full Text\n\nCooper TG, Noonan E, Von Eckardstein S, et al.: World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010; 16(3): 231–245. PubMed Abstract | Publisher Full Text\n\nNational Toxicology Program (NTP): Toxicology and Carcinogenesis Studies of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (CAS No. 1746-01-6) in Female Harlan Sprague-Dawley Rats (Gavage Studies). TR 521, Research Triangle Park, NC, USA. 2006.\n\nNational Toxicology Program (NTP): Toxicology and Carcinogenesis Studies of 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) (CAS No. 57117-31-4) in Female Harlan Sprague-Dawley Rats (Gavage Studies). TR 525, Research Triangle Park, NC, USA. 2006.\n\nVan den Berg M, Birnbaum LS, Denison M, et al.: The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol Sci. 2006; 93(2): 223–241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Global burden of disease regions used for WHO-CHOICE analyses. Cost effectiveness and strategic planning. 2015. Reference Source\n\nWild CP, Hall AJ: Primary prevention of hepatocellular carcinoma in developing countries. Mutat Res. 2000; 462(2–3): 381–393. PubMed Abstract | Publisher Full Text\n\nGibb H, Devleesschauwer B, Bolger PM, et al.: Dataset 1 in: World Health Organization estimates of the global and regional disease burden of four foodborne chemical toxins, 2010. F1000Research. 2015. Data Source"
}
|
[
{
"id": "11682",
"date": "05 Jan 2016",
"name": "Jonathan M. Spergel",
"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\nFor the estimate of Konzo , it was multiple by 10. I would suggest a range as it is an estimate based on poor reporting. Is there another disease to model off to get a better range?The rest of the article is acceptable.",
"responses": [
{
"c_id": "1781",
"date": "27 Jan 2016",
"name": "Herman Gibb",
"role": "Author Response",
"response": "In the section on cyanide in cassava, a range of 1 to 10-fold was reported: \"It was therefore decided to account for the uncertainty in the underreporting by applying an expansion factor ranging uniformly from 1 to 10 to the observed cases.\""
}
]
},
{
"id": "12086",
"date": "25 Jan 2016",
"name": "George M. Gray",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is a very useful addition to the goal of characterizing the disease burden from food contaminants. It applies appropriate, and for some contaminants state-of-the-art, analytic approaches. The fact that virtually all exposure to the four contaminants evaluated comes from food illustrates the importance of the exercise.\n\nThere are a few issues that deserve attention:It is difficult to tell from the manuscript whether the dose-response information for dioxins is from the epidemiologic study cited or from the animal studies. It is unfortunate that further information on the dose response refers to a manuscript in preparation and thus unavailable. What is the appropriate weighting for a substance that causes infertility? Presumably some number of affected individuals would want to reproduce and the exposure is effectively causing an entire lifetime of YLL for the child not born. I appreciate very much the effort to consider uncertainty in the projections from this analysis. However, it is very important not to imply greater characterization of uncertainty than has occurred. In this analysis the uncertainty bounds presented are primarily based on ranges for specific parameters in the models used to estimate YLL and YLD. Model uncertainty, for example, is not considered. Insofar as dose-response data for dioxins were generated from animal data (see point above) there is considerable quantitative uncertainty introduced by using animals as a model for humans. Similarly, in the case of aflatoxin it is recognized that a “bottom up” rather than “top down” model of analysis yields very different estimates of risk and uncertainty and it is not clear which is the better approach. Statements like that in paragraph 2 of the results section “The DALY estimates for aflatoxin and dioxin have the least uncertainty..” are likely to be misinterpreted. The smallest calculated uncertainty is not the same as the smallest range of actual uncertainty if all sources have not been considered.",
"responses": []
},
{
"id": "12410",
"date": "12 Feb 2016",
"name": "Mary A 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\n\nGibb et al. have made an important contribution to our understanding of the population health impacts of food-borne chemical exposures. I noted one minor data gap in the description of the approach taken for the dioxin analysis. In contrast to the other chemicals assessed, the authors did not report the disability weights (DWs) for dioxin outcomes in the main text; they are found only in the Supplementary material.\n\nMy main questions, however, relate to the conclusions. I feel that two of the paper’s bottom lines (on raising awareness of the impacts of food-borne chemicals and the need for better exposure data) deserve additional attention. On raising awareness: after reading the article, I was looking for some further characterization of the burden estimates. The estimates do seem substantial but what is the appropriate context for reference? The authors make reference to a Dutch National Institute for Public Health and the Environment assessment that made some comparisons of disease burdens for both chemicals and infectious agents in foods (listed as reference #12 Van Kreijl et al 2006). Perhaps that approach or some comparisons of the reported burden estimates to the total burden of the outcomes assessed could be made. Some further characterization of the burden estimates would assist the effort to raise awareness in the public health community. On better exposure data: The main text of the paper focuses largely on the outcomes or health effects related to the chemicals. Little is said about the exposure beyond an understanding that most exposures come from food. Description of the types of exposure data represented in the literature underlying the analysis would better set up the call for biomonitoring at the conclusion.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1393
|
https://f1000research.com/articles/4-1389/v1
|
02 Dec 15
|
{
"type": "Review",
"title": "Update on Neuromodulation for Treatment-Resistant Depression",
"authors": [
"Bettina Bewernick",
"Thomas E Schlaepfer",
"Bettina Bewernick"
],
"abstract": "About 30% of patients suffering from a major depressive disorder do not respond sufficiently to established pharmacological, psychotherapeutic, or somatic treatments. Advances in technology and emerging knowledge about the dysfunctional brain circuits underlying depression have led to the development of different neuromodulation techniques. The aim of the present review is to give an update on noninvasive techniques, such as electroconvulsive therapy (ECT), magnetic seizure therapy (MST), transcranial magnetic stimulation (TMS), and invasive techniques requiring brain surgery, such as vagus nerve stimulation (VNS) and deep brain stimulation (DBS). First, the clinical relevance for therapy-resistant depression, including the current level of evidence, are presented. Neuroethics is concerned with the ethical, legal and social policy implications of neuroscience. A second focus of the review is the application of fundamental ethical principles, such as patient autonomy, patient well-being and justice to neuromodulation therapies. Due to reduced availability and lacking long-term efficacy data, most patients with treatment-resistant depression face a trial-and-error approach to therapeutics. This contravenes the ethical criteria of patient autonomy and justice. In order to raise the level of evidence, financial support of long-term studies, including large samples and randomized control trials, are necessary.",
"keywords": [
"Depression",
"vagus nerve",
"seizures"
],
"content": "Introduction\n\nAbout 30% of patients suffering from a major depressive disorder do not respond sufficiently to established pharmacological, psychotherapeutic, or somatic treatments1. After nonresponse to two adequate treatment steps, a patient is described as having a treatment-resistant depression, which is associated with illness chronicity, a reduced quality of life, and a higher risk for suicide2. The grade of treatment resistance can be evaluated using different models, for example the antidepressant treatment history form3, or the Thase and Rush Model4. A substantial quota of patients with treatment-resistant depression have an anamnesis of multiple pharmacological and psychological treatment attempts and patients, as well as treating psychiatrists, are desperate for alternative approaches. Patients with treatment-resistant depression cannot be cured quickly5 and 20–80% of patients suffering from treatment-resistant depression face a relapse within 5 years, in spite of maintenance therapy1,6–9. It is therefore necessary to evaluate long-term effects (more than 5 years of treatment) in order to be able to assess the risk-benefit ratio for new treatment methods.\n\n\nNeuromodulation\n\nAdvances in technology and emerging knowledge about the dysfunctional brain circuits underlying depression have led to the development of different neuromodulation techniques. All these techniques attempt to change the brain’s neuronal activity in a more or less focal way. For treatment-resistant patients suffering from major depression, neuromodulation techniques offer a therapeutic option.\n\nIn this review, noninvasive techniques, such as electroconvulsive therapy (ECT), magnetic seizure therapy (MST), transcranial magnetic stimulation (TMS), and invasive techniques requiring brain surgery, such as vagus nerve stimulation (VNS) and deep brain stimulation (DBS), are described. The clinical relevance for therapy-resistant depression, including the current level of evidence, is discussed10.\n\n\nNeuroethics\n\nNeuroethics is concerned with the ethical, legal and social policy implications of neuroscience11. Fundamental ethical principles relevant for neuroethics are patient autonomy (i.e., the patient has a choice of treatment, gets information about different treatment options, as reflected in the informed consent procedure), patient well-being (physicians should prevent and remove harms, and weigh and balance possible benefits of an action against possible risks) and justice (priority should be given to patients who are most seriously impaired and who will benefit most from the intervention, patients should get access to the best treatment). These ethical criteria are analyzed in the context of the current clinical application of neuromodulation treatments in treatment-resistant depression.\n\n\nTreatments\n\nMethod. ECT was developed in 1938 and is the oldest and best evaluated neuromodulation therapy for treatment-resistant depression. An electrical current is administered to the brain through the scalp. Seizures are induced under general anesthesia and muscle relaxation. Usually, a series of seizures (9–12) are given over several weeks, generally 2–3 treatments per week.\n\nMechanism of action. The mechanism of action is not understood, but the induction of a generalized seizure and the postictal suppression12–17 are important factors contributing to the antidepressant effect.\n\nClinical application. ECT is highly effective in treatment-resistant depressive disorders, with 50–80% of patients achieving remission18,19, and is therefore the most effective acute treatment for major depressive disorder20,21.\n\nEfficacy. ECT has level I evidence for acute efficacy and relapse prevention, and level II for safety and tolerability22 (see Table 1). Transient cognitive side effects, such as postictal confusion and anterograde amnesia, are frequent and more pronounced in bilateral electrode placement as compared to unilateral electrode placement23,24. Up to 55% of patients report persistent negative cognitive side effects after ECT25.\n\nDBS, deep brain stimulation; ECT, electroconvulsive therapy; MDD, major depressive disorder; MST, magnetic seizure therapy; rTMS, repetitive transcranial magnetic stimulation; TMS, transcranial magnetic stimulation; VNS, vagus nerve stimulation.\n\nNote. Level of evidence according to 10:\n\nLevel 1 requires >2 randomized controlled trials and/or meta-analysis with narrow confidence interval;\n\nLevel 2 requires >1 randomized controlled trial and/or meta-analysis with wide confidence intervals;\n\nLevel 3 requires nonrandomized, controlled prospective studies, case series or retrospective studies.\n\nEthical aspects. Although very well investigated, effective and safe, ECT is still an underused treatment in treatment-resistant depression26 for several reasons. ECT is still stigmatized because of its different use in the past27,28 and cognitive side effects are often overestimated, in comparison to cognitive impairment due to depression itself. Clinical staff members are still inadequately trained and face prejudices, such as limitations on the use of ECT in elderly patients. This leads to a reduced availability in hospitals. The idea that every patient should have access to the treatment with the best prognosis is reflected in the ethical principle of justice. For ECT, the ethical principle of justice is not adequately met for the above mentioned reasons.\n\nCurrent research and outlook. Research has focused on maximizing antidepressant efficacy while minimizing cognitive side effects. Thus, administration techniques (unilateral vs. bilateral stimulation, ultra-brief pulse-width stimulation), the role of postictal depression, depth of anesthesia29,30, the separation of effects on cognition and depression31–33, and the best algorithm for maintenance therapy are current research questions.\n\nECT is established as a conventional treatment in treatment-resistant depression with few contraindications. ECT therefore often serves as treatment for the comparison group in studies as the “gold standard” for the evaluation of new treatments (e.g., MST).\n\nMethod. In MST, seizures are induced with magnetic pulses. The clinical procedure (general anesthesia, 9–12 sessions) is similar to ECT. The aim of the development of MST was to minimize cognitive side effects through a more focal induction of seizures34.\n\nMechanism of action. Similar to ECT, the exact mechanism of action is unknown. In MST, only the superficial cortex is exposed during seizure induction, but the seizure generalizes to broader brain regions35. Imaging studies have found evidence for changes in glucose metabolism in brain regions that have frequently been reported as dysfunctional in depression36–38.\n\nClinical application. Clinical application is limited to a few study centers worldwide, because a specially modified device is required.\n\nEfficacy. Only data from open-label pilot studies with small sample sizes in a few research sites are actually available. Efficacy seems to be similar to ECT, but possibly with a superior side-effect profile regarding cognition38–43 (see Table 1).\n\nEthical aspects. As long as MST is applied in clinical studies with careful patient selection and information about alternative treatment options is available, the ethical principles of patient autonomy and well-being are fulfilled.\n\nActually, research in MST is completely controlled by a few companies because special devices are required. In spite of attractive results from small samples, research activities have seemed to diminish. The fear of cognitive side effects is one major obstacle to encouraging patients to undergo seizure therapy. Thus, the development and availability of a potential treatment method with a possible superior side-effect profile is delayed. This contravenes the ethical principle of patient well-being and justice.\n\nCurrent research and outlook. Best stimulation parameters (e.g., finding the optimal stimulus intensity), the relevance of seizure threshold titration, and the development of devices and coils are current research foci. One controlled double-blind trial (n=20) is underway (Lisanby et al., see the Registry on ClinicalTrials.gov NCT00973934 https://clinicaltrials.gov/). Long-term blinded and controlled studies with larger samples, and studies into the evaluation of relapse rates and the role of maintenance MST are needed.\n\nMethod. Transcranial magnetic stimulation (TMS) is a noninvasive therapy option which can be applied to outpatients with treatment-resistant depression.\n\nDuring repetitive TMS, a fast series of brief pulses of strong magnetic stimuli are applied to the brain. Deep repetitive TMS is a modification of repetitive TMS which can reach deeper cortical regions with a special coil44. The H Coil45 is the only coil whose safety and effectiveness has been tested. This coil is able to change cortical excitability at a depth of up to 6 cm46.\n\nMechanism of action. Repetitive TMS and deep repetitive TMS produce changes in neuronal excitability. The magnetic field generated at the coil passes unimpeded through the scalp and skull. An electrical current is induced in the underlying tissue which modulates neural activity47. Depending on the parameters of stimulation, cortical excitability can be increased or decreased48.\n\nClinical application. After the identification of the motor threshold, the coil is moved from the motor cortex to the specific target cortical region. In the treatment of major depressive disorder, the target area is usually the left dorsolateral prefrontal cortex49–51. In contrast to ECT or MST, no general anesthesia is required. Patients and TMS operators should wear earplugs during TMS. Usually, 10–30 treatment sessions of 15–45 minutes are administered daily in an outpatient setting.\n\nEfficacy. There is evidence for repetitive TMS either as a mono- or add-on therapy for the treatment of moderate treatment-resistant depression (evidence level I)52. In 2008, repetitive TMS was approved by the FDA for the treatment of moderate treatment-resistant depression.\n\nSeveral studies have investigated the efficacy of deep repetitive TMS in patients suffering from treatment-resistant depression49–51,53–57. Deep repetitive TMS seems to be an effective and safe treatment for patients with treatment-resistant depression (see Table 1).\n\nSide effects. Overall, repetitive TMS is seen as safe without enduring side effects: no long-term neurological, cognitive, or cardiovascular side effects are reported58–61. Transient headache is the most common side effect after repetitive TMS.\n\nSimilar to repetitive TMS, deep repetitive TMS is considered a safe treatment. Scalp discomfort, transient headache and dizziness, insomnia, perceiving an odd smell, numbness in the right temporal and right cervical zone, and (in single cases) generalized seizures have been reported44.\n\nThere is no long-term evidence for either repetitive TMS or deep repetitive TMS because most studies are limited to 6–12 weeks (see 22 for a comprehensive review of TMS studies).\n\nEthical aspects. Although, the FDA has approved repetitive TMS (level I evidence for acute efficacy), this treatment is only available in special centers and patients do not have the opportunity to choose this therapy option even though its clinical evidence has been proven. Both repetitive TMS and deep repetitive TMS seem to have lower response rates in treatment-resistant depression as compared to ECT (Lipsman, Sankar et al. 2014), but the side-effect profile seems superior. In addition, TMS can be performed in an outpatient setting without anesthesia. Patients should be given the choice between a less effective but also less risky therapy, and a therapy with a higher risk for side effects and higher efficacy. This fits with the ethical criterion of justice and patient autonomy.\n\nCurrent research and outlook. Current research foci in TMS are the effect of low- and high-frequency stimulation and laterality issues, and optimizing TMS pulse and train parameters, as well as the influence of the characteristics of the TMS pulse itself (with the help of the controllable pulse TMS device). Little is known about combination therapy (e.g., pharmacotherapy, psychotherapy).\n\nMethod. VNS is an invasive brain stimulation method. A small electrical pulse is administered with an implanted neurostimulator to a bipolar electrode, surgically implanted at the left vagus. The pulse generator is implanted under the skin of the left chest. Intermittent electrical currents are sent from the generator to the vagus nerve and via the nucleus tractus solitarius to various regions of the brain. Usually, electrical pulses that last about 30 seconds are forwarded about every 5 minutes from the generator to the vagus nerve; other parameters consist of a current intensity of 0.20 to 2.50 mA, a pulse width of 500 ms and a pulse frequency of 20 Hz.\n\nMechanism of action. Brain imaging studies have demonstrated metabolic changes in the prefrontal cortex and in limbic structures relevant to mood regulation62, possibly through the modulation of monoaminergic neurotransmission63.\n\nClinical application. VNS, in its current form, is a chronic treatment. During the first months of treatment, the best stimulation parameters have to be selected; therefore, regular visits are required at the beginning of therapy. In the long-term, yearly checkups are advised to ensure the functioning of the device (e.g., battery exhaustion and lead connection) and to adjust parameters if necessary.\n\nEfficacy. In 2005, the FDA-approved VNS therapy for the adjunctive long-term treatment of chronic or recurrent depression for those patients who have not had an adequate response to two or more antidepressant treatments.\n\nLong-term effects were significantly superior by outcomes in comparison to patients receiving treatment as usual. However, VNS therapy is more effective in patients with moderate but not extreme levels of resistance64,65 (see Table 1).\n\nSide effects. Possible side-effects of VNS therapy are: an infection at the device, a hoarse voice, cough, and shortness of breath, as well as difficulties in swallowing64,65.\n\nEthical aspects. Although clinical efficacy has been proven and is superior to non-invasive treatments (e.g., repetitive TMS), VNS is not available for many patients as insurance companies only cover the cost for the surgery and not for the (psychiatric and neurosurgical) long-term treatment. For financial reasons, VNS is therefore unattractive to hospitals. This situation contravenes the criteria of justice, well-being and autonomy. In addition, the opportunity to conduct research is limited and important safety aspects (e.g., predictors of response and long-term side-effects) are not assessed sufficiently. This again contravenes the criterion of patient well-being.\n\nCurrent research and outlook. Research in VNS is limited because of the above mentioned financial restraints. Predictors of response (prior response to ECT, age, subtypes of depression etc.) and long-term safety (above 3 years) can only be inferred from its use in the treatment of epilepsy.\n\nMethod. DBS is the most invasive neuromodulation technique because it involves the stereotactic implantation of unilateral or bilateral electrodes in the brain, connected to a permanently implanted, battery-powered neurostimulator. Usually, a pair of electrodes are placed into a specific brain region assumed to be involved in mood regulation. Constant stimulation can be adjusted with the parameters of voltage, pulse width, frequency and shape of the electric field.\n\nMechanism of action. The effect of DBS on the brain is far from being understood. Stimulation parameters (frequency, amplitude, pulse width, duration) also clearly have an impact on the effect. With commonly used parameters, a relatively large volume of neural tissue is influenced66.\n\nFunctional neuroimaging data have demonstrated that DBS changes the activity of brain areas far beyond the targeted region. Thus complex neural networks are putatively modulated66–68.\n\nIn hypothesis-guided approaches, several brain structures are targets of DBS: the subgenual cingulate gyrus (Cg25)32,67,69, the anterior limb of the capsula interna (ALIC)70,71 and the nucleus accumbens (Nacc)68,72, and the supero-lateral branch of the medial forebrain bundle (slMFB)73.\n\nClinical application. DBS is only available for a highly selected group of patients suffering from very therapy-resistant depression in clinical studies in a few centers worldwide. Launching a DBS study requires a specialized multidisciplinary team, including a psychiatrist, psychologist, and neurosurgeon, and the possibility for a long-term follow-up.\n\nEfficacy. In small pilot studies, an antidepressant effect of DBS was described: a reduction of symptoms of greater than 50% was reached in about 50% of the patients after 12 months of DBS treatment70,74–78. First results have found superior response rates in the slMFB (more rapid effects and >70% response rates after 3 months79 and after 12 months79), but long-term data and larger samples are required for efficacy evaluation. First small studies with sham stimulation found conflicting results concerning placebo effects79,80 (see Table 1).\n\nSide effects. The adverse reactions caused by DBS can be differentiated in first effects related to the surgical implantation procedure itself (e.g., bleeding, infection) and second effects related to the stimulation which depend on the target site of stimulation (e.g., paresthesia, muscle contraction, dysarthria, and diplopia, hypomania, anxiety). The former are rare (i.e., risk of seizure 1–3%, of bleeding 1–5%, and of infection 2–25%), the latter are reversible with a parameter adjustment. DBS seems to have neutral-to-positive effects regarding cognition33,74,75.\n\nEthical aspects. Few treatment approaches in psychiatry have initiated as much ethical debate as DBS. Major issues concerning patient autonomy are: the manipulation of human personality with DBS81, a sudden disruption of the patient’s biography82, and the ability of patients with treatment-resistant depression to give informed consent83.\n\nRegarding well-being, the induction of new psychiatric symptoms (e.g., hypomania symptoms84 or high-risk behavior85) is debatable. Because DBS is a high-risk intervention, patients have to be carefully selected and, as long as the optimal target has not been established and efficacy is questionable, only patients resistant to all conventional treatment approaches (including ECT) should be selected for studies. Careful individual risk-benefit ratios are necessary to ensure the criterion of patient well-being.\n\nThe idea of possibly enhancing cognitive functions is important in terms of the criterion of justice, although in treatment-resistant depression, the amelioration of cognitive functions could be discussed in relation to a prior dysfunction in cognition caused by the depression81.\n\nDBS is only available in specialized centers for a few, highly selected, therapy-resistant patients. In addition, DBS is very expensive and dominated by a few companies, and little investment from the government exists. These factors restrict availability but should be seen in the light of patient well-being (e.g., to prevent harm from untrained staff, and random application before safety is assessed).\n\nCurrent research and outlook. DBS is a treatment method in the early phase of evaluation (level III). Therefore, efficacy and safety have to be assessed in sham stimulation control designs. Due to ethical reasons, it is difficult to install a randomized design. Current research questions are the best target site, parameter adjustment protocols, the predictive value of acute stimulation effects, and other predictors of response (e.g., depression subclusters, length and number of depressive episodes, former response to ECT). Furthermore, imaging studies are necessary to elucidate the mode of action.\n\n\nSummary and outlook\n\nIn the last two decades, many neuromodulation techniques have evolved at different levels of evidence. Noninvasive techniques (ECT, MST and TMS) and invasive techniques (VNS and DBS) with different safety profiles, as well as limited data on long-term efficacy and reduced availability, make it a challenge to select an appropriate treatment for patients with treatment-resistant depression.\n\nIllness chronicity, severity of the current episode, as well as nonresponse to other treatment approaches and fear of side effects, should be considered among other factors. After all, it is also the patient’s choice if an evaluated treatment with a very good short-term efficacy but inferior side-effect profile (e.g., ECT) is preferred, rather than a more experimental treatment with a possibly favorable side-effect profile (e.g., MST), or an experimental treatment with lower response rates (e.g., TMS). For more resistant courses of treatment-resistant depression (e.g., after non-response to TMS and ECT), VNS can be an option. At the current stage of research, DBS should only be offered to extremely treatment-resistant patients with limited psychiatric comorbidity within clinical studies in order to protect patient well-being.\n\nIf available, the treatment associated with the best side-effect profile and efficacy should be selected. Reality shows that, due to reduced availability and lacking long-term efficacy data, most patients with treatment-resistant depression face a trial-and-error approach to therapeutics. This contravenes the ethical criteria of patient autonomy and justice. There is minimal guidance for clinicians concerning long-term management of these complex patients. This is inefficient, costly, and associated with poor outcomes, and patients are facing a reduced quality of life. It is therefore necessary to support long-term research in neuromodulation for treatment-resistant depression and to conduct large sample randomized control trials. This only seems possible with public funding in addition to company-sponsored trials. This would allow us to raise the level of evidence for neuromodulation treatments as promising therapy options for treatment-resistant depression.\n\n\nAbbreviations\n\nDBS, deep brain stimulation; ECT, electroconvulsive therapy; MST, magnetic seizure therapy; slMFB, supero-lateral branch of the medial forebrain bundle; TMS, transcranial magnetic stimulation; VNS, vagus nerve stimulation.",
"appendix": "Competing interests\n\n\n\nNo funding specifically for conducting this review has been obtained. TS received partial funding for an investigator-initiated study on DBS for major depression from Medtronic Inc. TS is chair of the project group Deep Brain Stimulation in Psychiatry: Guidance for Responsible Research and Application, funded by the Volkswagen Foundation (Hanover, Germany). TS and BB are members of the working group Neuromodulation of the German Research Foundation.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nRush AJ, Trivedi MH, Wisniewski SR, et al.: Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR*D report. Am J Psychiatry. 2006; 163(11): 1905–17. PubMed Abstract | Publisher Full Text\n\nRush AJ, Warden D, Wisniewski SR, et al.: STAR*D: revising conventional wisdom. CNS Drugs. 2009; 23(8): 627–47. 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Publisher Full Text\n\nDougherty D, Carpenter L, Bhati M, et al.: 720-A Randomized Sham-Controlled Trial of DBS of the VC/VS for Treatment-Resistant Depression. Biol Psychiatry. 2012; 71(8): 230S.\n\nSynofzik M, Schlaepfer TE: Stimulating personality: ethical criteria for deep brain stimulation in psychiatric patients and for enhancement purposes. Biotechnol J. 2008; 3(12): 1511–20. PubMed Abstract | Publisher Full Text\n\nGisquet E: Cerebral implants and Parkinson's disease: a unique form of biographical disruption? Soc Sci Med. 2008; 67(11): 1847–51. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAppelbaum PS, Grisso T, Frank E, et al.: Competence of depressed patients for consent to research. Am J Psychiatry. 1999; 156(9): 1380–4. PubMed Abstract\n\nSynofzik M, Schlaepfer TE, Fins JJ: How Happy Is Too Happy? Euphoria, Neuroethics, and Deep Brain Stimulation of the Nucleus Accumbens. AJOB Neurosci. 2012; 3(1): 30–6. 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}
|
[
{
"id": "11408",
"date": "02 Dec 2015",
"name": "Pierre Blier",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11409",
"date": "02 Dec 2015",
"name": "Allan Young",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11410",
"date": "02 Dec 2015",
"name": "Michael E Thase",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1389
|
https://f1000research.com/articles/4-1380/v1
|
30 Nov 15
|
{
"type": "Review",
"title": "Current approaches to studying membrane organization",
"authors": [
"Thomas S. van Zanten",
"Satyajit Mayor"
],
"abstract": "The local structure and composition of the outer membrane of an animal cell are important factors in the control of many membrane processes and mechanisms. These include signaling, sorting, and exo- and endocytic processes that are occurring all the time in a living cell. Paradoxically, not only are the local structure and composition of the membrane matters of much debate and discussion, the mechanisms that govern its genesis remain highly controversial. Here, we discuss a swathe of new technological advances that may be applied to understand the local structure and composition of the membrane of a living cell from the molecular scale to the scale of the whole membrane.",
"keywords": [
"membrane",
"electron microscopy",
"proteins"
],
"content": "Introduction\n\n“The stone age did not end because we ran out of stones”1 …\n\nThere has always been a close association between technological advancement and new research questions. A more recent example is how the application of x-ray crystallography to studying protein structures has opened up the possibility to elucidate structure and relate it to function2–4. In particular, how can single molecules transfer the genetic code into chemical and structural information? Research on the structure and organization of the cell membrane is undergoing a similar revolution with the application of (nano-)technological tools for the observation of membrane structure and composition.\n\nThe outer membrane of the living cell is the interface that demarcates the cell and its environment. Communication in either direction takes place largely via the local arrangement of proteins and lipids at the plasma membrane. Decades of research on the mobility and spatial organization of components in the membrane by fluorescence microscopy and electron microscopy (EM), respectively, have suggested that the membrane is structured as a fluid lipid bilayer5. More recent studies indicate that the membrane of the cell is not a simple fluid where lipids form a well-behaved two-dimensional (2D) fluid and where proteins are solutes in this milieu. Instead, the plasma membrane is organized as a dynamic mosaic whose local assemblies can span nano- to mesoscopic scales6 (Figure 1a).\n\n(a) Image of membrane bilayer exhibits a patchwork mosaic of the distribution of lipids in the cell membrane and captures the lateral heterogeneity of the organization of membrane components in live cells. (b) This bilayer is anchored to the cortical actin meshwork as visualized by rapid-freeze deep-etch tomographic renderings of the cortical surface closest to the membrane. (c) Scanning electron microscopy image of a fibroblast cell. (d, i) The distribution of metabolically incorporated 15N-sphingolipids in the plasma membrane region indicated above represented as the detected sphingolipid-specific 15N-enrichment with NanoSIMS. Orange and yellow regions represent plasma membrane domains that are enriched with 15N-sphingolipids. (d, ii) The distribution of 18O-enrichment showing that the metabolically incorporated 18O-cholesterol is distributed relatively uniformly in the plasma membrane. Images (a), (b), and (c) and (d) are reproduced with permission from 6,19, and 35, respectively.\n\nIt is important to understand how this organization, whether caused by thermodynamic fluctuations7 or driven actively8,9, arises since it plays a significant role in the functioning of molecules embedded in this matrix. To build up a mechanistic understanding of how the cell effects this membrane organization, it is vital to have chemical, spatial, and temporal information of components in the cell membrane. Several technological advances are beginning to address these fundamental questions in more detail, and we will highlight how these are leading to a more complete picture of the cell membrane.\n\n\nHigh-resolution structural imaging\n\nAt the highest resolution, EM offers an unprecedented opportunity. With the development of cryo-EM tomography and of new detectors10, the possibilities of imaging molecular organization inside the cell are unparalleled. Macromolecular protein complexes of interest can be seen in the context of their natural environment with resolutions beyond the nanometer scale11–13. Contrast in EM, however, is chiefly dependent on electron density and has been directed to inquiries involving defined structures such as the cytoskeletal14,15, endocytic cups16,17, and adhesion plaques13,18. Noteworthy are several reports visualizing the interaction of the cortical actin with the membrane11,19 where actin meshwork-like structures (Figure 1b), aside from providing mechanical stability and shape, could impede membrane protein diffusion19. The size and dynamics of these structures might well prove to be important for membrane-related reactions, priming specific cell function20,21. EM becomes especially powerful when combined with chemical specificity in the form of genetically tagged contrast agents22 or through combination with fluorescence localization techniques23–25. However, obtaining chemically precise information with high resolution remains challenging.\n\n\nChemically parsed spatial localization\n\nThe plasma membrane of any animal cell is composed of over 1,000 different types of lipids and proteins, presumably each with a specific purpose. Together, this assortment of chemical species at the plasma membrane primes the cell to adjust and react to the external milieu and communicate information about its internal state. Composition of both protein and lipid of the plasma membrane changes dramatically, depending on cell type26, developmental stage27, and pathological state28,29.\n\nWith the advent of sensitive mass spectrometry, it is now possible to construct a quantitative map of the entire protein and lipid composition of biochemically purified membranes30,31. The resulting lipidome or proteome of the membrane is evidence of the different sets of molecules that work together in space and time to perform function and process information. Access to their localization can be achieved (a) by label-free methods and (b) via high-contrast imaging of individual species.\n\nArmed with a complete chemical composition of the membrane, obtaining the spatial organization of this information, preferably in real time and in a live cell, is the important next step. Matrix-assisted laser desorption ionization (MALDI) can locally vaporize material into ionized molecules or molecular fragments which are subsequently analyzed with a mass spectrometer. Raster-scanning the laser over a sample will generate an image with unprecedented chemical resolution, albeit at the rather low spatial scale of a few micrometers32.\n\nAt the expense of chemical bandwidth, magnetic sector secondary ion mass spectrometry (NanoSIMS) offers a typical spatial resolution of 100 nm on cell membranes33. In this method, a focused primary ion beam sputters neutral and ionized molecular fragments from the sample surface. These ejected secondary ions are subsequently collected and analyzed in the mass spectrometer. An additional benefit is a shallow sampling depth of 5 nm, which is due to the small secondary ion escape depth, making this technique exquisitely sensitive to the plasma membrane34. Because of the monoatomic and diatomic nature of the secondary ion component, identification is possible only if the molecules of interest contain distinct elements or isotopes33. For different lipids, this can be achieved by culturing cells in the presence of isotope-labeled precursors leading to their metabolic incorporation into the lipid of interest, which would have the same chemical structure as its unlabeled analogue.\n\nChemical mapping of the plasma membrane displayed 200 nm domains showing a significant sphingolipid enrichment34 (Figure 1c,d). These domains were further non-randomly assembled in patched regions that were about 3–10 μm in size34,35. Simultaneous chemical imaging of isotope-labeled cholesterol revealed that cholesterol, in contrast to sphingolipids, distributed in an apparent homogeneous fashion on the dorsal/upper membrane35 analogously to a recent dynamic study36. Despite the non-overlapping spatial distribution, cholesterol depletion did disperse the sphingolipid-enriched domains. Actin depolymerization had a more dramatic effect, suggesting a link between lipid organization and the actin architecture35,37. With the possibility to include specific protein labeling along with the mapping of lipid components at the nanometer scale, this technique will continue to contribute to our understanding of the spatial distribution of chemistry in the membrane38,39.\n\nThe benefit of label-free methods is avoiding the possible influence of an attached label on the behavior of the specific protein or lipid of interest; for any lipid, given the mass ratio of a fluorescent label to the mass of the lipid species, this perturbation is likely to be substantial. Nevertheless, to get more detailed knowledge of how a cell constructs complexes in a membrane, molecular recognition with a high signal-to-noise ratio in an aqueous scattering milieu, a feature that label-free methods still lack, is essential.\n\nIncrease of signal-to-noise ratio is attained by specifically targeting or labeling the molecule of interest with, for example, an antibody, genetically, or by chemically incorporating a contrast agent. If the labels are carrying electron-dense material22 or conjugated with gold nanoparticles40, they can be visualized with an electron microscope. In general, however, fluorescence light microscopy is used where individual targets of interest are coupled to fluorophores.\n\nFocusing of light, however, is inherently diffraction-limited. With lens-based optics, light cannot be focused better than about 200–300 nm and individual objects that are spaced closer cannot be distinguished as unique objects anymore. To overcome this concentration limit41, several approaches have come up in the last decade to either (temporally) dilute the observed molecules (stochastic super-resolution microscopy42,43) or decrease the observation volume (targeted super-resolution microscopy44,45). Single-molecule imaging46 has opened up a major avenue not only to observe the localization of single fluorophores at very high spatial resolution but also to study the biochemistry of individual species to derive ensemble properties of molecules inside a cell.\n\nOptically interrogating the dynamic behavior of single molecules in their highly concentrated presence on the plasma membrane is made feasible by isolating a fluorescently labeled representative. Although the membrane components still move in their natural environment, their dynamics can now be characterized by recording the motion of a number of such ‘single representatives’ on a camera. If the distance between the individual molecules in each image is larger than the diffraction limit, their positions can be determined with nanometer precision47,48. This accuracy of determining its center-of-mass is essentially inversely proportional to the square root of the number of photons emitted47. The positions of multiple fluorescent spots can be identified and related to their position in earlier images to build up their time trajectories49. The number of molecules can be tuned via the concentration of externally added specific markers, photo-activation of only a subset of fluorescent molecules50–52, or the photo-bleaching of a well-defined area followed by the sparse diffusion back in the observation area53,54. At the other end, technical advances in hyper-spectral detection should increase single-particle discrimination allowing an increase in concentrations of single molecular representatives55.\n\nRecording a sufficient number of tracks or a single molecule for a sufficiently long time builds up the statistical behavior of the membrane components in terms of the diffusion coefficient or type of mobility56. Individual trajectories pooled into a distribution of diffusion coefficients can then be related to the functional/affinity state of a receptor57,58. Local changes in the individual trajectory can be mapped out on the cell to indicate the nature of the area traversed59,60, in terms of diffusion50, confinement regions61, or local energetic changes62. Examining the individual tracks of receptors as they diffuse in the plasma membrane revealed that they could become obstructed by lipid domains63, protein-protein interaction64, tetraspanin network65, glycan structures59,66, or the actin cytoskeleton67–69 (Figure 2a,b).\n\n(a) Picket-fence model where transmembrane proteins anchored to the actin membrane skeleton meshwork effectively act as rows of pickets and temporarily confine the movement of lipids and proteins through steric hindrance and circumferential slowing (packing or frictional) effects. (b) Two representative trajectories of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids on a living cell membrane recorded at a time resolution of 25 μs (40,500 frames/s) for a period of 56 ms (2,250 frames) where plausible compartments are shown in different colors. (c) Schematic Voronoi lattices (purple) representative of actin-based compartment sizes together with simulated diffusion trajectories (cyan). Scale bar: 250 nm. (d) Dependency of the apparent diffusion coefficient of di-palmitoyl phosphoethanolamine (DPPE) lipids on the area of observation (blue line). Images reproduced with permission from 69 (a,b) and 135 (c,d).\n\nDetection of changes due to interactions or confinement within boundaries of a compartment is reflected in changes in the molecular diffusion coefficient, which in turn depends on interaction strengths, acquisition speed, and signal-to-noise ratio (localization accuracy)70. On the other hand, prior information on molecular mobility could facilitate teasing out a subset of molecules without effectively diluting the experiment. If the subset (of interest) moves significantly slower because of an activation event or substrate binding, it is possible to experimentally deconvolve out the contribution of individual players in the reduction of mobility60,71. Using relatively long exposure/integration times, fast-moving fluorescent molecules blur into the background while slower moving molecules emit photons from the same diffraction-limited volume and therefore can be localized.\n\nThe stochastic cycling of dyes between fluorescent on-states and non-fluorescent off-states is an unfavorable property for single-molecule tracking because the single molecule might become undetectable in several frames and get lost during the trace reconstruction. Alternatively, if tuned properly, this cycling between states can be used to temporarily dilute highly concentrated samples. The challenge is to have at each given time only a subset of molecules in the on-state and determining the center-of-mass for each molecule before they switch off again. If this process is repeated many times, all of the calculated positions can be used to reconstruct a “super-resolution” image72. This indeed is the concept of the localization techniques called stochastic optical reconstruction microscopy (STORM)73 and (fluorescent) photoactivatable localization microscopy, or (f)PALM42,43. Whereas STORM is essentially based on organic dyes that reversibly switch between on- and off-states74–77, PALM is based on engineered fluorescent proteins78–80.\n\nPhoto-localization-based super-resolution is excellent in determining sub-resolution complexes such as the endocytic clathrin-coated pits81,82, microtubular structures83–85, cytoskeletal structures86,87, and adhesion plaques88,89 (Figure 3). Quantitative analysis of super-resolved domains in the plasma membrane in terms of absolute number of molecules is challenging because of blinking90 and activation efficiency91. Nevertheless, analytical methods such the pair-correlation92 and Ripley’s K function93,94 have allowed a certain degree of quantification, but this is strongly dependent on the assumptions employed in applying these statistical analyses to the data.\n\n(a) Schematic model of the molecular architecture of focal adhesions. This model is based on the protein position measurement by interferometric photoactivatable localization microscopy (iPALM). The exquisite sensitivity of iPALM to register axial distances could determine the orientation of talin within focal adhesion. (b–e) Top and side views of iPALM images of focal adhesions (white boxes, top-view panels) and corresponding z histograms. Color encodes distance from the coverglass surface in nanometers. Placing the fluorescent probe at the two ends of the talin rod show that the N-terminus of talin rod localizes close to the cytoplasmic tails of integrin (b, c), whereas the C-terminus can localize up to 40 nm higher (d, e). Scale bars: 5 μm (b, d) and 500 nm (c, e). Images reproduced with permission from 89. Abbreviations: ECM, extracellular matrix.\n\nSince photo-localization-based super-resolution is based on a random spatial sampling of the structure, activating or switching light pulses are not necessarily required. In fact, it is possible to make use of the intrinsic trait of fluorophores to get temporarily trapped in a dark state, blinking. By engineering fluorophores that have longer dark states95 or exploiting the known on-off blinking of quantum dots96, the chances that nearby emitters are both in an on-state decrease. On the other hand, one could tune incorporation rates of fluorescent species to the membrane (proteins); in a bath of freely diffusing fluorescent ligands, only the temporarily bound ligands will be detected, essentially taking advantage of mobility difference between bound and unbound97–99.\n\nSuper-resolution imaging in the context of breaking Abbe’s diffraction limit has been achieved by decreasing the observation volume below the diffraction limit of light. This has been mainly accomplished with (a) use of near-field geometries or restricted physical apertures to confine the excitation volume or (b) the clever use of lasers to selectively deplete excited fluorophores in all except the very center of the optical volume to confine the emission volume.\n\n(a) Near-field optics. By physically confining the light inside a very small aperture of 50–150 nm in diameter, light propagation is discontinued and the electromagnetic fields become restricted to the aperture (i.e., to the near field). The light intensity exponentially decays away from the aperture, producing essentially a nanoscopic excitation source. For imaging purposes, such a sub-wavelength aperture is created in a tapered aluminum-coated optical fiber. The image can then be built up by raster-scanning the aperture in close proximity to the sample and in fact this was one of the first approaches to obtain super-resolution images45. Because the rendering of the image is independent of the photo-physical properties of the sample/fluorophore, it allows near-field scanning optical microscopy (NSOM) to obtain quantitative information at the nanometer scale. Additionally, the same physical aperture confines multiple wavelengths and the technique is therefore free from chromatic aberrations100,101. Colocalization102 among multiple chemical species (due to biochemical interaction), random scattering101 (due to the lack of interaction), and segregation103 are not the only modes of organization. Indeed, multicolor super-resolution imaging revealed multi-domain proximity on the order of 50–150 nm as another tendency101,104,105. Diffraction-limited techniques would erroneously identify such proximity as colocalization, showing the merit of any super-resolution technique. Proximity does not preclude interaction, and quantitative analysis showed that integrin activation could bias the glycosyl phosphatidylinositol-anchored protein (GPI-AP) organization to a more clustered state105. This more detailed quantification was granted by the possibility of having single-molecule sensitivity at the nanometer scale. Practical resolution of NSOM, however, is limited to approximately 50–70 nm, driving the field toward the use of optical antennas. Optical antennas, analogous to their radio frequency equivalent, convert freely propagating electromagnetic radiation into localized energy, and vice versa. Initial experiments have shown resolutions of 30–50 nm of proteins on a plasma membrane using a gold nano-particle106 or a sculpted monopole107 as photonic antenna. Recent advances in antenna design provided simultaneous multicolor localization accuracies well below 1 nm with low photon budgets108, showing tremendous potential for nanoscale sensing or imaging109. Near-field imaging or spectroscopy, however, is confined to sample surfaces that are accessible to the physical aperture/probes, making in vivo imaging inside cells a very difficult proposition.\n\n(b) Stimulated emission depletion. A different strategy toward true super-resolution is to confine the emission instead of the excitation110. Its principle is based on the positionally deterministic switching of the fluorophore state in contrast to the stochastic switching for localization-based super-resolution111,112. Stimulated emission depletion (STED) microscopy is founded on depleting the excited state of a fluorophore by stimulating the excited fluorophore to emit a photon of specified wavelength. By creating a highly intense donut-shaped emission depletion region around the confocal excitation volume, only the fluorophores in the center of the donut are spontaneously emitting in the detected wavelengths. By increasing the intensity of the depletion donut, the resolution of the microscope is increased. STED imaging has been used to identify and quantify cluster sizes in cell membranes59,113–115. The cluster sizes ranged from 50 to 160 nm, and STED experiments on membrane sheets indicated that the protein clusters were fine-tuned by electrostatic interactions and that these clusters are further assembled in relatively stable multi-protein assemblies116. Further development of the technique toward parallelization117,118, multicolor acquisition119–121, and different illumination schemes122,123 will definitely increase imaging capacity. Better spatial resolution, however, is accompanied by an increased on-off cycling load on the fluorophore during scanning, requiring further progress in fluorophore engineering.\n\nInstead of demanding the heavy burden of photo stability from the fluorophore during multiple rounds of irradiation in the course of image build-up, one could allow the molecule itself to diffuse through the observation volume. During this passage, a fluorescent molecule will cause an intensity burst that is inversely proportional to the number of molecules in the observation volume. When sufficient numbers of molecules have passed, the detected intensity fluctuations can be autocorrelated, designating the technique as fluorescence correlation spectroscopy (FCS). The time at which this autocorrelation function decays to half its original value corresponds to the characteristic timescale at which the molecules move through the observation volume.\n\nThere is a linear relationship between the diffusion time and the area of observation for 2D diffusion in the plasma membrane. The slope of this relation is inversely proportional to the apparent diffusion coefficient, and the time-axis intercept, obtained from extrapolation, is indicative of confinement124. According to this methodology, particles diffusing in the membrane can be divided in three major categories: (a) random diffusive, (b) domain interacting, and (c) meshwork constrained125,126. Exploitation of the FCS diffusion-law methodology found that sphingolipid- and cholesterol-dependent nanoscale domains are crucial for signaling127.\n\nSimilar methods of optically diluting the sample, as described above, can be employed for FCS128. More powerful is the combination of super-resolution techniques that confine the observation volume with FCS since this allows the registration of dynamics at the nanometer scale129–132. Mobility characteristics at the nanometer scale do not have to be extrapolated anymore125,126,133 but can be directly measured130,134,135 (Figure 2c,d). In fact, extensive research using the tunable nanoscopic observation volume provided by STED indicated that fast-moving lipid analogues exhibit distinct modes of mobility that can be divided in three classes130,136–139: (a) weak interactions of phosphoglycerolipids, (b) cholesterol-assisted binding mediated by the ceramide group, and (c) hydroxyl headgroup-assisted cholesterol-independent binding. In the future, bridging length scales with, for example, camera-based FCS140–142 or spatio-temporal image correlation spectroscopy143–146 should allow the visualization of how these fluctuating nanoscale assemblies can be stabilized to coalesce into functional signaling platforms7.\n\nWith the advent of reproducible nanofabrication techniques, a whole new field lies open for exploration. Engineered substrates can provide aperture-based134,147–150 or optical antenna-based151,152 nanofocusing of light on conventional microscopes. By virtue of the cells adhering to the substrate, the plasma membrane is brought in the near field of various nanoscopic excitation sources. Each of these excitation hotspots can now be addressed to locally probe membrane dynamics down to 20 nm152 or in a multicolor fashion150.\n\n\nMeasuring molecular interactions in the membrane\n\nMultiple components in the cell membrane work together and interact to effectuate signaling. Interacting molecules would diffuse together through the excitation volume. Measuring the fluorescence cross-correlation in an FCS setup will therefore display a correlation proportional to the interaction between the two particles153,154. Cross-correlation analysis among probes with different membrane anchoring units suggested domain formation but reiterated the notion of a complex underlying machinery155 that is not necessarily instructed by phase transitions156.\n\nAs an alternative to multi-particle tracking157 and cross-correlation spectroscopy158, a more direct path to uncover nanoscale multi-molecular mixing is Förster resonance energy transfer (FRET). Here, the energy of the excited state of a fluorophore (donor) is non-radiatively transferred to a neighboring fluorophore (acceptor). Upon returning to the ground state, the acceptor fluorophore subsequently emits a photon with different characteristics—lifetime159, polarization160, or Stokes-shifted161—as compared with an unperturbed donor fluorophore. Measurement of energy transfer between like fluorophores, homo-FRET, has been instrumental in the determination of small actively maintained nanoclusters8,162 (Figure 4a). The constant fraction of dense nanoclusters at a large range of concentrations163–165 together with large fluctuations in local density distribution165,166 of lipid-anchored proteins is inconsistent with thermal equilibrium. Recognizing that a cortical layer of actin and myosins can drive membrane components by the consumption of energy is reconcilable with non-equilibrium membrane organization. The resolution of the FRET signal is intrinsically limited by the optical resolution; however, combining super-resolution methods as those described above with FRET opens up using the information of molecular proximity at the nanometer scale with structure and organization at the tens of nanometers offered, for example, by NSOM (Figure 4b).\n\n(a) Quantitative analysis of the spatial distribution of glycosyl phosphatidylinositol-anchored proteins (GPI-APs). On flat regions of relatively constant fluorescence intensity (grayscale), the anisotropy images (pseudocolored, where low values indicate increased numbers of clusters) display a hierarchical distribution of GPI-AP (e.g., in the form of nanoclusters and characteristic distances between nanocluster-rich regions). Homo-Förster resonance energy transfer (h-FRET) imaging reports on the molecular proximity of like fluorophores at the 1- to 10-nm scale but imaging is still diffraction-limited and has no access to the region between 10 and 300 nm. (b) A super-resolution technique such as near-field scanning optical microscopy (NSOM) can provide access to these spatial scales and revealed GPI-AP nanoclusters to organize in 150- to 300-nm sized regions. Three-dimensional projection of a fluorescence intensity NSOM image with nanodomains (black arrows) and monomers (white arrows) of GPI-APs is shown. Contour dashed lines on the two-dimensional image illustrate the preference of nanodomains to concentrate on specific sites as hotspots. Images reproduced with permission from 165 (a) and 105 (b).\n\nIn such a composite, intermolecular connections in the membrane are being complemented by the interactions of each component with the underlying cortical actin9. Although inert particles are not influenced by the cortical actin165,166, interactions of passive particles to the actin can be either direct166 or indirect167. The formations of these local domains can subsequently be used as a signaling platform127,157,168. At the same time, the acto-myosin-dependent localization of domain creation and dissipation allows the cell to tune information-processing capacities of passive particles20,169. In this context, one can define a third class of membrane components as active: the membrane particles preoccupied with the tuning process. Examples of this last category are integrins170,171, G protein-coupled receptors172, and T-cell receptors173,174, all of which are intricately involved in cortical actin reshaping.\n\n\nOutlook on disentangling molecular function\n\nPrecise positional information of proteins, even in the context of their chemical and topographical environment, might not always be enough to tease out how a particular mechanism works. New tools such as sensors that can identify activation state will help elucidating the spatial patterns behind activating signals175–178. On the other hand, one could effectively influence the system via substrate-controlled calibration179, recruitment180,181, or perturbations182,183 and optogenetic regulation of protein (de)activation184–187. A completely different but nevertheless informative approach would be an in vitro assay to help untangle a mechanistic understanding of cellular behavior188–193. By taking the process out of the cell, one can rebuild step-by-step and investigate the minimal chemistry required to regain function194.\n\nA growing myriad and fruitful blend of interdisciplinary methodologies and technical improvements are shedding light on the spatio-temporal fluctuations of functional chemistry that underlies cellular behavior. This is captured in a 2D plot that maps the landscape of these possibilities (Figure 5). Of note is the observation that a large fraction of the area of this map is occupied, and only a few regions remain unpopulated by methods available today. It is only a matter of time until we can directly follow the evolution of nanoscale heterogeneities to microscale patterning of plasma membrane components after receptor activation.\n\nRepresentation of the landscape that current methods occupy in the space and time axes. Abbreviations: FCS, fluorescence correlation spectroscopy; FRET, Förster resonance energy transfer; NSOM, near-field scanning optical microscopy; PALM, photoactivatable localization microscopy; SPT, single particle tracking; STED, stimulated emission depletion; STICS, spatio-temporal image correlation spectroscopy; STORM, stochastic optical reconstruction microscopy.\n\n\nAbbreviations\n\n2D, two-dimensional; EM, electron microscopy; FCS, fluorescence correlation spectroscopy; FRET, Förster resonance energy transfer; NSOM, near-field scanning optical microscopy; PALM, photoactivatable localization microscopy; STED, stimulated emission depletion; STORM, stochastic optical reconstruction microscopy.",
"appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThomas S. van Zanten acknowledges the European Molecular Biology Organization for a long-term fellowship (ALTF1519-2013). Satyajit Mayor thanks the Human Frontier Science Program (RGP0027/2012) for program support and the Department of Science and Technology (Government of India) for a JC Bose fellowship.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nQuote originally attributed to former Saudi oil minister. Sheik Ahmed Zaki Yamani ca 1970s.\n\nWlodawer A, Minor W, Dauter Z, et al.: Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures. FEBS J. 2008; 275(1): 1–21. 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}
|
[
{
"id": "11380",
"date": "01 Dec 2015",
"name": "Mary L Kraft",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11381",
"date": "01 Dec 2015",
"name": "Paul W Wiseman",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11382",
"date": "01 Dec 2015",
"name": "Christian Eggeling",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1380
|
https://f1000research.com/articles/4-1377/v1
|
30 Nov 15
|
{
"type": "Research Note",
"title": "Metagenomic Chromosome Conformation Capture (3C): techniques, applications, and challenges",
"authors": [
"Michael Liu",
"Aaron Darling",
"Michael Liu"
],
"abstract": "We review currently available technologies for deconvoluting metagenomic data into individual genomes that represent populations, strains, or genotypes present in the community. An evaluation of chromosome conformation capture (3C) and related techniques in the context of metagenomics is presented, using mock microbial communities as a reference. We provide the first independent reproduction of the metagenomic 3C technique described last year, propose some simple improvements to that protocol, and compare the quality of the data with that provided by the more complex Hi-C protocol.",
"keywords": [
"Hi-C",
"3C",
"metagenomics"
],
"content": "Introduction\n\nMetagenomics has been proposed as a means to characterize the microbial communities that are pervasive in our environment (Handelsman, 2004). Current metagenomic protocols, however, fail to capture critical information on the organisation of genetic material in microbial communities, as the fine-scale structure of the community and linkage among DNA sequences is intentionally destroyed by cell lysis and DNA shearing steps prior to sequencing. Computational methods of sequence binning attempt to assign sequences to the species or strains that were present in the sample, thereby inferring the linkage information destroyed by sample processing, but these methods have limited resolution despite many years of development (Lindgreen et al., 2015; Peabody et al., 2015).\n\nChromosome conformation capture (3C) and related approaches offer an alternative strategy that allows the spatial organization of genetic material in a microbial community to be preserved and measured, either via high throughput sequencing or other assays. In 3C, the fine-scale structure of the sample is preserved via reversible crosslinking, typically by soaking the sample in formaldehyde immediately after collection (Dekker et al., 2002). The sample is then subjected to cell lysis and further steps are applied to interrogate the spatial structure in the sample.\n\nPublished protocols for coupling 3C with metagenomics involve restriction digestion, followed by a proximity ligation, followed by crosslink reversal, DNA collection, optional enrichment for ligation junctions, and sequencing library preparation (Beitel et al., 2014; Burton et al., 2014; Marbouty et al., 2014). The proximity ligation is a key step wherein a DNA ligation reaction is carried out under highly dilute conditions. The low concentration of sample material favors ligation events among DNA strands which are crosslinked together in the same molecular complex. Crucially, this allows separate DNA macromolecules, e.g. a chromosome and a plasmid, or two chromosomes that were co-bound in a protein complex, to become ligated to each other (Beitel et al., 2014). These ligation junctions can then be identified via high throughput sequencing. The rate at which such ligation events are observed in the data is highly correlated with the frequency at which the DNA was in close physical contact at the time of sample crosslinking (Lieberman-Aiden et al., 2009).\n\nSeveral other methods can support direct measurement or inference of linkage among metagenomic DNA sequences. We describe these below. Metagenomic 3C has several advantages relative to these other methods, along with some disadvantages.\n\nSingle cell sequencing methods can capture data on a relatively large fraction of the genetic material in a cell (10–80% depending on the whole genome amplification conditions). However single cell techniques are vulnerable to reagent and equipment contamination and depend on cells being readily separable, making them difficult to deploy widely. Moreover, single cell techniques gather data on only a small fraction of the cells in a sample rather than the entire population.\n\nThe Pacific Biosciences and Oxford Nanopore platforms implement sequencing technologies that can read DNA strands up to 100 kilobases (Laver et al., 2015) and possibly more. Long sequence reads capture more information about the arrangement of genes into chromosomes than is available in short (<1000nt) reads typical of other sequencing technologies. Single molecule sequence reads currently have accuracy ranging from 80–90%, which is sufficient for detecting genes but offers only limited ability to identify single nucleotide variants and indels (Quick et al., 2014). Consensus signal approaches such as Circular Consensus Sequencing can help to overcome the error in single molecule sequencing but do so at the expense of read length or throughput (Larsen et al., 2014). These methods read single molecules and therefore they are unable to identify relationships between plasmids and host chromosomes without being coupled to a library preparation method like 3C or Hi-C.\n\nThis strategy leverages the observation that genetic material present in the same species or strain changes in abundance over time & space in a highly correlated manner. By generating metagenomic data on an environment across multiple time points, sampling sites, or even different cell lysis treatments, it becomes possible to reconstruct linkage information by identifying sequences whose abundances are highly correlated across samples (Albertsen et al., 2013; Alneberg et al., 2014; Imelfort et al., 2014). The power to detect such associations grows with the number of samples and the extent of change across samples (Alneberg et al., 2014). This approach has the advantage of being relatively simple to implement, only requiring the additional effort to collect and process a larger number of samples. A potential drawback is that in recombining populations, the abundance of a particular gene, plasmid, or polymorphism may not correlate strongly with one particular host species' abundance, leading to a failure to correctly identify the linkage relationship. Plasmids and bacteriophage may have copy number dynamics that are independent of host chromosomes, potentially making some associations difficult to detect. Finally, this approach does not provide direct information to order & orient assembly contigs into genome-scale scaffolds, however the inferred linkage information could in principle be used to eliminate ambiguity in assembly graphs and so yield more contiguous assemblies.\n\n\nMetagenomic 3C\n\nMetagenomic 3C has thus far been implemented in two protocols. Text box 1 gives an overview of these protocols and Table 1 highlights the main differences in the quality of data generated by each protocol. The Hi-C approach was the first to be described in the context of metagenomics (Beitel et al., 2014; Burton et al., 2014), and involves steps that enrich the sample for proximity ligations. The basic metagenomic 3C approach has the advantage of being simpler to execute in the laboratory (Marbouty et al., 2014).\n\nWe have succeeded in implementing and extending the protocol first described by Marbouty et al., 2014 on a mock community to facilitate a detailed comparison of metagenomic 3C and Hi-C. Our extension of the original protocol adds a bead purification step following crosslink reversal and replaces the shearing & adapter ligation for sequencer library preparation with a tagmentation reaction. This in turn reduces input material requirements by several fold, enabling the reactions to be scaled down and reducing reagent cost. The details of the extended protocol and accession numbers for the associated data sets can be found in the Supplementary material.\n\nSeveral challenges emerge in applying 3C protocols to microbial communities. Samples often consist of heterogeneous cell types. The thick walls of some cells may affect the extent of crosslinking, causing some cells to crosslink more extensively than others. High formalin concentrations lead to reduced DNA recovery in later stages of the protocol. Data from experiments using a range of formalin concentrations on the same sample suggest that concentrations between 2 and 3% provide an optimal trade-off between proximity ligation rate in gram positive cells and DNA yield (see Supplementary material). However, these data reflect only a small number of species relative to the currently described microbial diversity.\n\nMicrobial communities can consist of organisms with a wide range of genomic G+C composition, and this must be considered when selecting a restriction enzyme to use in 3C and related protocols. Data on synthetic communities shows that density of restriction sites is directly proportional to the rate of observed proximity ligation events in metagenomic 3C data. For example, a library created using the enzyme HpaII (recognition site C^CGG) yields very few reads with proximity ligation junctions for S. aureus (32% G+C) but for P. aeruginosa (67% G+C) up to 6.5% of reads contain proximity ligation junctions. Therefore it may be advantageous to process samples in parallel with two or more enzymes having diverse recognition sites.\n\nChromosome conformation capture (3C) was first developed as a means to determine the average three dimensional chromosome structure in a population of cells, for a single species (Dekker et al., 2002). This general approach was later coupled with high throughput DNA sequencing (Lieberman-Aiden et al., 2009), providing a means to generate detailed 3D structure models of chromosomes. Many extensions of the 3C technique have been developed (Dekker et al., 2013).\n\nThe basic 3C protocol involves an initial step of reversible crosslinking, typically via formaldehyde at 1–3%. This step crosslinks proteins to each other and to DNA. The formaldehyde is then quenched and the cells are lysed either enzymatically or via physical disruption. Next, a restriction digestion is carried out using a 4- or 6-cutter that leaves a single-stranded overhang. Subsequently the sample is placed in a large volume DNA ligase reaction; yielding conditions that strongly favor the ligation of free ends that are co-bound in a protein complex. This step is referred to as proximity ligation. After proximity ligation, the crosslinks are reversed via heat incubation and the DNA is purified via proteinase K & RNAse digestion and EtOH precipitation. Finally, the purified DNA is ready for standard high throughput sequencing library preparation, for example via adapter ligation and enrichment PCR.\n\nHi-C extends the protocol described above by incorporating steps that enrich the final sequencing library for proximity ligation events. In Hi-C, the single stranded overhangs left after the restriction digest are filled with biotinylated nucleotides. The proximity ligation which follows is thus a blunt-end ligation and the junctions contain biotinylated nucleotides. Biotinylated nucleotides must be removed from any remaining unligated free ends. In the final steps of sequencing library preparation, fragments containing the biotinylated ligation junctions can be captured on streptavidin-coated magnetic beads, yielding a library substantially enriched for proximity ligations (Lieberman-Aiden et al., 2009).\n\nTable 1. Differences in the features of metagenomic 3C and Hi-C are listed. The proximity ligation read rate indicates the fraction of all reads that contain proximity ligation events. For Hi-C the rate varies widely in published data. The resolution limit is dictated by the density of restriction cut sites in the chromosome, which are typically more dense when using a 4-cutter (3C or Hi-C), than with a 6-cutter (Hi-C only). Marked ligation junctions are created as a by-product of the end-filling in Hi-C and can be identified as a tandem duplication of the overhang sequence in the data. The erroneous association rate is defined as the fraction of read pairs found to associate two different species or strains in mock community experiments.\n\n\nApplications of metagenomic 3C\n\nThe data produced by metagenomic 3C or Hi-C can be used to address a range of questions in microbial community analysis. Chief among these is reconstruction of the so-called population genomes of each species present in a microbial community. A population genome does not reflect the genome of an individual cell in the community, but rather is a consensus genome sequence describing the genetic material present in a collection of closely related cells, e.g. a population or species. The population genome may represent an amalgamation of many closely related strains each with their own strain-specific gene content and mutations. The extent of such microdiversity among strains has a strong influence on the ability of current sequence assembly algorithms to reconstruct a metagenomic assembly. Once recovered, the population genomes can support a range of downstream analysis such as metabolic network reconstruction for individual community members. Predicted metabolic networks can in turn be used to inform analysis of species interactions and help guide strategies for identifying and cultivating microbes of interest (Imelfort et al., 2014; Parks et al., 2015).\n\nCurrent approaches for reconstructing population genomes are relatively simplistic and involve a first step of mapping the 3C read pairs to the metagenomic assembly, counting the number of links found among each contig in the read pair data, and then using a clustering algorithm to group contigs by population/species. Several clustering algorithms have been explored for this task. Beitel et al., 2014 applied Markov clustering and found that use of a low inflation parameter in the algorithm led to clusters that accurately reflect population genomes. Marbouty et al., 2014 used Louvain clustering and were able to achieve similarly accurate results on simple test communities. Both of these algorithms have the advantage that prior knowledge of the number of population genomes is not required. Burton et al., 2014 applied a custom algorithm that requires the number of population genomes in the sample to be known a priori. This requirement is likely to pose a difficulty in cases where independent lines of evidence are unable to yield a reliable estimate of the number of population genomes in a sample.\n\nIn addition to its use in reconstructing population genome content, metagenomic 3C can in principle be used to guide the scaffolding of metagenomic assembly contigs. Hi-C data has already been demonstrated to facilitate chromosome-scale scaffolding of large eukaryotic genomes (Burton et al., 2013; Marie-Nelly et al., 2014). When scaffolding microbial genomes, the much greater resolution afforded by 4-cutters (as used in the basic metagenomic 3C protocol) is likely to be essential for accurately ordering & orienting contigs in population genomes. The signal available for scaffolding can be visualized using the contact map concept, as shown in Figure 1. When the contigs are correctly ordered and oriented the majority of contacts occur locally, obeying a distance-decay relationship dictated by polymer physics (Marie-Nelly et al., 2014). Figure 1 highlights an exception to this, where the strain used in the laboratory has undergone rearrangement relative to the finished reference genome.\n\nContact map of chromatin interactions identified by metagenomic 3C. A synthetic community of four bacterial isolates was subjected to metagenomic 3C and the resulting read data mapped back to reference chromosome assemblies. Heat intensity is proportional to the number of read pairs associating the two chromosome regions. In P. aeruginosa and B. subtilis, the two arms of the circular chromosome are colocalized, as reflected in the column of intense heat emanating from the middle of their chromosomes. Erroneous cross-species associations are seen to be rare (deep blue field).\n\nMetagenomic 3C offers the exciting possibility to quantify the frequency of association between mobile DNA such as plasmids and host chromosomes. In the simplest scenario, such data could be used in a purely descriptive capacity, to document the relationships between plasmids and hosts in various microbial ecosystems. Another possibility would be to characterise how the relationships between host chromosomes and plasmids change over time in response to external stimuli, for example antibiotic exposure. 3C-based protocols that employ 4-cutter enzymes are likely to be essential for such applications, since the use of a 4-cutter increases the likelihood that suitable cut sites will exist in small plasmids.\n\nIn principle a similar strategy could be applied to characterise relationships between host chromosomes and bacteriophage or other types of mobile DNA. Previous work in mouse models has suggested that bacteriophage in the mouse gut selectively transduce antibiotic resistance genes and broaden their host range in response to antibiotic treatment (Modi et al., 2013). Application of metagenomic 3C techniques in this context remains unexplored, although current protocols and computational techniques are adequate to support such applications.\n\n\nFuture directions\n\nMetagenomic 3C provides information on the spatial organisation of genetic material in microbial communities. This type of information is valuable and highly complementary to data generated by other strategies and technologies. In particular, the ability to link separate DNA polymers which are localized in the same cell creates opportunities for study that would be intractable with classic shotgun sequencing strategies, whether using long reads or not.\n\nSeveral barriers currently prevent ready application of metagenomic 3C and related methods to microbial communities. Naturally occurring microbial communities can harbour a milieu of live and dead cells, along with free DNA and protein. At the time of this writing, no application of the technique has yet been reported for a natural environmental sample. Marbouty et al., 2014 described an application to a sample sourced from Seine river sediments, however, that sample was subjected to an enrichment culture prior to formalin fixation. The enrichment culture presumably created a population of intact cells and reduced the prevalence of free DNA in the sample.\n\nClassic 3C and Hi-C protocols require large amounts of sample material, but microbial communities of interest can be of very limited biomass, for example subgingival dental plaques or individual soil particles. Improving the efficiency of the metagenomic 3C protocol will be essential before it can be applied to such sample types. Several possible avenues exist to improve the reaction efficiency, elements of which have already been described in the context of single-cell Hi-C experiments on mammalian cells (Nagano et al., 2013).\n\nA further major barrier to analysis of metagenomic 3C data is the presence of strain-level microdiversity in a sample. The existence of even just two strains with genomes around 98% average nucleotide identity is sufficient to cause extensive fragmentation in genome assemblies, depending on the assembly algorithm. The resulting assembly contigs can be too small to harbor restriction sites and therefore will fail to cluster into population genomes. In principle, advanced computational methods which operate directly on genome assembly string graphs (Myers, 2005) instead of their contig-based representations could solve this problem. However, such computational tools do not currently exist for metagenomic 3C data analysis. It is worth noting that this problem also impacts the use of other strategies for generating population genomes such as correlated coverage binning.\n\nHi-C data has been demonstrated to facilitate phasing human chromosomes (Selvaraj et al., 2013), and Beitel et al., 2014 showed that metagenomic Hi-C data had characteristics that would support resolution of the genotypes of two E. coli strains in a synthetic mixture. Much work remains before 3C or Hi-C could actually be applied to strain resolution, however. The number of genotypes present in a microbial community is unknown a priori, and the degree of divergence among genotypes is also unknown but has a major influence on the technique’s resolving power. Substantial investment will be required to develop tools for statistical inference on the genotypes present in samples characterized by metagenomic 3C sequencing. The fact that the number of genotypes and their divergences are unknown a priori will add significant complexity to the algorithms. It is likely the case that reconstructing the genotypes of individual cells in the sample will remain impossible, but inference algorithms may instead compute a probability distribution over cellular genotypes. Such a probability distribution could support testing & rejection of specific hypotheses, for example whether gene A and B are subject to an epistatic interaction, or whether population X is significantly more diverse than population Y. In the extreme case where strain genotypes are separated by just two variant sites in distant chromosomal locations, a very large amount of 3C data would be required to generate enough read pairs covering the two sites to estimate their frequency of linkage. This is due to nature of 3C data, and reflects the fact that distantly located sites rarely interact in the cell in most cases (Beitel et al., 2014; Marie-Nelly et al., 2014). This represents a fundamental limitation of metagenomic 3C and highlights a need for complementary strategies such as the single cell or correlated coverage techniques.",
"appendix": "Author contributions\n\n\n\nMichael Liu carried out the experiments, generated the data, and wrote material for the manuscript. Aaron Darling analysed the data and wrote material for the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Christopher W. Beitel for assistance with outlining the topics discussed and constructive comments on a draft manuscript, Carly Rosewarne for suggestions on implementing the metagenomic 3C protocol, and Matthew Z DeMaere for providing a script to generate heatmaps from mapped read pairs.\n\n\nSupplementary materials\n\nRaw, unnormalized rate of proximity ligation products in metagenomic 3C libraries, as a function of formalin concentration. A synthetic microbial community was subjected to metagenomic 3C library prep & sequencing at a range of formalin concentrations, and the fraction of read pairs mapping at distances >1000nt was taken as an estimate of the proximity ligation read rate.\n\nProtocol for the construction of metagenomic 3C (Meta3C) libraries, Illumina sequencing, analysis of metagenomic 3C data and analysis of the Burton et al., Hi-C 2014 data.\n\nClick here to access the data.\n\n\nReferences\n\nAlbertsen M, Hugenholtz P, Skarshewski A, et al.: Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat Biotechnol. 2013; 31(6): 533–38. PubMed Abstract | Publisher Full Text\n\nAlneberg J, Bjarnason BS, de Bruijn I, et al.: Binning metagenomic contigs by coverage and composition. Nat Methods. 2014; 11(11): 1144–46. PubMed Abstract | Publisher Full Text\n\nBeitel CW, Froenicke L, Lang JM, et al.: Strain- and plasmid-level deconvolution of a synthetic metagenome by sequencing proximity ligation products. PeerJ. 2014; 2: e415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurton JN, Adey A, Patwardhan RP, et al.: Chromosome-scale scaffolding of de novo genome assemblies based on chromatin interactions. Nat Biotechnol. 2013; 31(12): 1119–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBurton JN, Liachko I, Dunham MJ, et al.: Species-level deconvolution of metagenome assemblies with Hi-C-based contact probability maps. G3 (Bethesda). 2014; 4(7): 1339–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDekker J, Marti-Renom MA, Mirny LA: Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data. Nat Rev Genet. 2013; 14(6): 390–403. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDekker J, Rippe K, Dekker M, et al.: Capturing chromosome conformation. Science. 2002; 295(5558): 1306–11. PubMed Abstract | Publisher Full Text\n\nHandelsman J: Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev. 2004; 68(4): 669–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nImelfort M, Parks D, Woodcroft BJ, et al.: GroopM: an automated tool for the recovery of population genomes from related metagenomes. PeerJ. 2014; 2: e603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLarsen PA, Heilman AM, Yoder AD: The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms. BMC Genomics. 2014; 15(1): 720. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaver T, Harrison J, O’Neill PA, et al.: Assessing the performance of the Oxford Nanopore Technologies MinION. Biomolecular Detection and Quantification. 2015; 3: 1–8. Publisher Full Text\n\nLieberman-Aiden E, van Berkum NL, Williams L, et al.: Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009; 326(5950): 289–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLindgreen S, Adair KL, Gardner P: An evaluation of the accuracy and speed of metagenome analysis tools. bioRxiv. 2015. Publisher Full Text\n\nMarbouty M, Cournac A, Flot JF, et al.: Metagenomic chromosome conformation capture (meta3C) unveils the diversity of chromosome organization in microorganisms. Elife. 2014; 3: e03318. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarie-Nelly H, Marbouty M, Cournac A, et al.: High-quality genome (re)assembly using chromosomal contact data. Nat Commun. 2014; 5: 5695. PubMed Abstract | Publisher Full Text | Free Full Text\n\nModi SR, Lee HH, Spina CS, et al.: Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome. Nature. 2013; 499(7457): 219–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMyers EW: The fragment assembly string graph. Bioinformatics. 2005; 21(Suppl 2): ii79–85. PubMed Abstract | Publisher Full Text\n\nNagano T, Lubling Y, Stevens TJ, et al.: Single-cell Hi-C reveals cell-to-cell variability in chromosome structure. Nature. 2013; 502(7469): 59–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParks DH, Imelfort M, Skennerton CT, et al.: CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015; 25(7): 1043–1055. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPeabody MA, Van Rossum T, Lo R, et al.: Evaluation of shotgun metagenomics sequence classification methods using in silico and in vitro simulated communities. BMC Bioinformatics. 2015; 16: 363. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQuick J, Quinlan AR, Loman NJ: A reference bacterial genome dataset generated on the MinIONTM portable single-molecule nanopore sequencer. Gigascience. 2014; 3: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSelvaraj S, R Dixon J, Bansal V, et al.: Whole-genome haplotype reconstruction using proximity-ligation and shotgun sequencing. Nat Biotechnol. 2013; 31(12): 1111–18. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11375",
"date": "25 Jan 2016",
"name": "Mick Watson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nLiu and Darling present an excellent review of the application of 3C capture techniques to metagenomic data analysis and I recommend indexing.There are two issues I would have liked to have seen discussed/presented more:Often metagenomic samples undergo bead bashing in order to disrupt the gram+ cell walls, and this often results in highly fragmented DNA. It would be interesting to hear the thoughts of the authors on how this might affect the results from 3C capture techniques. Figure 1 shows a beautiful reconstruction of 4 genomes. However, many real environmental samples contain 1000+ genomes. The authors discuss this in great detail, and the problems that ensue; however, synthetic metagenomes exist consisting of more than 4 but less than 1000, and I wonder why nobody has applied 3C techniques to those synthetic communities?",
"responses": []
},
{
"id": "11377",
"date": "15 Mar 2016",
"name": "C. Titus 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 is a thorough discussion of using 3C for metagenome reconstruction. I was a little surprised by the elaborate review-style article together with some experimental data but I think it works well.I agree with the points made by the other reviewer (Mick Watson). It would be nice to see more complex metagenomes tackled!One final suggestion - the reference to graph genomes is poor and rather insufficient - there's been a lot of recent work in this area. I refer you to Dilthey et al, 2015, Improved genome inference in the MHC using a population reference graph for one reference.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1377
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https://f1000research.com/articles/4-1373/v1
|
30 Nov 15
|
{
"type": "Review",
"title": "Necrotizing enterocolitis: controversies and challenges",
"authors": [
"Augusto Zani",
"Agostino Pierro",
"Augusto Zani"
],
"abstract": "Necrotizing enterocolitis is a devastating intestinal disease that affects ~5% of preterm neonates. Despite advancements in neonatal care, mortality remains high (30–50%) and controversy still persists with regards to the most appropriate management of neonates with necrotizing enterocolitis. Herein, we review some controversial aspects regarding the epidemiology, imaging, medical and surgical management of necrotizing enterocolitis and we describe new emerging strategies for prevention and treatment.",
"keywords": [
"Gut",
"necrotizing enterocolitis",
"bowel"
],
"content": "Introduction\n\nNecrotizing enterocolitis is an inflammatory intestinal disorder primarily seen in premature infants, characterized by variable damage to the intestinal tract, ranging from mucosal injury to full-thickness necrosis and perforation. Reports of infants suffering from necrotizing enterocolitis are found in literature dating back to the nineteenth century1, but its pathology was described only in 1952, when Schmidt and Quaiser labeled it as enterocolitis ulcerosa necroticans2,3. Necrotizing enterocolitis became widely known in the 1960s following an epidemic occurring at the Babies Hospital in New York City between 1955 and 19664. The number of neonates collectively treated in New York helped to define the radiological and clinical presentation of this disease. Since then, the interest in and research into necrotizing enterocolitis has risen over the years (Figure 1), and several randomized controlled trials have been conducted to question or define various aspects of prevention and treatment of this condition. However, controversy still persists with regards to the most appropriate management of neonates with necrotizing enterocolitis. Herein, we review some controversial aspects regarding the epidemiology, imaging, medical and surgical management of necrotizing enterocolitis and we describe new emerging strategies for prevention and treatment.\n\n\nEpidemiology\n\nNecrotizing enterocolitis is considered to be the most common gastrointestinal emergency among neonates and affects primarily preterm infants. However, the true incidence of necrotizing enterocolitis is unknown, as it is difficult to establish and identify “mild” or “initial” cases, which correspond to stage I Bell’s classification [Table 1]5,6. For this reason, solid epidemiology data are lacking.\n\nDIC, disseminated intravascular coagulopathy; NEC, necrotizing enterocolitis; PVG, portal venous gas.\n\nIn 2010, Rees et al. reported the results of a national study based on a prospective cross-sectional survey administered to 158 level 2 and 3 neonatal intensive care units in the UK between 2005 and 20067. A total of 211 infants were diagnosed with necrotizing enterocolitis (45% Bell’s stage I, 21% stage II, and 33% stage III) for a period prevalence of 2% intensive care unit admissions. Data from the Canadian Neonatal Network reported that necrotizing enterocolitis (stage II and III) has an incidence of 5.1% in Canadian infants with a gestational age <33 weeks8. The incidence of necrotizing enterocolitis has increased in recent decades in Canada and the UK due to more preterm and low birth weight infants being born—a trend that will continue as more preterm infants are treated in neonatal intensive care units.\n\nThe development of necrotizing enterocolitis varies also according to geographical and ethnic distribution, with lower frequencies in Japan, Switzerland, and Austria, and higher frequencies in Northern America, the UK and Ireland8–14. It is still unknown whether this variability is due to actual genetic and/or environmental factors in the population or whether it is influenced by neonatal care strategies and/or ethnic background.\n\nThe overall mortality rate for necrotizing enterocolitis remains high (30–50%), despite advancements in neonatal care. Moreover, a significant proportion of the survivors, particularly those with stage III necrotizing enterocolitis, have profound neurodevelopmental delay14, resulting in reduced quality of life for the patient and family and in significant costs of ongoing treatment, calculated at between $500 million and $1 billion per year in the US15. The serious and life-long challenges necrotizing enterocolitis places on patient, family, and society indicate that therapeutic strategies to preserve and/or reconstitute the intestinal structure of necrotizing enterocolitis-affected neonates are urgently needed.\n\n\nEtiology\n\nAlthough the exact etiology of necrotizing enterocolitis remains imperfectly understood, it is considered multifactorial, with several contributing causes extensively analyzed over the past 40 years. Some factors are more frequently encountered in babies with necrotizing enterocolitis, thus constituting the classical triggering factors for necrotizing enterocolitis-like bowel damage16. Prematurity is the most recognized predisposing factor for necrotizing enterocolitis and it rarely occurs in full-term infants17, where it is usually secondary to congenital diseases, such as cardiac anomalies18,19. Conversely, the majority of infants with necrotizing enterocolitis are born preterm, and the risk of developing necrotizing enterocolitis is inversely related to gestational age and birth weight20–21. Prematurity implies immaturity of gut motility and digestion, intestinal circulatory regulation, gut barrier function, and immune defense. Another well-recognized predisposing factor is formula feeding. The mechanism of injury may be fluid shift from villus vessels to the bowel lumen, resulting in an ischemic insult to the mucosa. Therefore, not only hyperosmolar formulas but any hyperosmolar fluid, including oral medications using hyperosmolar vehicles or contrast media, could lead to mucosal injury in the bowel22,23. Hypoxia is a well-studied phenomenon that can lead to necrotizing enterocolitis: recurrent episodes of apnea, respiratory distress, assisted ventilation, and umbilical vessel catheterization, and can all contribute to hypoxic events in very low birth weight neonates24. Disruption of commensal bacteria, leading to deficient or abnormal microbial colonization of the gut, has been implicated as a key risk factor in the pathogenesis of necrotizing enterocolitis25. Whether bacterial infection has a primary inciting role in necrotizing enterocolitis or whether an initial intestinal mucosal injury allows secondary bacterial invasion is unclear. The most commonly identified organisms are: Escherichia Coli, Klebsiella pneumoniae, Proteus, Staphylococcus aureus, S. Epidermidis, Enterococcus spp., Clostridium perfringens, and Pseudomonas aeruginosa.\n\n\nImaging\n\nPlain radiography is the cornerstone of necrotizing enterocolitis diagnosis and staging (Table 1)5,6,26. The pathognomonic radiological finding of necrotizing enterocolitis is that of Pneumatosis intestinalis, defined as gas in the bowel wall originating from pathogenic bacteria. Other findings that are seen in more severe forms of necrotizing enterocolitis are ascites and portal venous gas. Pneumoperitoneum resulting from intestinal perforation is indicative of intestinal perforation and/or necrosis.\n\nOther imaging modalities could provide more information and help diagnosis and follow-up of infants with necrotizing enterocolitis. Doppler ultrasonography, especially aimed at measuring blood flow velocity in the coeliac trunk and superior mesenteric artery, has been used to identify patients at risk of developing necrotizing enterocolitis, as well as to assess the bowel viability in those infants with established necrotizing enterocolitis27–30. In particular, sonographic findings of free gas, focal fluid collection, increased bowel wall echogenicity, absent bowel perfusion, portal venous gas, bowel wall thinning or thickening, and Pneumatosis intestinalis, are associated with an adverse outcome. Conversely, the use of magnetic resonance imaging scans in human necrotizing enterocolitis was described only once in the literature and remains anecdotal so far31.\n\nNear-infrared spectroscopy is a noninvasive real-time method of measuring local tissue oxygenation that is being evaluated as a predictive diagnostic modality for necrotizing enterocolitis. Gay et al. demonstrated that, in a piglet model of necrotizing enterocolitis, splanchnic tissue oxygenation measurements are directly correlated with changes in intestinal blood flow and markedly reduced by necrotizing enterocolitis32. Patel et al. reported that abdominal near-infrared spectroscopy measurements are lower and have increased variability in preterm infants with necrotizing enterocolitis33.\n\n\nDiagnosis and disease progression\n\nInfants with necrotizing enterocolitis present with clinical symptoms of abdominal distension, feeding intolerance, bilious vomiting, and bloody stools, and with laboratory derangements characterized by neutropenia, thrombocytopenia, metabolic acidosis, and high C-reactive protein levels26.\n\nIn some infants, necrotizing enterocolitis progresses to peritonitis, intestinal perforation, septic shock, disseminated intravascular coagulation, and death. However, the majority of patients that require surgical intervention do not present with a fulminant course, resulting in disease progression34. Clinical parameters alone cannot accurately predict necrotizing enterocolitis progressing to surgical disease in over 40% of patients, and biomarkers have been used to evaluate disease extent and progression35. Novel biomarkers of necrotizing enterocolitis have been described in serum, urine, feces, buccal swab, or using noninvasive hemodynamic technique (heart rate activity)35–38.\n\nSeveral authors have reported the use of laparoscopy to diagnose necrotizing enterocolitis39–42. Pierro et al. reported that laparoscopy can provide important information regarding bowel viability, is feasible and tolerated even in infants weighing less than 1 kg, and can be safely performed on the intensive care unit39. Moreover, Numanoglu and Millar reported that when bowel ischemia is suspected, fluorescein laparoscopy could be useful to identify the necrotic segments41.\n\nIn 1956, Porter first described a 2-day-old infant who developed a spontaneous intestinal perforation (SIP)43. SIP most commonly presents as pneumoperitoneum without pneumatosis and, according to some studies, it has distinct, non-ischemic histopathology, different from that of necrotizing enterocolitis44,45. Gordon et al. were the first to demonstrate the deleterious relationship between early postnatal steroids and SIP in a retrospective cohort46. However, despite extensive literature on the subject, there has been an ongoing debate on whether this condition represents a mild form of necrotizing enterocolitis or a distinct entity. In the latter case, many studies on necrotizing enterocolitis could be “contaminated” by infants who instead had SIP.\n\n\nMedical management\n\nMost infants with suspected (Bell’s stage I) or confirmed (Bell’s stage II) necrotizing enterocolitis are managed non-operatively. Non-operative treatment includes withholding feeds, ventilatory support, fluid resuscitation, inotropic support, correction of acid-base imbalance, coagulopathy and/or thrombocytopenia, bowel rest, and antibiotics.\n\nCurrently, there is no consensus and no evidence in the literature on which antibiotic regimen should be prescribed for medically managed infants with necrotizing enterocolitis, and this is reflected by both an international survey and a Cochrane review48,49. Therefore, antibiotics are prescribed depending on institutional protocol and changed according to individual culture and sensitivity results. Similarly, the duration of bowel rest with no enteral feeds for medically treated infants with necrotizing enterocolitis is based on tradition and not on evidence-based treatment48.\n\n\nSurgical management\n\nA proportion of medically managed infants with necrotizing enterocolitis require acute surgical intervention, due to clinical deterioration or intestinal perforation. Whilst the latter indication is clearly identified with radiologic evidence of pneumoperitoneum, signs of clinical deterioration leading to surgery can be more subtle. These include requirement of inotropes, worsening abdominal findings, hemodynamic instability, worsening laboratory values (intractable acidosis, persistent thrombocytopenia, rising leukocytosis, or worsening leukopenia), and/or sonographic evidence of decreased or absent bowel perfusion.\n\nA laparotomy in high-risk neonates, especially if born with an extremely low weight, can result in serious morbidity or even mortality. To avoid this risk, in 1977 Ein et al. first described the percutaneous insertion of a peritoneal drain in five neonates with bowel perforation as a temporizing measure to delay laparotomy50. The authors noticed a clinical improvement of these infants within a week, so that they advocated the peritoneal drainage of small infants with perforated necrotizing enterocolitis. In support of this approach, a few years later, the same authors published a bigger series where they showed that 40% of neonates <1500 g treated with the peritoneal drain had complete resolution of their disease without requiring further surgery51. A similar experience with the peritoneal drain was later reported by other authors52–54. However, this surgical approach has been very controversial and two prospective randomized controlled trials comparing the use of peritoneal drain vs. laparotomy in infants with perforated necrotizing enterocolitis were carried out55,56. Interestingly, neither of the two trials was able to demonstrate an advantage of one treatment modality over the other55,56. Moreover, Rees et al. demonstrated that in neonates with <1000 g body weight and perforated necrotizing enterocolitis, peritoneal drainage was not a definitively effective procedure, as 74% of the infants required a rescue laparotomy57. It is still debatable whether there is a role for peritoneal drainage in the stabilization of a critically unwell child with perforated necrotizing enterocolitis and/or respiratory compromise, prior to the transfer to another center for laparotomy57.\n\nThe universal principles of surgery in necrotizing enterocolitis are to remove the necrotic intestine and control intra-abdominal sepsis while preserving as much intestinal length as possible57. Within these principles, there are different surgical options that surgeons favor on the basis of personal experience, rather than evidence-based literature58. The classical approach to necrotizing enterocolitis has been to resect all areas of the necrotic intestine and fashion a stoma to allow adequate time for healing and growth before restoring intestinal continuity at a later stage. However, stomas, and in particular jejunostomies, are poorly tolerated by preterm infants, as they predispose them to nutritional and metabolic disturbances and poor growth as a consequence of fluid and electrolyte depletion47. Therefore, some surgeons would resect necrotic bowel and perform a primary anastomosis, even in neonates weighing less than 1000 g58. To investigate which is the most effective operation for neonates with surgical necrotizing enterocolitis, a multicenter randomized controlled trial of resection with primary anastomosis vs. resection with stoma (STAT: Stoma or Intestinal Anastomosis Trial) is currently underway.\n\nMoreover, there is no consensus among surgeons on the type of stoma to fashion and where to locate it with regard to the surgical wound58. This is in line with the outcomes of a recent systematic review of the literature that showed no difference in the type or location of colostomy in children with colorectal disease59.\n\nAt laparotomy, some infants are found to have multifocal necrotizing enterocolitis and require multiple resections and multiple anastomoses. In 1996, Vaughan et al. described an alternative approach for such cases: the “clip-and-drop” technique60. According to this technique, the multiple necrotic areas are resected, the bowel ends are sealed with titanium clips or staples, and the clipped bowel loops are returned to the abdominal cavity. At a second-look laparotomy, the bowel loops can be reassessed and anastomoses can be performed. Since the first description, the “clip-and-drop” technique has been employed for infants with multifocal necrotizing enterocolitis by other authors60–62.\n\nWhen the vast majority of the intestine is affected by severe intestinal damage, the patient is considered to have pancolitis or NEC totalis. This is a very controversial scenario, as the resection of the necrotic bowel may involve almost the whole intestine. Options include closing the abdomen and withdrawing care, or creating a diverting jejunostomy. The latter has been described to rescue a proportion of neonates with extensive necrotizing enterocolitis and to result in enteral autonomy in most patients63.\n\n\nOutcome\n\nDespite advancement in medical and surgical treatment over the last 6 decades, the mortality for necrotizing enterocolitis is still very high, especially in extremely low birth weight (ELBW) infants64. Although the risk and absolute mortality of necrotizing enterocolitis decrease with higher birth weight, necrotizing enterocolitis has a relatively greater impact upon mortality at higher birth weight64. Moreover, mortality for necrotizing enterocolitis is high even in cases of minimal bowel involvement65.\n\nNecrotizing enterocolitis survivors are also at high risk of developing severe complications, related either to the intestinal or to the systemic insult. Morbidities include recurrent episodes of necrotizing enterocolitis, development of intestinal strictures, intestinal failure, parenteral nutrition-related complications, and neurodevelopmental disabilities. About 10% of infants who have previously undergone surgery for necrotizing enterocolitis develop a recurrent episode and this results in long-term parenteral nutrition dependency66. About a quarter of patients who have had necrotizing enterocolitis, especially if treated surgically, will develop one or more intestinal strictures67,68. Such strictures are investigated with contrast studies, typically in the form of enemas, due to the higher incidence in the colon, and usually require surgical resection. Strictures are the result of a vascular occlusion or spasm following the initial ischemic episode, as confirmed at histology by Kosloske et al.68. In this seminal paper, Kosloske et al. reported the death of an infant due to the late diagnosis of post-necrotizing enterocolitis strictures and recommended a barium enema for all infants who had necrotizing enterocolitis about 6 weeks after the acute episode68.\n\nLonger-term outcome is related to the remaining intestinal length and its capacity for adequate nutrient absorption. The incidence of intestinal failure among infants undergoing surgical treatment for necrotizing enterocolitis is high, and many factors characteristic of severe necrotizing enterocolitis (such as low birth weight, antibiotic use, ventilator use, and greater extent of bowel resection) are associated with the development of intestinal failure69. However, according to a recent study on a large cohort of children with intestinal failure one year after diagnosis, the diagnosis of necrotizing enterocolitis proved to be a significant predictive factor of enteral autonomy, possibly due to the greater capacity for late adaptation than the residual intestine after resection for other etiologies70.\n\nFinally, it is being increasingly recognized that approximately 50% of the neonates who developed necrotizing enterocolitis have a deleterious neurodevelopmental effect, although the mechanisms by which this develops is still poorly understood14,71. It is known that infants with necrotizing enterocolitis are at greater risk of motor impairment, and this seems to be mediated by white matter abnormalities on magnetic resonance imaging at term72.\n\n\nEmerging strategies\n\nEfforts to improve necrotizing enterocolitis outcome are directed towards prevention and treatment of the disease.\n\nProbiotics are live microorganisms that increase natural intestinal defenses by regulating inflammatory response, cellular proliferation, and apoptosis. Several studies have demonstrated the efficacy and safety of prophylactic enteral probiotic administration in the prevention of necrotizing enterocolitis in infants with very low birth weight. A Cochrane review on this topic analyzed 24 trials and demonstrated that enteral probiotics supplementation significantly reduced the incidence of severe necrotizing enterocolitis and mortality73. The probiotics preparations that were found to be effective contained lactobacillus either alone or in combination with bifidobacterium73. New studies on probiotics in necrotizing enterocolitis are now directed to assess the most effective preparations, timing, and length of therapy to be utilized.\n\nOther preventive strategies are to supplement formulas with prebiotics or synbiotics. Prebiotics are indigestible fiber compounds that stimulate the activity and growth of healthy bacteria within the intestine, whereas synbiotics are a combination of both prebiotics and probiotics, which exert a synergistic effect. Several studies report the beneficial effect of prebiotics or synbiotics on necrotizing enterocolitis incidence in preterm infants with variable outcomes74–77.\n\nNovel treatment strategies have been tested in experimental models of necrotizing enterocolitis. These include Captopril78, platelet-activating factor antagonists79, heparin-binding epidermal growth factor-like growth factor80, granulocyte colony-stimulating factor and erythropoietin81. One promising maneuver that was initially tested in animals and then confirmed in a safety and feasibility trial on human infants is moderately controlled hypothermia82–87. In animal studies, controlled hypothermia resulted in prolonged survival, prevention of liver energy failure, reduction in neutrophil infiltration of lungs and intestine, attenuation of the derangement in cardiac oxidative metabolism, attenuation of histological damage to the intestine and attenuation of the pro- and anti-inflammatory cytokine response in the portal vein and the systemic circulation82–86. In the first trial on preterm neonates with severe necrotizing enterocolitis, controlled hypothermia proved to be feasible and safe for 48 hours87.\n\nStem cell therapy, which is nowadays a therapeutic option for refractory inflammatory bowel disease88, has recently been proposed as a novel strategy for infants with necrotizing enterocolitis. In a neonatal rat model of necrotizing enterocolitis, amniotic fluid stem cells, injected intraperitoneally, proved to integrate into the bowel wall, improve survival, reduce necrotizing enterocolitis incidence, decrease gut damage and improve intestinal function89–92. Amniotic fluid stem cell administration was associated with the migration of cyclooxygenase-2 (COX-2) expressing stromal cells from the lamina propria of the small intestinal villi to a position near the base of the intestinal crypts90. The beneficial effects of amniotic fluid stem cells on the development of necrotizing enterocolitis were blocked by the administration of a selective COX-2 inhibitor, suggesting that the migration of COX-2 cells was involved in the protective effects of the amniotic fluid stem cells. Moreover, the same beneficial effect exerted by amniotic fluid stem cells on rat survival was also obtained when a conditioned medium (supernatant) from amniotic fluid stem cells was administered90. These findings suggest that amniotic fluid stem cells act via a paracrine mechanism, by secreting factors that stimulate bowel regeneration. Future studies, aimed at identifying those factors secreted by amniotic fluid stem cells, could pave the way for a novel pharmacological therapy for infants with necrotizing enterocolitis.",
"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\nAcknowledgments\n\nThis work was supported by the endowment of the Robert M. Filler Chair of Surgery, The Hospital for Sick Children.\n\n\nReferences\n\nObladen M: Necrotizing enterocolitis--150 years of fruitless search for the cause. Neonatology. 2009; 96(4): 203–10. PubMed Abstract | Publisher Full Text\n\nSchmid KO: [A specially severe form of enteritis in newborn, enterocolitis ulcerosa necroticans. I. Pathological anatomy]. Osterr Z Kinderheilkd Kinderfuersorge. 1952; 8(2): 114–136. PubMed Abstract\n\nQuaiser K: [A specially severe form of enteritis in newborn, enterocolitis ulcerosa necroticans. II. Clinical studies]. Osterr Z Kinderheilkd Kinderfuersorge. 1952; 8(2): 136–152. PubMed Abstract\n\nTouloukian RJ, Berdon WE, Amoury RA, et al.: Surgical experience with necrotizing enterocolitis in the infant. J Pediatr Surg. 1967; 2(5): 389–401. Publisher Full Text\n\nBell MJ, Ternberg JL, Feigin RD, et al.: Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging. Ann Surg. 1978; 187(1): 1–7. PubMed Abstract | Free Full Text\n\nWalsh MC, Kliegman RM: Necrotizing enterocolitis: treatment based on staging criteria. Pediatr Clin North Am. 1986; 33(1): 179–201. PubMed Abstract\n\nRees CM, Eaton S, Pierro A: National prospective surveillance study of necrotizing enterocolitis in neonatal intensive care units. J Pediatr Surg. 2010; 45(7): 1391–7. PubMed Abstract | Publisher Full Text\n\nYee WH, Soraisham AS, Shah VS, et al.: Incidence and timing of presentation of necrotizing enterocolitis in preterm infants. Pediatrics. 2012; 129(2): e298–304. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSankaran K, Puckett B, Lee DS, et al.: Variations in incidence of necrotizing enterocolitis in Canadian neonatal intensive care units. J Pediatr Gastroenterol Nutr. 2004; 39(4): 366–72. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHolman RC, Stoll BJ, Curns AT, et al.: Necrotising enterocolitis hospitalisations among neonates in the United States. Paediatr Perinat Epidemiol. 2006; 20(6): 498–506. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKawase Y, Ishii T, Arai H, et al.: Gastrointestinal perforation in very low-birthweight infants. Pediatr Int. 2006; 48(6): 599–603. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSchmolzer G, Urlesberger B, Haim M, et al.: Multi-modal approach to prophylaxis of necrotizing enterocolitis: clinical report and review of literature. Pediatr Surg Int. 2006; 22(7): 573–80. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nGuner YS, Friedlich P, Wee CP, et al.: State-based analysis of necrotizing enterocolitis outcomes. J Surg Res. 2009; 157(1): 21–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRees CM, Pierro A, Eaton S: Neurodevelopmental outcomes of neonates with medically and surgically treated necrotizing enterocolitis. Arch Dis Child Fetal Neonatal Ed. 2007; 92(3): F193–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeu J, Walker WA: Necrotizing enterocolitis. N Engl J Med. 2011; 364(3): 255–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLin PW, Stoll BJ: Necrotising enterocolitis. Lancet. 2006; 368(9543): 1271–83. PubMed Abstract | Publisher Full Text\n\nNg S: Necrotizing enterocolitis in the full-term neonate. J Paediatr Child Health. 2001; 37(1): 1–4. PubMed Abstract | Publisher Full Text\n\nBolisetty S, Lui K, Oei J, et al.: A regional study of underlying congenital diseases in term neonates with necrotizing enterocolitis. Acta Paediatr. 2000; 89(10): 1226–30. PubMed Abstract | Publisher Full Text\n\nMcElhinney DB, Hedrick HL, Bush DM, et al.: Necrotizing enterocolitis in neonates with congenital heart disease: risk factors and outcomes. Pediatrics. 2000; 106(5): 1080–7. PubMed Abstract\n\nGuthrie SO, Gordon PV, Thomas V, et al.: Necrotizing enterocolitis among neonates in the United States. J Perinatol. 2003; 23(4): 278–85. PubMed Abstract | Publisher Full Text\n\nHsueh W, Caplan MS, Qu X, et al.: Neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts. Pediatr Dev Pathol. 2003; 6(1): 6–23. PubMed Abstract | Publisher Full Text\n\nTuladhar R, Daftary A, Patole SK, et al.: Oral gastrografin in neonates: a note of caution. Int J Clin Pract. 1999; 53(7): 565. PubMed Abstract\n\nTravadi J, Patole S, Simmer K: Gastrointestinal contrast studies in high-risk neonates with suspected necrotising enterocolitis--a note of caution. J Perinat Med. 2003; 31(6): 523–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPalmer SR, Thomas SJ, Cooke RW, et al.: Birthweight-specific risk factors for necrotising enterocolitis. J Epidemiol Community Health. 1987; 41(3): 210–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatel RM, Denning PW: Intestinal microbiota and its relationship with necrotizing enterocolitis. Pediatr Res. 2015; 78(3): 232–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKim SS, Albanese CT: Necrotizing enterocolitis. In: O'Neill JA, Coran AG, Fonkalsrud E, Grosfeld JL. Pediatric Surgery. 6th ed. St. Louis: MO Mosby; 2003; 1427–1452.\n\nFaingold R, Daneman A, Tomlinson G, et al.: Necrotizing enterocolitis: assessment of bowel viability with color doppler US. Radiology. 2005; 235(2): 587–94. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMurdoch EM, Sinha AK, Shanmugalingam ST, et al.: Doppler flow velocimetry in the superior mesenteric artery on the first day of life in preterm infants and the risk of neonatal necrotizing enterocolitis. Pediatrics. 2006; 118(5): 1999–2003. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSilva CT, Daneman A, Navarro OM, et al.: Correlation of sonographic findings and outcome in necrotizing enterocolitis. Pediatr Radiol. 2007; 37(3): 274–82. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nYikilmaz A, Hall NJ, Daneman A, et al.: Prospective evaluation of the impact of sonography on the management and surgical intervention of neonates with necrotizing enterocolitis. Pediatr Surg Int. 2014; 30(12): 1231–40. PubMed Abstract | Publisher Full Text\n\nMaalouf EF, Fagbemi A, Duggan PJ, et al.: Magnetic resonance imaging of intestinal necrosis in preterm infants. Pediatrics. 2000; 105(3 Pt 1): 510–4. PubMed Abstract\n\nGay AN, Lazar DA, Stoll B, et al.: Near-infrared spectroscopy measurement of abdominal tissue oxygenation is a useful indicator of intestinal blood flow and necrotizing enterocolitis in premature piglets. J Pediatr Surg. 2011; 46(6): 1034–40. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPatel AK, Lazar DA, Burrin DG, et al.: Abdominal near-infrared spectroscopy measurements are lower in preterm infants at risk for necrotizing enterocolitis. Pediatr Crit Care Med. 2014; 15(8): 735–41. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nJi J, Ling XB, Zhao Y, et al.: A data-driven algorithm integrating clinical and laboratory features for the diagnosis and prognosis of necrotizing enterocolitis. PLoS One. 2014; 9(2): e89860. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSylvester KG, Ling XB, Liu GY, et al.: A novel urine peptide biomarker-based algorithm for the prognosis of necrotising enterocolitis in human infants. Gut. 2014; 63(8): 1284–92. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStone ML, Tatum PM, Weitkamp JH, et al.: Abnormal heart rate characteristics before clinical diagnosis of necrotizing enterocolitis. J Perinatol. 2013; 33(11): 847–50. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nEvennett N, Cerigioni E, Hall NJ, et al.: Smooth muscle actin as a novel serologic marker of severe intestinal damage in rat intestinal ischemia-reperfusion and human necrotising enterocolitis. J Surg Res. 2014; 191(2): 323–30. PubMed Abstract | Publisher Full Text\n\nNg PC, Ma TP, Lam HS: The use of laboratory biomarkers for surveillance, diagnosis and prediction of clinical outcomes in neonatal sepsis and necrotising enterocolitis. Arch Dis Child Fetal Neonatal Ed. 2015; 100(5): F448–52. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPierro A, Hall N, Ade-Ajayi A, et al.: Laparoscopy assists surgical decision making in infants with necrotizing enterocolitis. J Pediatr Surg. 2004; 39(6): 902–6; discussion 902–6. PubMed Abstract | Publisher Full Text\n\nLeva E, Di Cesare A, Canazza L, et al.: The role of laparoscopy in newborns affected by NEC. J Laparoendosc Adv Surg Tech A. 2010; 20(2): 187–9. PubMed Abstract | Publisher Full Text\n\nNumanoglu A, Millar AJ: Necrotizing enterocolitis: early conventional and fluorescein laparoscopic assessment. J Pediatr Surg. 2011; 46(2): 348–51. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSmith J, Thyoka M: What role does laparoscopy play in the diagnosis and immediate treatment of infants with necrotizing enterocolitis? J Laparoendosc Adv Surg Tech A. 2013; 23(4): 397–401. PubMed Abstract | Publisher Full Text\n\nPorter A: Spontaneous pneumoperitoneum in the newborn; report of a case. N Engl J Med. 1956; 254(15): 694–6. PubMed Abstract | Publisher Full Text\n\nAschner JL, Deluga KS, Metlay LA, et al.: Spontaneous focal gastrointestinal perforation in very low birth weight infants. J Pediatr. 1988; 113(2): 364–7. PubMed Abstract | Publisher Full Text\n\nPumberger W, Mayr M, Kohlhauser C, et al.: Spontaneous localized intestinal perforation in very-low-birth-weight infants: a distinct clinical entity different from necrotizing enterocolitis. J Am Coll Surg. 2002; 195(6): 796–803. PubMed Abstract | Publisher Full Text\n\nGordon PV, Young ML, Marshall DD: Focal small bowel perforation: an adverse effect of early postnatal dexamethasone therapy in extremely low birth weight infants. J Perinatol. 2001; 21(3): 156–60. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHall NJ, Eaton S, Pierro A: Royal Australasia of Surgeons Guest Lecture. Necrotizing enterocolitis: prevention, treatment, and outcome. J Pediatr Surg. 2013; 48(12): 2359–67. PubMed Abstract | Publisher Full Text\n\nZani A, Eaton S, Puri P, et al.: International survey on the management of necrotizing enterocolitis. Eur J Pediatr Surg. 2015; 25(1): 27–33. PubMed Abstract | Publisher Full Text\n\nShah D, Sinn JK: Antibiotic regimens for the empirical treatment of newborn infants with necrotising enterocolitis. Cochrane Database Syst Rev. 2012; 8: CD007448. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nEin SH, Marshall DG, Girvan D: Peritoneal drainage under local anesthesia for perforations from necrotizing enterocolitis. J Pediatr Surg. 1977; 12(6): 963–7. PubMed Abstract | Publisher Full Text\n\nJanik JS, Ein SH: Peritoneal drainage under local anesthesia for necrotizing enterocolitis (NEC) perforation: a second look. J Pediatr Surg. 1980; 15(4): 565–6. PubMed Abstract | Publisher Full Text\n\nLessin MS, Luks FI, Wesselhoeft CW Jr, et al.: Peritoneal drainage as definitive treatment for intestinal perforation in infants with extremely low birth weight (<750 g). J Pediatr Surg. 1998; 33(2): 370–2. PubMed Abstract | Publisher Full Text\n\nRovin JD, Rodgers BM, Burns RC, et al.: The role of peritoneal drainage for intestinal perforation in infants with and without necrotizing enterocolitis. J Pediatr Surg. 1999; 34(1): 143–7. PubMed Abstract | Publisher Full Text\n\nGoyal A, Manalang LR, Donnell SC, et al.: Primary peritoneal drainage in necrotising enterocolitis: an 18-year experience. Pediatr Surg Int. 2006; 22(5): 449–52. PubMed Abstract | Publisher Full Text\n\nMoss RL, Dimmitt RA, Barnhart DC, et al.: Laparotomy versus peritoneal drainage for necrotizing enterocolitis and perforation. N Engl J Med. 2006; 354(21): 2225–34. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nRees CM, Eaton S, Kiely EM, et al.: Peritoneal drainage or laparotomy for neonatal bowel perforation? A randomized controlled trial. Ann Surg. 2008; 248(1): 44–51. PubMed Abstract | Publisher Full Text\n\nPierro A, Eaton S, Rees CM, et al.: Is there a benefit of peritoneal drainage for necrotizing enterocolitis in newborn infants? J Pediatr Surg. 2010; 45(11): 2117–8. PubMed Abstract | Publisher Full Text\n\nHall NJ, Curry J, Drake DP, et al.: Resection and primary anastomosis is a valid surgical option for infants with necrotizing enterocolitis who weigh less than 1000 g. Arch Surg. 2005; 140(12): 1149–51. PubMed Abstract | Publisher Full Text\n\nvan den Hondel D, Sloots C, Meeussen C, et al.: To split or not to split: colostomy complications for anorectal malformations or hirschsprung disease: a single center experience and a systematic review of the literature. Eur J Pediatr Surg. 2014; 24(1): 61–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nVaughan WG, Grosfeld JL, West K, et al.: Avoidance of stomas and delayed anastomosis for bowel necrosis: the 'clip and drop-back' technique. J Pediatr Surg. 1996; 31(4): 542–5. PubMed Abstract | Publisher Full Text\n\nRon O, Davenport M, Patel S, et al.: Outcomes of the \"clip and drop\" technique for multifocal necrotizing enterocolitis. J Pediatr Surg. 2009; 44(4): 749–54. PubMed Abstract | Publisher Full Text\n\nPang KK, Chao NS, Wong BP, et al.: The clip and drop back technique in the management of multifocal necrotizing enterocolitis: a single centre experience. Eur J Pediatr Surg. 2012; 22(1): 85–90. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nThyoka M, Eaton S, Kiely EM, et al.: Outcomes of diverting jejunostomy for severe necrotizing enterocolitis. J Pediatr Surg. 2011; 46(6): 1041–4. PubMed Abstract | Publisher Full Text\n\nFitzgibbons SC, Ching Y, Yu D, et al.: Mortality of necrotizing enterocolitis expressed by birth weight categories. J Pediatr Surg. 2009; 44(6): 1072–5; discussion 1075–6. PubMed Abstract | Publisher Full Text\n\nThyoka M, de Coppi P, Eaton S, et al.: Advanced necrotizing enterocolitis part 1: mortality. Eur J Pediatr Surg. 2012; 22(1): 8–12. PubMed Abstract | Publisher Full Text\n\nThyoka M, Eaton S, Hall NJ, et al.: Advanced necrotizing enterocolitis part 2: recurrence of necrotizing enterocolitis. Eur J Pediatr Surg. 2012; 22(1): 13–6. PubMed Abstract | Publisher Full Text\n\nSchwartz MZ, Hayden CK, Richardson CJ, et al.: A prospective evaluation of intestinal stenosis following necrotizing enterocolitis. J Pediatr Surg. 1982; 17(6): 764–70. PubMed Abstract | Publisher Full Text\n\nKosloske AM, Burstein J, Bartow SA: Intestinal obstruction due to colonic stricture following neonatal necrotizing enterocolitis. Ann Surg. 1980; 192(2): 202–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDuro D, Kalish LA, Johnston P, et al.: Risk factors for intestinal failure in infants with necrotizing enterocolitis: a Glaser Pediatric Research Network study. J Pediatr. 2010; 157(2): 203–208.e1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDemehri FR, Stephens L, Herrman E, et al.: Enteral autonomy in pediatric short bowel syndrome: predictive factors one year after diagnosis. J Pediatr Surg. 2015; 50(1): 131–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHintz SR, Kendrick DE, Stoll BJ, et al.: Neurodevelopmental and growth outcomes of extremely low birth weight infants after necrotizing enterocolitis. Pediatrics. 2005; 115(3): 696–703. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nShah DK, Doyle LW, Anderson PJ, et al.: Adverse neurodevelopment in preterm infants with postnatal sepsis or necrotizing enterocolitis is mediated by white matter abnormalities on magnetic resonance imaging at term. J Pediatr. 2008; 153(2): 170–5, 175.e1. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAlFaleh K, Anabrees J: Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2014; 4: CD005496. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAsmerom M, Crowe L, Marin T: Understanding the Biologic Therapies of Probiotics, Prebiotics, and Synbiotics: Exploring Current Evidence for Use in Premature Infants for the Prevention of Necrotizing Enterocolitis. J Perinat Neonatal Nurs. 2015; 29(3): 240–7; quiz E2. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPatel R, DuPont HL: New approaches for bacteriotherapy: prebiotics, new-generation probiotics, and synbiotics. Clin Infect Dis. 2015; 60(Suppl 2): S108–21. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTimofeev VA: Elektronnomikroskopicheskie dannye ob èkskretsii lipidov Triaenophorus nodulosus (Pall). Tsitologiia. 1967; 9: 1413–5.\n\nArmanian AM, Sadeghnia A, Hoseinzadeh M, et al.: The Effect of Neutral Oligosaccharides on Reducing the Incidence of Necrotizing Enterocolitis in Preterm infants: A Randomized Clinical Trial. Int J Prev Med. 2014; 5(11): 1387–95. PubMed Abstract | Free Full Text | Faculty Opinions Recommendation\n\nZani A, Eaton S, Leon FF, et al.: Captopril reduces the severity of bowel damage in a neonatal rat model of necrotizing enterocolitis. J Pediatr Surg. 2008; 43(2): 308–14. PubMed Abstract | Publisher Full Text\n\nLu J, Pierce M, Franklin A, et al.: Dual roles of endogenous platelet-activating factor acetylhydrolase in a murine model of necrotizing enterocolitis. Pediatr Res. 2010; 68(3): 225–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWei J, Besner GE: M1 to M2 macrophage polarization in heparin-binding epidermal growth factor-like growth factor therapy for necrotizing enterocolitis. J Surg Res. 2015; 197(1): 126–38. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEl-Ganzoury MM, Awad HA, El-Farrash RA, et al.: Enteral granulocyte-colony stimulating factor and erythropoietin early in life improves feeding tolerance in preterm infants: a randomized controlled trial. J Pediatr. 2014; 165(6): 1140–1145.e1. PubMed Abstract | Publisher Full Text\n\nStefanutti G, Pierro A, Parkinson EJ, et al.: Moderate hypothermia as a rescue therapy against intestinal ischemia and reperfusion injury in the rat. Crit Care Med. 2008; 36(5): 1564–72. PubMed Abstract | Publisher Full Text\n\nVejchapipat P, Williams SR, Proctor E, et al.: Moderate hypothermia ameliorates liver energy failure after intestinal ischaemia-reperfusion in anaesthetised rats. J Pediatr Surg. 2001; 36(2): 269–75. PubMed Abstract | Publisher Full Text\n\nVejchapipat P, Proctor E, Ramsay A, et al.: Intestinal energy metabolism after ischemia-reperfusion: Effects of moderate hypothermia and perfluorocarbons. J Pediatr Surg. 2002; 37(5): 786–90. PubMed Abstract | Publisher Full Text\n\nVinardi S, Pierro A, Parkinson EJ, et al.: Hypothermia throughout intestinal ischaemia-reperfusion injury attenuates lung neutrophil infiltration. J Pediatr Surg. 2003; 38(1): 88–91; discussion 88-91. PubMed Abstract | Publisher Full Text\n\nStefanutti G, Vejchapipat P, Williams SR, et al.: Heart energy metabolism after intestinal ischaemia and reperfusion. J Pediatr Surg. 2004; 39(2): 179–83; discussion 179-83. PubMed Abstract | Publisher Full Text\n\nHall NJ, Eaton S, Peters MJ, et al.: Mild controlled hypothermia in preterm neonates with advanced necrotizing enterocolitis. Pediatrics. 2010; 125(2): e300–8. PubMed Abstract | Publisher Full Text\n\nIrhimeh MR, Cooney J: Management of inflammatory bowel disease using stem cell therapy. Curr Stem Cell Res Ther. 2015. PubMed Abstract\n\nZani A, Cananzi M, Eaton S, et al.: Stem cells as a potential treatment of necrotizing enterocolitis. J Pediatr Surg. 2009; 44(3): 659–60. PubMed Abstract | Publisher Full Text\n\nZani A, Cananzi M, Fascetti-Leon F, et al.: Amniotic fluid stem cells improve survival and enhance repair of damaged intestine in necrotising enterocolitis via a COX-2 dependent mechanism. Gut. 2014; 63(2): 300–9. PubMed Abstract | Publisher Full Text\n\nZani A, Cananzi M, Lauriti G, et al.: Amniotic fluid stem cells prevent development of ascites in a neonatal rat model of necrotizing enterocolitis. Eur J Pediatr Surg. 2014; 24(1): 57–60. PubMed Abstract | Publisher Full Text\n\nEaton S, Zani A, Pierro A, et al.: Stem cells as a potential therapy for necrotizing enterocolitis. Expert Opin Biol Ther. 2013; 13(12): 1683–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11371",
"date": "30 Nov 2015",
"name": "David Wesson",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11372",
"date": "30 Nov 2015",
"name": "Atsuyuki Yamataka",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11373",
"date": "30 Nov 2015",
"name": "Paul Tam",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1373
|
https://f1000research.com/articles/4-1366/v1
|
27 Nov 15
|
{
"type": "Review",
"title": "The Surgical Approach to Differentiated Thyroid Cancer",
"authors": [
"Iain J. Nixon"
],
"abstract": "The incidence of thyroid cancer is increasing rapidly. A large percentage of new cases identified fall into a low-risk category. As the incidence has increased, clinical experience has confirmed that the majority of patients will have excellent outcomes and that those at risk of doing badly can be reliably identified. Treatment for thyroid cancer is predominantly surgical. The decision about how aggressively this disease should be managed has remained controversial due to the excellent outcomes irrespective of the nature of surgical procedure chosen. This article reviews the developments in our understanding of the biology of thyroid cancer and the evidence that supports the approach to management.",
"keywords": [
"Differentiated thyroid cancer",
"Thyroid surgery",
"Lymph node surgery",
"Thyroid lobectomy",
"Cervical lymphadenectomy",
"Primary thyroid surgery"
],
"content": "Introduction\n\nDifferentiated thyroid cancer is the most common endocrine malignancy and rates are increasing worldwide1–8. Patients and clinicians are increasingly aware of the diagnosis and, with ever more cases being reported, an understanding of the most appropriate way of managing this condition is essential.\n\nOptimal management of this disease is controversial. The reasons for the controversy include the low rates of disease-specific mortality, relatively low morbidity from therapy, and the gradual evolution in our understanding of the biology of the disease.\n\nLow mortality rates have been a challenge for investigators with an interest in differentiated thyroid cancer. Less than 10% of patients will die of the disease within 10 years of presentation. Indeed, even recurrence rates are low, particularly in the most controversial low-risk groups. As such, randomized controlled trials of therapy have not been considered feasible9. The lack of prospective trials leads clinicians to rely on retrospective data, which has inherent flaws, no matter how it is collected.\n\nThe mainstay of therapy for differentiated thyroid cancer is surgery. Although when initially attempted, thyroidectomy was associated with high rates of mortality, improvements in anaesthetic and surgical technique have resulted in thyroid surgery being extremely safe. Death following thyroid surgery is reported in <0.5% of cases10. In contrast, rates of injury to the recurrent laryngeal nerve and parathyroid glands are more common (2% nerve palsy and 6% need for calcium supplements at follow up10). These complications, while not life threatening, result in voice change (and occasionally tracheostomy in cases of bilateral recurrent laryngeal nerve injury) and the need for long term calcium supplementation.\n\nThe initial surgical approach to thyroid cancer was radical. Total thyroidectomy and bilateral radical neck dissection achieved macroscopic disease clearance at a cost. In particular, the cervical lymphadenectomy was associated with significant functional and cosmetic impact. The recognition that histology could predict the biological behaviour of tumors was made in the mid-20th century. Good outcomes were described for patients with differentiated lesions of follicular cell origin (papillary carcinoma/follicular carcinoma/Hurthle cell carcinoma) in comparison with medullary or anaplastic carcinoma11. This observation led to a significant change in surgical approach, with a move away from aggressive neck surgery in patients with differentiated thyroid cancer.\n\nThe reports of large institutions’ retrospective case series provided further insight into disease biology with the recognition of age, tumor size, presence of extra thyroid extension, and distant metastases as risk factors within the differentiated thyroid cancer patient group12–16. These observations provided a framework for clinicians to risk stratify their patient group into those at low, intermediate, and high risk of disease-specific death (abbreviated to AMES system of risk stratification).\n\nAlong with the interest in predicting features of patients and their tumors, which predicted the biology of disease, came scrutiny of outcomes related to management of the disease itself. Until this point, no universally agreed approach had been accepted. In 1977, a report of outcomes for 576 patients with papillary thyroid cancer (the most common differentiated thyroid cancer) recorded in the US Air Force Central Tumor Registry found that total thyroidectomy and post-operative radioactive iodine therapy were associated with lower rates of recurrence and higher survival17. Clinicians would use this evidence to support an approach of total thyroidectomy and radioactive iodine for all cases of differentiated thyroid cancer. The report also found high rates of surgical morbidity following neck surgery with no impact on outcome. This finding supported an approach of primary thyroid surgery without neck dissection in those patients without evidence of regional metastases.\n\nIn contrast, other groups found that, in lower risk patients, outcomes were similar following total thyroidectomy and thyroid lobectomy18,19. The conflicting findings of the impact of the extent of initial therapy on outcome continue to this day. The surgical debate has focused on the need for aggressive primary thyroid surgery (total thyroidectomy versus lobectomy)20–25 and the approach to the clinically uninvolved central neck (prophylactic dissection versus observation)26–33.\n\nMuch research has followed since the early reports. However, prospective trials are still lacking and most recommendations are based upon retrospective data, which have been analysed by expert authors with long standing biases.\n\nThe aim of this article is to examine the contemporary approach to surgical management of differentiated thyroid cancer by analysing arguments for primary thyroid surgery and cervical lymphadenectomy.\n\n\nThe aims of surgical management of differentiated thyroid cancer\n\nThe primary aim of surgical oncology is to prevent death from disease. However, for the overwhelming majority of patients with differentiated thyroid cancer, the risk of death is minimal. In addition, for many patients, occult disease will be present even after successful treatment. So the goals of therapy must be seen in the appropriate context. Risk stratification is paramount. High risk patients are treated aggressively, while low risk patients may be suitable for a less aggressive approach, and some select patients with the lowest risk disease (micropapillary carcinoma distant from the recurrent nerve or trachea) may even be candidates for an observational approach. In addition to preventing disease specific death, minimizing the chance of recurrence and preventing iatrogenic injury are key objectives for the treatment team.\n\n\nPrimary thyroid surgery\n\nThe most important aim of primary surgery is to achieve complete macroscopic disease clearance and to minimize the chance that ipsilateral thyroid bed surgery will ever be required again. This requires a thyroid lobectomy as a minimum (other than for the occasional patient with isolated isthmic disease). An extra capsular thyroidectomy with preservation of the recurrent laryngeal nerve and parathyroid glands should be the standard. This will achieve disease clearance and minimize the risk of thyroid bed recurrence.\n\nA second aim of primary surgery is to render the patient suitable for adjuvant radioactive iodine by removing all thyroid tissue. The approach to adjuvant therapy is now changing. Previously, radioactive iodine was recommended for the majority of patients with tumors of 1cm or greater. Particularly in health care systems that do not biopsy lesions smaller than 1cm, this meant that all patients were candidates for total thyroidectomy. More recently, however, the role of radioactive iodine in intermediate and low risk patients has been questioned. There is a trend away from the blanket approach of total thyroidectomy and radioactive iodine for all towards a risk-adapted, individualized approach. Although radioactive iodine is still recommended in patients with aggressive primary lesions or metastatic disease to the neck or beyond, low risk patients with small-volume primary disease and without evidence of spread have little to gain from adjuvant therapy34.\n\nTherefore, appropriately selected patients are suitable for thyroid lobectomy rather than total thyroidectomy. While preserving excellent oncological outcomes, this approach has significant potential benefits. Rates of recurrent laryngeal nerve injury, hypocalcaemia, and tracheostomy are significantly lower following such unilateral surgery.\n\nThe ideal candidate for such an approach is a young patient with uninodular disease limited to the thyroid. The risk of permanent post-operative hypocalcaemia and tracheostomy is more or less 0% following thyroid lobectomy. Recurrent nerve injury is most commonly a temporary palsy, but is permanent in around 2% of cases10. Operating on one side rather than both is, unsurprisingly, associated with lower rates of morbidity35. However, these patients, by definition, have a residual lobe following treatment. They are not suitable for radioactive iodine and require monitoring of the contralateral lobe in the post-operative period by ultrasound. In the long term, 5–10% of such patients will require completion thyroidectomy at some point during follow up. This is mainly due to the development of nodular disease in the residual lobe. Such disease is malignant approximately half of the time21.\n\nA number of factors make the decision of which primary procedure to offer complex. Many expert authors report extremely low complication rates following total thyroidectomy. In contrast, reports from a community setting suggest that complication rates are significantly higher for the majority of patients who are operated on outside centers of excellence35,36. Following initial therapy, the tumor marker thyroglobulin can be used during follow up to detect recurrence. This tumor marker is produced both from native thyroid tissue and persistent disease. Therefore it is less useful in patients who have had thyroid lobectomy. Due to high rates of multifocal disease within the thyroid, most authors do not recommend thyroid lobectomy if there are nodules in the contralateral lobe, even if they appear benign. This is particularly relevant in areas with a high incidence of multinodular thyroid disease and is an issue increasingly encountered due to improvements in ultrasound imaging, which now detect nodular disease in over 50% of otherwise healthy individuals37.\n\nWhen making a decision about primary thyroid surgery, the clinician must consider a number of factors. Risk stratification should be performed for each patient and used as a guide to selection of therapy. Tumor factors are critical, and total thyroidectomy remains the treatment of choice for those high-risk patients who will be candidates for adjuvant radioactive iodine. Surgical factors must also be considered. A patient presenting to a high-volume thyroid surgeon in a center of excellence has a significantly lower risk of suffering a complication than one who presents to a surgeon with little experience. Although an ideal solution would be the centralization of thyroid surgery to minimize the number of surgeons performing this procedure, this is not feasible for most patients. Patient factors must also be considered. Differentiated thyroid cancer commonly affects young women. This patient group is often well read, motivated, and anxious. Patients may have an idea of what they consider the treatment of choice long before they arrive in the surgical clinic.\n\nFor many patients, total thyroidectomy is the treatment of choice. When performed well, it provides excellent oncological outcomes safely. It facilitates radioactive iodine if required, allows optimal follow up using thyroglobulin as a tumor marker, and addresses concerns about multifocal disease within the gland.\n\nClinicians should also be aware that thyroid lobectomy offers equal oncological outcomes in appropriately selected patients. There are significant advantages, particularly when patients are managed outside high-volume surgical departments. In addition, although the use of thyroglobulin in follow up is less accurate, it can still be used effectively. It should be remembered that “low-risk” patients have a mortality rate of <5% at 20 years and recurrences are extremely rare, so the value of a highly accurate tumor marker is questionable.\n\nDisease management teams should consider the issues listed above when counselling patients with differentiated thyroid cancer. By balancing tumor, clinician, and patient factors, an individualized plan can be tailored for each patient using a risk-adapted approach to optimize outcome on a case by case basis (Figure 1).\n\n\nLymph node surgery\n\nDifferentiated thyroid cancer and in particular papillary thyroid cancer commonly metastasizes to the neck. The most common site of metastasis is the central neck (levels VI and VII), which surrounds the thyroid gland. The second echelon of lymph nodes is the lateral neck (most commonly levels III and IV). As stated earlier, radical neck dissection was considered the treatment of choice at one point38. This was an operation that was relatively quick, safe, and resulted in macroscopic disease clearance. However, it was associated with high rates of morbidity. A vogue for a much less aggressive “berry picking” approach to the removal of macroscopically involved neck nodes has largely been abandoned due to unacceptably high recurrence rates. As experience in neck surgery has improved, a compartment oriented neck dissection is recommended by most authors as the operation of choice for patients with evidence of neck disease39.\n\nCritically, the surgeon must ensure the neck has been properly evaluated prior to embarking on surgery. Ultrasound is a reliable way of assessing the lateral neck and is also the investigation of choice for the thyroid. If lateral nodal disease is encountered, imaging of the central neck may be considered using CT or MRI. Cross sectional imaging is preferable to ultrasound in assessing the central neck, particularly the mediastinal component (level VII), which is poorly visualized using ultrasound.\n\nThose patients considered N1a or N1b (metastatic disease in the central or lateral neck respectively) following investigation should have surgery planned to remove all involved levels, and any other levels considered at risk (therapeutic neck dissection). So, those patients with disease in the central neck alone should have a central neck dissection (almost always with a total thyroidectomy as radioactive iodine is likely to be indicated).\n\nThose patients with lateral neck involvement should have clearance of levels II-V, which are at the highest risk of metastatic involvement38. Involvement of the neck above the accessory nerve and in the submental/submandibular region (level I) is uncommon, so these levels are routinely spared. In addition, differentiated thyroid cancer rarely presents with aggressive nodal disease and extra nodal extension. Therefore, in almost all patients, the sternocleidomastoid muscle, internal jugular vein, and accessory nerve can be spared. This significantly limits the morbidity of surgery and has become the standard of care for patients with lateral neck disease.\n\nIn contrast, the approach to the clinically negative neck is highly controversial. The reasons for the controversy are multiple, and again, without prospective evidence may never be resolved.\n\nThere are authors who recommend prophylactic lateral neck dissection (surgery without pre-operative evidence of involved nodes)28. However, they are in the minority and the vast majority do not consider the morbidity of lateral neck surgery worth the “benefit”39,40. Despite this, if one chooses to dissect the apparently uninvolved lateral neck, metastatic disease will often be found on histology28. This disease rarely manifests and if it does it can safely be salvaged at a later date. In addition, entering the lateral neck requires an extended incision and places structures at risk that are not routinely encountered in thyroid and central neck surgery (accessory nerve, marginal mandibular nerve, carotid sheath and thoracic duct).\n\nIn contrast, when performing a thyroidectomy, the central neck is, by definition, entered. The recurrent laryngeal nerves and parathyroid glands are encountered during the dissection and revision central neck surgery carries higher risks than primary procedures.\n\nAuthors who argue for prophylactic central neck surgery highlight the fact that the central neck is exposed during primary thyroid surgery. They also cite high rates of occult histopathological metastases and that such metastases “upstage” patients when identified, which gives the treating team an effective way of further risk stratifying patients to rationalize the approach to adjuvant radioactive iodine. There is some evidence that excision of the involved nodes in the central neck results in lower post-operative thyroglobulin levels, which may result in lower recurrence rates. No author has ever proven that prophylactic central neck dissection results in improved survival, as almost no patient without metastatic disease dies during follow up.\n\nIn contrast, those authors who do not support prophylactic surgery highlight the higher surgical morbidity of the procedure versus thyroidectomy alone, and the fact that patients who have observation rather than central neck dissection have extremely good outcomes with low rates of recurrence and extremely low rates of death. With such good outcomes enjoyed by this group of low-risk patients, the need for radioactive iodine is questionable. In addition, the approach to a central neck dissection is probably variable. Prophylactic central neck surgery involves removing tissue that lies between the recurrent laryngeal nerves. However, those centers with experience of re-operative central neck surgery find high rates of disease in areas not normally included in prophylactic surgery, such as dorsal to the recurrent laryngeal nerve or low at the thoracic inlet, which are high-risk areas for dissection and hence not included in the primary surgical field41.\n\nThe controversy has resulted in ambiguity in international guidelines39,40. Such documents recommend an individualization of approach dependant on risk factors for involvement. Patients with large volume tumors and those with evidence of extra thyroid extension are at higher risk and, in such patients, guidelines recommend considering prophylactic central neck surgery without definite evidence that such an approach results in improved outcomes. In general, there is a move away from an aggressive approach where central neck dissection should be considered for all patients (with papillary thyroid cancer) and towards selecting on a case by case basis, recognizing that the degree of potential benefit cannot be calculated.\n\nAgain, in making a decision about the surgical approach to the central neck, patient, tumor, and surgeon factors must be considered. Older male patients with large volume, multicentric disease and extra thyroid extension are at higher risk of metastatic disease to the central neck, even if it is not evident on pre-operative investigation. Such patients may be considered for prophylactic central neck surgery. However, the experience of the surgeon involved should also be weighed in the decision. Morbidity for high-volume neck surgeons is lower than that for those with less experience. The potential for benefit is small, and this must be weighed against the increased risks of damage to the parathyroids and recurrent laryngeal nerves when giving advice to patients without evidence of metastatic disease in the lymph nodes (cN0).\n\n\nConclusions\n\nOutcomes for patients with differentiated thyroid cancer are excellent in comparison with other human cancers. Those at high risk can be easily identified from patient and tumor factors. Based upon a risk-stratified approach to managing differentiated thyroid cancer, therapeutic decisions in regard to surgery can be broken down into primary thyroidectomy and regional lymphadenectomy.\n\nTotal thyroidectomy is an operation associated with high cure rates and has been considered the gold standard internationally for years. However, thyroid lobectomy is now recognized as equally oncologically effective and is associated with lower morbidity in properly selected patients.\n\nCompartment-oriented neck dissection has replaced both radical neck dissection and berry picking as the favoured approach to therapeutic lymphadenectomy. Outcomes for patients who have no evidence of regional metastases are now recognized as excellent, irrespective of the surgical approach. This has led to a less aggressive prophylactic surgical approach to the central neck in recent guidelines. This move away from routine prophylactic surgery has continued with the updated 2015 American Thyroid Association guidelines34.\n\nAlthough many treatment recommendations are now available, definitive prospective evidence to guide the thyroid surgeon is lacking in most cases. For this reason the surgical approach to this increasingly common disease remains controversial. By understanding the pros and cons of more and less aggressive approaches to the thyroid gland and the regional lymph nodes, with an understanding of the disease biology, and by applying risk stratification to all patients with differentiated thyroid cancer, appropriate decisions can be made that result in excellent oncological and surgical outcomes for the treatment team and their patients.",
"appendix": "Competing interests\n\n\n\nThe author completed his head and neck surgical training in Memorial Sloan Kettering Cancer Center, an institution renowned for championing a conservative approach to the management of differentiated thyroid cancer.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAmphlett B, Lawson Z, Abdulrahman GO Jr, et al.: Recent trends in the incidence, geographical distribution, and survival from thyroid cancer in Wales, 1985–2010. Thyroid. 2013; 23(11): 1470–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAschebrook-Kilfoy B, Ward MH, Sabra MM, et al.: Thyroid cancer incidence patterns in the United States by histologic type, 1992–2006. Thyroid. 2011; 21(2): 125–34. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChen AY, Jemal A, Ward EM: Increasing incidence of differentiated thyroid cancer in the United States, 1988–2005. Cancer. 2009; 115(16): 3801–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDavies L, Welch HG: Increasing incidence of thyroid cancer in the United States, 1973–2002. JAMA. 2006; 295(18): 2164–7. PubMed Abstract | Publisher Full Text\n\nEnewold L, Zhu K, Ron E, et al.: Rising thyroid cancer incidence in the United States by demographic and tumor characteristics, 1980–2005. Cancer Epidemiol Biomarkers Prev. 2009; 18(3): 784–91. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nKilfoy BA, Zheng T, Holford TR, et al.: International patterns and trends in thyroid cancer incidence, 1973–2002. Cancer Causes Control. 2009; 20(5): 525–31. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMcNally RJ, Blakey K, James PW, et al.: Increasing incidence of thyroid cancer in Great Britain, 1976–2005: age-period-cohort analysis. Eur J Epidemiol. 2012; 27(8): 615–22. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOlaleye O, Ekrikpo U, Moorthy R, et al.: Increasing incidence of differentiated thyroid cancer in South East England: 1987–2006. Eur Arch Otorhinolaryngol. 2011; 268(6): 899–906. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nCarling T, Carty SE, Ciarleglio MM, et al.: American Thyroid Association design and feasibility of a prospective randomized controlled trial of prophylactic central lymph node dissection for papillary thyroid carcinoma. Thyroid. 2012; 22(3): 237–44. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChadwick D, Kinsman R, Walton P: The British Association of Endocrine & Thyroid Surgeons Fourth National Audit Report. Dendrite Clinical Systems Ltd, The Hub, Station Road, Henley-on-Thames, Oxfordshire RG9 1AY, United Kingdom. 2012. Reference Source\n\nGraev M, Panunzi CA: [Findings on one hundred cases of thyroid carcinoma; incidence, frequency of various histological types, prognosis on the basis of histology, survival]. Arch De Vecchi Anat Patol. 1957; 27(1): 31–63. PubMed Abstract\n\nCady B, Sedgwick CE, Meissner WA, et al.: Risk factor analysis in differentiated thyroid cancer. Cancer. 1979; 43(3): 810–20. PubMed Abstract | Publisher Full Text\n\nMcConahey WM, Hay ID, Woolner LB, et al.: Papillary thyroid cancer treated at the Mayo Clinic, 1946 through 1970: initial manifestations, pathologic findings, therapy, and outcome. Mayo Clin Proc. 1986; 61(12): 978–96. PubMed Abstract | Publisher Full Text\n\nShah JP, Loree TR, Dharker D, et al.: Prognostic factors in differentiated carcinoma of the thyroid gland. Am J Surg. 1992; 164(6): 658–61. PubMed Abstract | Publisher Full Text\n\nByar DP, Green SB, Dor P, et al.: A prognostic index for thyroid carcinoma. A study of the E.O.R.T.C. Thyroid Cancer Cooperative Group. Eur J Cancer. 1979; 15(8): 1033–41. PubMed Abstract | Publisher Full Text\n\nHay ID, Bergstralh EJ, Goellner JR, et al.: Predicting outcome in papillary thyroid carcinoma: development of a reliable prognostic scoring system in a cohort of 1779 patients surgically treated at one institution during 1940 through 1989. Surgery. 1993; 114(6): 1050–7; discussion 1057–8. PubMed Abstract\n\nMazzaferri EL, Young RL, Oertel JE, et al.: Papillary thyroid carcinoma: the impact of therapy in 576 patients. Medicine (Baltimore). 1977; 56(3): 171–96. PubMed Abstract | Publisher Full Text\n\nHay ID, Grant CS, Taylor WF, et al.: Ipsilateral lobectomy versus bilateral lobar resection in papillary thyroid carcinoma: a retrospective analysis of surgical outcome using a novel prognostic scoring system. Surgery. 1987; 102(6): 1088–95. PubMed Abstract\n\nShah JP, Loree TR, Dharker D, et al.: Lobectomy versus total thyroidectomy for differentiated carcinoma of the thyroid: a matched-pair analysis. Am J Surg. 1993; 166(4): 331–5. PubMed Abstract | Publisher Full Text\n\nBilimoria KY, Bentrem DJ, Ko CY, et al.: Extent of surgery affects survival for papillary thyroid cancer. Ann Surg. 2007; 246(3): 375–81; discussion 381–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNixon IJ, Ganly I, Patel SG, et al.: Thyroid lobectomy for treatment of well differentiated intrathyroid malignancy. Surgery. 2012; 151(4): 571–9. PubMed Abstract | Publisher Full Text\n\nVaisman F, Momesso D, Bulzico DA, et al.: Thyroid Lobectomy Is Associated with Excellent Clinical Outcomes in Properly Selected Differentiated Thyroid Cancer Patients with Primary Tumors Greater Than 1 cm. J Thyroid Res. 2013; 2013: 398194. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMendelsohn AH, Elashoff DA, Abemayor E, et al.: Surgery for papillary thyroid carcinoma: is lobectomy enough? Arch Otolaryngol Head Neck Surg. 2010; 136(11): 1055–61. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAdam MA, Pura J, Goffredo P, et al.: Impact of extent of surgery on survival for papillary thyroid cancer patients younger than 45 years. J Clin Endocrinol Metab. 2015; 100(1): 115–21. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMatsuzu K, Sugino K, Masudo K, et al.: Thyroid lobectomy for papillary thyroid cancer: long-term follow-up study of 1,088 cases. World J Surg. 2014; 38(1): 68–79. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nNixon IJ, Ganly I, Patel SG, et al.: Observation of clinically negative central compartment lymph nodes in papillary thyroid carcinoma. Surgery. 2013; 154(6): 1166–72; discussion 1172–3. PubMed Abstract | Publisher Full Text\n\nMonchik JM, Simon CJ, Caragacianu DL, et al.: Does failure to perform prophylactic level VI node dissection leave persistent disease detectable by ultrasonography in patients with low-risk papillary carcinoma of the thyroid? Surgery. 2009; 146(6): 1182–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHartl DM, Leboulleux S, Al Ghuzlan A, et al.: Optimization of staging of the neck with prophylactic central and lateral neck dissection for papillary thyroid carcinoma. Ann Surg. 2012; 255(4): 777–83. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nYwata de Carvalho A, Chulam TC, Kowalski LP: Long-term Results of Observation vs Prophylactic Selective Level VI Neck Dissection for Papillary Thyroid Carcinoma at a Cancer Center. JAMA Otolaryngol Head Neck Surg. 2015; 141(7): 599–606. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSosa JA: Is routine prophylactic central neck dissection indicated for low-risk papillary thyroid cancer: can we determine cost-effectiveness if we are unsure about its effectiveness and safety? Surgery. 2013; 154(6): 1146–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHartl DM, Mamelle E, Borget I, et al.: Influence of prophylactic neck dissection on rate of retreatment for papillary thyroid carcinoma. World J Surg. 2013; 37(8): 1951–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDralle H: [Prophylactic central lymph node dissection improves prognosis for papillary thyroid cancer]. Chirurg. 2013; 84(2): 149. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nZanocco K, Elaraj D, Sturgeon C: Routine prophylactic central neck dissection for low-risk papillary thyroid cancer: a cost-effectiveness analysis. Surgery. 2013; 154(6): 1148–55; discussion 1154–5. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHaugen BR Md, Alexander EK, Bible KC, et al.: 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2015. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nZerey M, Prabhu AS, Newcomb WL, et al.: Short-term outcomes after unilateral versus complete thyroidectomy for malignancy: a national perspective. Am Surg. 2009; 75(1): 20–4. PubMed Abstract | Faculty Opinions Recommendation\n\nRosato L, Avenia N, Bernante P, et al.: Complications of thyroid surgery: analysis of a multicentric study on 14,934 patients operated on in Italy over 5 years. World J Surg. 2004; 28(3): 271–6. PubMed Abstract | Publisher Full Text\n\nEzzat S, Sarti DA, Cain DR, et al.: Thyroid incidentalomas. Prevalence by palpation and ultrasonography. Arch Intern Med. 1994; 154(16): 1838–40. PubMed Abstract | Publisher Full Text\n\nStack BC Jr, Ferris RL, Goldenberg D, et al.: American Thyroid Association consensus review and statement regarding the anatomy, terminology, and rationale for lateral neck dissection in differentiated thyroid cancer. Thyroid. 2012; 22(5): 501–8. PubMed Abstract | Publisher Full Text\n\nAmerican Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid Cancer, Cooper DS, Doherty GM, et al.: Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid. 2009; 19(11): 1167–214. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPerros P, Boelaert K, Colley S, et al.: Guidelines for the management of thyroid cancer. Clin Endocrinol (Oxf). 2014; 81(Suppl 1): 1–122. PubMed Abstract | Publisher Full Text\n\nClayman GL, Agarwal G, Edeiken BS, et al.: Long-term outcome of comprehensive central compartment dissection in patients with recurrent/persistent papillary thyroid carcinoma. Thyroid. 2011; 21(12): 1309–16. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11367",
"date": "27 Nov 2015",
"name": "Brendan Stack",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11368",
"date": "27 Nov 2015",
"name": "Martin Schlumberger",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11369",
"date": "27 Nov 2015",
"name": "Sebastiano Bonventre",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1366
|
https://f1000research.com/articles/2-99/v1
|
28 Mar 13
|
{
"type": "Research Article",
"title": "Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa",
"authors": [
"Érica L. Fonseca",
"Ana Carolina Paulo Vicente",
"Ana Carolina Paulo Vicente"
],
"abstract": "The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (Pc), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position impacts the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.",
"keywords": [
"DNA",
"mRNA",
"ribosomes"
],
"content": "Introduction\n\nClass 1 integrons are capable of inserting, excising and rearranging gene cassettes by a site-specific recombination mechanism. These assembly platforms can also act as expression systems due to the presence of a promoter region (Pc), which drives the expression of genes captured by integron1. Moreover, naturally occurring integrons may have a second promoter (P2), which is activated by the insertion of three G residues between -35 and -10 hexamers1. Gene cassettes are generally promoterless units associated with a recombination site (attC or 59-be), which confers the ability of each structure to be mobilized independently2. In addition, attC sites regulate the translation of downstream cassettes due to their peculiar sequences composed by imperfect inverted repeats. The formation of stem-loop structures by attC sites prevents ribosome progression throughout mRNA, reflecting in a decreased expression of more distal genes regarding Pc3. Although rare, fused cassettes may be generated by partial or total loss of the first attC, retaining both complete coding regions and, therefore, creating permanent gene arrays comparable to bacterial operons4. The functionality of such structures has been indirectly inferred by the resistance profile of transformants carrying the fusion5; however, the transcription itself has never been verified.\n\nThis study showed the dynamics of fused cassette mobilization, the co-transcription of the gcu14-bla GES-1/aacA4 cassette array and the effect of cassette position on transcription levels in Pseudomonas aeruginosa wild lineages carrying class 1 integrons. Moreover, the presence of translation signals in this gene cassette array was determined.\n\n\nMaterial and methods\n\nAn unknown Open Reading Frame (ORF), gcu14, followed by the fused cassette blaGES-1/aacA4, created by partial loss of GES-1 attC were present in integrons from clinical P. aeruginosa isolates (PS1 and PS26)6. Total RNA was extracted and purified according to the manufacturers instructions with the SV 96 Total RNA Isolation System (Promega). Northern blot using 7 μg of total RNA from PS1 and PS26 was performed in order to detect the transcript originated from gcu14-blaGES-1/aacA4 cassette array. After electrophoresis in a denaturing-formaldehyde 1.5% agarose gel, the total RNA was transferred to the Hybond-N+ nylon membrane (GE Healthcare) by upward capillary transfer. An amplicon of 519bp corresponding to part of the blaGES-1 gene was used as a probe (Table 1) in hybridization assay. The GES probe was labeled with the AlkPhos Direct Labelling kit (GE Healthcare) and hybridized with the target RNA immobilized on the Hybond-N+ membrane as recommended. The chemiluminescence was detected with the CDP-Star detection reagent (GE Healthcare) according to manufactures. Immediately after applying the detection reagents, the blot was drained, incubated five minutes at room temperature and exposed to the Hyperfilm ECL (GE Healthcare) for 60 minutes at room temperature.\n\na Gcu RSQ – AACA4 FSQ and GES RSQ – AACA4 FSQ were used in combination to obtain the full length and the blaGES-1/aacA4 fused cassette circular forms, respectively.\n\nIn order to verify whether the relative position of gene cassettes on the variable region plays a role in transcription level, real-time RT-PCR reactions using the TaqMan System (Applied Biosystems) were performed with primers and probes detailed in Table 1. The single-copy ribosomal rpsL gene of the P. aeruginosa chromosome was amplified by PCR (Table 1) and used as a reference gene for normalization. The relative quantification (RQ) results were presented as ratios of gene transcription between the target gene (cassettes) and the reference gene (rpsL), which were obtained according to the following equation: RQ=2-ΔCT, where CT is the value corresponding to the crossing point of the amplification curve with the threshold and ΔCT=CT target gene minus CT reference gene. The effect of cassette position on gene transcription was considered significant when the ratios obtained between RQ values (RQ value of cassette 1/RQ value of cassette 2) were ≥2.0, taking into account the standard deviation intervals.\n\nIn order to induce cassette excision from integrons, PS1 and PS26 strains6 were submitted to thermal stress during the log growth phase to induce integrase activity. Cells were grown on Luria-Bertani (LB) broth medium (OXOID) at 37ºC for two hours. subsequently, the bacterial cultures were submitted to a heat shock at 4ºC for 30 minutes and immediately incubated at 42ºC for another 30 minutes. Briefly, the total DNA from PS1 and PS26 cultured under thermal stress were obtained with the Wizard Genomic DNA purification kit (Promega) following manufacturer recommendations and used as templates in inverse PCR reactions. The inverse PCR was performed with primers facing outwards towards the ends of gcu14, blaGES-1 and aacA4 so that only circular gene cassette configurations would be amplified. The reactions target the circular forms of gcu14, blaGES-1, aacA4 and blaGES-1/aacA4 fusion by using the primers and combinations described in Table 1. The inverse PCR was performed using Platinum Taq DNA Polymerase reagents (Invitrogen), and the following components were added to a sterile 0.2-mL tube: 5 µL of 10X PCR buffer (1X final concentration); 1 µL of 10mM dNTP mixture (0.2 mM each); 1.5 µL of 50mM MgCl2 (1.5 mM final concentration); 2 µL of 15 µM of each primer (30 µM each); 100 ng of template DNA; 0.3 µL of Platinum Taq DNA Polymerase (1U final concentration). The tubes were incubated in the Eppendorf MasterCycler (Eppendorf) at 94°C for 2 minutes and PCR amplification was performed in 40 cycles consisting of: 94°C for 30 seconds; 55 °C for 30 seconds; and 72°C for 3 minutes. The amplicons generated with the inverse PCR were purified using Wizard SV Gel and PCR Clean-Up system kit (Promega) and directly sequenced on both strands. Sequencing reactions were performed with Big Dye Terminator RR Mix (Applied Biosystems) in an ABI 3730 XL DNA Analyzer (Applied Biosystems). Nucleotide sequences were compared to those available in the GenBank database accessible on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). All primers used in PCR, sequencing and real time RT-PCR are described in Table 1.\n\nAnalyses in silico were performed to search for a potential promoter for gcu14 gene cassette. The 5’UTR from gcu14 were submitted to the promoter predictor programs Neural Network for Promoter Prediction version 2.2 (Berkeley Drosophila Genome Project, http://www.fruitfly.org/index.html) and BPROM (SoftBerry, http://linux1.softberry.com/berry.phtml). Results with the highest scores were selected as candidates for a putative promoter.\n\n\nResults and discussion\n\nThe integrons analyzed in this study harbored a rare weak Pc configuration (PcWTGN-10), which presents a C to G mutation 2 bp upstream of the −10 hexamer that causes an abrupt decrease in promoter strength as reported previously7. Considering that transcription initiates from the Pc promoter placed upstream the gcu14-blaGES-1/aacA4 cassette array, both monocistronic and full length polycistronic transcripts could be identified. In fact, Northern blot and hybridization assays revealed a unique signal of approximately 2,300 bases which corresponds to the co-transcription of the entire array (gcu14-blaGES-1/aacA4 ) (Figure 1). This result is in agreement with previous work in which the occurrence of transcripts containing more than one gene cassette was observed by Northern blot analysis1. Moreover, this finding gives support to the lack of attC function in terminating transcription of downstream gene cassettes as demonstrated previously3.\n\nThe full length transcript (2,300 bases), corresponding to the entire gene cassette array, hybridized with the GES probe (arrow). The fragment sizes of the RNA marker (Promega) used in RNA electrophoresis are indicated.\n\nThis fusion retained both entire coding regions and suffered partial loss of 91 bp at the GES-1 attC site (DQ236170)6. Previous studies demonstrated that the attC region flanked by Left Hand (LH) and Right Hand (RH) domains, which is an imperfect inverted repeat and is missing in the blaGES-1 recombination site, is crucial for cassette mobilization8. Taking into account that the region responsible for stem-loop formation was missing in GES-1 attC and the participation of this site in terminating translation3, our findings indirectly suggested that blaGES-1 and aacA4 translation is occurring in a unique step.\n\nGene cassettes can be found inserted in integrons or in other secondary sites or free in the cytoplasm as a closed circle, in which the 5’ end (5’ UTR) and the attC recombination site are covalently linked9. As demonstrated previously, several stress conditions could evoke the activation of the SOS response resulting in integron-integrase expression10. Therefore, under stress, the integrase activity increases, favoring the occurrence of integration/excision/rearrangements events. Since the excision event depends on the recognition of the LH and RH domains of the attC site, and that these regions are missing in GES-1 attC, it is expected that the blaGES-1/aacA4 excision occurs only at the aacA4 attC site, and that this structure is excised together as a unique cassette.\n\nPositive results were obtained for the gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 circular forms, but not for blaGES-1 and aacA4 alone, showing that the GES-1 attC is not functional and that the fused gene cassette is excised as a unique cassette. Sequencing assessed the recombination point where excision occurred, confirming the occurrence of free circular forms. The lack of activity of a truncated attC had also been observed before when associated with aadA1011. However, Ramirez and colleagues12 showed that the integrase was able to recognize and mediate excision of a truncated site associated to aadA1, indicating that the genetic context of such truncated sites could influence their role in IntI1 recognition and mobilization.\n\nThe relative quantification performed by real time RT-PCR revealed that PS1 and PS26 presented very similar RQ values for gcu14-blaGES-1/aacA4 transcription (Figure 2). This result was expected since integrons from these two strains have the same backbone, including the Pc promoter, and are at the same genetic environment6.\n\nThe relative quantification values obtained by real time RT-PCR are indicated for each gene cassette and for the fusion blaGES-1/aacA4 when considered as a unique gene.\n\ngcu14, the first cassette in integrons with the weak PcWTGN-10 configuration, presented approximately two-fold higher transcription when compared to blaGES-1 and aacA4 separately or when the fused cassette blaGES-1/aacA4 was considered (Figure 2). The same RQ value obtained for blaGES-1, aacA4 and the fusion reveals that these two ORFs are transcribed as a unique gene. The lower transcript amount of blaGES-1/aacA4 compared to gcu14 lies on the distance between these gene cassettes and Pc, which is one of the determinants influencing cassette transcription1,10, and it shows the effect of cassette position on expression levels.\n\nA putative promoter for gcu14 (-35 TTGATG [17 bp] -10 TGTTAC) was found 45 bp upstream from its start codon. Therefore, the increased gcu14 transcription could have resulted from a synergistic effect of this putative promoter with Pc. Considering that this putative promoter was found at attI1, which is highly conserved among class 1 integrons, it can be suggested that such a promoter is able to drive transcription of any cassette placed in the first integron position. Moreover, the ORF-11, which enhances the translation efficiency of downstream TIR (translation initiation region)-deficient cassettes inserted in integrons13, was found at the attI1 region preceding the TIR-deficient gcu14 gene cassette. This ORF contained its own Shine-Dalgarno (SD) sequence placed 8 bp upstream of the ATG codon. The ribosome at the ORF-11 stop codon could therefore be carried along the mRNA by lateral diffusion, reinitiating translation at the gcu14 start codon. A potential SD sequence was identified 10 bp upstream of the fused cassette blaGES-1/aacA4. In addition, the loss of the GES-1 attC region, which is involved in stem-loop formation, may enhance the chances of aacA4 translation, since this attC no longer constitutes a physical barrier to ribosome progression3. This deletion also then brings the second gene (aacA4) closer to Pc14. Together, these findings create a scenario for the occurrence of gcu14-blaGES-1/aacA4 expression in PS1 and PS26, which then provides a possible explanation for their resistance profile to β-lactams and aminoglycosides that has been observed elsewhere6.\n\n\nConclusions\n\nFused cassettes have been found in class 1 integrons5,11,12,15–18 ; however, the transcription of such structures has never been addressed. This work showed the transcription pattern of a fused cassette as a polycistronic mRNA and that these unusual structures are excised as a unique cassette.\n\nThe sequence of the cassette array composed by the fusion has been deposited in the GenBank database under accession number DQ236170.",
"appendix": "Author contributions\n\nELF and ACPV conceived the study and designed the experiments. ELF carried out the research and prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\nNo relevant competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the CNPq and FAPERJ fellowship and Oswaldo Cruz Institute Grant.\n\n\nAcknowledgements\n\nWe thank those involved in PDTIS platform to enable us to do the sequencing and real-time relative quantification analysis work.\n\n\nReferences\n\nCollis CM, Hall RM: Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother. 1995; 39: 155–162.\n\nCollis CM, Hall RM: Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase. J Bacteriol. 1992; 174: 1574–1585.\n\nJacquier H, Zaoui C, Sanson-le Pors MJ, et al:Translation regulation of integrons gene cassette expression by the attC sites. Mol Microbiol. 2009; 72: 1475–1486.\n\nRecchia GD, Hall RM: Gene cassettes: a new class of mobile element. Microbiology. 1995; 141: 3015–3027.\n\nMendes RE, Toleman MA, Ribeiro J, et al:Integron carrying a novel metallo-β-lactamase gene, blaIMP-16, and a fused form of aminoglycoside-resistant gene aac(6')-30/aac(6')-Ib': report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother. 2004; 48: 4693–4702.\n\nda Fonseca EL, Vieira VV, Cipriano R, et al:Emergence of blaGES-5 in clinical colistin-only-sensitive (COS) Pseudomonas aeruginosa strain in Brazil. J Antimicrob Chemother. 2007; 59: 576–577.\n\nJové T, Da Re S, Denis F, et al:Inverse correlation between promoter strength and excision activity in class 1 integrons. PLoS Genet. 2010; 6: e1000793.\n\nStokes HW, O'Gorman DB, Recchia GD, et al:Structure and function of 59-base element recombination sites associated with mobile gene cassettes. Mol Microbiol. 1997; 26: 731–745.\n\nRecchia GD, Hall RM: Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the incQ plasmid RSF1010. Mol Microbiol. 1995; 15: 179–187.\n\nHocquet D, Llanes C, Thouverez M, et al:Evidence for induction of integron-based antibiotic resistance by the SOS response in a clinical setting. PLOS Pathog. 2012; 8: e1002778.\n\nPartridge SR, Collis CM, Hall RM: Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151. Antimicrob Agents Chemother. 2002; 46: 2400–2408.\n\nRamirez MS, Parenteau TR, Centron D, et al:Functional characterization of Tn1331gene cassettes. J Antimicrob Chemother. 2008; 62: 669–673.\n\nHanau-Berçot B, Podglajen I, Casin I, et al:An intrinsic control element for translational initiation in class 1 integrons. Mol Microbiol. 2002; 44: 119–130.\n\nPartridge SR, Tsafnat G, Coeira E, et al:Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol Rev. 2009; 33: 757–784.\n\nPoirel L, Le Thomas I, Naas T, et al:Biochemical sequence analyses of GES-1, a novel class A extended-spectrum β-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob Agents Chemother. 2000; 44: 622–632.\n\nCéntron D, Roy PH: Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion. Antimicrob Agents Chemother. 2002; 46: 1402–1409.\n\nDubois V, Poirel L, Marie C, et al:Molecular characterization of a novel class 1 integron containing bla(GES-1) and a fused product of aac3-Ib/aac6'-Ib' gene cassettes in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2002; 46: 638–645.\n\nWalsh TR, Toleman MA, Hryniewicz W, et al:Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from SENTRY antimicrobial surveillance program. J Antimicrob Chemother. 2003; 52: 116–119.\n\n\n\n\n"
}
|
[
{
"id": "920",
"date": "02 May 2013",
"name": "Sally Partridge",
"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 looks at expression of genes in a gene cassette array that includes a fused cassette and at the excision of cassette from this array. Polycistronic transcripts from cassette arrays and the excision of other fused cassettes have previously been noted by others, as cited in this paper. The sequences in Appendix 1 need to be annotated properly, rather than just the results of searches given, and the point illustrated by each sequence needs to be explained to make it possible to assess whether they support the conclusions drawn in the paper. Some reorganisation (moving some information in the Results and Discussion to the Introduction) would help to make the paper easier to follow and the Conclusions section is very short and could be expanded. The English could also be improved and the manuscript checked for typos etc. A few specific points also need correcting or clarifying:Abstract Line 5- fused cassettes do not always have fused orfs (e.g. aacA1/orfG)Abstract 6th Line from end and Results and Discussion p. 5 1st Line of 2nd paragraph – what is in the gcu14-aacA4 circular form? Should this be blaGES-1/aacA4? Materials and Methods Line 1 – this needs rewording to explain that gcu is a gene cassette of unknown function.Results and Discussion p. 4 – the fusion here is the type where part of the blaGES-14 attC site is replaced by part of the attI1 site (see ref. 14). The LH and RH domains are part of the attC site, rather than flanking it. The 5′ UTR contains 6 bp of the attC site. Results and Discussion p. 5 – most of the 1L core site and the 1R core site of the blaGES attC site are present. Results and Discussion p. 6 – the deletion of part of the attC site doesn’t bring the aacA4 gene much closer to Pc and ref. 14 makes slightly different point (that expression of the downstream cassette may be enhanced).Formatting “14” of gcu14 and “A4” of aacA4 should be in italics.Transposon, gene and species names etc are not correctly formatted in the references.",
"responses": [
{
"c_id": "1499",
"date": "07 Sep 2015",
"name": "Erica Fonseca",
"role": "Author Response",
"response": "The sequences in Appendix 1 need to be annotated properly, rather than just the results of searches given, and the point illustrated by each sequence needs to be explained to make it possible to assess whether they support the conclusions drawn in the paper.Response: The sequence resulted from inverse PCR product, showing the circular form of the gcu14-blaGES-1-aacA4 array, was submitted to GenBank under accession number KT336477. This information was included in the text. Moreover, we provided a new figure showing a schematic representation of primer targeting sites used in inverse PCR in order to make easier for the reviewers comprehend our strategy and results. Some reorganisation (moving some information in the Results and Discussion to the Introduction) would help to make the paper easier to follow and the Conclusions section is very short and could be expanded. The English could also be improved and the manuscript checked for typos etc.Response: We agree that some points from results and discussion section would be more adequate in the introduction section, and they were moved in the new version of the manuscript (Page 3, lines 59-60 and 65-70; page 4, lines 71-76 in the new version).The English was reviewed by a native spoken-English, and the conclusion was expanded. A few specific points also need correcting or clarifying: Abstract Line 5- fused cassettes do not always have fused orfs (e.g. aacA1/orfG)Response: We agree and it was modified: We affirmed that only in some cases fused orfs can be created. Abstract 6th Line from end and Results and Discussion p. 5 1st Line of 2nd paragraph – what is in the gcu14-aacA4 circular form? Should this be blaGES-1/aacA4?Response: The presence of the gcu14-aacA4 circular form indicates that this strain harbours a second integron composed by the gcu14-aacA4 arrangement. However, this result does not invalidate our main conclusion, which is that the truncated GES-1 attC site is not functional and this gene is only mobilized when recombination occurs in aacA4 attC. A brief explanation was included in the text (Results section). Materials and Methods Line 1 – this needs rewording to explain that gcu is a gene cassette of unknown function.Response: It was included in the text. Results and Discussion p. 4 – the fusion here is the type where part of the blaGES-1 attC site is replaced by part of the attI1 site (see ref. 14). The LH and RH domains are part of the attC site, rather than flanking it. The 5′ UTR contains 6 bp of the attC site.Response: We change the text emphasizing that the attC was replaced by part of attI1. The referee is right; LH and RH domains are part of the attC site, rather than flanking it. This general idea concerning attC site and its domains was modified throughout the text. Results and Discussion p. 5 – most of the 1L core site and the 1R core site of the blaGES attC site are present.Response: In fact, only the 1L core site is present in the truncated attC. We included a figure (figure 2) in this new version showing this. Results and Discussion p. 6 – the deletion of part of the attC site doesn’t bring the aacA4 gene much closer to Pc and ref. 14 makes slightly different point (that expression of the downstream cassette may be enhanced).Response: The referee is right. This statement was removed. Formatting “14” of gcu14 and “A4” of aacA4 should be in italics. Transposon, gene and species names etc are not correctly formatted in the references.Response: All formatting errors were properly corrected."
}
]
},
{
"id": "961",
"date": "21 May 2013",
"name": "Béatrice Berçot",
"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 work is conducted to address the transcription of the fused gene cassette blaGES-1/aacA4. This arrangement is rare but not exceptional in a class 1 integron. In this work, the experimentation confirmed that the three gene cassettes harbored in this class 1 integron were expressed together from a unique polycistronic transcript.It seems that the authors have observed a circular form containing the gcu14 gene cassette associated with the aacA4 gene cassette. Could they explain this arrangement? Is it possible that the Pseudomonas isolates harbored another class 1 integron containing the gcu14 gene followed by the aacA4 gene?The authors have to provide in the paper the classical length of the blaGES-1 attC site, which is 110bp and contained the 1L, 2L, 2R and 1R sequences-shown in bold. TTGACGCCC GTCTAAC AATTC GTTCAAGC CGACGTTGCTTCGTGGCGGCGCTTGCGTGCTACGCTAAGCTTCGCACGCCGCTTGCCACTGCGCACCGCG GCTTAAC TCAGGC GTTAGNCIn the Pseudomonas SP26 and PS1 isolates, the blaGES- 1 attC site is reduced to the 6 bp of 1 L box.TTGACGCCC GTCTAA AACAAAGTTAGGCATCACAAAGTACAGCATC GTGSo, the distance from of the stop of blaGES-1 to the start codon of the aacA4 gene is 46bp. The translational start codon of aacA4 gene is erroneous in the Genbank data base (DC236170). Indeed, it has been determined by N-terminal amino acid sequencing to be a GTG (in italic in the sequence above) and the beginning of the cassette was 24 bp before [Hanau-Bercot B. et al., 2002].-Could the Genbank annotation be modified as explained above?-The Appendex is not necessary and should be deleted.-In the sentence page 4, line 13, “The single copy of rpsL gene of Pseudomonas...”, should be replaced by “the rpsL gene of Pseudomonas (see Bodilis et al., 2012)-In the conclusion, page 6, change “never” to “rarely”.",
"responses": [
{
"c_id": "1498",
"date": "07 Sep 2015",
"name": "Erica Fonseca",
"role": "Author Response",
"response": "It seems that the authors have observed a circular form containing the gcu14 gene cassette associated with the aacA4 gene cassette. Could they explain this arrangement? Is it possible that the Pseudomonas isolates harbored another class 1 integron containing the gcu14 gene followed by the aacA4 gene?Response: Yes, the presence of the gcu14-aacA4 circular form indicates that this strain harbours a second integron composed by the gcu14-aacA4 arrangement. However, this result does not invalidate our main conclusion, which is that the truncated GES-1 attC site is not functional and this gene is only mobilized when recombination occurs in aacA4 attC. A brief explanation was included in the text (Results section). The authors have to provide in the paper the classical length of the blaGES-1 attC site, which is 110bp and contained the 1L, 2L, 2R and 1R sequences-shown in bold.Response: A figure showing the truncated and the complete canonical attC from GES1 was included in the new version. So, the distance from of the stop of blaGES-1 to the start codon of the aacA4 gene is 46bp. The translational start codon of aacA4 gene is erroneous in the Genbank data base (DC236170). Indeed, it has been determined by N-terminal amino acid sequencing to be a GTG (in italic in the sequence above) and the beginning of the cassette was 24 bp before [Hanau-Bercot B. et al., 2002].-Could the Genbank annotation be modified as explained above?Response: We performed this modification and the updated sequence regarding the beginning of aacA4 gene is already accessible on GenBank database under the same accession number (DQ236170). -The Appendex is not necessary and should be deleted.Response: The appendix is only for helping referees in their evaluation. It will not be published. -In the sentence page 4, line 13, “The single copy of rpsL gene of Pseudomonas...”, should be replaced by “the rpsL gene of Pseudomonas (see Bodilis et al., 2012).Response: this was modified in the new version of the manuscript. In the conclusion, page 6, change “never” to “rarely”.Response: this was modified in the new version of the manuscript."
}
]
}
] | 1
|
https://f1000research.com/articles/2-99
|
https://f1000research.com/articles/4-1360/v1
|
26 Nov 15
|
{
"type": "Research Article",
"title": "Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples",
"authors": [
"Cristina E. Stanciu",
"M. Katherine Philpott",
"Ye Jin Kwon",
"Eduardo E. Bustamante",
"Christopher J. Ehrhardt",
"Cristina E. Stanciu",
"M. Katherine Philpott",
"Ye Jin Kwon",
"Eduardo E. Bustamante"
],
"abstract": "The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface – one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100%) of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x103 and ~1x105) could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence.",
"keywords": [
"forensic science",
"flow cytometry",
"extracellular DNA",
"epidermal cell",
"touch DNA"
],
"content": "Introduction\n\n‘Touch’ or trace DNA samples represent a significant portion of evidence submitted to forensic caseworking laboratories. Understanding the mechanisms of DNA transfer through touch and developing methods to maximize the level of DNA recovery from contact surfaces is a continuing priority for the forensic science community1. Historically, the quantity of DNA found in a contact sample was thought to be primarily based on the number of cells that people shed naturally from the outermost layer of skin2. This concept continues to be perpetuated in the forensic community and analysts still testify to this effect3–5.\n\nHowever, recent studies have shown that touch samples can also contain ‘cell-free’ or extracellular nucleic acids (referred to as CNAs, eDNA, or cfDNA; in contrast to intracellular DNA or iDNA) that could be derived from a variety of sources such as sweat and oil secretions6–10. Additionally, it has been suggested that small amounts of saliva can be transferred through touch which may be a source of both cell-free DNA and intracellular DNA (via nucleated buccal cells) to a contact sample11.\n\nAlthough there are many possible sources of genetic material in touch evidence, the proportion of cellular and extracellular components is currently unclear. A recent survey of casework samples reported that more than 70% of contact samples contained extracellular DNA, which often provided an added value to the short tandem repeat (STR) profile generated from the pelleted cellular material6. The study also found that the relative proportion of extracellular DNA to the total amount of DNA in each sample varied considerably.\n\nIn addition to understanding their relative contributions to contact samples, the forensic community would also benefit from determining whether different factors affect the deposition and persistence of epidermal cells and extracellular DNA on touched surfaces. Addressing these issues has important implications for optimizing DNA collection techniques as well as developing alternative analytical strategies for processing caseworking samples (e.g., 12).\n\nTherefore, the goal of this study was to investigate the relative contributions of extracellular and intracellular DNA and their relationship to the quantity of cells recovered from touch samples under controlled conditions, and assess how the transfer and recovery of each type of biological material may be influenced by particular actions of the individual contributor. To accomplish this, we used flow cytometry for precise and non-destructive measurements of touch samples that were simultaneously processed using standard caseworking techniques for DNA analyses.\n\n\nMethods\n\nFor initial imaging studies, two individuals were asked to hold a sterile conical tube (P/N: 229421; Celltreat Scientific) in one hand for five minutes. Samples were collected from the tube surface with one sterile, pre-wetted swab (P/N: 22037924; Fisher Scientific) followed by one dry swab. To elute the cells into solution, the swabs were manually stirred then vortexed for 15 seconds in 1 mL of Sterile DNAse-Free, Protease-Free Water (P/N: BP24701; Fisher Scientific). All procedures for participant solicitation and consent for human subject research were approved by the VCU-Institutional Review Board (ID# HM20000454_CR).\n\nFor experiments involving comparisons of cell and DNA yields between washed and unwashed hands, two sets of two samples (one tube in each hand) were collected from eight individuals using the protocol described above: the first set of 16 was collected before hand washing, and the second after washing hands with soap and water for 20 seconds and air drying. A 20μL aliquot of each 1mL cell solution was used in subsequent flow studies (including cell enumeration), and the remaining 980μL was used for DNA studies.\n\nAnother 20 samples were collected without any control for hand washing from these eight donors, along with three additional donors, using the protocol described above. The entirety of each of these samples was processed for DNA.\n\nIn order to separate intact cells from debris and cellular fragments for imaging purposes, after passing cell suspensions through a 100 µm mesh filter, Fluorescence-Activated Cell Sorting (FACS) was performed on the BD FACSAria™ Ilu (Becton Dickinson) flow cytometer using a 488 ηm Coherent solid-state laser. Channel voltages were set as follows: FSC, 50V; SSC, 200V. Events falling into gate “K” (see Figure 1) were sorted into a new tube, then imaged using the Amnis® Imagestream X MK II Software (EMD Millipore) by activating the Bright Field channel. Pictures were analyzed and exported with the IDEAS® Software v6.1 (EMD Millipore).\n\nCell events fall into two distinct populations along the Forward Scatter (FSC) and Side Scatter (SSC) axes: intact cells (‘K’) and cell debris (‘D’). Right insets show images of individual events within the K population. Scale bar=7 µm.\n\nCell suspensions were passed through a 100 µm mesh filter prior to flow cytometry analysis on the BD FACSCanto™ II analyzer (Becton Dickinson) using 488 ηm and 633 ηm lasers. The channel voltages were set as follows: FSC, 150V; SSC, 200V; FITC, 335V; PE, 233V; PE-Cy5, 300V; PE-Cy7, 400V; and APC, 250V. Data acquisition was performed using the FACSDIVA Software v8.0.1 (Becton Dickinson) and analyzed using FCS Express 4.0 (DeNovo).\n\nIn order to precisely quantify the cells in our samples during flow analysis, we spiked our cell solutions with a known concentration of 123 eBeads (01-1234-42; Affymetrix eBioscience), fluorescently-labeled microparticle standards that are 7 µm and easily distinguishable from our target cell population both in size and fluorescence (FITC, PE, and APC channels). The ratio of cells to beads was then used to determine the concentration of cells in the sample through the following formula:\n\nAbsolute Count(cells/μL)=(Cell Count×eBead Volume)(eBead Concentration)¯(eBead Count×Cell Volume)\n\nThe concentration of cells in the 20µL aliquot was then used to estimate the total number of cells present in the entire volume of eluent (1mL) recovered from the collection swabs. Flow cytometry analysis of bead standards was conducted on events detected within the ‘K’ gate for each sample. The procedures for detecting and differentiating eBeads from target cells followed the manufacturer's suggested protocol (http://www.ebioscience.com/media/pdf/tds/01/01-1234.pdf).\n\nOnce an aliquot was removed for cell quantification, the remaining cell suspension was transferred to a new 2mL collection tube and centrifuged at 10,000 xg for five minutes at room temperature. The supernatant was added to a pre-washed Amicon filter (UFC210024; EMD Millipore). The remaining pellet was washed with 500 µL of sterile water twice, each time adding the supernatant to the Amicon filter. The combined supernatant fraction was then centrifuged at 3,220 ×g for 30 minutes, followed by a wash step in 2 mL 1xTE Buffer (P/N 50-843-203; Teknova). The DNA was collected by inverting the filter and centrifuging at 1,000 xg for 2 minutes at room temperature. The final volume of the eluted retentate was approximately 20 µl.\n\nAdditionally, in order to maximize the recovery of cell material and/or DNA, the wet swab tips were placed in a spin basket (P/N 19597; Investigator Lyse & Spin Basket Kit; Qiagen) immediately following the initial elution, and centrifuged at 10,000 ×g for 5 minutes at room temperature (absent any additional reagents). The resulting liquid eluent that passed through the spin basket was added to the supernatant solution (prior to Amicon filtration) and remaining cell pellet in the spin basket was dissolved in ~50 µl of sterile water, combined with its respective fraction, and subjected to DNA extraction using the following protocol. The cell pellet material was incubated with 500 µL Cell Lysis Buffer (P/N BDB559759; BD Pharmigen) and 10 µL Proteinase K (P/N EO0491; Fisher Scientific) in a 56°C water bath for 17 hours. The sample was then centrifuged at 10,000 ×g for 5 minutes at room temperature. The supernatant was purified with an equal volume of UltraPure Phenol:Chloroform:Isoamyl Alcohol (P/N 15593-031; Life Technologies (25:24:1, v/v)), then 1xTE Buffer, and finally concentrated to a final volume of 20–40 µL using a pre-washed Amicon filter.\n\nDNA quantitation was performed using the Investigator Quantiplex® Human Kit (P/N 387016, Qiagen) coupled with the ABI Prism 7500 Sequence Detection System (Applied Biosystems). A 25 µl reaction was used for all samples following manufacturer’s suggested protocol (‘Investigator Quantiplex Handbook’, www.qiagen.com).\n\n\nResults\n\nInitial characterizations of touch samples with flow cytometry showed two distinct size fractions (‘K’ and ‘D’ populations in Figure 1). Size and morphological information derived from AMNIS images of the K fraction from two touch samples revealed that this population was consistent with fully differentiated keratinocytes (i.e., corneocytes) ~20–40 µm in diameter, while the D fraction was consistent with cellular debris/fragments. Other epithelial cell types (e.g., buccal cells >60 µm) were not observed among the AMNIS images captured (Figure S1, Figure S2).\n\nCell counts and DNA yields were compared across 31 touch samples generated from eight different individuals that used both dominant and non-dominant hands to hold the substrate. To investigate the effect of hand washing on the transfer of cellular and extracellular components of a touch sample, half of these samples were collected after donors had washed their hands and the other half without immediate hand washing.\n\nAn estimated ~5×103 to ~1×105 cells were recovered from washed hand samples, versus ~1×103 to ~8×104 cells from unwashed hand samples (Figure 2; Table S1). Overall, we observed greater transfer of cells in the washed hand samples than the unwashed hand samples (median of 2.5×104 cells vs. 8.6×103 cells, respectively). Despite the often high recovery of cells from touch samples, DNA recovery from the cell pellet was consistently low, whether from washed or unwashed hands. DNA was detected in the cell pellet of one unwashed hand sample (0.220 ng) and three washed hands samples (0.049, 0.042, 0.060 ng). No DNA was detected in any of the other cell pellets.\n\nFor each graph, the Y axis represents the number of “K events” (cells) detected in solution from collection swabs (unwashed hands in a and b; washed hands in c and d), while the X axis represents the number of nanograms of DNA recovered (from supernatant (a) and cell pellet (b) of unwashed hands, and from supernatant (c) and cell pellet (d) of washed hands).\n\nIn contrast, consistent differences were observed in eDNA recovery from samples generated from washed versus unwashed hands. Little to no DNA was recovered from the extracellular fraction of touch samples left by donors who had washed their hands, with quantitation values ranging from zero to 0.242 ng (Figure 2c). In samples from unwashed hands, extracellular DNA recovery varied between zero and 4.646 ng (Figure 2a). There was no apparent correlation between the number of cells and the quantity of DNA recovered from the samples (either eDNA or cell pellet). Neither could DNA recovery with or without hand washing be correlated to hand dominance, in contrast to findings by others13.\n\nThe additional 20 samples tested for relative quantity of eDNA versus intracellular DNA produced results that are consistent with the above findings (Table 1, compilation of all samples without hand washing (n = 35)). In samples where DNA was detected, the total proportion of eDNA ranged from 84–100% with the majority of the samples at or near 100%.\n\nND=below the limit of detection, ~1 pg/µl. Samples refer to individual donors. Each row within a single donor shows results from replicate experiments performed on different days.\n\n\nDiscussion\n\nOur results contribute to the forensic community’s growing body of knowledge on touch samples. We found that the vast majority (~84–100%) of nuclear DNA recovered from touch samples collected under the conditions described above is extracellular. Amplifiable DNA from the pelleted cellular fraction was detected in only eight of the 51 touch samples analyzed (Figure 2, Table 1).\n\nAlthough this finding is generally consistent with other recent studies suggesting the significance of extracellular DNA in touch evidence6,8, the prevalence and proportion of extracellular DNA relative to the total DNA yield shown in Table 1 was higher than observed in other studies6. It is possible that the multiple wash steps performed on the pelleted cell material for this study removed more eDNA than efforts utilizing a single wash. In a separate analysis of seven replicate samples, we found that additional eDNA was often recovered with additional wash steps, and concurrently, that a clear systematic cell loss at each wash step was not observed—a Student’s t-test on cell counts before and after three wash steps yielded an average p-value of 0.28 with only two of the individual replicates yielding p-values less than 0.01 (Table S2). This suggests that while some cells may have been unintentionally removed from some cell pellets by our methodology, this phenomenon is unlikely to explain the consistent increased DNA recovery in the supernatant with additional washes across samples.\n\nThe nature of the samples likely played a role as well, as there may have been more opportunities to pick up nucleated cells for some casework samples described in other research6 than our controlled conditions. The fact that the “typical” or “standard” touch sample evades definition poses a challenge when designing studies to better understand these kinds of samples. It has been suggested that saliva, which contains buccal cells, may be an important (i.e., DNA rich) component of some touch samples11. We observed no evidence of such cells – which generally appear larger than corneocytes (>60 µm for buccal cells versus 20–40 µm for corneocytes) – in microscopic surveys of individual cells within two touch samples (Figure S1, Figure S2). However, this does not preclude the possibility that non epidermal cells were present, since only a portion of the sample was surveyed, and because deformed or fragmented cells from different tissues may be indistinguishable from corneocytes. Future work could explicitly test for the presence of buccal cells in touch samples through, e.g., antibody hybridizations targeting tissue specific surface antigens coupled with flow cytometry.\n\nThe mechanism of touching could also affect the proportion of eDNA to iDNA in touch samples; our preliminary data from touch samples deposited by rubbing suggest that this action may result in considerably higher cell pellet yields than samples deposited by holding, perhaps by exposing deeper (i.e., undifferentiated) layers of cells. However, in these preliminary experiments we also observed that the amount of eDNA left by rubbing the substrate was similar to levels of eDNA left by holding. This suggests that the transfer of eDNA may not be as affected by the manner in which a substrate was handled as iDNA transfer.\n\nIn any case, our results lend further support to the concept that extracellular DNA is particularly crucial to the analysis of touch samples. Measures should be explored to exploit this source of information to the greatest extent possible. For sample collection and processing purposes, this may dictate that touch samples be treated differently than other types of forensic biological sample. To avoid the significant loss of DNA that may be associated with extraction, it may make sense to process the eDNA-containing supernatant separately via direct amplification; our results suggest that care should be taken to maximize the amount of eDNA washed into the supernatant.\n\nOur finding that the number of cells in touch samples was uncorrelated to the amount of extracellular DNA or the total DNA yield suggests that not only is the recoverable DNA primarily extracellular but that it is not immediately derived from the large numbers of epidermal cells that are shed daily. DNA was not detected in the cell pellet of samples that contained more than 100,000 cells, while samples comprised of far fewer cells (~2000) yielded DNA. Our extraction methodology likely had some impact on overall DNA yield14; we have found in other experiments that other extraction methodologies (e.g., DNA IQ) resulted in low (<80pg) but quantifiable DNA yields in samples that yielded no DNA after processing with the extraction method utilized here. However, this does not change the fact that a considerable portion of DNA from the touch samples that we analyzed was extracellular, and that the number of cells shed was not a reliable indicator of DNA yield. These results are compatible with previous medical research showing that corneocytes from the outermost epidermal layer (i.e., stratum corneum) have little to no genomic DNA owing to the controlled degradation of intracellular components during differentiation15.\n\nAccordingly, epidermal cells – even when present in large quantities – may make a fairly insignificant contribution to either intra- or extracellular DNA recovery from touch samples. Consistent with recent studies that found no evidence of fragmented DNA in the epidermal layers (in contrast to sebaceous cell sources)10, the majority of extracellular DNA in touch samples is likely derived from alternate sources such as oil and sweat secretions, or saliva8,11. Where intracellular (i.e., cell pellet) DNA levels from touch samples are considerably higher than those observed in this study, a nucleated cell source (i.e., non-epidermal, or more basal epidermal) may be implicated, though certain skin conditions are known to result in the aberrant retention of nuclear DNA in corneocytes15.\n\nAlthough hand washing resulted in the transfer and subsequent recovery of little to no eDNA, we found that cells were nonetheless transferred. In fact, we observed greater levels of cellular transfer among washed hand samples than unwashed hand samples. It is possible that the act of hand washing loosens or sloughs off corneocytes, and that these cells (perhaps because of their flattened morphology) are more likely to persist through the washing process than eDNA. Regardless of the explanation, an estimated thousands to hundreds of thousands of cells survived the hand washing process to be transferred from the palmar surface by simple touching.\n\nConsistent with Locard’s principle, while these shed corneocytes may not contain sufficient levels of nuclear DNA to generate a probative STR profile, there is the possibility that other, non-genetic signatures could be analyzed, so that the most challenging touch samples (i.e. those that contain little to no DNA) may provide forensically relevant information. For example, the average size of individual corneocytes has been shown to vary with source factors such as age, sex, and anatomical region16–18, as does the composition of intracellular cytokeratin components19. While further research is of course necessary to assess the degree of inter- and intra-individual variance in particular cellular features, determining such source attributes from unknown contributors could potentially provide leads or exclude suspects in specific types of investigations, e.g., sexual assault, molestation. Further, the absence of amplifiable nuclear DNA in corneocytes does not necessarily preclude the presence of sufficient levels of mitochondrial DNA to permit typing. Combining techniques to sort epidermal cells into donor populations (e.g., using factors described above) and typing the mtDNA of those populations is an avenue that warrants further exploration.\n\nOverall, our observations suggest that many traditional explanations of DNA analysis from touch samples used in expert testimony – which often seek to explain the quantity and quality of DNA detected (or lack thereof) in terms of an individual’s inherent or circumstantial susceptibility to shed epidermal cells – may need to be modified to reflect fundamental shifts in the forensic community’s understanding of touch evidence. Future research efforts should continue to examine the relationship between the transfer of eDNA, iDNA, and intact corneocytes onto touch surfaces by testing other types of depositional circumstances, e.g., different substrate material or touch samples from multiple donors.",
"appendix": "Author contributions\n\n\n\nCE, CS, and KP conceived the study. CE, CS, EB, and YK designed the experiments. CS, EB, YK carried out the research. KP assisted with data analysis and provided expertise in the area of forensic DNA casework. CE, CS, and KP prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis project was funded by the National Institute of Justice Award number 2013-DN-BX-K033 (PI: Ehrhardt). Flow cytometry services in support of the project were provided by the VCU Massey Cancer Center, supported in part with funding from NIH-NCI P30CA016059.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors gratefully acknowledge Daniel Conrad, Julie Farnsworth, Qingzhao Zhang, and Tracey Dawson Cruz for providing technical assistance for this project.\n\n\nSupplementary materials\n\nFigure S1. Supplemental imaging results for individual events within the large cell fraction of touch samples. All cells were collected from the ‘K’ population shown in Figure 1. Scale bar=7 µm.\n\nClick here to access the data.\n\nFigure S2. Supplemental imaging results for individual events within the small cell fraction of touch samples. All cells were collected from the ‘D’ population shown in Figure 1. Scale bar=7 µm.\n\nClick here to access the data.\n\nTable S1. Source data showing DNA yield and cell counts for all analyzed touch samples. DNA quantitation and cell enumeration was performed as described in the Methods section. Unique sample names (e.g., ‘E14’) correspond to individual donors. ‘N/A’ indicates that cell counts were not performed on that sample. ‘ND’ indicates that DNA concentration was below the limit of detection, ~1pg/µl.\n\nClick here to access the data.\n\nTable S2. Source data showing cell counts from touch samples before and after three water wash steps. Cell enumeration was performed using fluorescently labeled microparticle standards (See Methods). Unique sample names (e.g., ‘E14’) correspond to individual donors. Replicate experiments using the same donors are designated by 'exp1' and 'exp2'.\n\nClick here to access the data.\n\n\nReferences\n\nMeakin G, Jamieson A: DNA transfer: review and implications for casework. Forensic Sci Int Genet. 2013; 7(4): 434–43. PubMed Abstract | Publisher Full Text\n\nAditya S, Bhattacharryya CN, Chaudhuri K: Generating STR profile from \"Touch DNA\". J Forensic Leg Med. 2011; 18(7): 295–8. PubMed Abstract | Publisher Full Text\n\nMinor J: Touch DNA: From the Crime Scene to the Crime Laboratory. Forensic Magazine. 2013. Reference Source\n\nPalmer R: Boston Marathon Crime Scene: How Forensic Techniques Might Point Toward the Perpetrator. International Business Times. 2013. Reference Source\n\nWilliamson AL: Touch DNA: Forensic Collection and Application to Investigations. The Association for Crime Scene Reconstruction. 2012. Reference Source\n\nVandewoestyne M, Van Hoofstat D, Franssen A, et al.: Presence and potential of cell free DNA in different types of forensic samples. Forensic Sci Int Genet. 2013; 7(2): 316–20. PubMed Abstract | Publisher Full Text\n\nKita T, Yamaguchi H, Yokoyama M, et al.: Morphological study of fragmented DNA on touched objects. Forensic Sci Int Genet. 2008; 3(1): 32–6. PubMed Abstract | Publisher Full Text\n\nQuinones I, Daniel B: Cell free DNA as a component of forensic evidence recovered from touched surfaces. Forensic Sci Int Genet. 2012; 6(1): 26–30. PubMed Abstract | Publisher Full Text\n\nLinacre A, Pekarek V, Swaran YC, et al.: Generation of DNA profiles from fabrics without DNA extraction. Forensic Sci Int Genet. 2010; 4(2): 137–41. PubMed Abstract | Publisher Full Text\n\nZoppis S, Muciaccia B, D'Alessio A, et al.: DNA fingerprinting secondary transfer from different skin areas: Morphological and genetic studies. Forensic Sci Int Genet. 2014; 11: 137–43. PubMed Abstract | Publisher Full Text\n\nWarshauer DH, Marshall P, Kelley S, et al.: An evaluation of the transfer of saliva-derived DNA. Int J Legal Med. 2012; 126(6): 851–61. PubMed Abstract | Publisher Full Text\n\nDean L, Kwon YJ, Philpott MK, et al.: Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS). Forensic Sci Int Genet. 2015; 17: 8–16. PubMed Abstract | Publisher Full Text\n\nPhipps M, Petricevic S: The tendency of individuals to transfer DNA to handled items. Forensic Sci Int. 2007; 168(2–3): 162–8. PubMed Abstract | Publisher Full Text\n\nDoran AE, Foran DR: Assessment and mitigation of DNA loss utilizing centrifugal filtration devices. Forensic Sci Int Genet. 2014; 13: 187–90. PubMed Abstract | Publisher Full Text\n\nEckhart L, Fischer H, Tschachler E: Mechanisms and emerging functions of DNA degradation in the epidermis. Front Biosci (Landmark Ed). 2012; 17: 2461–75. PubMed Abstract | Publisher Full Text\n\nPlewig G, Marples RR: Regional differences of cell sizes in the human stratum corneum. I. J Invest Dermatol. 1970; 54(1): 13–8. PubMed Abstract\n\nPlewig G: Regional differences of cell sizes in the human stratum corneum. II. Effects of sex and age. J Invest Dermatol. 1970; 54(1): 19–23. PubMed Abstract\n\nLee SH, Lee S: Regional and sexual differences in corneocytes among young Korean adults. Yonsei Med J. 1986; 27(3): 213–8. PubMed Abstract | Publisher Full Text\n\nOender K, Trost A, Lanschuetzer C, et al.: Cytokeratin-related loss of cellular integrity is not a major driving force of human intrinsic skin aging. Mech Ageing Dev. 2008; 129(10): 563–71. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11361",
"date": "10 Dec 2015",
"name": "Ram Gopal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 ‘’Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples’’ by Stanciu CE et al. describes the genetic (nuclear DNA) and biological components (Cells and Cell debris) in samples created by touch or contact with a surface, both in controlled and non controlled situations. The question to understand and characterize the components of touch sample is of paramount importance in forensic science research. The manuscript attempts to decipher the mechanism of DNA transfer in touch samples and suggests methods to maximize the recovery of DNA from the touch samples. On the cellular components side, the FACS coupled with microscopy technique was employed to explore the number and identity of the cells in touch samples. The cells in touch samples were found to be consistent with the keratinocyte morphology and size. This work also shows that after washing hands there is tendency to shed more cells in touch samples but the increase in the cell number is not correlated with the increase in DNA yield, rather there was no or very little DNA recovered after washing hands. Thus, this study provides evidence that there is no or very little DNA associated with the cellular component of the touch samples. It makes a strong case for the next important question to ask. How we can differentiate keratinocytes from different individuals? As the authors suggests that other biological information still may be present on the keratinocytes, it opens up the possibilities of a new field for characterization of keratinocytes from touch samples. The manuscript’s conclusion that the source of majority of DNA in touch samples comes from extracellular components, not from the cellular components, is in well agreement of previous studies but the proportion of extracellular DNA is found to be higher in this study than the earlier reports. The manuscript is well written and presents the data in a logical way. Overall, the manuscript adds further knowledge to the body of knowledge existing in this field and I recommend this manuscript for indexation. However I have following minor suggestions/comments to improve the manuscript.No profiles, conventional or LCN, are shown to show the quality of DNA recovered. It will be interesting to explore the contributions (both of DNA as well as Cells components) made by normal flora of human skin. ‘’....flow cytometer using a 488 ηm Coherent solid-state laser.... ‘’ in this line and elsewhere the nanometer symbol should be written as ‘nm’.",
"responses": []
},
{
"id": "12080",
"date": "25 Jan 2016",
"name": "Antonio Filippini",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 “Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples” by Stanciu CE et al. is focused on the “Touch DNA” topic and is aimed at investigating the relative contributions of extracellular and intracellular DNA and their relationship to the quantity of cells recovered from touch samples under controlled conditions. It also assesses how the transfer and recovery of each type of biological material may be influenced by particular actions of the individual contributor.To this aim, the Authors used flow cytometry for precise and non-destructive measurements of touch samples that were simultaneously processed using standard caseworking techniques for DNA analyses. The well described analytical methods and results show that there was no apparent correlation between the number of cells and the quantity of DNA recovered from the samples, neither could DNA recovery with or without hand washing be correlated to hand dominance.According to recent studies, they found that the vast majority (~84–100%) of nuclear DNA recovered from touch samples collected under the conditions described above is extracellular (i.e. oil and sweat secretions, saliva), and suggest that future work could explicitly test for the presence of buccal cells in touch samples through, e.g., antibody hybridizations targeting tissue specific surface antigens coupled with flow cytometry.They also highlighted the peculiarities, and consequently the analytical challenges, of touch samples, thus suggesting these samples to be treated differently than other types of forensic biological sample as for sample collection and processing purposes; e.g. to avoid the significant loss of DNA that may be associated with extraction, it may make sense to process the eDNA-containing supernatant separately via direct amplification.An interesting and innovative element represented in the manuscript is the possibility that non-genetic signatures could be analyzed, so that the most challenging touch samples (i.e. those that contain little to no DNA) may provide forensically relevant information. To this regard, it is reported that “… the average size of individual corneocytes has been shown to vary with source factors such as age, sex, and anatomical region, as does the composition of intracellular cytokeratin components … combining techniques to sort epidermal cells into donor populations (e.g., using factors described above) and typing the mtDNA of those populations is an avenue that warrants further exploration …”. Finally, as an interested researcher in the field of “Touch DNA”, I appreciate the efforts of the Authors in preparing this interesting manuscript, that is well written and follows a logical structure. I just have a minor suggestion to improve the manuscript, that is to include some STR profiles in order to show the quality of the DNA recovered from the analyzed samples.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1360
|
https://f1000research.com/articles/4-1359/v1
|
26 Nov 15
|
{
"type": "Review",
"title": "Transversus abdominal plane (TAP) block for postoperative pain management: a review",
"authors": [
"Jan G. Jakobsson",
"Liselott Wickerts",
"Sune Forsberg",
"Gustaf Ledin",
"Liselott Wickerts",
"Sune Forsberg",
"Gustaf Ledin"
],
"abstract": "Transversus abdominal plane (TAP) block has a long history and there is currently extensive clinical experience around TAP blocks. The aim of this review is to provide a summary of the present evidence on the effects of TAP block and to provide suggestions for further studies.There are several approaches to performing abdominal wall blocks, with the rapid implementation of ultrasound-guided technique facilitating a major difference in TAP block performance. During surgery, an abdominal wall block may also be applied by the surgeon from inside the abdominal cavity.Today, there are more than 11 meta-analyses providing a compiled evidence base around the effects of TAP block. These analyses include different procedures, different techniques of TAP block administration and, importantly, they compare the TAP block with a variety of alternative analgesic regimes.The effects of TAP block during laparoscopic cholecystectomy seem to be equivalent to local infiltration analgesia and also seem to be beneficial during laparoscopic colon resection. The effects of TAP are more pronounced when it is provided prior to surgery and these effects are local anaesthesia dose-dependent. TAP block seems an interesting alternative in patients with, for example, severe obesity where epidural or spinal anaesthesia/analgesia is technically difficult and/or poses a risk. There is an obvious need for further high-quality studies comparing TAP block prior to surgery with local infiltration analgesia, single-shot spinal analgesia, and epidural analgesia. These studies should be procedure-specific and the effects should be evaluated, both regarding short-term pain and analgesic requirement and also including the effects on postoperative nausea and vomiting, recovery of bowel function, ambulation, discharge, and protracted recovery outcomes (assessed by e.g., postoperative quality of recovery scale).",
"keywords": [
"TAP-block",
"Transversus abdominal-plane block",
"ultra-sound",
"pain managment"
],
"content": "Introduction\n\nThere is an increasing interest in abdominal wall block. The ultrasound-guided technique has improved the performance and success rate. The effects, advantages and potential disadvantages as compared to alternative pain management are, however, not obvious. There are several alternatives and the best technique is not clear. The ultrasound guidance has made this block more attractive. There are today several videos describing anatomy and how to perform the TAP block effectively (https://www.youtube.com/watch?v=9TIHDn7uBZI, https://www.youtube.com/watch?v=CzMDdrPbLEM). Not introducing the needle too close to the probe is important in order to visualise the needle reaching the layer between the internal oblique and transverse abdominal muscle (https://www.youtube.com/watch?v=6E3ynIn6Ud4).\n\nOur aim in this paper is to present the benefits and merits of TAP block in adults. It will provide the readers a review of techniques and outcomes from available studies. There are more than 11 meta-analyses available compiling studies assessing TAP blocks. This paper will provide summaries of these meta-analyses and suggest best practice. It will also suggest areas where there is a need for further high-quality studies.\n\n\nWhat we know now: meta-analyses and systematic reviews\n\nThere are 11 published meta-analyses around the effects of TAP block. The most recent was published in September 20151 and the first was a Cochrane review by Charlton et al. published in 20102. They assess the effects of various abdominal blocks, most provided by ultrasound-guided technique. The studies are, however, hard to compare as different approaches, local anaesthetic concentrations, and volumes have been used. The abdominal block has been compared to “placebo” or to other anaesthetic techniques, for example, local infiltration analgesia or intrathecal anaesthesia. The most recent meta-analysis by Baeriswyl et al. was published in the September 2015 issue of Anaesthesia and Analgesia1. It included 31 controlled trials and 611 adult patients in all. Its primary focus was on the opioid-sparing effects, and the cumulative morphine consumption at 6 hours postoperatively, and its secondary objectives were 24-hour morphine consumption, pain ratings, and postoperative nausea and vomiting. It showed that the ultrasound-guided TAP block was associated with a reduced IV morphine consumption at 6 hours postoperatively by a mean difference of 6 mg, independent of the type of surgical anaesthesia (general anaesthesia, spinal anaesthesia with or without intrathecal long-acting opioid). The beneficial effect of cumulative morphine consumption was also seen at 24 hours (mean 11 mg). Pain ratings were reduced at 6 hours postoperatively but no effect was seen in the incidence of postoperative nausea and/or pruritus, either at rest or during movement. The authors conclude, “Ultrasound-guided TAP block provides marginal postoperative analgesic efficacy after abdominal laparotomy or laparoscopy and cesarean delivery. However, it does not provide additional analgesic effect in patients who also received spinal anesthesia containing a long-acting opioid”. Thus the result of this most recent review is in line with the ones previously published.\n\nCharlton et al. published a Cochrane systematic review in 2010 assessing the effect of TAP block for pain relief after abdominal surgery2. They included 8 prospective randomised studies. A clear opioid-sparing effect was found as compared to placebo or “no block”. Compared with no TAP block or saline placebo, TAP block resulted in significantly less postoperative requirement for morphine at 24 hours (mean difference -21.95 mg) and 48 hours (cumulative difference -28.50 mg). No effect was found on nausea and vomiting. The authors requested further studies comparing TAP block with alternative local anaesthesia techniques, for example, local infiltration and single-shot intrathecal anaesthesia.\n\nSiddiqui et al. published a second meta-analysis in 2011 around the efficacy of the TAP block3. Four studies were included; laparoscopic cholecystectomy, caesarean section with Pfannenstiel incisions, total abdominal hysterectomy, and large bowel resection midline incision. It was found that patients that were randomised to active TAP block had a significantly lower cumulative morphine need during the first 24 hours post surgery (P<0.001), a significantly longer time until they needed rescue morphine (P<0.001), as well as less pain up to 24 hours post surgery. No significant effects from the TAP block were noticed in postoperative nausea and vomiting. The most profound TAP block effects were noticed for the caesarean section and colon surgery.\n\nMishriky et al. published a third meta-analysis in 2012, looking at analgesia after caesarean section. Nine studies were included4. They found that TAP block significantly reduced opioid consumption (mg morphine equivalents) after caesarean section. The mean difference in opioid need was -10.18, -13.83, and -20.23 mg at 6, 12, and 24 hours respectively. TAP block also reduced pain during the first 12 hours and reduced nausea among the patients who did not have intrathecal morphine. The combination of TAP block and spinal morphine was associated with a small reduction in pain during movement in the first 6 hours after surgery. Intrathecal morphine was, however, more effective; it was associated with a lower pain score and opioid consumption at 24 hours after surgery. The intrathecal morphine group also had a longer time before the first rescue analgesia request. The intrathecal morphine caused more morphine-related side effects.\n\nJohns et al. conducted a fourth meta-analysis published in 2012 looking at the analgesic effects of TAP block after abdominal surgery5. In all, 9 studies representing both published and unpublished results were analysed including 413 patients; 205 that had a TAP block and 208 control patients. TAP block was found to be safe and effective and was associated with a significantly lower morphine need both 24 and 48 hours after surgery, -23.71 mg (P<0.002) and -38.08 mg (P<0.0001) respectively, and also a lower incidence of postoperative nausea and vomiting. Pain scores did not differ significantly.\n\nAbdallah et al. published a meta-analysis in 2012 assessing TAP block for postoperative analgesia after caesarean delivery performed under spinal anaesthesia6. They analysed the results for 5 studies including 312 patients. TAP block was found to reduce the mean first 24-hour post surgery cumulative morphine need by 24 mg when intrathecal morphine had not been used. TAP block also lowered pain scores (0.8/10) and morphine related adverse effects. The effects of TAP block were not significantly different from intrathecal morphine. It was concluded that TAP block can reduce morphine need during the first 24 hours after surgery when intrathecal morphine is not used.\n\nAbdallah et al. published another meta-analysis in 2013 focusing on the difference between two TAP block approaches (the posterior and the lateral) and their effect on the duration of pain relief after lower abdominal surgery incision7. In all, 12 randomised studies were included in the analysis (641 patients); 4 studies with a posterior TAP technique and 8 with a lateral technique. They found the posterior approach was associated with a significantly lower morphine need both 12 to 24 hours and 24 to 48 hours after surgery; a mean difference of 9.1 mg (P<0.02) and 5 mg (P<0.03), respectively. The posterior TAP block also had significant effects on pain, reducing pain scores at rest and during movement at 24, 36, and 48 hours after surgery. The lateral TAP was not associated with any significant differences.\n\nDe Oliviera et al. published a 7th meta-analysis assessing TAP block analgesic effects after laparoscopic surgery8. They included 10 randomised studies covering 633 patients. They found TAP block to lower pain at rest -2.41/10 during the first 4 hours postoperatively and -1.33/10 at 24 hours post surgery. TAP also reduced IV morphine need (weighed mean -5.74 mg morphine equivalents). It was also found that TAP block administered preoperatively was more effective on pain, and reduced postoperative morphine consumption when compared with blocks placed postoperatively. No local anaesthesia toxicity was reported.\n\nZhao et al. published an 8th meta-analysis assessing TAP block for postoperative analgesia after laparoscopic surgery9. In all, 14 studies with a total of 905 patients were included in this analysis. TAP block resulted in significantly less postoperative analgesic consumption at 24 hours (mean difference = -25.46, P<0.00001), and less patients requiring analgesic postoperatively (P=0.03). TAP block reduced pain; pain scores were significantly different at 2 hours (mean difference = -1.55, P<0.00001). A borderline difference between the active TAP block and control was seen at 6 hours (mean difference = -1.13, P=0.05). TAP block had no effect on pain at 24 hours. TAP block was associated with significantly more postoperative nausea and vomiting (odds ratio 2.04, P=0.34). The authors concluded TAP reduced 24-hour analgesic requirements, had minor effects on early pain, and may increase the risk of postoperative nausea and vomiting.\n\nYu et al. published the 9th meta-analysis in December 2014 assessing TAP block as compared to local wound infiltration analgesia in patients undergoing lower abdominal surgery10. In all, 4 randomised studies were included in this analysis; 96 patients having a TAP block and 100 patients having local infiltration analgesia. The TAP block reduced pain, pain scores were lower both at rest and during movement as compared to local infiltration analgesia at 24 hours postoperatively; weighed mean difference -0.67 (P<0.01) and -0.89 (P<0.01) respectively. Postoperative 24-hour morphine need, incidence of postoperative nausea and vomiting, and pain assessed by the visual analogue scale score at 2 and 4 hours did not differ between the TAP and local infiltration analgesia groups of patients.\n\nRipollés et al. published an update and summary of the existing evidence in early 2015 and indeed supports TAP block’s beneficial effects11. The analysis was based on prospective randomised studies published between 2007 and 2013 in English or Spanish with a Jadad score of >1. Studies in adult patients including ultrasound-guided blocks compared to other analgesic techniques were assessed. In all, 28 randomised clinical trials were included in the analysis. There was a huge heterogeneity in study design. The studies used different TAP techniques, local anaesthetic concentrations as well as volumes, and also comparators differed. Most studies compared the TAP block against placebo but there were also studies comparing the TAP block against epidural analgesia, local infiltration, and ileoinguinal-ileohipogastric block. Outcomes studied were opioid consumption, and pain at rest or during movement. However, the results were not entirely congruent, although most studies did see some beneficial effects. These authors did, however, conclude that TAP block is an effective technique for reducing opioid use postoperatively following colorectal surgery, caesarean section, cholecystectomy, hysterectomy, appendectomy, donor nephrectomy, retropubic prostatectomy, and bariatric surgery. They did see obvious gaps: the data found in available randomised clinical trials was not considered fully conclusive. These authors suggest that there is a need to develop new and well-designed randomised clinical trials, with enough statistical power to compare different approaches, drugs, doses, and volumes for the same intervention, aiming to answer the current questions and assess the effect of TAP-block effects in routine clinical practice.\n\n\nDiscussion\n\nThere has been an increasing interest in the transversal abdominal plane block during the last decade. It seems reasonable to conclude that TAP block is a safe technique; no significant side effects have been reported (Table 1). The block provides an opioid-sparing effect, but the effects on opioid-related side effects, postoperative nausea and vomiting, and bowel function are not fully consistent. The effects on early postoperative pain and reduced opioid consumption 24–48 hours after surgery are seemingly similar to single-shot spinal anaesthetic with intrathecal morphine and more or less equal to local infiltration analgesia.\n\nThere are obvious factors to consider before the implementation of TAP block in routine clinical practice; patient- and surgery-related factors, alternatives, and the technique to be used (see Table 2). The introduction of the ultrasound-guided block technique has made the TAP block an interesting option as part of multimodal postoperative pain management, partly because of its technical simplicity. The ultrasound technique has made the TAP block easier to perform but it is at present not possible to provide any firm data showing a higher efficacy for the ultrasound-guided techniques. Ultrasound-guided bilateral TAP block is commonly performed with a high-frequency linear ultrasound probe and an in-plane needle guidance technique12. The TAP block provides effective analgesia with opioid-sparing effects. Disadvantages include the need for a bilateral block for midline incisions and the absence of effectiveness for visceral pain13. The effect of the block is dependent on the technique used and patient anatomy. Støving et al. studied the effect of TAP block in healthy volunteers. They found huge inter-individual variability in objective sensory block and duration of effect14. The TAP block is a volume block and is performed by injecting local anaesthetic solutions in the transverse abdominis plane without specific reference to the nerves responsible for the innervations of the abdomen wall. It is not surprising that in these conditions the quality of a TAP block is dependent on the approach and the volume administered. There are more specific approaches: blocks specifically blocking the involved innervation of the abdomen include ilioinguinal, iliohypogastric, and/or intercostal blocks that can be used alone and/or in combination depending on the surgical incision. Early reduced opioid consumption is a consistent finding associated with the TAP block. These effects are seemingly most pronounced in transverse incisions, when the block is provided prior to surgery, and there is also a dose effect. Kokulu et al. showed that TAP block provided preoperatively was associated to a reduction of desflurane need and thus was cost effective during elective cholecystectomy15. It is somewhat surprising that the effects on postoperative nausea and vomiting are not entirely conclusive: one meta-analysis even suggested there was no benefit and another a potential increase in postoperative nausea and vomiting in the TAP groups.\n\nThe long-term effects require further studies. Keller et al. found, studying 200 consecutive patients who underwent a laparoscopic colorectal resection, that adding TAP blocks to an enhanced recovery pathway facilitated shorter length of hospital stay with lower readmission and reoperation rates, when compared to previously published series. This suggested TAP blocks might be an efficient, cost-effective method for improving laparoscopic colorectal surgery results16. Similar positive experiences were reported by Favuzza et al.17. Both these studies compared a TAP block performed at the end of surgery with the laparoscope still in place, actually visualising the muscle layers of the structures from the “inside”, thus not performed by ultrasound technique but by the surgeon. The surgeon performed the block from the outside, passing the needle through the skin, mid-axillary, approximately half the distance between the iliac crest and the costal margin. The traditional two pops technique, passing two fascia borders, was used and the injection of 30 ml of 2.5 mg/ml bupivacaine was injected under surveillance of the laparoscope, imaging the spread at the place for the transversus abdominis muscle. It should be acknowledged that these studies were not randomised or blinded.\n\nThe analgesic effect has a duration related to the local anaesthetic administered. The analgesic duration has been shown to be longer when dexamethasone is added to local anaesthesia18. There are also positive results from the use of liposomal bupivacaine: the total opioid use in the first 72 hours after injection was significantly decreased in the group that received liposomal bupivacaine compared to non-liposomal bupivacaine. Patients in the liposomal bupivacaine group had significantly lower maximal pain scores at all time periods studied, as well as a decreased incidence of nausea/vomiting. There was a trend toward decreased length of stay in the liposomal bupivacaine group19. The addition of sufentanil to bupivacaine did not provide longer or more effective effects as compared to bupivacaine alone for pain management following laparoscopic cholecystectomy20.\n\nToday, there are several meta-analyses around the effects of TAP block, but still, the exact place for TAP block requires further studies. The last meta-analysis by Ripollés et al. concludes that the data found in randomised clinical trials are not conclusive and, as a result, it is necessary to develop new and well-designed randomised clinical trials, with enough statistical power to compare different approaches, drugs, doses, and volumes for the same intervention, aiming to answer the current questions and monitoring their effects in routine clinical practice. The TAP block as part of a multimodal analgesic strategy, as compared to local wound infiltration analgesia, and spinal/intrathecal analgesia (IT morphine), not only improved morphine consumption during the first 24 to 48 hours but also quality of recovery assessed in a broader and more protracted/long-term time perspective, for example, assessed by postoperative quality of recovery scale21. It seems it would also be of value to conduct studies comparing the TAP block to paravertebral block. Chelly et al. have shown that the paravertebral block was effective for pain management following open radical retropubic prostatectomy22. There is also a need to better explore the place for TAP block in paediatric perioperative care23.\n\n\nConclusion\n\nIn conclusion, TAP block is a safe and interesting block that may be provided by ultrasound-guided technique or intraoperatively by the surgeon, providing postoperative analgesia, and a reduced need for morphine analgesia during the first 24 to 48 hours following abdominal procedures. TAP block administered prior to surgery reduces not only postoperative opioid requirements but also intraoperative anaesthetic needs. There is, however, a need for further high-quality studies assessing the effects of TAP block as part of multimodal analgesia, and as compared to local infiltration analgesia and intrathecal morphine, assessed in a more protracted time perspective of quality of recovery. Studies performed should also be procedure specific.\n\n\nAbbreviations\n\nTAP block, transversus abdominal plane block.",
"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\nBaeriswyl M, Kirkham KR, Kern C, et al.: The Analgesic Efficacy of Ultrasound-Guided Transversus Abdominis Plane Block in Adult Patients: A Meta-Analysis. Anesth Analg. 2015; 121(6): 1640–1654. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCharlton S, Cyna AM, Middleton P, et al.: Perioperative transversus abdominis plane (TAP) blocks for analgesia after abdominal surgery. Cochrane Database Syst Rev. 2010; (12): CD007705. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSiddiqui MR, Sajid MS, Uncles DR, et al.: A meta-analysis on the clinical effectiveness of transversus abdominis plane block. J Clin Anesth. 2011; 23(1): 7–14. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMishriky BM, George RB, Habib AS: Transversus abdominis plane block for analgesia after Cesarean delivery: a systematic review and meta-analysis. Can J Anaesth. 2012; 59(8): 766–78. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nJohns N, O'Neill S, Ventham NT, et al.: Clinical effectiveness of transversus abdominis plane (TAP) block in abdominal surgery: a systematic review and meta-analysis. Colorectal Dis. 2012; 14(10): e635–42. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAbdallah FW, Halpern SH, Margarido CB: Transversus abdominis plane block for postoperative analgesia after Caesarean delivery performed under spinal anaesthesia? A systematic review and meta-analysis. Br J Anaesth. 2012; 109(5): 679–87. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAbdallah FW, Laffey JG, Halpern SH, et al.: Duration of analgesic effectiveness after the posterior and lateral transversus abdominis plane block techniques for transverse lower abdominal incisions: a meta-analysis. Br J Anaesth. 2013; 111(5): 721–35. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nDe Oliveira GS Jr, Castro-Alves LJ, Nader A, et al.: Transversus abdominis plane block to ameliorate postoperative pain outcomes after laparoscopic surgery: a meta-analysis of randomized controlled trials. Anesth Analg. 2014; 118(2): 454–63. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nZhao X, Tong Y, Ren H, et al.: Transversus abdominis plane block for postoperative analgesia after laparoscopic surgery: a systematic review and meta-analysis. Int J Clin Exp Med. 2014; 7(9): 2966–75. PubMed Abstract | Free Full Text | F1000 Recommendation\n\nYu N, Long X, Lujan-Hernandez JR, et al.: Transversus abdominis-plane block versus local anesthetic wound infiltration in lower abdominal surgery: a systematic review and meta-analysis of randomized controlled trials. BMC Anesthesiol. 2014; 14: 121. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRipollés J, Mezquita SM, Abad A, et al.: Analgesic efficacy of the ultrasound-guided blockade of the transversus abdominis plane - a systematic review. Braz J Anesthesiol. 2015; 65(4): 255–80. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEl-Dawlatly AA, Turkistani A, Kettner SC, et al.: Ultrasound-guided transversus abdominis plane block: description of a new technique and comparison with conventional systemic analgesia during laparoscopic cholecystectomy. Br J Anaesth. 2009; 102(6): 763–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLissauer J, Mancuso K, Merritt C, et al.: Evolution of the transversus abdominis plane block and its role in postoperative analgesia. Best Pract Res Clin Anaesthesiol. 2014; 28(2): 117–26. PubMed Abstract | Publisher Full Text\n\nStøving K, Rothe C, Rosenstock CV, et al.: Cutaneous Sensory Block Area, Muscle-Relaxing Effect, and Block Duration of the Transversus Abdominis Plane Block: A Randomized, Blinded, and Placebo-Controlled Study in Healthy Volunteers. Reg Anesth Pain Med. 2015; 40(4): 355–62. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nKokulu S, Bakı ED, Kaçar E, et al.: Effect of transversus abdominis plane block on cost of laparoscopic cholecystectomy anesthesia. Med Sci Monit. 2014; 20: 2783–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nKeller DS, Ermlich BO, Delaney CP: Demonstrating the benefits of transversus abdominis plane blocks on patient outcomes in laparoscopic colorectal surgery: review of 200 consecutive cases. J Am Coll Surg. 2014; 219(6): 1143–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFavuzza J, Delaney CP: Outcomes of discharge after elective laparoscopic colorectal surgery with transversus abdominis plane blocks and enhanced recovery pathway. J Am Coll Surg. 2013; 217(3): 503–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nAkkaya A, Yildiz I, Tekelioglu UY, et al.: Dexamethasone added to levobupivacaine in ultrasound-guided tranversus abdominis plain block increased the duration of postoperative analgesia after caesarean section: a randomized, double blind, controlled trial. Eur Rev Med Pharmacol Sci. 2014; 18(5): 717–22. PubMed Abstract | F1000 Recommendation\n\nHutchins J, Delaney D, Vogel RI, et al.: Ultrasound guided subcostal transversus abdominis plane (TAP) infiltration with liposomal bupivacaine for patients undergoing robotic assisted hysterectomy: A prospective randomized controlled study. Gynecol Oncol. 2015; 138(3): 609–13. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSaliminia A, Azimaraghi O, Babayipour S, et al.: Efficacy of transverse abdominis plane block in reduction of postoperation pain in laparoscopic cholecystectomy. Acta Anaesthesiol Taiwan. 2015; pii: S1875-4597(15)00059-4. PubMed Abstract | Publisher Full Text | F1000 Recommendation\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\nChelly JE, Ploskanych T, Dai F, et al.: Multimodal analgesic approach incorporating paravertebral blocks for open radical retropubic prostatectomy: a randomized double-blind placebo-controlled study. Can J Anaesth. 2011; 58(4): 371–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMai CL, Young MJ, Quraishi SA: Clinical implications of the transversus abdominis plane block in pediatric anesthesia. Paediatr Anaesth. 2012; 22(9): 831–40. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11356",
"date": "26 Nov 2015",
"name": "Anne M Lynn",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11357",
"date": "26 Nov 2015",
"name": "Jacques Chelly",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1359
|
https://f1000research.com/articles/4-1356/v1
|
26 Nov 15
|
{
"type": "Review",
"title": "Varicella Zoster Virus in the Nervous System",
"authors": [
"Don Gilden",
"Maria Nagel",
"Randall Cohrs",
"Ravi Mahalingam",
"Nicholas Baird",
"Maria Nagel",
"Randall Cohrs",
"Ravi Mahalingam",
"Nicholas Baird"
],
"abstract": "Varicella zoster virus (VZV) is a ubiquitous, exclusively human alphaherpesvirus. Primary infection usually results in varicella (chickenpox), after which VZV becomes latent in ganglionic neurons along the entire neuraxis. As VZV-specific cell-mediated immunity declines in elderly and immunocompromised individuals, VZV reactivates and causes herpes zoster (shingles), frequently complicated by postherpetic neuralgia. VZV reactivation also produces multiple serious neurological and ocular diseases, such as cranial nerve palsies, meningoencephalitis, myelopathy, and VZV vasculopathy, including giant cell arteritis, with or without associated rash. Herein, we review the clinical, laboratory, imaging, and pathological features of neurological complications of VZV reactivation as well as diagnostic tests to verify VZV infection of the nervous system. Updates on the physical state of VZV DNA and viral gene expression in latently infected ganglia, neuronal, and primate models to study varicella pathogenesis and immunity are presented along with innovations in the immunization of elderly individuals to prevent VZV reactivation.",
"keywords": [
"Varicella zoster virus",
"VZV",
"Latency",
"Vasculopathy",
"Giant Cell Arteritis",
"Immunization"
],
"content": "Introduction\n\nVaricella zoster virus (VZV) is an exclusively human alphaherpesvirus. Primary infection causes varicella (chickenpox), after which the virus becomes latent in neurons of cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis. In elderly and immunocompromised individuals, a decline in VZV-specific cell-mediated immunity results in VZV reactivation, typically manifesting as herpes zoster (shingles) in a dermatomal distribution. The annual medical care cost of treating approximately one million cases of zoster in the US is estimated at $1.1 billion, most of which is used to treat immunocompetent adults who are at least 50 years old1. Since the risk of zoster increases with age and since the population that is at least 65 years old is expected to increase to 72 million in 2030, zoster and its attendant serious neurological complications will continue to be a significant health-care burden.\n\nZoster is often complicated by chronic pain (postherpetic neuralgia). VZV reactivation can also produce cranial nerve palsies, meningoencephalitis, VZV vasculopathy, myelopathy, and ocular disease with or without associated rash. Recently, the spectrum of VZV vasculopathy has expanded to include giant cell arteritis (GCA), which is the most common systemic vasculitis in the elderly and which frequently results in vision loss. Herein, we review the clinical, laboratory, imaging, and pathological features of neurological complications of VZV reactivation as well as diagnostic tests to verify VZV infection of the nervous system. We also discuss VZV latency, neuronal and primate models to study varicella pathogenesis and immunity, and immunization of elderly individuals to prevent VZV reactivation.\n\n\nNeurological complications of VZV reactivation\n\nMost cases of VZV reactivation manifest as zoster (dermatomal distribution pain and rash). Rash and pain usually occur within hours to days of each other, although pain may precede rash by weeks to months2. Since VZV is latent in ganglia along the entire neuraxis, zoster can develop anywhere on the body. Risk factors for the development of zoster include advanced age3 and immunosuppression, such as in individuals with AIDS or cancer, organ transplant recipients, and individuals who received immunosuppressive medications4. Zoster in an otherwise healthy young person may be the first manifestation of HIV infection5; zoster in children or adolescents is also seen in individuals who acquired primary VZV infection in utero or in the first year of life; these people are 20.9 times more likely to develop zoster before age 206.\n\nSince VZV is latent in all cranial nerve ganglia and in autonomic (ciliary and otic) ganglia in the head, multiple cranial nerve palsies can develop after zoster. Ophthalmoplegia from involvement of cranial nerve 3, 4, or 6 or any combination thereof is a well-documented complication. Another is weakness or paralysis of facial muscles on one side of the face, associated with vesicles in the ipsilateral external auditory canal (zoster oticus), or on the tympanic membrane, the ipsilateral anterior two thirds of the tongue, or hard palate. The combination of peripheral facial palsy and zoster oticus constitutes the Ramsay Hunt syndrome (RHS). Because the facial nerve is adjacent to the eighth cranial nerve in the facial canal, patients with RHS often have tinnitus, hearing loss, nausea, vomiting, vertigo, or nystagmus. Cranial neuropathies usually occur days to weeks after zoster. The temporal relationship most likely reflects the time needed for the virus to spread transaxonally along trigeminal and other ganglionic afferent fibers, thereafter replicating in small arteries with resultant micro-infarction of cranial nerves (as occurs in patients with diabetes) in the same manner that produces VZV vasculopathy in larger arteries. Importantly, cranial neuropathies produced by VZV can occur in the absence of rash, virologically verified by the detection of VZV DNA or anti-VZV IgG antibody in cerebrospinal fluid (CSF).\n\nZoster paresis is characterized by weakness in the arm or diaphragm after cervical distribution zoster or in the leg after lumbar or sacral distribution zoster. Thoracic zoster has been associated with abdominal muscle weakness and hernia. Pathological features of zoster include inflammation and hemorrhagic necrosis with associated neuritis, localized leptomeningitis, unilateral segmental poliomyelitis, and degeneration of related motor and sensory roots7. Demyelination may be seen in areas with mononuclear cell (MNC) infiltration and microglial proliferation. In acutely infected ganglia, intranuclear inclusions, viral antigen, and herpesvirus particles are present. Oral antiviral drugs speed healing of zoster rash and shorten the duration of acute pain. Immunocompromised patients and patients with ophthalmic-distribution zoster should receive intravenous acyclovir (10 to 15 mg/kg three times daily for 5 to 7 days).\n\n\nPostherpetic neuralgia\n\nPostherpetic neuralgia (PHN) is defined as pain persisting for more than 3 months after zoster. Age is the single most important predictor, and more than 40% of zoster patients who are more than 60 years old develop PHN. Pathological analysis of ganglia from an early case of PHN of 2.5 months’ duration revealed diffuse and focal infiltration by chronic inflammatory cells, a finding confirmed by the detection of prominent collections of lymphocytes in ganglia from a patient with PHN of 2 years’ duration8. A possible explanation is that chronic inflammation reflects prolonged viral infection, a notion supported by the detection of VZV DNA in blood MNCs of many patients with PHN (presumably by MNCs trafficking through ganglia productively infected with VZV) and from the favorable response of some patients with PHN to antiviral treatment9. Symptomatic treatment for PHN is challenging. Tricyclic antidepressants, gabapentin, and pregabalin are used as first-line therapies. Many patients with PHN also require topical capsaicin cream, lidocaine patches, capsaicin 8% patches, tramadol, or opioids or a combination of these to help alleviate debilitating pain. Nerve blocks and ablation, as well as nerve stimulators, have variable effectiveness. The potential role of antiviral medications as a treatment for PHN awaits analyses in larger clinical studies.\n\n\nCentral nervous system disease caused by VZV reactivation\n\nVZV meningitis, meningoencephalitis, meningoradiculitis, cerebellitis, myelopathy, and vasculopathy may develop after zoster. Importantly, all may develop in the absence of rash, as confirmed by the detection of VZV DNA or anti-VZV antibody (or both) in CSF. VZV myelitis commonly presents as frank invasion of the spinal cord by the virus. Disease is usually progressive and infrequently fatal. Magnetic resonance imaging reveals longitudinal serpiginous-enhancing lesions10. Early diagnosis and aggressive treatment with intravenous acyclovir are beneficial, even in immunocompromised patients11; VZV myelitis may recur, even in immunocompetent patients10. Aside from myelitis, VZV can produce spinal cord infarction from occlusion of spinal arteries12.\n\nVZV vasculopathy occurs following productive infection of cerebral arteries and pathological vascular remodeling. The most common manifestations are ischemic stroke, but hemorrhagic stroke, aneurysm, with and without subarachnoid and intracerebral hemorrhage, dolichoectasia, dissection, and venous sinus thrombosis may also be produced by VZV. Although the exact incidence of VZV vasculopathy is unknown, epidemiological studies from Taiwan, Denmark, and the UK have all revealed that the risk of stroke is increased after zoster, particularly when zoster is in the ophthalmic distribution of the trigeminal nerve13, and that antiviral therapy may reduce stroke risk. Retrospective analysis of 30 subjects with VZV vasculopathy, confirmed virologically, revealed that 63% had associated rash, 67% had a CSF pleocytosis, 97% had abnormalities on brain imaging, and 70% had abnormalities on angiographic studies14. Of the 30 subjects, 50% had both large and small artery involvement, 37% had exclusively small artery involvement, and 13% had exclusively large artery involvement. Because 93% of the subjects had intrathecal synthesis of anti-VZV antibodies and only 30% had VZV DNA in CSF, the detection of anti-VZV IgG antibody is the best test for diagnosis consistent with a prior study15. Importantly, since there was no zoster rash in one third of subjects, VZV DNA is absent in 70% of CSF, and there is a 4.2-month delay from onset of rash to neurological disease, thus the diagnosis of VZV vasculopathy is often missed.\n\nThe proposed mechanism for VZV vasculopathy involved virus reactivation from cranial nerve ganglia followed by transaxonal spread of the virus to the outermost adventitial layer of cerebral arteries whereupon productive virus infection induces pathological vascular remodeling through a direct effect or an indirect effect of virus-induced inflammation. Immunohistochemical studies of VZV-infected cerebral and temporal arteries from patients with VZV vasculopathy show that arteries contain a thickened intima composed of myofibroblasts, loss of medial smooth muscle cells, and a disrupted/duplicated internal elastic lamina16. Examination of the inflammatory infiltrate in VZV-infected arteries reveals CD4 and CD8 T cells, macrophages, and rare B cells predominantly located in the adventitia and to a lesser extent in the thickened intima17. In early VZV vasculopathy, neutrophils are present in the adventitia; no neutrophils were detected in late VZV. Consistent with studies of pathological remodeling in coronary and pulmonary arteries, the thickened intima was associated with inflammation in the underlying adventitia, supporting the notion that inflammatory cells secrete soluble factors that contribute to vascular remodeling. A recent study also revealed that VZV-infected brain vascular adventitial fibroblasts produce elevated levels of matrix metalloproteinases that degrade extracellular matrix and may contribute to migration of cells to the lumen and thrombosis as well as to aneurysm formation and hemorrhage18.\n\n\nAssociation of VZV with giant cell arteritis\n\nEvidence that VZV infection triggers the inflammatory cascade characteristic of GCA came from analysis of formalin-fixed, paraffin-embedded GCA-positive temporal artery (TA) biopsies and normal TA biopsies from subjects who are more than 50 years of age for the presence and distribution of VZV antigen. VZV antigen was found in 61 out of 82 (74%) GCA-positive TAs compared with 1 out of 13 (8%) normal TAs, and most GCA-positive TAs contained viral antigen in skip areas19. VZV antigen was present mostly in adventitia, followed by the media and intima (Figure 1). Hematoxylin-and-eosin staining revealed VZV antigen in 12 out of 32 (38%) skeletal muscles adjacent to VZV antigen-positive TAs. Despite formalin fixation, polymerase chain reaction (PCR) detected VZV DNA in 18 out of 45 (40%) GCA-positive VZV antigen-positive TAs, in 6 out of 10 (60%) VZV antigen-positive skeletal muscles, and in one VZV antigen-positive normal TA. Electron microscopy revealed VZV virions in a GCA-positive TA. GCA pathology in sections adjacent to those containing VZV was seen in 89% of GCA-positive TAs but in none of 18 adjacent sections from normal TA. Most GCA-positive TAs contained VZV in skip areas that correlated with adjacent GCA pathology, supporting the notion that VZV triggers GCA immunopathology.\n\nHematoxylin-and-eosin staining of the temporal artery from a 90-year-old man with classic giant cell arteritis (A). Note extensive inflammation in the adventitia, media and intima, disruption of the media, a thickened intima, and nearly occluded arterial lumen; inset denotes a giant cell. Immunohistochemical staining with mouse anti-VZV gE antibody revealed VZV antigen (red) in the adventitia and media of the temporal artery (B) that was not seen when mouse isotype IgG1 antibody was substituted for mouse anti-VZV gE antibody (C).\n\nThe association between GCA and TA infection by VZV was further analyzed by immunohistochemistry in archived TAs of patients with clinically suspected GCA whose TAs were negative histopathologically, and in normal TAs removed postmortem from age-matched subjects20. VZV antigen was detected in 45 out of 70 (64%) GCA-negative TAs compared with 11 out of 49 (22%) normal TAs, and extension of our earlier study revealed VZV antigen in 68 out of 93 (73%) GCA-positive TAs compared with 11 out of 49 (22%) normal TAs. VZV antigen was more likely to be present in adventitia of both GCA-negative TAs and GCA-positive TAs than in normal TA adventitia. In GCA-negative subjects whose TAs contained VZV antigen, adventitial inflammation was seen in 36% adjacent to viral antigen but not in any normal TAs. Overall, the prevalence of VZV in TAs of subjects with clinically suspected GCA was independent of whether they were GCA-negative or -positive pathologically.\n\n\nVZV latency\n\nDuring primary infection, VZV disseminates hematogenously and infects ganglionic neurons along the entire neuraxis. Neurons may also become infected by retrograde axonal transport of the virus from skin vesicles. In non-neuronal cells, VZV replicates and causes cell death, whereas VZV does not replicate in or kill neurons; rather, transcription of virtually all viral genes is silenced21. Serologic evidence shows that more than 95% of the world’s population is exposed to VZV22, and most autopsy studies have found latent VZV DNA in trigeminal ganglia from more than 87% of individuals23. Whereas latent VZV is innocuous, reactivation results in significant disease (see above). Thus, understanding molecular aspects of virus latency is necessary to design therapeutic interventions to inhibit virus reactivation.\n\nIn latently infected human trigeminal ganglia, one to five copies of VZV DNA are present in 1.5% of the approximately 27,000 neurons24. Whereas VZV double-stranded DNA is linear in infectious virus particles, VZV DNA is ‘endless’, most likely existing as circular episomes in the nucleus of latently infected neurons25. VZV encodes about 70 genes, all of which are transcribed during productive infection26, but in latently infected human ganglia, only VZV gene 63 RNA is found in the first 9 hours after death27 and fewer than 12 VZV genes are transcribed in the next 15 hours28,29. Regulation of VZV genes during latency, an active area of research, most likely involves post-translational modification of histone proteins that comprise nucleosomes bound to promoters of virus genes. Nucleosomes containing histone protein 3 (H3) post-transcriptionally modified by acetylation on lysine 9 (K9ac) are present on promoters of actively transcribed cell genes30. Analysis of VZV DNA in human trigeminal ganglia shows that H3K9ac-containing nucleosomes are also present on the promoters of actively transcribed virus genes (VZV genes 21 and 63) but not on virus genes that are transcriptionally silent (VZV genes 14 and 36)31, findings which suggest that latent VZV gene transcription is, in part, regulated epigenetically. Thus, novel therapies that inhibit specific histone modifications may reduce virus reactivation32. Although the initial steps in virus reactivation may involve histone modifications on the latent virus that increase the number of VZV genes transcribed27, the end result of virus reactivation is assembly and release of infectious virions, with extensive cell death and induction of major histocompatibility complex (MHC) class I and II protein with CD4 and CD8 T-cell recruitment33–35.\n\nBecause VZV is an exclusively human pathogen, studies of VZV latency have been limited to analysis of human ganglia obtained postmortem. However, molecular analysis of latently infected primate ganglia with simian varicella virus (SVV) has provided useful information about varicella latency, and recent in vitro studies of neurons experimentally infected with VZV offer promise to study mechanisms of VZV reactivation (see below). The immediate future of research on VZV latency will involve correlative analyses of latently infected human ganglia, primate ganglia latently infected with SVV, and in vitro models of neurons experimentally infected with VZV, which can be exploited to prevent serious neurological, ocular, and visceral diseases produced by VZV reactivation.\n\n\nEfforts to produce VZV latency and reactivation in animals\n\nAnimal models developed for VZV latency using rodents and primates do not fulfill the criteria for VZV latency, which include detection of VZV DNA exclusively in ganglia, detection of latent VZV only in neurons, restricted transcription of the virus genome, and ability to reactivate the virus. Inoculation of VZV into guinea pigs36, mice37, and rats38 resulted only in seroconversion. In intradermally inoculated guinea pigs, VZV nucleic acids and proteins were still found in enteric ganglia, stomach, ileum, and colon of guinea pigs 1 month later39. In non-human primates, experimental inoculation of VZV produced a humoral and cell-mediated immune response, but the virus did not become latent in ganglia40.\n\nHowever, clinical, virological, immunological, and pathological features of SVV infection in non-human primates closely parallel VZV infection in humans41,42, and SVV becomes latent exclusively in ganglionic neurons43,44. SVV-specific transcription (open reading frames 21, 62, 63, and 66) is limited in ganglia, and the virus can be reactivated by immunosuppression45. SVV antigens are present in macrophages and dendritic cells during reactivation46. Primate models of varicella pathogenesis have revealed that the virus enters ganglia hematogenously before the appearance of skin rash47, memory T cells disseminate SVV to lung and ganglia during primary infection48, and T-cell infiltration correlates with expression of CXCL10, a chemokine that recruits activated T cells and natural killer (NK) cells, in ganglia at the time of zoster49. Pro-inflammatory cytokines and chemokines and anti-inflammatory mediators are elevated at the time of zoster in immunosuppressed monkeys50. All T-cell subsets decreased during immunosuppression and peaked (except for CD8 T cells) 2 weeks before zoster51.\n\n\nNeuronal models for VZV latency\n\nIn latently infected human ganglia, the entire VZV genome is present with termini that are covalently linked (i.e., either circular or concatemeric (end-to-end))25, viral transcription is severely restricted27,29,52,53, and the virus can reactivate. An in vitro model of VZV latency that satisfies all three of these conditions has not yet been developed.\n\nNeuroblastoma cell lines, including SH-SY5Y, are malignant cell lines of neurogenic origin that can be induced to differentiate in vitro to a neuron-like cell54. However, differentiation into neurons is inefficient (only approximately 70% are differentiated as determined by phase-contrast microscopy) and infection of the remaining non-neuronal cells leads to productive infection.\n\nInduced pluripotent stem cells (iPSCs) are fibroblasts that have been de-differentiated to a stem cell-like state and then induced to differentiate into neurons. Infection of iPSCs with a low dose of VZV results in a slowly spreading productive infection that produces viral DNA, transcripts, protein, and infectious virus55,56. A cytopathic effect does not develop until about 4 weeks after infection57, whereas similarly infected fibroblasts die in 7 to 10 days. Although this is promising, additional studies are necessary to suppress viral DNA replication, transcription, and virion production to mimic VZV latency in vivo.\n\nTwo other human stem cells have also been used to study the VZV-neuron relationship in vitro: neural stem cells (hNSCs) and embryonic stem cells (hESCs). At 9 weeks’ gestation, hNSCs were purified from fetal brain, resulting in approximately 90% neuron purity as indicated by staining for the neuronal markers MAP2a and β-tubulin58. Two weeks after infection with low-dose VZV, no cytopathic effect was evident, although viral DNA, transcripts, and protein were all detectable. hESCs are derived from the inner cell mass of in vitro fertilized human embryos and can be maintained undifferentiated for at least 8 months in culture59. After differentiation in vitro, VZV infection of hESC-derived neurons results in a lytic infection60,61. Recently, Markus and colleagues62 claimed to have developed an in vitro model of latency with reactivation of VZV. Although the data in their report do not recapitulate what is known of VZV latency in human ganglia (see above), their model does advance the VZV field since it represents long-term (over a 7-week period), low-level infection, which can be stimulated with various pharmacological agents to boost viral biogenesis (DNA replication, transcription, and translation).\n\nNormal human neural progenitor cells can be grown on inert microbeads inside NASA-designed rotary vessels to form tissue-like assemblies (TLAs) that resemble the structure of human ganglia obtained at autopsy63. Cells in the TLA express both progenitor and mature neuronal markers and can be maintained for at least 6 months. VZV infection of TLAs results in virus transcription and DNA replication during the initial approximately 18 days of infection, after which a steady-state characterized by persistent infection with sporadic release of virus is reached.\n\nOverall, the iPSCs, hESCs, and TLA cells have provided prolonged VZV infection of neurons before lytic infection develops but have not produced VZV latency. Further studies involving the use of cytokines, antivirals, and other compounds are needed to possibly achieve latency in vitro.\n\n\nVaccination to prevent varicella and zoster\n\nIn 1995, varicella vaccine became available in the US. The Centers for Disease Control and Prevention (CDC) recommends immunization at 1 year of age and a second dose at age 4 to 6. Contraindications to the vaccine include life-threatening allergy to previous administration, gelatin or neomycin, pregnancy, and severe immunosuppression. Among states that accurately reported the incidence of varicella, disease declined by 79% overall between 2000 and 2010 and this was likely due to vaccine.\n\nIn 2005, the Shingles Prevention Study, a randomized placebo-controlled trial, provided evidence that the herpes zoster vaccine reduced the incidence of zoster and PHN in the elderly64. In that study, 38,546 healthy adults at least 60 years of age (median of 69 years) were randomly assigned to receive a single dose of attenuated VZV with 14-fold higher potency than varicella vaccine or placebo. The higher-potency vaccine was required to increase VZV-specific cell-mediated immunity in latently infected older adults. The incidence of zoster as well as the burden of illness due to zoster (total pain and discomfort) and the incidence of PHN were determined. Average follow-up of 3.13 years in a total of 19,270 people who received zoster vaccine and 19,276 who received placebo revealed 957 confirmed cases of zoster (315 in vaccine recipients and 642 in placebo recipients). In both groups, more than 93% of the subjects with zoster were positive for wild-type VZV DNA by PCR; none had vOka DNA. Zoster vaccine reduced the incidence of zoster by 51.3% (63.9% in people 60 to 69 years old but only 37.6% in people at least 70 years old). The burden of disease was reduced by 61.1% (65.5% in people 60 to 69 years old and 55.4% in people at least 70 years old), and the duration of pain and discomfort among subjects with zoster was shorter in vaccinated candidates compared with placebo recipients. PHN was reduced by more than 65% for both age groups, and the most benefit was in the age group at least 70 years old. The study also showed that vaccination reduced the adverse impact of zoster on patients’ capacity to perform daily life activities and on health-related quality of life.\n\nZoster vaccine is safe. Rates of serious adverse events, systemic adverse events, hospitalizations, and deaths were low in vaccine recipients in the study and comparable to those in placebo recipients. During the first 42 days after vaccination, there were 24 cases of zoster in placebo recipients and 7 cases in the vaccination group, but none was caused by vOka. Unlike prophylactic vaccines such as those against varicella and measles, zoster vaccine is a therapeutic vaccine aimed at preventing reactivation of latent VZV in humans already infected before vaccination and thus with substantial immunity.\n\nZostavax can be given to patients with leukemia, to liver and gastrointestinal transplant recipients, and to patients with HIV infection, but is contraindicated in patients with bone marrow or lymphatic malignancy and patients receiving more than 20 mg/day of prednisone. Zoster vaccine was licensed by the US Food and Drug Administration (FDA) for healthy adults at least 50 years old to prevent zoster and its complications, mainly PHN. Post-licensure studies have confirmed the vaccine’s safety and efficacy. Unfortunately, participation in the zoster vaccine program has been low and this is likely due to cost and failure to recognize the importance of preventing infectious diseases in older adults. The Shingles Prevention Study demonstrated efficacy for 3 years after vaccination, whereas subsequent studies indicated efficacy for 8 years65. Currently, the CDC does not recommend booster doses of zoster vaccine, but may in the future.\n\nRecently, a liposome-based subunit vaccine, HZ/su, containing the VZV glycoprotein E and the adjuvant ASO1B was created. Studies revealed that two doses of HZ/su containing 50 µg of recombinant VZV glycoprotein E administered at 1- or 2-month intervals were well tolerated and induced much more robust VZV-specific and VZV glycoprotein E-specific CD4+ T-cell and antibody responses than did vOka66,67. A randomized placebo-controlled study of 15,411 subjects at least 50 years of age revealed a remarkable 97.2% efficacy in preventing zoster for a 3.2-year period that did not diminish with increasing age68. Systemic adverse reactions were 2.2-fold greater in the vaccine group compared with the placebo group. Because HZ/su does not replicate, it will be safe for immunosuppressed patients. The duration of its effect is unknown. HZ/su vaccine is not yet FDA-approved.",
"appendix": "Author contributions\n\n\n\nAll authors conceived the study, designed the experiments, carried out the research, and contributed to the design of the study. All authors were involved in preparing the first draft of the manuscript and in the revision of the draft manuscript, and all have agreed to the final content.\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 in part by Public Health Service grants AG032958 (MAN, RJC, RM, and DG), NS093716 (DG), NS067070 (MAN), and NS082228 (RJC) from the National Institutes of Health. NLB was supported by training grant NS007321 to DG from the National Institutes of Health. The authors thank Marina Hoffman for editorial assistance and Cathy Allen for word processing and formatting of the manuscript.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nYawn BP, Itzler RF, Wollan PC, et al.: Health care utilization and cost burden of herpes zoster in a community population. Mayo Clin Proc. 2009; 84(9): 787–94. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGilden DH, Dueland AN, Cohrs R, et al.: Preherpetic neuralgia. Neurology. 1991; 41(8): 1215–8. PubMed Abstract | Publisher Full Text\n\nHarnisch JP: Zoster in the elderly: clinical, immunologic and therapeutic considerations. J Am Geriatr Soc. 1984; 32(11): 789–93. PubMed Abstract | Publisher Full Text\n\nGilden DH, Cohrs RJ, Mahalingam R: Clinical and molecular pathogenesis of varicella virus infection. Viral Immunol. 2003; 16(3): 243–58. 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PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDukhovny A, Sloutskin A, Markus A, et al.: Varicella-zoster virus infects human embryonic stem cell-derived neurons and neurospheres but not pluripotent embryonic stem cells or early progenitors. J Virol. 2012; 86(6): 3211–8. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMarkus A, Lebenthal-Loinger I, Yang IH, et al.: An in vitro model of latency and reactivation of varicella zoster virus in human stem cell-derived neurons. PLoS Pathog. 2015; 11(6): e1004885. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nGoodwin TJ, McCarthy M, Osterrieder N, et al.: Three-dimensional normal human neural progenitor tissue-like assemblies: a model of persistent varicella-zoster virus infection. PLoS Pathog. 2013; 9(8): e1003512. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOxman MN, Levin MJ, Johnson GR, et al.: A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med. 2005; 352(22): 2271–84. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMorrison VA, Johnson GR, Schmader KE, et al.: Long-term persistence of zoster vaccine efficacy. Clin Infect Dis. 2015; 60(6): 900–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTseng HF, Smith N, Harpaz R, et al.: Herpes zoster vaccine in older adults and the risk of subsequent herpes zoster disease. JAMA. 2011; 305(2): 160–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLangan SM, Smeeth L, Margolis DJ, et al.: Herpes zoster vaccine effectiveness against incident herpes zoster and post-herpetic neuralgia in an older US population: a cohort study. PLoS Med. 2013; 10(4): e1001420. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLal H, Cunningham AL, Godeaux O, et al.: Efficacy of an adjuvanted herpes zoster subunit vaccine in older adults. N Engl J Med. 2015; 372(22): 2087–96. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11349",
"date": "26 Nov 2015",
"name": "Kamel Khalili",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11350",
"date": "26 Nov 2015",
"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": "11351",
"date": "26 Nov 2015",
"name": "Howard Lipton",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1356
|
https://f1000research.com/articles/4-1353/v1
|
25 Nov 15
|
{
"type": "Review",
"title": "Pathogenesis of Dengue: Dawn of a New Era",
"authors": [
"Scott B. Halstead"
],
"abstract": "Dengue virus (DENV) infections of humans were long thought to be self-limited and of low mortality. Beginning in the 1950s, at the time when four different DENVs were discovered, a lethal variant of dengue emerged. Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) initially observed in Southeast Asia now has spread throughout the world. Two risk factors for DHF/DSS are well-established: severe disease occurs during a second heterotypic DENV infection or during a first DENV infection in infants born to dengue-immune mothers. A large number of hypotheses have been proposed to explain severe dengue disease. As discussed, few of them attempt to explain why severe disease occurs under the two different immunological settings. New experimental evidence has demonstrated that DENV non-structural protein 1 (NS1) is toll-receptor 4 agonist that stimulates primary human myeloid cells to produce the same cytokines observed during the course of severe dengue disease. In addition, NS1 directly damages endothelial cells. These observations have been repeated and extended to an in vivo mouse model. The well-established phenomenon, antibody-dependent enhancement of DENV infection in Fc-receptor-bearing cells, should similarly enhance the production of DENV NS1 in humans, providing a unitary mechanism for severe disease in both immunological settings",
"keywords": [
"Dengue virus",
"hemorrhagic fever",
"dengue shock syndrome",
"dengue vascular permeability syndrome",
"Dengue pathogenesis",
"viral toxicosis"
],
"content": "Introduction\n\nDuring the first half of the 20th century, human responses to dengue virus (DENV) infection were described in multiple studies on hundreds of adult volunteers in different parts of the world1–9. On the basis of these case descriptions, many earlier 19th and 20th century outbreaks were identified as dengue fever (DF). This large historical experience failed to prepare the scientific community for a surprise in 1956. DENVs were identified as the cause of a fatal hemorrhagic fever in Southeast Asian children with few features of DF10,11. Ever since, the question has been “why did dengue turn deadly?”\n\nIn the 1960s, dengue research programs in Southeast Asia found that children were dying of a new clinical syndrome, an acute febrile disease accompanied by a complex of physiologic abnormalities affecting multiple organ systems including the liver, blood coagulation, complement, hematopoiesis, serum proteins, and the vascular system that reach maximal expression at defervescence. Initially, this entity was named the dengue shock syndrome (DSS) and sub-shock, dengue hemorrhagic fever (DHF)12–16. However, the disease described in some detail in Box 1 is best identified as the dengue vascular permeability syndrome (DVPS).\n\nThere is an abrupt onset of fever accompanied by malaise, vomiting, headache, anorexia, abdominal pain and upper respiratory symptoms. Two to five days later the patient may rapidly deteriorate and collapse. At or near defervescence, the patient may have cold, clammy extremities, slow venous filling, a warm trunk, flushed face, circumoral and peripheral cyanosis, diaphoresis, restlessness, irritability, mid-epigastric pain, decreased urinary output and hypovolemia. There may be scattered petechiae on the forehead and extremities or spontaneous ecchymoses, bruising and bleeding at sites of venipuncture. Respirations are rapid. The pulse is weak, rapid, and thready and the heart sounds are faint. The liver may enlarge to 4–6 cm below the costal margin and is usually firm and somewhat tender. Laboratory findings during acute stage illness include thrombocytopenia, elevated liver enzymes, activated complement with high levels of C3a and C5a, fibrin split products, low fibrinogen, prolonged bleeding time, prolonged APTT, low serum albumin and elevated hematocrit13,17,85,86.\n\nA critical loss of fluid and smaller macromolecules through damaged endothelium may result in reduced blood volume and an increase in hematocrit. A hematocrit that is 20% or greater than a convalescent value denotes cardiovascular instability. Vascular leakage can be detected directly by x-ray or sonography. Pleural effusions are best detected by lateral view chest x-ray. Abdominal sonograms may detect gall bladder wall thickening and serosal effusions87. Approximately 20–30% of cases of DVPS develop shock with an onset that can be subtle, arising in patients who are fully alert, and accompanied by increased peripheral vascular resistance. Shock is not due to congestive heart failure but to hypovolemia. With increasing cardiovascular compromise, diastolic pressure rises towards systolic and pulse pressure narrows to less than 20 mm Hg. Fewer than 10% of patients have gross ecchymosis or gastrointestinal bleeding, usually after a period of uncorrected shock.\n\nAfter a 24- to 36-hr period of crisis, the vascular leak self-heals and convalescence is fairly rapid and complete. Encephalopathy may be seen during this period. Rare complications are myocarditis and hepatitis. Bradycardia and ventricular extrasystoles are common during convalescence87.\n\nEtiological studies discovered that hospitalized DVPS occurs in two immunological settings: 1) approximately 90% with secondary-type DENV antibody responses were shown epidemiologically to accompany a second heterotypic DENV infection, and 2) approximately 5% of cases were infants born to dengue-immune mothers who had primary DENV antibody responses17–19. Important caveats have been discovered for each of these two immunological risk factors. Hospitalized DVPS accompanying a second heterotypic dengue infection is a rare event. Approximately 2% to 4% of secondary dengue infections resulted in hospitalized DHF/DSS20–23. Young children are inherently at greater risk of developing DVPS during a second heterotypic dengue infection than are older children or adults24. When sequential DENV infections are closely spaced, there is significant cross-protection25. This cross-protection initially prevents infection by a second DENV but it persists in partial form, preventing DVPS as a component of the disease response for at least two years26,27. As the interval between sequential DENV infections increases beyond two years, an ever larger fraction of second heterotypic DENV 2 and 3 infections have culminated in severe DVPS28,29. DVPS in infants usually occurs during the second half of the first year of life. Passive antibodies from mothers, known to have been infected by multiple DENV earlier in life, protect against DENV infections for a period of months, then mediate DVPS and finally disappear at around 12 months18,30. DVPS occurs more frequently during DENV infections of infants with passively acquired DENV antibodies than in older children accompanying a second DENV infection30,31.\n\nIn the decades after DHF and DSS were first described, numerous observations have been made in dengue-infected individuals of all ages, and hypotheses have been put forward to explain the mechanism of severe and fatal dengue. Roughly in chronological order, those attracting the most attention are the following:\n\nDuring a second heterotypic dengue infection, the simultaneous circulation of anamnestic IgG dengue antibodies and dengue viral antigens activates complement via the C3 activator and by initiating the C1, C4, C2 cascade, contributing to a reduced level of C3. The resulting increased levels of C3a and C5a anaphylatoxins are thought to mediate vascular permeability14,15,32.\n\nThis phenomenon was discovered when it was observed that DENV readily grew in vitro in cultures of peripheral blood monocytes obtained from dengue-immune monkeys or humans but less well in monocytes from non-immunes33,34. Soon after, it was discovered that this phenomenon was readily mediated by dengue antibodies that were diluted above the neutralization endpoint, added to dengue viruses, and grown in cultured monocytes from seronegative donors35,36. In rhesus monkeys, enhanced viremias were observed in vivo during secondary compared with primary DENV 2 infections37. Enhanced DENV 2 viremias were also produced in susceptible monkeys sensitized with a small intravenous dose of dengue-immune human cord blood serum38.\n\nVascular permeability has been attributed to cytokines, such as interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNFα), released by cohorts of overactive T cells that accompany immune responses during a second heterotypic DENV infection39,40.\n\nThe concept of “virulent” or “non-virulent” dengue viruses developed when it was observed that the American genotype of DENV 2 did not produce a large outbreak of DHF/DSS in Iquitos, Peru, during a 1995 outbreak in a population that was highly immune to DENV 141,42. Also, pronounced differences in clinical expression of infections caused by a genotype of DENV 2 were observed in outbreaks on different Pacific islands43.\n\nIn the extensive experimental literature describing observations in mouse models, it is proposed that DVPS is a short-lived autoimmune disease resulting from destructive tissue responses to pathogenic antibodies raised to DENV NS1 proteins. These antibodies cross-react with host endothelial cells, blood-clotting proteins, and liver cells. Mimetic antibodies are thought to reach pathological levels during secondary DENV infections44–46.\n\nAnalysis of the functional phenotypes of CD8+ T cells in DHF cases revealed that recognition between different DENV peptides was associated with reduced cytolytic potential without reducing cytokine production47–49. Activation of both CD4+ and CD8+ T cells with peptide variants induced different sets of cytokines50. Pathogenic heterologous T-cell responses or selectively defective T-cell responses (original antigenic sin) result in cytokines and chemokines (“cytokine storm”) that produce vascular permeability leading to DHF/DSS. T-cell responses enhance the severity of DENV infections by an in vitro process that has not been demonstrated in vivo47,51–53.\n\nA host of in vitro studies suggest that vascular permeability results from cytokines or other factors generated by DENV infection of myeloid cells, including mast cells54–59.\n\nDengue viruses readily grow in primary human endothelial cell explants, generating products that increase vascular permeability60. Transcriptional activity, protein production, and cell surface protein expression by endothelial cells are significantly altered by DENV infection in vitro. Several pathways identified in DHF/DSS, including inflammation, apoptosis, and coagulation, are affected61,62. Apoptosis of endothelial cells has been demonstrated in mice and has been proposed to be the mechanism of vascular leakage63.\n\n\nWhich is the more relevant pathogenic mechanism?\n\nOnly one of these hypotheses satisfies the requirement of “Occam’s razor” (among competing hypotheses, the one with the fewest assumptions should be selected), providing a hypothesis that offers the simplest explanation why DVPS occurs in persons who are actively or passively dengue-immune. One hypothesis, antibody-dependent enhancement of dengue infection (ADE) (#2), satisfies this requirement, whereas hypotheses #1 (acute immune complex disease), #3 (exaggerated T-cell response), #5 (heterophile immunity), and #6 (original antigenic sin) do not. These hypotheses are unable to explain why DVPS accompanies a primary DENV infection in infants who circulate passively acquired dengue antibodies. Hypothesis #4 (virulent DENV) suggests that it is the innate properties of different genotypes or strains of dengue viruses that control the outcome of disease. DVPS, carefully documented, has not accompanied DENV infections in naïve populations. Dengue viral contributions to DVPS are conditioned by pre-illness dengue antibodies. This is not to say that differences between DENV strains or genotypes may not interact with antibodies to profoundly change biological outcomes. This important possibility is partially discussed at greater length elsewhere64,65. This pathogenesis-relevant antibody-dependent phenomenon is best described as “fitness” and not as “virulence”. Hypothesis #7, when confined to experimental studies on the implications of direct infection, predicts that any DENV infection, regardless of immunological status, may result in DVPS. Hypothesis #8 attributes DVPS to the direct viral infection of endothelial cells. The problem here is the paucity of high-quality studies. The reagents needed to identify sites of DENV replication in human autopsy tissues are scarce, present difficult quality-control challenges, and are not standardized throughout the dengue research community. In studies using anti-NS3 staining, DENV antigens have been localized to focal endothelial cells in several organs. But the distribution and intensity of staining is unlike that observed with the direct infection of endothelial cells in hantaviral pulmonary syndrome associated with severe localized vascular permeability66–68. A recent effort made to identify endothelial cells stained with DENV antigens was negative69.\n\n\nDengue pathogenesis: ADE and dengue viral toxicosis\n\nAll infectious diseases are kinetic, consisting of the invasion of the microorganism (afferent phase) followed by the host response (efferent phase), including disease and elimination of the organism. The setting of DENV infections is unique in that pre-infection events may control afferent phenomena. During the afferent phase of dengue infections, DENV infectious immune complexes regulate infection of Fc-receptor-bearing cells. Antibodies, whether passively or actively acquired, on some, but not all occasions, intervene with infection dynamics to produce an expanded infected cell mass. As predicted by experimental ADE studies, early acute-phase illness sera from children with second DENV infections were found to have higher peak viremia and antigenemia titers prior to the onset of DHF/DSS than did similarly timed sera from children who developed a milder illness70–72. More recently, Vietnamese and Sri Lankan researchers observed the circulation of NS1 at higher titers and longer intervals during severe disease73,74. Afferent ADE has been successfully modeled in vivo. DENV 2, 3, and 4 infections in type I and II interferon receptor-deficient mice produce a non-paralytic lethal disease accompanied by many features of DVPS, high levels of virus in tissues and circulating in blood and efferent phenomena, a cytokine storm, low platelet counts, elevated hematocrit, increased vascular permeability, and intestinal hemorrhage75–77. Mice transfused with enhancing concentrations of dengue antibodies prior to infection with a sub-lethal dose of mouse-adapted DENV 2 developed lethal vascular permeability with TNF release78,79. It is now known that dengue immune complex infection of human monocytes/macrophages boosts DENV replication approximately 100-fold in association with the suppression of type I interferon or increased production of IL-10 or both. This phenomenon is called intrinsic ADE (iADE)80,81. Infectious immune complexes also achieve a threefold infection advantage in FcR-bearing cells compared with DENV alone (extrinsic ADE– eADE)82.\n\nWhat causes the efferent signs and symptoms of DVPS? Missing is the “smoking gun” that produces liver injury, vascular permeability, activation of complement, and alteration of hemostasis. Very recently, Paul Young’s group observed an analogy between the cellular biology of bacterial lipopolysaccharides (LPS) and that of DENV NS183. Each of these compounds interacts with Toll-like receptor 4 (TLR 4) on the surface of monocytes, macrophages, and endothelial cells, inducing the release of a range of cytokines and chemokines. These are the same mediators identified in the blood of patients with DHF/DSS. In vitro, NS1 resulted in the disruption of endothelial cell monolayer integrity. The authors conclude that DSS may be a viral protein toxicosis. NS1-mediated cytokine release was inhibited by the TLR4 antagonist LPS-Rhodobacter sphaeroides, suggesting an avenue for therapeutic intervention. Crucially, this same observation has been confirmed in an in vivo model. The Harris laboratory has shown that DENV 2 NS1 inoculated intravenously at physiologically relevant concentrations in sub-lethal DENV 2-infected IFNAR−/−C57BL/6 mice produced lethal vascular permeability84. In vitro, NS1, when added to cultured endothelial cells, resulted in endothelial permeability. Vaccination of mice with DENV 2 NS1 protected against endothelial leakage and death due to lethal DENV 2 challenge. Mice immunized with DENV 2 NS1 protein were completely protected against homologous DENV 2 challenge, and immunization with DENV 1, 3, and 4 NS1 proteins partially protected against heterologous DENV 2 challenge.\n\nDENV NS1 blood levels and therefore NS1 toxicosis are efferent mechanisms directly controlled by ADE. It is not clear yet whether NS1 alone, NS1-induced cytokines, virus replication-induced damage, or activated complement is responsible for the in vivo efferent DVPS phenomenon. There is a delay of several days between the early occurrence of peak blood levels of NS1 and defervescence-associated organ pathology. This is not fully understood. It is clear, however, that DENV NS1 toxicosis introduces a new era to dengue pathogenesis research.",
"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\nCleland JB, Bradley B, McDonald W: On the Transmission of Australian Dengue by the Mosquito Stegomyia fasciata. Med J Aust. 1916; 2(10): 179–184. Reference Source\n\nChandler AG, Rice L: Observations on the etiology of dengue fever. Am J Trop Med Hyg. 1923; s1–3(3): 233–262. Reference Source\n\nSiler JF, Hall MW, Hitchens AP: Dengue: Its history, epidemiology, mechanism of transmission, etiology, clinical manifestations, immunity, and prevention. The Philippine Journal of Science. 1926; 29: 1–304.\n\nSimmons JS, St John JH, Reynolds FHK: Experimental Studies of Dengue. Philipp J Sci. 1931; 44(1–2): 1–252. Reference Source\n\nSnijders EP, Dinger EJ, Schuffner WAP: On the transmission of dengue in Sumatra. Am J Trop Med Hyg. 1931; s1–11(3): 171–197. 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PubMed Abstract | Publisher Full Text\n\nMathew A, Kurane I, Green S, et al.: Predominance of HLA-restricted cytotoxic T-lymphocyte responses to serotype-cross-reactive epitopes on nonstructural proteins following natural secondary dengue virus infection. J Virol. 1998; 72(5): 3999–4004. PubMed Abstract\n\nRothman AL, Ennis FA: Immunopathogenesis of Dengue hemorrhagic fever. Virology. 1999; 257: 1–6. PubMed Abstract | Publisher Full Text\n\nRico-Hesse R, Harrison LM, Salas RA, et al.: Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology. 1997; 230(2): 244–51. PubMed Abstract | Publisher Full Text\n\nRico-Hesse R: Microevolution and virulence of dengue viruses. Adv Virus Res. 2003; 59: 315–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSteel A, Gubler DJ, Bennett SN: Natural attenuation of dengue virus type-2 after a series of island outbreaks: a retrospective phylogenetic study of events in the South Pacific three decades ago. Virology. 2010; 405(2): 505–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFalconar AK: The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis. Arch Virol. 1997; 142(5): 897–916. PubMed Abstract\n\nLin YS, Yeh TM, Lin CF, et al.: Molecular mimicry between virus and host and its implications for dengue disease pathogenesis. Exp Biol Med (Maywood). 2011; 236(5): 515–23. PubMed Abstract | Publisher Full Text\n\nWan SW, Lin CF, Yeh TM, et al.: Autoimmunity in dengue pathogenesis. J Formos Med Assoc. 2013; 112(1): 3–11. PubMed Abstract | Publisher Full Text\n\nMongkolsapaya J, Dejnirattisai W, Xu XN, et al.: Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever. Nat Med. 2003; 9(7): 921–7. PubMed Abstract | Publisher Full Text\n\nMongkolsapaya J, Duangchinda T, Dejnirattisai W, et al.: T cell responses in dengue hemorrhagic fever: are cross-reactive T cells suboptimal? J Immunol. 2006; 176(6): 3821–9. PubMed Abstract | Publisher Full Text\n\nDong T, Moran E, Vinh Chau N, et al.: High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection. PLoS One. 2007; 2(12): e1192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMangada MM, Rothman AL: Altered cytokine responses of dengue-specific CD4+ T cells to heterologous serotypes. J Immunol. 2005; 175(4): 2676–83. PubMed Abstract | Publisher Full Text\n\nRothman AL: Cellular immunology of sequential dengue virus infection and its role in disease pathogenesis. Curr Top Microbiol Immunol. 2010; 338: 83–98. PubMed Abstract | Publisher Full Text\n\nPang T, Cardosa MJ, Guzman MG: Of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS). Immunol Cell Biol. 2007; 85(1): 43–5. PubMed Abstract | Publisher Full Text\n\nRothman AL: Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nat Rev Immunol. 2011; 11(8): 532–43. PubMed Abstract | Publisher Full Text\n\nCarr JM, Hocking H, Bunting K, et al.: Supernatants from dengue virus type-2 infected macrophages induce permeability changes in endothelial cell monolayers. J Med Virol. 2003; 69(4): 521–8. PubMed Abstract | Publisher Full Text\n\nLuplertlop N, Missé D, Bray D, et al.: Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction. EMBO Rep. 2006; 7(11): 1176–81. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLuplertlop N, Missé D: MMP cellular responses to dengue virus infection-induced vascular leakage. Jpn J Infect Dis. 2008; 61(4): 298–301. PubMed Abstract\n\nDejnirattisai W, Duangchinda T, Lin CL, et al.: A complex interplay among virus, dendritic cells, T cells, and cytokines in dengue virus infections. J Immunol. 2008; 181(9): 5865–74. PubMed Abstract | Publisher Full Text\n\nMichels M, Djamiatun K, Faradz SM, et al.: High plasma mid-regional pro-adrenomedullin levels in children with severe dengue virus infections. J Clin Virol. 2011; 50(1): 8–12. PubMed Abstract | Publisher Full Text\n\nSt John AL, Rathore AP, Raghavan B, et al.: Contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage. Elife. 2013; 2: e00481. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSrikiatkhachorn A: Plasma leakage in dengue haemorrhagic fever. Thromb Haemost. 2009; 102(6): 1042–9. PubMed Abstract | Publisher Full Text\n\nAvirutnan P, Malasit P, Seliger B, et al.: Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol. 1998; 161(11): 6338–46. PubMed Abstract\n\nHuang YH, Lei HY, Liu HS, et al.: Dengue virus infects human endothelial cells and induces IL-6 and IL-8 production. Am J Trop Med Hyg. 2000; 63(1–2): 71–5. PubMed Abstract\n\nChen H, Hofman FM, Kung JT, et al.: Both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage. J Virol. 2007; 81(11): 5518–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalstead SB: In Clinical Insights: Dengue fever: transmission, diagnosis & surveillance. J Whitehorn, J Farrar, Eds. Future Medicine, London, 2014; 84–101.\n\nHalstead SB: Dengue Antibody-Dependent Enhancement: Knowns and Unknowns. Microbiol Spectr. 2014; 2(6). PubMed Abstract | Publisher Full Text\n\nBalsitis SJ, Coloma J, Castro G, et al.: Tropism of dengue virus in mice and humans defined by viral nonstructural protein 3-specific immunostaining. Am J Trop Med Hyg. 2009; 80(3): 416–24. PubMed Abstract\n\nPóvoa TF, Alves AM, Oliveira CA, et al.: The pathology of severe dengue in multiple organs of human fatal cases: histopathology, ultrastructure and virus replication. PLoS One. 2014; 9(4): e83386. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSrikiatkhachorn A, Spiropoulou CF: Vascular events in viral hemorrhagic fevers: a comparative study of dengue and hantaviruses. Cell Tissue Res. 2014; 355(3): 621–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAye KS, Charngkaew K, Win N, et al.: Pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from Myanmar. Hum Pathol. 2014; 45(6): 1221–33. PubMed Abstract | Publisher Full Text\n\nVaughn DW, Green S, Kalayanarooj S, et al.: Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis. 2000; 181(1): 2–9. PubMed Abstract | Publisher Full Text\n\nLibraty DH, Endy TP, Houng HH, et al.: Differing influences of virus burden and immune activation on disease severity in secondary dengue-3 virus infections. J Infect Dis. 2002; 185(9): 1213–21. PubMed Abstract | Publisher Full Text\n\nLibraty DH, Young PR, Pickering D, et al.: High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis. 2002; 186(8): 1165–8. PubMed Abstract | Publisher Full Text\n\nParanavitane SA, Gomes L, Kamaladasa A, et al.: Dengue NS1 antigen as a marker of severe clinical disease. BMC Infect Dis. 2014; 14: 570. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTricou V, Minh NN, Farrar J, et al.: Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with dengue. PLoS Negl Trop Dis. 2011; 5(9): e1309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShresta S, Sharar KL, Prigozhin DM, et al.: Murine model for dengue virus-induced lethal disease with increased vascular permeability. J Virol. 2006; 80(20): 10208–17. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nSarathy VV, White M, Li L, et al.: A lethal murine infection model for dengue virus 3 in AG129 mice deficient in type I and II interferon receptors leads to systemic disease. J Virol. 2015; 89(2): 1254–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMilligan GN, Sarathy VV, Infante E, et al.: A Dengue Virus Type 4 Model of Disseminated Lethal Infection in AG129 Mice. PLoS One. 2015; 10(5): e0125476. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZellweger RM, Prestwood TR, Shresta S: Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe. 2010; 7(2): 128–39. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBalsitis SJ, Williams KL, Lachica R, et al.: Lethal antibody enhancement of dengue disease in mice is prevented by Fc modification. PLoS Pathog. 2010; 6(2): e1000790. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nChareonsirisuthigul T, Kalayanarooj S, Ubol S: Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells. J Gen Virol. 2007; 88(Pt 2): 365–75. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHalstead SB, Mahalingam S, Marovich MA, et al.: Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis. 2010; 10(10): 712–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoonnak K, Dambach KM, Donofrio GC, et al.: Cell type specificity and host genetic polymorphisms influence antibody-dependent enhancement of dengue virus infection. J Virol. 2011; 85(4): 1671–83. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nModhiran N, Watterson D, Muller DA, et al.: Dengue virus NS1 protein activates cells via Toll-like receptor 4 and disrupts endothelial cell monolayer integrity. Sci Transl Med. 2015; 7(304): 304ra142. PubMed Abstract | Publisher Full Text\n\nBeatty PR, Puerta-Guardo H, Killingbeck SS, et al.: Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination. Sci Transl Med. 2015; 7(304): 304ra141. PubMed Abstract | Publisher Full Text\n\nWills BA, Oragui EE, Stephens AC, et al.: Coagulation abnormalities in dengue hemorrhagic Fever: serial investigations in 167 Vietnamese children with Dengue shock syndrome. Clin Infect Dis. 2002; 35(3): 277–85. PubMed Abstract | Publisher Full Text\n\nTrung DT, Thao le TT, Hien TT, et al.: Liver involvement associated with dengue infection in adults in Vietnam. Am J Trop Med Hyg. 2010; 83(4): 774–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition. World Health Organization, Geneva, 2009. PubMed Abstract"
}
|
[
{
"id": "11346",
"date": "25 Nov 2015",
"name": "Eng Eong Ooi",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11347",
"date": "25 Nov 2015",
"name": "Wei-Kung 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": "11348",
"date": "25 Nov 2015",
"name": "Aravinda de Silva",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1353
|
https://f1000research.com/articles/4-1349/v1
|
25 Nov 15
|
{
"type": "Research Note",
"title": "Transcription factor regulation as a mechanism of confounding effects between distinct human traits",
"authors": [
"Milos Pjanic",
"Clint L. Miller",
"Thomas Quertermous",
"Clint L. Miller",
"Thomas Quertermous"
],
"abstract": "Genome-wide association studies (GWAS) to date have discovered thousands of genetic variants linked to human diseases and traits, which hold the potential to unravel the mechanisms of complex phenotypes. However, given that the majority of these associated variants reside in non-coding genomic regions, their predicted cis and trans-regulatory functions remain largely undefined. Here we show that correlation between human diseases and traits can follow geographical distribution of human populations, and that the underlying mechanism is at least partly genetically based. We report two Type 2 Diabetes (T2D) GWAS variants (rs7903146 and rs12255372) in the TCF7L2 locus that regulate expression in skin tissues but not lymphoblastoid or adipose tissues, of the KITLG gene that encodes an important regulator of melanogenesis and light hair color in European populations. We also report extensive binding events of TCF7L2 protein in the promoter region, immediate upstream region and first intron of the KITLG gene, which supports a trans-interaction between TCF7L2 and KITLG. We further show that both light hair color and T2D genetic variants are correlated with geographic latitude. Taken together, our observations suggest that natural variation in transcription factor loci in European human populations may be an underlying and confounding factor for the geographical correlation between human phenotypes, such as type 2 diabetes and light hair color. We postulate that transcription factor regulation may confound the correlation between seemingly diverse human traits. Furthermore, our findings demonstrate the importance of dissecting the genomic architecture of GWAS loci using multiple genetic and genomic datasets.",
"keywords": [
"rs7903146",
"rs12255372",
"GWAS",
"Type 2 diabetes",
"TCF7L2",
"KITLG",
"transcription factor regulation"
],
"content": "Introduction\n\nA recent publication1 has demonstrated the potential causative mechanism of a genome-wide association study (GWAS) locus for the development of blond hair. Through a series of elegant in vivo experiments in mice, the study's findings strengthen association of single nucleotide polymorphism (SNP) rs12821256, initially discovered as one of the top GWAS hits in European populations2, with light hair color development. This work implicates a mechanism of long-range regulation of a gene on chromosome 12, termed KITLG that encodes the ligand for a receptor-type protein-tyrosine kinase, and is located 350kb away from the variant. Further, using data generated by the ENCODE consortium, the study reveals a molecular mechanism by which SNP rs12821256 confers the blond hair phenotype via directly altering a canonical binding site for transcription factor TCF7L23. This may shed light on possible cis- and trans-acting mechanisms responsible for the association of rs12821256 with the quantitative trait of light hair color.\n\nOn the other hand, the TCF7L2 locus on chromosome 10 is well-known for its strong association with type 2 diabetes (T2D) and glycemic traits from several GWAS studies4,5. It confers the strongest effect on T2D to date, with a per-allele odds ratio of 1.396. Lead risk-associated SNPs from the TCF7L2 locus include two intronic SNPs (rs7903146 and rs4506565). The majority of SNPs from the TCF7L2 locus are non-coding and may alter the levels of expression or affect alternative splicing of TCF7L2, while SNPs located in TCF7L2 exons give rise to alternate protein isoforms. In addition, numerous SNPs from this locus that are in linkage disequilibrium (LD) with GWAS lead SNPs could be candidates for the causal variant(s). Given these reports, it seems likely that specific TCF7L2 expression levels or the composition of its 13 or more transcripts (UCSC annotation) and isoforms in pancreatic beta cells confer risk for T2D, while in melanocytes the composition of TCF7L2 variants and levels may influence trans TCF7L2 protein binding to SNP rs12821256 to alter expression of the downstream KITLG gene, an important regulator of melanogenesis.\n\nTCF7L2 is expressed in a variety of human tissues, where it plays a critical role in the Wnt signaling pathway. In skin tissues TCF7L2 reaches moderate expression levels with RPKM (Reads Per Kilobase of transcript per Million mapped reads) values between 10 and 20, which are higher than that observed in pancreas (<10)7.\n\n\nMain body\n\nIn addition to binding to rs12821256, we report here that TCF7L2 binds to the promoter region of the KITLG gene (as shown in the ENCODE ChIP-Seq data sets), as well as throughout the first intron and immediate upstream region, and overlaps the active enhancer histone modification mark H3K27ac (Figure 1A), which further implicates its role in the regulation of KITLG expression. When we queried the Genotype-Tissue Expression (GTEx) database or eQTL resources from the Gilad/Pritchard group there were no SNPs from the TCF7L2 locus detected as expression quantitative trait loci (eQTL SNPs) for KITLG (search terms in Supplementary Table S1), nor when we investigated HapMap data through the GENEVAR (GENe Expression VARiation) platform. However, a significant eQTL association between TCF7L2 SNPs (rs7903146 and rs12255372) and KITLG was observed in skin tissues in data from the MuTHER (Multiple Tissue Human Expression Resource) healthy female twin studies8 (Figure 1B, p=0.0089 and 0.0349, respectively), implicating a strong trans-eQTL interaction in skin tissues compared to Lymphoblastoid cell lines (LCL) or adipose tissues where either the absence of, or weak eQTL association was found.\n\nA. TCF7L2 protein binding at the KITLG promoter, upstream of the KITLG promoter and multiple binding events in the first intron of KITLG gene. TCF7L2 binding sites overlap regulatory histone mark H3K27ac, implying their functionality in gene regulation. Data were taken from the ENCODE consortium. B. eQTL analysis of two T2D SNPs from TCF7L2 locus (rs7903146 and rs12255372) and KITLG gene in multiple tissues: skin, lymphoblastoid cell line (LCL) and adipose. Data from MuTHER healthy female twin studies.\n\nAs demonstrated by the International Diabetes Federation data for 20149, T2D is less prevalent in northern European populations (compared to, e.g. southern Europeans). Data of the frequency of T2D patients in Europe shows that southern European countries, i.e. Spain (7.9%), Portugal (9.6%), Balkan countries (9.8%) and Turkey (14.8%) have the highest percentage of T2D patients in Europe (average 10.2%). On the other hand, northern European countries like Britain (3.9%), Sweden (4.5%), Norway (5.2), Baltic countries (3.8%, 5.0% and 5.7%), and Iceland (3.2%) have lower percentage of diabetics compared to the rest of Europe (average 3.9%). This difference in disease prevalence could be attributed to differences in dietary or other environmental factors, but also could reflect differences in allele frequency of disease-associated alleles. In fact, the frequencies of SNP rs12821256 and light hair color are more common in northern European populations, e.g., blond and light brown hair reaching 75% in Icelandic populations and rs12821256 MAF reaching its frequency maximum of 0.19 in Iceland (Supplementary Figure S1)1,2. Similarly, using data from ALFRED (ALlele FRequency Database), we found an inverse correlation of population’s geographic latitude and frequency of TCF7L2 SNP rs7903146 (Figure 2A), and another TCF7L2 SNP rs12255372 also showed a similar trend (Figure 2B). Thus, it is intriguing to speculate whether TCF7L2 protein isoforms may give rise to light hair color via binding to rs12821256 and regulating the KITLG gene in one cell type (melanocytes), while in pancreatic beta cells they may act as risk factors for the development of diabetes (Figure 3), through TCF7L2 gene regulation and potential cross-composition of TCF7L2 isoforms.\n\nMaximal geographical latitude of the population and T2D SNP minor allele frequency (MAF) were taken from Alfred (Allele Frequency Database) and plotted as a heatmap. A. SNP rs7903146 B. SNP rs12255372.\n\nT2D SNP rs7903146 from TCF7L2 locus is shown as eQTL SNP for KITLG gene in skin tissues. eQTL association is lost in other tissues, indicating regulation of KITLG gene by TCF7L2 isoforms explicitly in skin tissues.\n\n\nConclusion\n\nIn summary, the putative trans-eQTL interaction in skin tissues we report here implicates natural genetic variation in the T2D locus, TCF7L2, to regulate expression of KITLG, a gene linked to light hair color development. We postulate that this could be the underlying genetic mechanism accounting for the association between hair color and T2D risk in European populations. Our observations here strengthen the hypothesis of a genetically determined correlation between diseases and traits in human population, as also demonstrated in a recent publication with the inversely correlated height and coronary artery disease (CAD) phenotypes, where height-associated variants were associated with an increase of 13.5% in the risk of CAD10. Furthermore, these observations illustrate how investigating the genetic architecture underlying complex traits and diseases may inform appropriate risk stratification in diverse human populations.",
"appendix": "Author contributions\n\n\n\nMP conceived the study. MP, CLM, and TQ jointly analyzed the results and wrote and revised the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors have no conflicts of interest or competing interests to disclose.\n\n\nGrant information\n\nWe acknowledge support from the following grants: HL109512 (Quertermous), R21HL120757 (Quertermous), K99 HL125912 (Miller) and a grant from the LeDucq 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\nSupplementary information\n\nSupplementary Figure S1. Correlation of geographic latitude and rs12821256 minor allele frequency.\n\nMaximal geographic latitude of the population and rs12821256 minor allele frequency (MAF) were taken from Alfred (Allele Frequency Database) and plotted as a heatmap.\n\nClick here to access the data.\n\nSupplementary Table S1. Query results from the Genotype-Tissue Expression (GTEx) database and eQTL resources from the Gilad/Pritchard group.\n\nThere were no SNPs from the TCF7L2 locus that could be detected as expression quantitative trait loci SNPs (eQTL SNPs) for KITLG gene using GTEx database, neither TCF7L2 locus (500Kb from chr10:114553649-115062602) could be linked to any trans eQTL SNP-gene interaction using eQTL resources from the Gilad/Pritchard group.\n\nClick here to access the data.\n\n\nReferences\n\nGuenther CA, Tasic B, Luo L, et al.: A molecular basis for classic blond hair color in Europeans. Nat Genet. 2014; 46(7): 748–52. PubMed Abstract | Publisher Full Text\n\nSulem P, Gudbjartsson DF, Stacey SN, et al.: Genetic determinants of hair, eye and skin pigmentation in Europeans. Nat Genet. 2007; 39(12): 1443–52. PubMed Abstract | Publisher Full Text\n\nENCODE Project Consortium: An integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414): 57–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcCarthy MI, Zeggini E: Genome-wide association studies in type 2 diabetes. Curr Diab Rep. 2009; 9(2): 164–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrant SF, Thorleifsson G, Reynisdottir I, et al.: Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet. 2006; 38(3): 320–3. PubMed Abstract | Publisher Full Text\n\nPalomaki GE, Melillo S, Marrone M, et al.: Use of genomic panels to determine risk of developing type 2 diabetes in the general population: a targeted evidence-based review. Genet Med. 2013; 15(8): 600–11. PubMed Abstract | Publisher Full Text\n\nGTEx Consortium: The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013; 45(6): 580–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNica AC, Parts L, Glass D, et al.: The architecture of gene regulatory variation across multiple human tissues: the MuTHER study. PLoS Genet. 2011; 7(2): e1002003. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFederation ID: IDF Diabetes Atlas. 6th edn. Brussels, Belgium: International Diabetes Federation, 2013. Reference Source\n\nNelson CP, Hamby SE, Saleheen D, et al.: Genetically determined height and coronary artery disease. N Engl J Med. 2015; 372(17): 1608–18. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11334",
"date": "07 Jan 2016",
"name": "Gregory Gibson",
"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\nPjanic, Miller and Quertermous suggest a genetic mechanism that may account for the correlation of latitude, frequency of blond hair color and prevalence of type 2 diabetes (T2D) in European populations. The mechanism involves TCF7L2 and its regulation of KITLG in skin tissue. Specifically, they describe (i) that rs12821256 (SNP associated with blond hair color) alters a binding site for TCF7L2, (ii) that KITLG contains other binding sites for TCF7L2 in its vicinity (Fig1A in the manuscript) and (iii) that rs7903146 in TCF7L2 (the strongest known risk SNP for T2D) acts as a trans-eQTL for KITLG in skin tissue (Fig 1B in the manuscript). The manuscript does not provide new evidence, but connects disparate evidence from GWAS, ENCODE, eQTL databases and molecular studies to put forward a very interesting hypothesis. However, and even if the pieces do fit together, more clarifying evidence is needed to validate this hypothesis: Fig 1B shows that C-allele increases expression of KITLG in skin tissue (this is T2D protective allele, but this is not indicated anywhere in the text). According to the authors’ mechanism, C-allele should also lead to higher expression of TCF7L2 and consequently lower expression of KITLG in individuals with “blond” allele at rs12821256. However, and here lies the main caveat of the study, the evidence for a trans-eQTL is minuscule. The authors need to explain how much of the variance is explained at each step (for instance, variance in expression levels of KITLG in skin tissue that can be explained by rs7903146). Even if the proposed mechanism is interesting, a minor contribution by rs7903146 could not “explain” the core hypothesis. The authors should go beyond SNP associations and validate this mechanism by looking at TCF7L2 expression levels (and its correlation with KITLG levels among genotypes) in skin samples from GTEx. There are >200 such individuals available, which should be enough to validate this point. Moreover, other association evidence from GWAS could strengthen the hypothesis. For instance, rs7903146 should have a suggestive p-value for being a “blond hair” SNP and rs12821256 blond allele should in turn have a suggestive p-value for being a T2D protective allele (as blond people should be overrepresented among T2D cases). The authors could check publicly available GWAS p-value files to check this point (preferably within cohorts of a very homogeneous genetic background, e.g. Iceland). Are there studies on the extent to which the lower prevalence of T2D in Northern Europeans is genetic in origin? Is there any evidence from less heterogeneous sources at the environmental level? For instance, do European Americans of Northern European ancestry have less T2D than other European Americans? Prevalence data is suggestive, but more evidence would reinforce the authors’ point about a pleiotropic mechanism between blond hair color and genetic protection to T2D. At the very least, the authors should discuss more about this part of the hypothesis. Related to the previous point, do the authors think this molecular mechanism explains the lower prevalence of T2D in Northern Europeans, or it is one of many other causal factors? This aspect should be made more clear for readers. Fig 2B is redundant",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1349
|
https://f1000research.com/articles/4-1341/v1
|
24 Nov 15
|
{
"type": "Research Article",
"title": "Validating the use of Medicare Australia billing data to examine trends in skin cancer",
"authors": [
"Eshini Perera",
"Neiraja Gnaneswaran",
"Marlon Perera",
"Rodney Sinclair",
"Neiraja Gnaneswaran",
"Marlon Perera",
"Rodney Sinclair"
],
"abstract": "Background: Epidemiological data surrounding non-melanomatous skin cancer (NMSC) is highly variable, in part due to the lack of government cancer registries. Several studies employ the use of Medical Australia (MA) rebate data in assessing such trends, the validity of which has not been studied in the past. Conversely, melanoma skin cancer is a notifiable disease, and thus, MA and cancer registry data is readily available. The aim of the current study is to assess the use of MA for epidemiological measures for skin cancers, by using melanoma as a disease sample. Methods: Following ethics approval, data from MA and Victorian Cancer Registry (VCR) from 2004-2008 were extracted. Incidence of MA and VCR unique melanoma cases were compared and stratified by age and local government area (LGA). Regression and a paired-samples t-test were performed. Results: During the study period; 15,150 and 13,886 unique melanoma patients were identified through VCR and MA data sources respectively. An outlier in the >80 year age group was noted between MA and VCR data. When stratified by age, significant correlation between MA and VCR was observed for all patients (gradient 0.91, R²= 0.936) and following exclusion of >80 patients (gradient 0.96, R²= 0.995). When stratified by LGA, a high degree of observation was observed for all patients (gradient 0.94, R²= 0.977) and following exclusion of >80 patients (gradient 0.996, R²= 0.975). Conclusion: Despite the inclusion of outlier data groups, acceptable correlation between MA and VCR melanoma data was observed, suggesting that MA may be suitable for assessing epidemiological trends. Such principals may be used to validate the use of MA data for similar calculations assessing NMSC trends.",
"keywords": [
"Nonmelanoma skin cancer",
"Dermatology incidence",
"epidemiology",
"melanoma",
"medicare"
],
"content": "Introduction\n\nNon-melanoma skin cancers (NMSC) are the most commonly diagnosed cancer in Australia. In Australia, excluding Tasmania, no government cancer registries record information regarding NMSC1. Incidence and prevalence data surrounding NMSC is difficult to collect and results are highly variable2–4. Previously in Australia, NMSC epidemiological data has been obtained through large-scale prospective surveys and clinical examinations1,3–21. Recently, studies examining the costs and rates of NMSC services have employed the use of the Medicare Australia (MA) database of item numbers billed2,22. Furthermore, studies examining other cancer trends also examine MA data in a similar way23–25. The MA data source has the potential to provide a very large amount of information concerning skin cancer trends in Australia. No previous study has validated the use of MA data for epidemiological cancer calculations. Thus there is a need to demonstrate that the level of ascertainment of cases captured by MA is sufficient to allow for meaningful research of skin cancer using this dataset.\n\nIn Australia, melanoma is a notifiable cancer, with state-based cancer registry data26. Further, MA billing data exists for the management of malignant melanoma. The availability of such data provides the possibility of validating the use of MA data for epidemiological measures of melanoma. The aim of this study was to assess the accuracy of MA’s Medical Benefits Schedule (MBS) rebate data for incidence and patterns of melanoma skin cancer in Australia. The methodology used to validate the use of MA to calculate incidence may represent the possibility of using MA data to examine other types of skin cancer trends in Australia.\n\n\nMethodology\n\nThe Human Resources and Ethics Committee (HREC) granted approval to the Cancer Council of Victoria (CCV) on 1/10/2008 by the Department of Health and Ageing and was given the reference number 2008/CO004599. Ethics approval was for the use of MA data to be examined by the CCV for epidemiological purposes. Access to the Victorian Cancer Registry (VCR) dataset was released under the ‘Memorandum of Understanding.’ The data release was approved by the Director of the VCR on 9/7/2013. HREC approval was not required because only aggregated de-identified data was requested.\n\nThe following item numbers pertaining to excision of malignant melanoma and in situ melanoma were extracted for use from the MA dataset: 31300, 31305, 31310, 31315, 31320, 31325, 31330, 31335. The data available for analysis included age group, gender, local government area (LGA) in which the patient was located, grouped location of tumour removal and tumour sizing (grouped as < 10mm, = 10mm or > 20mm). The MA criteria for these item numbers includes excision of both malignant and in situ melanoma (labelled as ‘Hutchinson’s melanotic freckle’ in the MA definition)27,28. Melanomas that were both malignant and in situ were extracted from the VCR database for the period of 2004 to 2008. Melanoma data registered with the VCR over the years 2004 to 2008 was also extracted for use in the study. Data available included age of patient, gender, LGA, in situ or malignant tumour and thickness level of tumour. Lastly, population data, from the Australian Bureau of Statistics (ABS) was used. Population data for the years studied was extracted for each LGA in Victoria.\n\nThe number of unique patients, that is, the number of different individuals requiring one or more melanoma treatment(s) was determined for the MA and VCR dataset. The number of unique patients in each LGA was then examined in the following components: VCR and MA dataset; VCR and MA dataset stratified by year; VCR and MA dataset stratified by gender; and VCR and MA dataset stratified by age group.\n\nOrdinary least squares’ (OLS) regression was used to analyse both datasets in each of the stratifications. In all of the OLS regressions performed, the y-axis intercept (b) was never significantly different from zero. Therefore the simplest formula y = mx was used to describe the relationship between VCR and MA data across LGAs. Using the OLS regression method, the standard uncertainty in the gradient (u(m)) and the Pearson’s correlation coefficient squared (R2) values were obtained.\n\nIncidence rates were calculated for each LGA with ABS data. The mean difference in incidence rates between the following ‘pairs’ were calculated across LGAs: VCR vs. MA incidence for each of the study years between 2004 and 2008; VCR vs. MA total population; VCR vs. MA incidence for males and females; and VCR vs MA incidence for each of the stratified age group: 0–19, 20–29, 30–39, 40–49, 50–59, 60–69, 70–79 and >80.\n\nThe paired sample t-test was used to test whether the mean difference in incidence between each of these pairs was greater than zero. The mean difference for each VCR-MA pair calculated and the 95% confidence interval for each pair was produced. A 2-tail significance test was performed to determine if the mean difference was statistically significant. All statistical analysis was carried out by IBM SPSS v.20.\n\n\nResults\n\nA total of 15,150 unique patients with malignant melanoma and in situ melanomas were registered with the VCR between 2004 and 2008. During the same time period, MA was billed for 13,886 patients requiring melanoma treatment services. During the study period, the number of unique cases registered remained relatively stable for both datasets.\n\nDuring preliminary analysis it was noted that the number of melanoma cases did not correlate closely between both datasets in the 80+ year age group. Table 1 summarises the number of unique cases registered with VCR and MA resolved by gender and including and excluding patients in the 80+ year age group. Comparisons of both datasets stratified by age group revealed that the 80+ year age group was a clear visible outlier (Figure 1). The standard of uncertainty was significant at ± 5% (u(m) ±0.046). Despite the inclusion of the 80+ year age group, the correlation between both datasets remained relatively high at 94% (i.e R²= 0.936). The regression in the population stratified by age group after the exclusion of the 80+ year age group demonstrated an improvement to 99% correlation between both datasets.\n\nNumber of unique patients for all age groups and excluding the 80+ year age group are listed.\n\n(a) including all patients (b) excluding patients >80 years old.\n\nWhen stratified by LGA, comparisons of the VCR and MA datasets showed a close mapping when OLS regression was performed using both datasets in their entirety and when stratified by gender (Figure 2). The gradient remained close to unity (m=0.976 for the total dataset, m=0.905 for males and m=0.972 for females) with only a small standard of uncertainty (≈ u(m)±0.01). The R² values of 0.977, 0.979 and 0.967 respectively indicated a 97% correlation between the two datasets when all unique patients and cases by males and females were compared.\n\n(a) all patients (b) males (c) females.\n\nStatistical analysis was repeated on the entire dataset after excluding the 80+ year age group. Comparisons of the VCR and MA datasets after the exclusion of the 80+ year age group demonstrated a closer association between both datasets (Figure 3). The gradient (m=0.985) showed a closer degree of equivalence between the two datasets after this exclusion (Figure 3). The R² were largely unaffected by the exclusion of the age group demonstrating that the correlation was high in both the male and female populations, regardless of the exclusion of the 80+ age group.\n\n(a) all patients (b) males (c) females.\n\nThe mean incidence data for the age groups 20–29, 30–39, 40–49 and 60–69 showed no statistical significance in the mean difference. The magnitude of mean difference for the remaining pairs was relatively low for patients in the 0–19, 50–59 and 70–79 year age group (Figure 4).\n\nEach data point represents the difference between the VCR and MA mean values for melanoma incidence. The uncertainty bars represent the 95% confidence interval.\n\n\nDiscussion\n\nExamining the ascertainment level MA MBS billing data for melanoma treatment is one step towards exploiting the vast body of epidemiological information collected on skin cancers by MA. In addition it has the potential for exploring other diseases which do not have mandatory reporting. The MA and VCR databases were examined looking at melanoma skin cancer. The findings of this study illustrate that the number of cases picked up by MA is comparable to the number of melanomas (malignant and in situ) registered with the VCR in patients above the age of 19 and below the age of 80 years old. Incidence values were also found to be similar in both datasets. This suggests that MA data may potentially be useful in examining melanoma trends. Furthermore, the findings represent the possibility of using this billing data to examine other types of skin cancer trends in Australia.\n\nThis study employed the use of VCR data, a large dataset for melanomas in Victoria. Mandatory reporting of melanomas is required in Australia and incidence and patterns are almost completely captured by population-based cancer registries26. The large sample size of this dataset and variety of parameters (gender, age and LGA) strengthens the evaluation of the MA dataset. Overall, both the VCR and MA datasets showed a correlation when compared by year, gender and age. The 80+ year age group however was identified as a clear outlier. A potential reason for the discrepancy is the number of patients over the age of 80 who are billed by the Department of Veterans Affairs (DVA). Veterans, members of the Australian Defence Force and their spouses are eligible for a DVA health card which pays benefits for health care, pharmaceutical therapies and travel29. Information regarding melanoma benefits paid by DVA was not included in the MA dataset.\n\nThis study is limited by the broad definitions of the MA item numbers. The melanoma item numbers used in this study covered the excision of the following: malignant melanomas, in situ melanomas (listed as Hutchinson’s freckle), appendageal carcinomas, malignant fibrous tumours of skin and Merkel cell carcinomas (MCC)27. However the incidence rates of appendageal carcinomas, malignant fibrous tumours and MCC are low as these cancers are relatively rare30,31. Whilst no data exists on the exact figures of MCC Australia wide or within Victoria, estimates of 1 per 105 men and 0.63 per 105 women have been produced in a study in Western Australia30. These low rates would not significantly affect the statistical results of this study and consequently the comparison between the MA item numbers and the VCR data was justified. However, the rates of MCC, fibrous tumours, malignant fibrous tumours and appendage carcinomas rise with age30. In patients above the age of 85 the rates of MCC are much higher than the general population, occurring in 15.5 in 105 people30. The increase in these rarer skin cancers in the older age groups may also explain the discrepancy in the 80+ year age group. The analysis was conducted after excluding the 80+ year age group and this yielded a higher correlation. Furthermore, while melanomas are required to be histologically confirmed by MA, NMSC are not. GPs are required to obtain histological confirmation, in the case of an excision. Specialists, however, are permitted to bill MA without histological confirmation. This could potentially result in an over-estimate of lesions. Similarly, lesions that are treated with cryotherapy do not require histological verification, and this may also have resulted in an over-estimation by both GPs and specialists.\n\nSince reporting of melanoma is mandatory by law, the data captured by cancer registries is therefore assumed to be sufficiently accurate to use for comparison purposes. Several epidemiological studies examining cancer databases compared capture rates to central cancer registries to determine ascertainment levels32,33. There are no figures published on the completeness of the VCR data. However, a study on the National Cancer Registry in Britain, which used a two-source capture-recapture method to estimate the number of cases in Britain and the fraction of registered patients, determined that the registry was 97–98% complete for melanoma registration; the assumption is that the capture rate in Australia would be similar32.\n\nMA has the potential to be used in order to examine NMSC trends. This potential could be explored further. NMSC are captured by registry within Tasmania and there is possibility of comparing MA data to NMSC registry data within Tasmania to further establish the ascertainment of MA1. There is a paucity of data on incidence trends in pre-cancerous lesions such as actinic keratosis (AK)34. Analysis of item numbers pertaining to the treatment of AK may potentially provide insight into these rates34. Furthermore, longitudinal analysis of these lesions may help identify whether the introduction of newer agents including field treatments are more cost effective in treating AK.\n\n\nConclusion\n\nThe current study explores the capture rate of MA for determining melanoma rate. The findings suggest that despite the inclusion of outlying patient groups, MA rebate data correlates closely with VCR data when assessing incidence calculation and other epidemiological measures for melanoma. NMSCs are not currently required to be reported to Australian cancer registries and thus use of such data may be used to capture NMSC cases, which represents a cost-effective method to establish trends. Longitudinal studies examining incidence trends and trends in residual and recurrent NMSC over several decades can examine the effectiveness of public health campaigns and consequential savings for future governments.\n\n\nData availability\n\nRaw datasets for MA and VCR are not available as they contain confidential information that cannot be deidentified. This data can be obtained by applying to the registry (MA or VCR) for access.\n\nSome data about melanoma for the specific time period analysed is available from: http://medicarestatistics.humanservices.gov.au/statistics/mbs_item.jsp. This data includes the number of times an item number is billed, but does not provide unique identifiers (such as date of birth or location).",
"appendix": "Author contributions\n\n\n\nEP: drafting of manuscript, data cleaning and analysis; NG: drafting of manuscript; MP: drafting of manuscript, review of analysis; RS: supervisor of project and review of manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKaldor J, Shugg D, Young B, et al.: Non-melanoma skin cancer: ten years of cancer-registry-based surveillance. Int J Cancer. 1993; 53(6): 886–91. PubMed Abstract | Publisher Full Text\n\nFransen M, Karahalios A, Sharma N, et al.: Non-melanoma skin cancer in Australia. Med J Aust. 2012; 197(10): 565–8. PubMed Abstract | Publisher Full Text\n\nStaples MP, Elwood M, Burton RC, et al.: Non-melanoma skin cancer in Australia: the 2002 national survey and trends since 1985. Med J Aust. 2006; 184(1): 6–10. PubMed Abstract\n\nStaples M, Marks R, Giles G: Trends in the incidence of non-melanocytic skin cancer (NMSC) treated in Australia 1985–1995: are primary prevention programs starting to have an effect? Int J Cancer. 1998; 78(2): 144–8. PubMed Abstract | Publisher Full Text\n\nMarks R, Staples M, Giles GG: Trends in non-melanocytic skin cancer treated in Australia: the second national survey. Int J Cancer. 1993; 53(4): 585–90. PubMed Abstract | Publisher Full Text\n\nGiles GG, Marks R, Foley P: Incidence of non-melanocytic skin cancer treated in Australia. Br Med J (Clin Res Ed). 1988; 296(6614): 13–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGreen A, Beardmore G, Hart V, et al.: Skin cancer in a Queensland population. J Am Acad Dermatol. 1988; 19(6): 1045–52. PubMed Abstract | Publisher Full Text\n\nGoodman GJ, Marks R, Selwood TS, et al.: Non-melanotic skin cancer and solar keratoses in Victoria--clinical studies II. Australas J Dermatol. 1984; 25(3): 103–6. PubMed Abstract | Publisher Full Text\n\nMarks R, Ponsford MW, Selwood TS, et al.: Non-melanotic skin cancer and solar keratoses in Victoria. Med J Aust. 1983; 2(12): 619–22. PubMed Abstract\n\nMarks R, Jolley D, Dorevitch AP, et al.: The incidence of non-melanocytic skin cancers in an Australian population: results of a five-year prospective study. Med J Aust. 1989; 150(9): 475–8. PubMed Abstract\n\nSilverstone H, Campbell CB, Hosking CB, et al.: Regional studies in skin cancer. First report: North-Western Queensland. Med J Aust. 1963; 50(1): 312–5. PubMed Abstract\n\nCarmichael GG, Silverstone H: The epidemiology of skin cancer in Queensland: the incidence. Br J Cancer. 1961; 15: 409–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarmichael GG: The epidemiology of skin cancer in Queensland: the significance of premalignant conditions. Br J Cancer. 1961; 15: 425–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStenbeck KD, Balanda KP, Williams MJ, et al.: Patterns of treated non-melanoma skin cancer in Queensland--the region with the highest incidence rates in the world. Med J Aust. 1990; 153(9): 511–5. PubMed Abstract\n\nKricker A, English DR, Randell PL, et al.: Skin cancer in Geraldton, Western Australia: a survey of incidence and prevalence. Med J Aust. 1990; 152(8): 399–407. PubMed Abstract\n\nKricker A, Armstrong BK, Parkin DM: Measurement of skin cancer incidence. Health Rep. 1993; 5(1): 63–6. PubMed Abstract\n\nEnglish DR, Kricker A, Heenan PJ, et al.: Incidence of non-melanocytic skin cancer in Geraldton, Western Australia. Int J Cancer. 1997; 73(5): 629–33. PubMed Abstract | Publisher Full Text\n\nGreen A, Battistutta D, Hart V, et al.: Skin cancer in a subtropical Australian population: incidence and lack of association with occupation. The Nambour Study Group. Am J Epidemiol. 1996; 144(11): 1034–40. PubMed Abstract\n\nRaasch BA, Buettner PG: Multiple nonmelanoma skin cancer in an exposed Australian population. Int J Dermatol. 2002; 41(10): 652–8. PubMed Abstract | Publisher Full Text\n\nRichmond-Sinclair NM, Pandeya N, Ware RS, et al.: Incidence of basal cell carcinoma multiplicity and detailed anatomic distribution: longitudinal study of an Australian population. J Invest Dermatol. 2009; 129(2): 323–8. PubMed Abstract | Publisher Full Text\n\nSaint-Yves IF, Honari M: Skin cancers in Australia's Northern Territory 1981–85. J R Soc Health. 1988; 108(2): 69–74. PubMed Abstract | Publisher Full Text\n\nOlsen CM, Williams PF, Whiteman DC: Turning the tide? Changes in treatment rates for keratinocyte cancers in Australia 2000 through 2011. J Am Acad Dermatol. 2014; 71(1): 21–6.e1. PubMed Abstract | Publisher Full Text\n\nCooper GS, Yuan Z, Jethva RN, et al.: Use of Medicare claims data to measure county-level variation in breast carcinoma incidence and mammography rates. Cancer Detect Prev. 2002; 26(3): 197–202. PubMed Abstract | Publisher Full Text\n\nCooper GS, Yuan Z, Stange KC, et al.: The sensitivity of Medicare claims data for case ascertainment of six common cancers. Med Care. 1999; 37(5): 436–44. PubMed Abstract | Publisher Full Text\n\nMcBean AM, Babish JD, Warren JL: Determination of lung cancer incidence in the elderly using Medicare claims data. Am J Epidemiol. 1993; 137(2): 226–34. PubMed Abstract\n\nAIHW & AACR: Cancer in Australia: an overview 2012. Canberra, Australia: Australian Institute of Health and Welfare. Contract No.: Cat.no. CAN70, 2012. Reference Source\n\nMedicare Australia: MBS Online Medicare Benefits Schedule. 2014. Reference Source\n\nAustralian Bureau of Statistics: Australia's Population. 2014. Reference Source\n\nDepartment of Veterans Affairs: Benefits & Services. 2014. Reference Source\n\nGirschik J, Thorn K, Beer TW, et al.: Merkel cell carcinoma in Western Australia: a population-based study of incidence and survival. Br J Dermatol. 2011; 165(5): 1051–7. PubMed Abstract | Publisher Full Text\n\nLaureano A, Cunha D, Pernandes C, et al.: Dermoscopy in Merkel cell carcinoma: a case report. Dermatol Online J. 2014; 20(2): pii: doj_21543. PubMed Abstract\n\nKroll ME, Murphy MF, Carpenter LM, et al.: Childhood cancer registration in Britain: capture-recapture estimates of completeness of ascertainment. Br J Cancer. 2011; 104(7): 1227–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreeman JL, Zhang D, Freeman DH, et al.: An approach to identifying incident breast cancer cases using Medicare claims data. J Clin Epidemiol. 2000; 53(6): 605–14. PubMed Abstract | Publisher Full Text\n\nPerera E, McGuigan S, Sinclair R: Cost for the treatment of actinic keratosis on the rise in Australia [version 2; referees: 2 approved]. F1000Res. 2014; 3: 184. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11331",
"date": "03 Dec 2015",
"name": "Mohamed Badawy Hassan Tawfik Abdel-Naser",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is well designed and the results are convincing.The authors mentioned that the time period of data collection is from 2004 to 2008 which means 5 years (year 2008 is included). However in the table and figures the mentioned period was from 2004 to 2007 which means 4 year. Perhaps authors need to clarify this issue.",
"responses": []
},
{
"id": "12365",
"date": "11 Feb 2016",
"name": "Herbert Hönigsmann",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 is well-written and appears sound. It is quite difficult to find appropriate statistic material on skin cancers in many countries. I see no problems.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1341
|
https://f1000research.com/articles/4-1339/v1
|
24 Nov 15
|
{
"type": "Research Article",
"title": "Maternal age and intracytoplasmic sperm injection outcome in infertile couples at Khartoum, Sudan",
"authors": [
"Mohamed Ahmed",
"Osama Shareef",
"Ishag Adam",
"Duria Rayis",
"Mohamed Ahmed",
"Osama Shareef",
"Duria Rayis"
],
"abstract": "BackgroundIntracytoplasmic sperm injection (ICSI) was considered as the mainstay of treatment for male infertility. Nowadays, the scope of ICSI has been widened to include other causes of infertility. There are few published data on ICSI in countries with low incomes.AimsA cross-sectional study was conducted at Saad AbuAlla and Banoun Centers, Khartoum, Sudan to investigate outcomes of ICSI and to determine the parameters that might predict pregnancy success rate following ICSI.MethodsThe study included 191 infertile couples who underwent 296 ICSI cycles between 1st April 2013 and 31 March 2014.ResultsOne hundred and ninety one couples (comprising 296 cycles of ICSI) were enrolled to the study. The mean (SD) number of retrieved oocytes was 9.7 (7.5). The mean (SD) number of transferred embryos was 2.9 (1.0). Out of these, 50 (26.2%) and 40 (20.9%) had chemical and clinical pregnancy, respectively. Thirty–six couples (18.8%) and five couples (2.6%) had miscarriage and had ectopic pregnancy, respectively. Under logistic regression, younger age (OR = 0.8, 95% CI= 0.81 ─ 0.96, P = 0.004) and endometrial thickness (OR = 1.3, 95% CI= 1.07─1.60, P = 0.009) were the significant predictors for the success of ICSI in inducing pregnancy.Conclusion\n\nThe rates of successful fertilisation and pregnancy-to-term rates in this setting depend mainly on the maternal age.",
"keywords": [
"age",
"ICSI",
"intracytoplasmic sperm injection",
"infertility",
"Sudan"
],
"content": "Introduction\n\nIn vitro fertilization (IVF) is recognized as the last treatment option for infertile couples who want biological children, and has been widely accepted as the most important and efficient treatment for infertility (Khalaf et al., 2008). Intracytoplasmic sperm injection (ICSI) is the gold-standard technique for the treatment of male factor infertility (Oehninger et al., 2002). However, ICSI or IVF is also recommended to patients with tubal factor infertility (Staessen et al., 1999), as well as treatment of infertile couples with unexplained infertility and some polycystic ovary syndrome (PCOS) cases (Van der Westerlaken et al., 2005; Youn et al., 2011). Unfortunately due to the high cost, IVF/ICSI services are not widely available at both public and private health institutions in developing countries (ESHRE, 2008). However, in countries with lower incomes, the utility of infertility treatment is not well-established and there are few existing private IVF/ICSI centers, and those that exist are associated with a high cost; beyond the reach of most couples (Giwa-Osagie, 2004; Otubu et al., 2006). Because ICSI has a high cost to both the treatment-seeking couple and the health care system, it is necessary to assess its efficacy in different settings. Research in the IVF/ICSI field is of importance for both the treating physicians and the healthcare policy makers and will yield data necessary for patients' counseling. Different success rates/outcomes of ICSI have also been observed in different settings. There are few published data on the outcome of ICSI in countries with low income and there is no published data on ICSI in Sudan. The current study was conducted at Khartoum, Sudan to investigate ICSI outcome and to determine the parameters that might predict pregnancy success rate resulting from ICSI. Different causes of infertility, and both male and female infertility were observed in Sudan (Elussein et al., 2008).\n\n\nMethods\n\nA cross-sectional study was conducted during the period of 1st April 2013 through to 31 March 2014 at Saad AbuAlla and Banoun Centers, Khartoum, Sudan to investigate ICSI outcome and to determine the parameters that might predict pregnancy success rate following ICSI.\n\nAfter signing an informed consent form, a questionnaire was used to gather information about age, parity, menstrual history, duration of infertility, type of infertility (male infertility, failure of ovulation, tubal infertility, unexplained infertility, endometriosis and PCO), cause of infertility, number of previous cycle, endometrial thickness, number of embryos transferred, and the outcome of ISCI (pregnancy rate, rate of miscarriage and ectopic pregnancy).\n\nCouples where males had testicular atrophy, and/or females had uterine anatomical abnormalities, were aged > 44 years, had experience uterine fibroids and/or ICSI failure more than three times were excluded from the study.\n\nIn female participants, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were measured on day 3 of the cycle; preceding ovarian stimulation which was performed followed the short GnRH agonist protocol (Ergenoğlu et al., 2012).\n\nAfter the workup was done (physical examination, blood group, complete hemogram, viral screening for HIV, HBV and HCV) in the previous cycles, pituitary down-regulation started on the second day of the cycle by daily subcutaneous injection of gonadotrophins and continued until ≥ 3 follicles were present that measured ≥ 17 mm when a 10,000 IU dose of human chorionic gonadotrophin (hCG) was given. Oocyte pickup was scheduled 34–35 hours after the hCG injection. The dose of hCG ranged between 150–450 IU, depending upon the patient's age, and in response to ovarian stimulation in previous ICSI procedures. Transvaginal ultrasound was done on the day of stimulation to exclude ovarian cysts, and on cycle day seven and every other day to monitor follicle size. E2 (17 beta-estradiol) level was measured on cycle day two and when follicle maturation was achieved. In poor respondents, stimulation was stopped at 20th day of the cycle.\n\nOvum pickup was done under general anesthesia using a laryngeal mask airway using propofol lipuro 1% (10 mg/ml) 20 ml IV, plus atropine 0.5 mg IV, plus 4 mg dexamethasone as needed to prevent laryngeal spasm, in addition to the anesthetic gas, Nitrous oxide. Fentanyl IV was given as analgesic. Follicles were flushed using flush media from Origio (SynVitro™ Flush, Denmark) using a double lumen needle from (Origio®) if the number of follicles was ≤ 5; otherwise, a single lumen needle from (Wallace®, Wallace Ltd, Colchester, England) was used, without flushing. Embryo transfer was done without anesthesia or sedation using a soft catheter from (Wallace®). Briefly, under sterile condition, vaginal parts were cleaned with saline and draped and a Cusco speculum inserted to expose cervix. Cervical mucus was aspirated. The embryos were deposited in uterine cavity under ultrasound guidance at a position approximately 1cm shorter than the fundus. The catheter was then checked under a dissecting microscope for retained embryos. If these were found, they were reloaded and transferred again (repeat transfer). The patients were asked to remain in bed for 15–30 min following transfer.\n\n\nStatistics\n\nThe data were entered into computer using SPSS for Windows version 16.0. The mean (SD) of the ICSI variables (age and BMI) were compared between the women who had clinical pregnancy and women who had not using a Student’s t-test. These variables were compared between the different age groups using one–way ANOVA for continuous variables and Pearson’s chi-squared (X2) test for the proportions of the pregnancy rate, ectopic pregnancies and miscarriage. Logistic regression was performed where induction of clinical pregnancy was the dependent variable and the ICSI variables (age, type and duration of infertility, endometrial thickness and the number of oocytes retrieved and their stage of maturation) were the independent variables. A P value < 0.05 was considered significant.\n\n\nEthics\n\nThe study received ethical clearance from the Research Board at Department of Obstetrics and Gynecology, Faculty of Medicine, University of Khartoum, Sudan.\n\n\nResults\n\nOne-hundred and ninety-one couples were enrolled to the study, comprising 296 total cycles of ICSI. Out of these 191 couples; 82 (42.9%), 48 (25.1%), seven (3.7%) and 54 (28.3%) had male, female, combined and unexplained infertility, respectively. The vast majority (160; 83.8%) of these 191 couples had primary infertility (failure to achieve pregnancy after one year of unprotected intercourse) and the rest (31; 16.2%) had secondary infertility (failure to achieve pregnancy after one year of unprotected intercourse with previous pregnancy(ies) regardless of its outcome). The mean (SD) duration of infertility was 6.6 (4.4) years. Maternal age range was 18–44 years and the mean (SD) was 32.7 (6.2) years.\n\nThe mean (SD) number of retrieved oocytes was 9.7 (7.5). The mean (SD) number of transferred embryos was 2.9 (1.0). The number of retrieved, fertilized oocytes and the transferred embryos was significantly higher in women with age < 30 years (Table 1; Figure 1).\n\nOut of these 50 (26.2%) and 40 (20.9%) had chemical and clinical pregnancy, respectively. Thirty-six (18.8%) and five (2.6%) had miscarriage and ectopic pregnancy, respectively. The rate of induction of pregnancy was significantly higher in women of < 30 years of age (Table 2; Figure 2).\n\nWhile the mean (SD) of the age [29.8 (4.7) vs. 33.5 (6.3) years, P = 0.001] was significantly higher, the endometrial thickness [11.1 (2.2) vs. 10.2 (1.7) mm, P = 0.005] was significantly higher in the women with clinical pregnancy (n=40) than in women who had no pregnancy (n=151). Seventeen (42.05%) out of the 40 couples who experienced successful ICSI had male factor infertility, whereas 65 couples (43.0%; P = 0.767) in which ICSI were unsuccessful had male factor infertility (Table 3).\n\nIn logistic regression, younger age (OR = 0.8, 95% CI = 0.81–0.96, P = 0.004) and endometrial thickness (OR = 1.3, 95% CI = 1.07–1.60, P = 0.009) were the significant predictors for the success of ICSI treatment (Table 4). Raw dataset available in Dataset 1.\n\n\nDiscussion and conclusions\n\nThe main findings of the current study were that the number of eggs retrieved, fertilized ovum, the number of embryos successfully transferred and the rate of successful induction of pregnancy depend on age of the woman and endometrial thickness. The pregnancy rate (20.9%) in this study was lower than the rates recently reported in Nigeria (30%; Orhue et al., 2012); Tunisia (32.4%; Fourati et al., 2009), Vienna, Austria (27.3%; Nouri et al., 2015) and in Singapore (Tan et al., 2014). It is worth mentioning, however, that all of these studies (with exception of Fourati et al.) report the pregnancy rate following IVF/ICSI and not the rate following ICSI alone, as in our study.\n\nIn the current study, ICSI outcomes such as eggs retrieved, fertilized ovums, embryos transferred and the rate of successful induction of pregnancy depend primarily on age of the woman, where the optimal outcomes were observed in women < 30 years of age. This is consistent with Tan et al.’s (2014) findings where optimal IVF outcomes (the number of oocytes retrieved) was highest among women aged < 30 years, with a reduced number of oocytes retrieved per cycle, lower pregnancy and live birth rates seen among women of older age groups. Likewise, Nouri et al. (2015) observed that age was an independent factor for pregnancy rate following IVF/ICSI. The decreasing ovarian reserve (Speroff, 1994), poor oocyte quality (Simpson et al., 2000), higher embryo implantation failure (Navot et al., 1991), ovulatory dysfunction due to poor hormonal environment (Hull et al., 1996; Sherman et al., 1976) and uterine problems (Faddy et al., 1992; Scwartz & Mayaux, 1982) were the postulated effects of the aging process that could have a detrimental effect on the efficacy of IVF/ICSI.\n\nIn the current study the pregnancy rate was associated with endometrial thickness. This agrees with the several previous studies which have shown a significant correlation between pregnancy rate and endometrial thickness (Al-Ghamdi et al., 2008; Kasius et al., 2014; Okohue et al., 2009). Endometrial thickness <7 mm was reported to have a significant reduction in the implantation rate and pregnancy rate. It has recently been shown that (systematic review and meta-analysis) probability of clinical pregnancy for an endometrial thickness ≤7 mm was significantly lower compared with cases with endometrial thickness >7 mm which investigated for pregnancy outcomes after IVF (Kasius et al., 2014).\n\nWe conclude that the fertilization and pregnancy rates in this setting depend mainly on maternal age.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for Ahmed et al., 2015 'Maternal age and intracytoplasmic sperm injection outcome in infertile couples at Khartoum, Sudan', 10.5256/f1000research.7386.d107727",
"appendix": "Author contributions\n\n\n\nMAA - data collection, laboratory work, manuscript preparation. OS - study design, data analysis, and manuscript preparation. IA - data collection, data analysis. DAR- data collection, manuscript preparation. All authors have read and approved the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no funding was involved in supporting this work.\n\n\nSupplementary materials\n\nQuestionnaire for collection of ICSI data.\n\nAfter signing an informed consent form, the questionnaire was used to gather information about age, parity, menstrual history, duration of infertility, type of infertility (male infertility, failure of ovulation, tubal infertility, unexplained infertility, endometriosis and PCO), cause of infertility, number of previous cycle, endometrial thickness, number of embryos transferred, and the outcome of ISCI (pregnancy rate, rate of miscarriage and ectopic pregnancy).\n\nClick here to access the data.\n\n\nReferences\n\nAhmed M, Shareef O, Adam I, et al.: Dataset 1 in: Maternal age and intracytoplasmic sperm injection outcome in infertile couples at Khartoum, Sudan. F1000Research. 2015. Data Source\n\nAl-Ghamdi A, Coskun S, Al-Hassan S, et al.: The correlation between endometrial thickness and outcome of in vitro fertilization and embryo transfer (IVF-ET) outcome. Reprod Biol Endocrinol. 2008; 6: 37–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nElussein EA, Magid YM, Omer MM, et al.: Clinical patterns and major causes of infertility among Sudanese couples. Trop Doct. 2008; 38(4): 243–4. PubMed Abstract | Publisher Full Text\n\nErgenoğlu MA, Yeniel AÖ, Akdoğan A, et al.: The effects of GnRH analogs on serum and follicular fluid leptin levels and pregnancy outcomes in short protocols of assisted reproductive technology. J Turk Ger Gynecol Assoc. 2012; 13(2): 91–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nESHRE Task Force on Ethics and Law, Pennings G, de Wert G, et al.: ESHRE Task Force on Ethics and Law 14: equity of access to assisted reproductive technology. Hum Reprod. 2008; 23(4): 772–4. PubMed Abstract | Publisher Full Text\n\nFaddy MJ, Gosden RG, Gougeon A, et al.: Accelerated disappearance of ovarian follicles in mid-life: implications for forecasting menopause. Hum Reprod. 1992; 7(10): 1342–6. PubMed Abstract\n\nFourati S, Chaker A, Fadhlaoui A, et al.: [A retrospective study of 339 ICSI cycles: assesment of the two first years of activity of the Assisted Reproductive Technology Center of Aziza Othmana Hospital]. Tunis Med. 2009; 87(3): 173–9. PubMed Abstract\n\nGiwa-Osagie OF: The need for infertility services in the developing world: the WHO point of view. Gynecol Obstet Invest. 2004; 57(1): 58–67. PubMed Abstract\n\nHull MG, Fleming CF, Hughes AO, et al.: The age-related decline in female fecundity: a quantitative controlled study of implanting capacity and survival of individual embryos after in vitro fertilization. Fertil Steril. 1996; 65(4): 783–90. PubMed Abstract\n\nKasius A, Smit JG, Torrance HL, et al.: Endometrial thickness and pregnancy rates after IVF: a systematic review and meta-analysis. Hum Reprod Update. 2014; 20(4): 530–541. PubMed Abstract | Publisher Full Text\n\nKhalaf Y, El-Toukhy T, Coomarasamy A, et al.: Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and postintervention study. BJOG. 2008; 115(3): 385–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNavot D, Bergh PA, Williams MA, et al.: Poor oocyte quality rather than implantation failure as a cause of age-related decline in female fertility. Lancet. 1991; 337(8754): 1375–7. PubMed Abstract | Publisher Full Text\n\nNouri K, Tempfer CB, Walch K, et al.: Predictive value of the time interval between embryo loading and transfer for IVF/ICSI success: a prospective cohort study. Reprod Biol Endocrinol. 2015; 13: 51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOehninger S, Gosden RG: Should ICSI be the treatment of choice for all cases of in-vitro conception? No, not in light of the scientific data. Hum Reprod. 2002; 17(9): 2237–2242. PubMed Abstract | Publisher Full Text\n\nOkohue JE, Onuh SO, Ebeigbe P, et al.: The effect of endometrial thickness on in vitro fertilization (IVF)-embryo transfer/intracytoplasmic sperm injection (ICSI) outcome. Afr J Reprod Health. 2009; 13(1): 113–21. PubMed Abstract\n\nOrhue AA, Aziken ME, Osemwenkha AP, et al.: In vitro fertilization at a public hospital in Nigeria. Int J Gynaecol Obstet. 2012; 118(1): 56–60. PubMed Abstract | Publisher Full Text\n\nOtubu JAM: Infertility. In: Agboola A editor, Textbook of Obstetrics and Gynecology for Medical Students. Ibadan, Heineman Educational Books, 2006; 128–38.\n\nScwartz D, Mayaux MJ: Female fecundity as a function of age: results of artificial insemination in 2193 nulliparous women with azoospermic husbands. Federation CECOS. N Engl J Med. 1982; 306(7): 404–6. PubMed Abstract | Publisher Full Text\n\nSherman BM, West JH, Korenman SG: The menopausal transition: analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women. J Clin Endocrinol Metab. 1976; 42(4): 629–36. PubMed Abstract | Publisher Full Text\n\nSimpson JL, Lobo RA, Kelsey J, et al.: Genetic programming in ovarian development and oogenesis. Menopause: biology and pathobiology. San Diego, Academic Press. 2000; 77–94. Publisher Full Text\n\nSperoff L: The effect of aging on fertility. Curr Opin Obstet Gynecol. 1994; 6(2): 115–20. PubMed Abstract | Publisher Full Text\n\nStaessen C, Camus M, Clasen K, et al.: Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen. Hum Reprod. 1999; 14(10): 2474–2479. PubMed Abstract | Publisher Full Text\n\nTan TY, Lau SK, Loh SF, et al.: Female ageing and reproductive outcome in assisted reproduction cycles. Singapore Med J. 2014; 55(6): 305–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan der Westerlaken L, Helmerhorst F, Dieben S, et al.: Intracytoplasmic sperm injection as a treatment for unexplained total fertilization failure or low fertilization after conventional in vitro fertilization. Fertil Steril. 2005; 83(3): 612–617. PubMed Abstract | Publisher Full Text\n\nYoun JS, Cha SH, Park CW, et al.: Predictive value of sperm motility characteristics assessed by computer-assisted sperm analysis in intrauterine insemination with superovulation in couples with unexplained infertility. Clin Exp Reprod Med. 2011; 38(1): 47–52. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "12588",
"date": "10 Mar 2016",
"name": "Frank H de Jong",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, results of an ICSI programme in Khartoum, Sudan are reported. The amount of new information is limited: as indicated in the discussion, a host of published papers already concluded that maternal age is an important factor determining the success of ICSI or IVF procedures.The manuscript might be improved on the following aspects:There is hardly information on the male partners. Data on hormone levels and sperm quality might be as important as the data on hormone levels, endometrial thickness and age in the female partners. There is no information on sperm retrieval, ICSI procedure or embryo culture. On page 3, right-hand column, line 6 \"HCG\"should probably read \"FSH\". What happens when pregnancy results are normalized for numbers of embryos transferred? Can the difference in numbers of pregnancies in the different age groups be explained on basis of this number? It might be better to replace \"ovum\" (line 2 under Discussion and Conclusions) and \"ovums\" (line 2 in right-hand column on page 6) with \"ova\".",
"responses": []
},
{
"id": "13995",
"date": "26 May 2016",
"name": "Akmal El-Mazny",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title is appropriate for the content of the article, and the abstract represents a suitable summary of the work. The design, methods, and analysis of the results from the study have been explained and are appropriate for the study. The conclusions are sensible, balanced, and justified on the basis of the results, and enough information has been provided in a usable format to be able to replicate the study.\nRecommendations:\nMethods:\nAdequately describe inclusion and exclusion criteria. Mention appropriate details of ovarian stimulation and ICSI technique.\n\nResults:\nThe text should complement the tables and figures (do not duplicate). Clarify the effect size for the main outcome.\n\nDiscussion:\nMention the differences from other studies. Mention the limitations of the study. Suggest clinical implications.",
"responses": []
},
{
"id": "14119",
"date": "14 Jun 2016",
"name": "Silke J. Dyer",
"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 giving us the opportunity to review this article. The authors report on female factors impacting outcome and pregnancy success rate following ICSI for treatment of infertility in a low income setting.\nWe congratulate and thank the authors on publishing findings on the effectiveness of ART in their country, as data pertaining to the availability, utilization, effectiveness and safety of ART in sub-Saharan Africa are overall scant and much needed.\nThe paper is generally well written. Data are presented clearly, and the tables and illustrations are relevant. The study design is appropriate, and the analysis and interpretation of data are overall sound. The conclusions derived are balanced and justified. Although the main finding, especially in relation to age is not novel, the paper contributes relevant information on the status of ART in Sudan.\n\nThe paper could be further strengthened if (1) information on biochemical pregnancies was excluded and simply considered under “not pregnant”; (2) data on live births and on complications of ART especially multiple pregnancies were included or, if not available, acknowledged as a study limitation; (3) more information on semen quality and prevalence of severe male factor infertility in the study group were presented; (4) a section on study strengths and limitations was included.\n\nWe have the following additional major and minor observations/ suggestions in order of appearance in the manuscript:\nMajor:\nAccording to current evidence ICSI should be considered first line intervention for severe male factor infertility. The use for other indications is of no proven benefit according to our understanding of current evidence, although we acknowledge that it is used for other indications as indeed in the index study (see ICMART World report 2008-2009-2010 and accompanying Editorial). The authors may wish to stay away from a discussion on the role and indications of ICSI in the introduction as in our opinion it weakens the paper; a comment under study limitations may suffice. Instead, it would be of interest to briefly expand on the current situation of ART in Sudan in the introduction.\n\nIn table 3, number of eggs fertilized and numbers of eggs transferred also differed between the two groups (p<0.05). It is not clear if these variables were included in the regression analysis- and if not, why. Either way it justifies some attention in both the result and discussion section.\n\nMinor:\nThe title is somewhat misleading and could be rephrased. Perhaps “Factors predicting pregnancy following ART with ICSI in Sudan” or similar would capture the study better?\n\nThe conclusion in the abstract is not strictly based on the results as no information pertaining to fertilisation are presented in the abstract.\n\nPage 3 column 2; line 1 and 2 should be revised. At present the reader gets the impression that gonadotrophins rather than GnRH agonists are used for pituitary down-regulation. In ‘The dose of hCG ranged between 150 – 450IU’ presumably FSH and not hCG is meant. In addition, the entire section on methods could perhaps be shortened, at least if we assume that readers are familiar with the basic principles of ART.\n\nPresumably “pregnancy” in table 2 and figure 2 refers to “clinical pregnancies’ only. If the authors follow our recommendation to include biochemical pregnancies under “not pregnant” no changes are required; otherwise an asterix in both the table and figure is needed to specify clinical versus biochemical pregnancy.\n\nThe choice of age groups is not in keeping with age categories used by regional/international data registries. This limits valuable comparisons. It would be advantageous to consider these age categories in future work.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1339
|
https://f1000research.com/articles/4-1160/v1
|
28 Oct 15
|
{
"type": "Opinion Article",
"title": "Quality of histone modification antibodies undermines chromatin biology research",
"authors": [
"Goran Kungulovski",
"Albert Jeltsch",
"Goran Kungulovski"
],
"abstract": "Histone post-translational modification (PTM) antibodies are essential research reagents in chromatin biology. However, they suffer from variable properties and insufficient documentation of quality. Antibody manufacturers and vendors should provide detailed lot-specific documentation of quality, rendering further quality checks by end-customers unnecessary. A shift from polyclonal antibodies towards sustainable reagents like monoclonal or recombinant antibodies or histone binding domains would help to improve the reproducibility of experimental work in this field.",
"keywords": [
"histone modifications",
"antibodies",
"recombinant proteins",
"quality control"
],
"content": "\n\nThe lack of reproducibility is widely recognized as a serious issue in contemporary research (see (Buck, 2015; Freedman & Inglese, 2014; Freedman et al., 2015; McNutt, 2014a; McNutt, 2014b) and the Nature special “Challenges in irreproducible research” April 2, 2013). In molecular biology, the quality of antibodies has been identified and highlighted as one of the most recurring stumbling blocks that undermine the quality and validity of experimental results (Baker, 2015; Bordeaux et al., 2010; Bradbury & Pluckthun, 2015). This issue is even more pervasive in the field of molecular epigenetics and chromatin biology, where antibodies for various types of histone post translational modifications (PTMs) have been single-handedly used to translate the language of histone modifications into experimentally observable properties. Hence most of what we know about the distribution, role and function of histone modifications so far has been passed through an antibody as essential mediator.\n\nRaising a specific histone modification antibody is not a trivial task; this is mostly due to the hypermodified state of the histone tail, coupled with the minute size and the chemical relatedness of many histone modifications and similarities in the amino acid sequence surrounding the modified residues. The antibody has to be able to discriminate between the unmodified and the modified state of the targeted amino acid residue, as well as between different forms of modifications (e.g. acetylations of different lysine residues, mono-, di- and trimethylation of lysine residues, or symmetric and asymmetric methylation of arginine residues). Moreover, the presence of an adjacent modification might prevent binding of an antibody to the target modification, causing false negative results. Another difficulty is that some histone modifications such as methylation or acetylation of H3K9 and H3K27 lie within an identical amino acid context (ARKS motif), which makes the readout of the target peptide sequence outside of this central motif very important as well.\n\nIn spite of the intricate task of producing histone modification antibodies and their crucial role in chromatin biology, surprisingly, they remain to be insufficiently characterized. In line with this, numerous scientific groups have alarmingly raised concerns about the promiscuous behavior of some histone modification antibodies and the undocumented effects of secondary modifications (Bock et al., 2011; Egelhofer et al., 2011; Hattori et al., 2013; Kungulovski et al., 2014; Nishikori et al., 2012; Rothbart et al., 2015). As mentioned above, the situation in chromatin biology is exceptional, because of the outstanding role of histone PTM antibodies as the sole research tool in this field. As a consequence, elaborate quality control criteria for histone PTM antibodies were put forward to ensure the integrity of research (Egelhofer et al., 2011; Kungulovski et al., 2015; Landt et al., 2012). To increase transparency, at least two databases for deposition of antibody quality data by researchers were put in place (http://compbio.med.harvard.edu/antibodies/; http://www.histoneantibodies.com/) (Egelhofer et al., 2011; Rothbart et al., 2015). However, in spite of being heroic attempts, these and similar databases have only a limited value, because most of the antibodies used in chromatin biology are polyclonal, and lab experience over the last years has demonstrated that the specificity data obtained for one batch of antibody do not necessarily reflect the properties of another one (Kungulovski et al., 2014), a caveat which is still often ignored by naïve end-users.\n\nThe necessary quality control steps for histone modification antibodies (Egelhofer et al., 2011; Kungulovski et al., 2015; Landt et al., 2012) currently burden the individual antibody user with high costs and workload. Given that antibodies are expensive reagents, which are of no use without appropriate quality documentation, these efforts must be redirected from the end-customer to the manufacturers of antibodies. Herein, we urgently ask the vendors and manufacturers of antibodies to provide the necessary product sheets for all types of antibodies on a regular basis, including quality control documentation for each batch of polyclonal and each catalog number of recombinant or monoclonal antibodies. The following information must be provided:\n\n1. Combinatorial profiling of specificity with peptide arrays or similar high-throughput methods. If possible, profiling of specificity with barcoded nucleosomes harboring different modifications.\n\n2. Western blot results with native (as positive control) and recombinant histones (as negative control).\n\n3. Western blot results with native histones or nuclear extracts from cells where the responsible histone modifying enzyme has been deleted or depleted (mammalian cells) or mutant histones (yeast).\n\n4. Reproducibility of ChIP-seq data and high correlation with similar validated ChIP-seq datasets.\n\nAs proposed by others (Bradbury & Pluckthun, 2015) end-users should consider boycotting companies not complying with this demand, or at least stay away from products lacking a proper lot-specific documentation.\n\nThe batch-to-batch variability of critical properties like cross reactivity or inhibition by secondary marks makes the application of polyclonal antibodies intrinsically unsustainable, because experiments cannot be reproduced after the corresponding batch of an antibody is sold out. As a consequence of this, rigorously speaking, large data sets in chromatin biology exist in a “grey” area outside of natural science, since it is impossible to repeat the underlying experiments. In a long-term perspective, a shift away from polyclonal antibodies towards alternative reagents, which can be produced at constant quality, would help to reduce the necessary costs and workload associated with quality control of polyclonal antibodies and ensure sustainability. This applies to high quality monoclonal antibodies, recombinant antibodies (Hattori et al., 2013) or analogous recombinant reading domains (Kungulovski et al., 2014). This will not only help to reduce costs in chromatin research in the long run (once obtained, the documentation will be valid for all lots) but also help to standardize the affinity reagents used and ease the lab-to-lab comparison of data. Of note, in chromatin biology native reading domains designed by nature to specifically recognize relevant histone PTM marks are available as an alternative to antibodies (Kungulovski et al., 2014), which is an advantage over other fields, where recombinant production of antibodies is the only technical solution to the issue of reproducible performance and long term availability of these essential research reagents.",
"appendix": "Author contributions\n\n\n\nGK and AJ wrote the paper. 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\nWork in the authors’ lab has been supported by the DFG JE 252/26-1.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBaker M: Reproducibility crisis: Blame it on the antibodies. Nature. 2015; 521(7552): 274–276. PubMed Abstract | Publisher Full Text\n\nBock I, Dhayalan A, Kudithipudi S, et al.: Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays. Epigenetics. 2011; 6(2): 256–263. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBordeaux J, Welsh A, Agarwal S, et al.: Antibody validation. Biotechniques. 2010; 48(3): 197–209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBradbury A, Pluckthun A: Reproducibility: Standardize antibodies used in research. Nature. 2015; 518(7537): 27–29. PubMed Abstract | Publisher Full Text\n\nBuck S: Solving reproducibility. Science. 2015; 348(6242): 1403. PubMed Abstract | Publisher Full Text\n\nEgelhofer TA, Minoda A, Klugman S, et al.: An assessment of histone-modification antibody quality. Nat Struct Mol Biol. 2011; 18(1): 91–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreedman LP, Cockburn IM, Simcoe TS: The Economics of Reproducibility in Preclinical Research. PLoS Biol. 2015; 13(6): e1002165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreedman LP, Inglese J: The increasing urgency for standards in basic biologic research. Cancer Res. 2014; 74(15): 4024–4029. PubMed Abstract | Publisher Full Text\n\nHattori T, Taft JM, Swist KM, et al.: Recombinant antibodies to histone post-translational modifications. Nat Methods. 2013; 10(10): 992–995. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKungulovski G, Kycia I, Tamas R, et al.: Application of histone modification-specific interaction domains as an alternative to antibodies. Genome Res. 2014; 24(11): 1842–1853. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKungulovski G, Mauser R, Jeltsch A: Affinity reagents for studying histone modifications and guidelines for their quality control. Epigenomics. 2015; in press.\n\nLandt SG, Marinov GK, Kundaje A, et al.: ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res. 2012; 22(9): 1813–1831. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcNutt M: Journals unite for reproducibility. Science. 2014a; 346(6210): 679. PubMed Abstract | Publisher Full Text\n\nMcNutt M: Reproducibility. Science. 2014b; 343(6168): 229. PubMed Abstract | Publisher Full Text\n\nNishikori S, Hattori T, Fuchs SM, et al.: Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay. J Mol Biol. 2012; 424(5): 391–399. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRothbart SB, Dickson BM, Raab JR, et al.: An Interactive Database for the Assessment of Histone Antibody Specificity. Mol Cell. 2015; 59(3): 502–511. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "10961",
"date": "02 Nov 2015",
"name": "Andrew Bradbury",
"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\nParagraph 2 should also indicate that antibodies purportedly recognizing PTMs at specific sites, need to have their recognition specificity also tested against the same PTM at different sites, in the same protein, or others, and with the same core sequence or others. Many so-called specific phosphotyrosine antibodies actually recognize the phosphotyrosine modification independently of its sequence context.\"In spite of the intricate task of producing histone modification antibodies and their crucial role in chromatin biology, surprisingly, they remain to be insufficiently characterized\" should be changed to \"In spite of the intricate task of producing histone modification antibodies and their crucial role in chromatin biology, surprisingly, they remain insufficiently characterized\"\"the specificity data obtained for one batch of antibody do not necessarily reflect the properties of another one\" Voskuil (Commercial antibodies and their validation. Version 2. F1000Res. 2014 Oct 2 [revised 2014 Oct 15];3:232. doi: 10.12688/f1000research.4966.2. eCollection 2014) describes the relatively unknown practice of some antibody manufacturers to use historical data sheets for antibodies to which they do not apply. This should also be mentioned and cited.The recommendations in paragraph 4 are commendable. However, if manufacturers are expected to carry this out on every lot they sell, the author must acknowledge that the cost of antibodies will have to increase. \"As proposed by others (Bradbury & Pluckthun, 2015) end-users should consider boycotting companies not complying with this demand, or at least stay away from products lacking a proper lot-specific documentation.\" Actually, this was not our main proposal. Our main point (amplified in Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents. Bradbury AR, Plückthun A. Protein Eng Des Sel. 2015 Oct;28(10):303-5. doi: 10.1093/protein/gzv051) was that we should move away from the use of polyclonal antibodies altogether and use only well characterized sequenced recombinant antibodies. Only in this way can we ensure antibody reproducibility. In the last paragraph, the author indicates that recombinant antibodies (or other proteins) may solve this problem. However, this will only be the case if such binders can be unequivocally identified, which will only occur if sequences can be referred to unambiguously. Otherwise, as antibody companies are bought and sold, catalog numbers will change and it may become difficult to reproduce experiments, because it will not be clear which original antibody was used.",
"responses": [
{
"c_id": "1693",
"date": "24 Nov 2015",
"name": "Albert Jeltsch",
"role": "Author Response",
"response": "“Paragraph 2 should also indicate that antibodies purportedly recognizing PTMs at specific sites, need to have their recognition specificity also tested against the same PTM at different sites, in the same protein, or others, and with the same core sequence or others. Many so-called specific phosphotyrosine antibodies actually recognize the phosphotyrosine modification independently of its sequence context.”Reply: We agree. This point was mentioned in paragraph 2 on p. 2 using methylation at H3K9 and H3K27 as an example. “Another difficulty is that some histone modifications such as methylation or acetylation of H3K9 and H3K27 lie within an identical amino acid context (ARKS motif), which makes the readout of the target peptide sequence outside of this central motif very important as well.” We have modified this paragraph to make the point clearer and better reflect what the reviewer was asking for: “Moreover, the antibody should bind the modified amino acid residue only at defined modification sites on the target protein, which implies that not only the modification but also the amino acid sequence must be recognized. This is particularly difficult for some histone modifications such as methylation or acetylation of H3K9 and H3K27 which occur within an identical amino acid context (ARKS motif) and make the readout of the target peptide sequence outside of this central motif vital as well.” \"In spite of the intricate task of producing histone modification antibodies and their crucial role in chromatin biology, surprisingly, they remain to be insufficiently characterized\" should be changed to \"In spite of the intricate task of producing histone modification antibodies and their crucial role in chromatin biology, surprisingly, they remain insufficiently characterized\"Reply: This has been changed as proposed. \"the specificity data obtained for one batch of antibody do not necessarily reflect the properties of another one\" Voskuil (Commercial antibodies and their validation. Version 2. F1000Res. 2014 Oct 2 [revised 2014 Oct 15];3:232. doi: 10.12688/f1000research.4966.2. eCollection 2014) describes the relatively unknown practice of some antibody manufacturers to use historical data sheets for antibodies to which they do not apply. This should also be mentioned and cited.Reply: Thank you. This point and reference has been added. “The recommendations in paragraph 4 are commendable. However, if manufacturers are expected to carry this out on every lot they sell, the author must acknowledge that the cost of antibodies will have to increase.”Reply: Please note in the original manuscript on p. 2 we stated that a shift to sustainable reagents (i.e. away from polyclonal antibodies) “would help to reduce the necessary financial and workload efforts associated with quality control of polyclonal antibodies and ensure sustainability”, which partially addressed this point. We now added an additional sentence stating “While one may expect that better quality control will increase the prices of commercial antibodies, end-customers will not be forced to conduct their own quality control and they will not waste money for non-functional antibodies, so that the overall final costs may not be much higher.” to incorporate this request more explicitly. \"As proposed by others (Bradbury & Pluckthun, 2015) end-users should consider boycotting companies not complying with this demand, or at least stay away from products lacking a proper lot-specific documentation.\" Actually, this was not our main proposal. Our main point (amplified in Getting to reproducible antibodies: the rationale for sequenced recombinant characterized reagents. Bradbury AR, Plückthun A. Protein Eng Des Sel. 2015 Oct;28(10):303-5. doi: 10.1093/protein/gzv051) was that we should move away from the use of polyclonal antibodies altogether and use only well characterized sequenced recombinant antibodies. Only in this way can we ensure antibody reproducibility.”Reply: We have added the citation to this very insightful paper now also at the corresponding place in our manuscript and also added the Prot. Eng. Des. Sel. reference. “As already proposed, in a long-term perspective, a shift away from polyclonal antibodies towards alternative reagents, which can be produced at constant quality, would help to reduce the necessary financial and workload efforts associated with quality control of polyclonal antibodies and ensure sustainability (Bradbury & Plückthun, 2015, Nature 518, 27-29; Bradbury & Plückthun, 2015, Prot. Eng. Des. Sel. 28, 303-305).” “In the last paragraph, the author indicates that recombinant antibodies (or other proteins) may solve this problem. However, this will only be the case if such binders can be unequivocally identified, which will only occur if sequences can be referred to unambiguously. Otherwise, as antibody companies are bought and sold, catalog numbers will change and it may become difficult to reproduce experiments, because it will not be clear which original antibody was used.”Reply: This is a valid point. We have added on sentence to stress this: “The recombinant reagents would be particularly promising, because their sequences can be published, which ensures full transparency and reproducibility.”"
}
]
},
{
"id": "10993",
"date": "03 Nov 2015",
"name": "Scott B. Rothbart",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAntibody reliability in biomedical research is of utmost importance. The quality of these reagents in chromatin biology applications is of particular concern given their position as essential tools for most techniques characterizing the cellular abundance and genomic distribution of histone post-translational modifications (PTMs). I agree with the Kungulovski and Jeltsch that increased accountability needs to be demanded from companies who sell histone PTM antibodies, and their four recommended quality control measures are reasonable expectations. In addition, it should also be noted that the practice of retaining catalog numbers for new polyclonal antibody lots is unacceptable and misleading. Moreover, and particularly in light of the increased awareness of antibody concerns in the field, experimentalists and epigenome consortium leaders (e.g., ENCODE, BLUEPRINT) should be more rigorous in their own evaluation of histone PTM antibodies when choosing a reagent for their study. Antibody specificity data and lot numbers used should also be standard requests from journal editors and reviewers. Accountability clearly needs to come from all parties if we are to continue benefiting from the use of these affinity tools in chromatin research.",
"responses": [
{
"c_id": "1692",
"date": "24 Nov 2015",
"name": "Albert Jeltsch",
"role": "Author Response",
"response": "“I agree with the Kungulovski and Jeltsch that increased accountability needs to be demanded from companies who sell histone PTM antibodies, and their four recommended quality control measures are reasonable expectations. In addition, it should also be noted that the practice of retaining catalog numbers for new polyclonal antibody lots is unacceptable and misleading.”Reply: We have added the sentence to the paper “that the practice of retaining catalog numbers for new polyclonal antibody lots is unacceptable and misleading”. Thanks a lot for this helpful suggestion. “Moreover, and particularly in light of the increased awareness of antibody concerns in the field, experimentalists and epigenome consortium leaders (e.g., ENCODE, BLUEPRINT) should be more rigorous in their own evaluation of histone PTM antibodies when choosing a reagent for their study. Antibody specificity data and lot numbers used should also be standard requests from journal editors and reviewers. Accountability clearly needs to come from all parties if we are to continue benefiting from the use of these affinity tools in chromatin research.”Reply: We like to mention, that in our view more responsibility in structured product documentation lies at the side of the supplier. Quality checks done by end-customers are an emergency action, but they will not solve the problem of long-term and lab-to-lab reproducibility. Also they put all financial pressure on the end customer or a product, which is not common practice in other parts of the economy."
}
]
},
{
"id": "10962",
"date": "19 Nov 2015",
"name": "Shohei Koide",
"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 succinctly reviews antibody-related problems that have been widely recognized in the biological and biomedical community, in a specific context of anti-histone posttranslational modification (PTM) antibodies and their uses in epigenetic research. The authors correctly emphasize the large negative impact of the batch-to-batch variation of anti-histone PTM antibodies and its consequence ('large data sets in chromatin biology exist in a “grey” area outside of natural science, since it is impossible to repeat the underlying experiments.'). I generally agree with the current challenges described in this paper, but it could be improved by addressing the following points.An important omission in the paper is the limited recognition by users of potentially large effects of the mismatch between validation methods and real applications. We can perhaps agree that antibodies should ideally be validated in a manner that closely mimics how it is used in the actual application. Validation methods can be divided into two general types based on their formats: one in which antibodies are immobilized and captured antigens are detected (\"immunoprecipation (IP) type\") and the other in which antigens are immobilized and captured antibodies are detected (\"blotting type\"). Peptide arrays and Western blotting fall into the latter blotting-type validation methods, in which peptides or denatured proteins are localized, often at high density, on a solid support and binding of dilute antibody samples is detected. To state the obvious, IP-type methods are suited for validating antibodies for IP-type applications (IP, ChIP), whereas blotting-type methods are suited for validating antibodies for blotting-type applications (Western, immunostaining).Potential problems arise when the validation format is different from the application format. It is not easy to predict whether antibodies validated using blotting-type methods perform well in IP-type applications such as ChIP, and vise versa. Egelhofer et al. (2011) reported that more than 20% of antibodies that have been validated to be specific in peptide blots still fail in ChIP experiments. In typical IP applications where an antibody is immobilized on a solid support, antibody affinity is a critical parameter. In contrast, affinity is not critical in blotting type applications, because the bivalent format of the conventional antibody (i.e. two antigen-binding sites per molecule) helps boost binding (the so-called avidity effect). Indeed, it has been found that an antibody that looked good on peptide arrays performed poorly in IP and conversely another antibody that did not look good on peptide arrays performed exceedingly well in IP (Nishikori et al., 2012). Similarly, an antibody (Active Motif 39156) performed well in IP validation using semi-synthetic nucleosomes (\"IceChIP\"), although it appeared cross-reactive in peptide-array validation and in Western blotting (Rothbart et al., 2015). Further complications arise from the fact that spot intensities in array-type experiments are not quantitatively correlated with the strengths of the measured interactions (see, for examples, Stiffler et al., 2006; Hause et al. 2012).Another omission is that validation and actual results depend on experimental conditions and accordingly an antibody validated under one set of conditions may not perform as well under a different set of conditions. Practitioners of immunoblotting are all familiar with the need for \"optimizing\" conditions for their own experiments. Similar optimizations are needed for other types of applications for which desired outcomes are less obvious. Furthermore, the abundance of the antigen of interest relative to off targets influences the outcome. Even a highly selective and potent antibody may not sufficiently enrich extremely rare antigens. Accordingly, for IP-type applications, mass spectroscopy-based validation using IP with input materials similar to those used in actual experiments (Peach et al., 2012; Hattori et al., 2013; Marcon et al. 2015) and IP of semi-synthetic nucleosomes (Grzybowski et al., 2015) are better suited as validation methods.This paper should emphasize more that the end user must critically evaluate limitations of validation methods caused by format mismatches and/or variations in experimental conditions. I do not agree with this paper's recommendations that a single set of information be provided with any antibody regardless of its intended use.",
"responses": [
{
"c_id": "1705",
"date": "11 Jan 2016",
"name": "Albert Jeltsch",
"role": "Author Response",
"response": "We agree with the general conclusions of this reviewer that validation methods and protocols need to be adjusted to the planned applications. However, it should be noticed that the main aim of this short opinion paper was to further raise concerns and contribute to a move towards better documentation and sustainable reagents in chromatin research. The current state is that antibody documentation is widely insufficient and the reagents are often not sustainable. We propose to improve this situation step by step. It would be an important advance for the field to have a panel of standardized quality documentation data available for each antibody lot. This includes data in different formats, which will allow users to select the most relevant information for their experiments. To take up the point of this reviewer, we included a hint that validation methods must reflect the intended application of antibodies.Defining the best format of quality checks will be an ongoing challenge for the entire field. We already expressed a preferences for the application of recombinant modified nucleosomes as validation method in chromatin biology in the original version of the manuscript. Another very important improvement for the field would be to have more recombinant affinity reagents, which are fully sustainable.However, we like to mention that we are not fully convinced by the statement that the differences in technical formats (blotting vs. pull-down) is the only or main reason of differences in antibody performance between certain assays. One additional, very critical, difference is that short peptides are used in many validation assays (for practical reasons) but real applications deal with the pull-down of full histone tails. Moreover, we are not convinced that the development of special conditions by each lab is an advisable development, because in many cases these may not be fully documented which - again - would undermine reproducibility. While it is self-evident that the researchers are ultimately responsible for the validity of all their results, lab internal quality certifications in our view are not the best way to proceed in an ever growing experimental field."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1160
|
https://f1000research.com/articles/4-1337/v1
|
24 Nov 15
|
{
"type": "Case Report",
"title": "Case Report: Delayed presentation of penile epidermoid cyst following reconstruction for Peyronie’s disease",
"authors": [
"Luriel I. Smith-Harrison",
"Jacques Farhi",
"Raymond A. Costabile",
"Ryan P. Smith",
"Jacques Farhi",
"Raymond A. Costabile"
],
"abstract": "Penile masses are a concerning finding for both patient and clinician upon initial presentation. There is a wide differential for penile masses from the benign (fibrous plaques, cysts, ulcerative lesions, benign penile pearly papules, etc.) to more concerning malignant lesions. A proper history and physical is the first step to determining the etiology of the mass and any future clinical interventions. In this paper, we review a case of a 73-year-old male who is found to have an enlarging mass during work-up for possible placement of inflatable penile prosthesis. Fortunately, the mass was determined to be a benign epidermoid cyst presenting thirty years after reconstruction for Peyronie’s disease using dermal penile skin graft. With this unique presentation we review the scant literature on penile mass formation following Peyronie’s repair.",
"keywords": [
"Penile mass",
"Epidermoid cyst",
"Peyronie’s Disease"
],
"content": "Introduction\n\nPeyronie’s disease is a common urologic entity with multiple options for definitive surgical repair. Plaque excision with grafting is a known and accepted method for reconstruction. There are multiple options for graft material, each carrying its own specific risk for complications and comorbidities1. In this particular case, we discuss Peyronie’s disease treated with plaque excision and dermal skin grafting.\n\n\nCase presentation\n\nA 73-year-old man was referred to our clinic in surgical consultation for possible placement of inflatable penile prosthesis due to progressively worsening erectile dysfunction. At his initial visit, he was found to have a non-tender rapidly growing mass in the distal penile shaft, which prohibited him from using his vacuum erection device. His past medical history was significant for type 2 diabetes and Peyronie’s disease. Thirty-two years prior, he underwent corrective surgery for Peyronie’s disease. Operative and clinical notes from that period could not be obtained, though the patient reported the procedure included plaque excision and use of a dermal penile skin graft. Following the procedure, he only reported mild residual penile desensitization.\n\nOn exam, we noted a well-healed surgical scar and a 3 cm nodule arising from the left lateral aspect of the distal shaft. Moderate corporal fibrosis was also noted. We did not appreciate any concerning erythema, tenderness, drainage or ulceration. Importantly, the size and location of the mass prevented the patient from using a vacuum erection device. With the atypical presentation of this mass, the decision was made to proceed with further work-up prior to discussing any interventions for his erectile dysfunction.\n\nFrom this point, we proceeded with pelvic magnetic resonance imaging (MRI) without contrast (Figure 1). This revealed a 2.4 × 2.8 × 4.1 cm, rim-enhancing, hemorrhagic mass without internal solid components. The mass was abutting and mildly compressing the left corpus cavernosum. The mass did not invade the corpus cavernosum and the tunica albuginea was intact, though thickened. The right corpus cavernosum and corpus spongiosum were normal.\n\na: T-2 MRI reveals non-enhancing 4.1 cm lesion abutting the left corpus cavernosa and exerting mild compression on the left corpus cavernosum. b: T-1 MRI without contrast shows a homogenous rim enhancing lesion without solid components. c: Subtraction MRI of lesion. Differential for this lesion based on imaging is proteinaceous fluid versus subacute hemorrhage.\n\nPenile duplex ultrasound, performed after an injection of 10 mcg alprostadil into the right corpora, revealed a 4 cm mass with complex internal echoes without Doppler flow. Compression of the corpora was seen with a moderate wasting deformity opposite of the mass. Approximately 15 degrees of mild leftward deviation was noted. Arterial peak flow was estimated at 12 cm/sec and resistive indices were 0.6 bilaterally. In addition, plaque without calcification was seen in the mid-shaft.\n\nGiven the constellation of residual penile curvature, erectile dysfunction which was non-responsive to phosphodiesterase inhibitors, and the presence of a penile mass, the patient elected for placement of a three-piece inflatable penile prosthesis in conjunction with excision of the mass. We reviewed the possibility of other adjunct procedures such as penile modeling, grafting and plication. Penile prosthesis placement was declined by the patient’s insurance and mass excision was pursued alone. An incision was made over the site of the mass which was removed in its entirety without complication. During dissection, previous sutures from the dermal graft were appreciated. The corporal body was left intact. The pathology report was consistent with a benign inclusion cyst and his post-operative recovery was unremarkable with discharge to home immediately following surgery. Upon close follow-up, his penile curvature is stable, as determined by clinical exam and he has resumed using a combination of phosphodiesterase inhibitors and a vacuum erection device.\n\n\nDiscussion\n\nThere are multiple options for surgical management of Peyronie’s disease. Part of the treatment algorithm includes a number of options for grafting. It is well-known that skin grafts carry a greater risk of transplanting apocrine glands and hair follicles to the donor site. Due to this, it is incumbent upon the surgeon to pick a graft best suited for the operative site and graft intent. The surgeon must also weigh the risks and benefits of each possible donor site. Although this principle is followed in reconstructive surgery for Peyronie’s disease, there is a paucity of case reports documenting cyst formation after dermal graft inlay procedures. The authors most commonly use small intestinal submucosa or tunica vaginalis grafts.\n\nTo our knowledge, there are two case reports describing this complication2,3. One case report describes a middle-aged male who presented with a unilateral enlarging penile lesion 2 years after having a dermal graft procedure for Peyronie’s disease. Upon exploration, a fluid filled keratin mass containing hair was removed2. The other case report described an elderly man who had dermal graft repair for a dorsal plaque3. The graft was harvested from a site devoid of hair, the abdominal wall near the left flank. Four years later the patient developed a swelling at the dorsum of the penis. In these two cases, the cyst presented less than 5 years after the operation.\n\nIn our case, the inclusion cyst presented more than 30 years after the operation, suggesting that cyst formation can be sporadic and yet rapid. The latency of cyst formation could be due to more extensive de-epithelialization of the graft in our case compared to the other cases. Still rapidly expanding soft tissue penile masses could be concerning for a neoplasm, albeit extremely rare. Therefore rapidly expanding soft tissue penile masses should be investigated with MRI to rule out a neoplasm and to further classify the lesion and location, which could prove to be valuable in surgical planning4,5. However, Peyronie’s disease is not thought to be a predisposition to a penile neoplastic lesion and to date the literature is devoid of a penile neoplasm after a dermal graft procedure6.\n\n\nConclusions\n\nThe development of any penile mass should be concerning and warrants a full work-up by the appropriate medical provider. As this case shows, benign epidermoid cysts must be considered in those patients with a history of prior skin graft to the penis. Options for management of erectile dysfunction should not be limited following excision of an epidermoid cyst.\n\n\nConsent\n\nWritten, informed consent for publication of clinical details and images was sought and obtained from the patient.",
"appendix": "Author contributions\n\n\n\nRyan P Smith, MD – Primary attending. Responsible for work-up, medical/surgical management and follow-up. Also worked in editing process.\n\nRaymond A Costabile, MD – Surgeon for excision of mass and consulted for management.\n\nLI Smith-Harrison, MD – Primary author of the manuscript. Reviewed previous literature on the subject.\n\nJack Farhi, BA – assisted in writing the manuscript, editing and literature review.\n\nAll authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors have declared no competing interests.\n\n\nGrant information\n\nThe authors declared that no funding was involved in supporting this work.\n\n\nReferences\n\nZaid UB, Alwaal A, Zhang X, et al.: Surgical management of Peyronie’s disease. Curr Urol Rep. 2014; 15(10): 446. PubMed Abstract | Publisher Full Text\n\nLiou LS, Montague DK, Angermeier KW: Dermal graft repair of peyronie’s disease complicated by epidermoid cyst. J Urol. 2003; 169(2): 617–8. PubMed Abstract | Publisher Full Text\n\nSavoca G, Ciampalini S, De Stefani S, et al.: Epidermoid cyst after dermal graft repair of Peyronie’s disease. BJU Int. 1999; 84(9): 1098–9. PubMed Abstract | Publisher Full Text\n\nSuwa M, Takeda M, Bilim V, et al.: Epidermoid cyst of the penis: a case report and review of the literature. Int J Urol. 2000; 7(11): 431–3. PubMed Abstract | Publisher Full Text\n\nUsta MF, Adams DM, Zhang JW, et al.: Penile epithelioid sarcoma and the case for a histopathological diagnosis in Peyronie’s disease. BJU Int. 2003; 91(6): 519–21. PubMed Abstract | Publisher Full Text\n\nDevine CJ Jr, Horton CE: Surgical treatment of Peyronie’s disease with a dermal graff. J Urol. 1974; 111(1): 44–9. PubMed Abstract"
}
|
[
{
"id": "11315",
"date": "30 Nov 2015",
"name": "James Hotaling",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well done article on a rare entity that urologists might encounter once or twice in a career. Penile masses are certainly challenging and there is little data on their management.",
"responses": []
},
{
"id": "11314",
"date": "14 Dec 2015",
"name": "Alexander W. Pastuszak",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a much needed perspective on penile masses in the setting of Peyronie's disease after surgical repair. The manuscript is well written and appropriately identifies an overall paucity of studies examining this complication, summarizing the extent of our knowledge in these specific situations in a total of 2 other available case reports. As such, this work is a very useful and important contribution to the literature. There are two points on which additional information would be useful, however. First, with regards to the patient case, it would be useful to include any serum studies that were performed, which may be salient particularly in the setting of a mass that is not benign. Second, a short discussion of the rarity (i.e. incidence / prevalence) of malignant penile masses in this type of setting would be helpful.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1337
|
https://f1000research.com/articles/4-8/v1
|
12 Jan 15
|
{
"type": "Review",
"title": "Nutrition transition in South Asia: the emergence of non-communicable chronic diseases",
"authors": [
"Ghose Bishwajit"
],
"abstract": "Overview: South Asian countries have experienced a remarkable economic growth during last two decades along with subsequent transformation in social, economic and food systems. Rising disposable income levels continue to drive the nutrition transition characterized by a shift from a traditional high-carbohydrate, low-fat diets towards diets with a lower carbohydrate and higher proportion of saturated fat, sugar and salt. Steered by various transitions in demographic, economic and nutritional terms, South Asian population are experiencing a rapidly changing disease profile. While the healthcare systems have long been striving to disentangle from the vicious cycle of poverty and undernutrition, South Asian countries are now confronted with an emerging epidemic of obesity and a constellation of other non-communicable diseases (NCDs). This dual burden is bringing about a serious health and economic conundrum and is generating enormous pressure on the already overstretched healthcare system of South Asian countries. Objectives: The Nutrition transition has been a very popular topic in the field of human nutrition during last few decades and many countries and broad geographic regions have been studied. However there is no review on this topic in the context of South Asia as yet. The main purpose of this review is to highlight the factors accounting for the onset of nutrition transition and its subsequent impact on epidemiological transition in five major South Asian countries including Bangladesh, India, Nepal, Pakistan and Sri Lanka. Special emphasis was given on India and Bangladesh as they together account for 94% of the regional population and about half world’s malnourished population.Methods: This study is literature based. Main data sources were published research articles obtained through an electronic medical databases search.",
"keywords": [
"South Asia",
"Nutrition Transition",
"Epidemiological Transition",
"Non-communicable diseases",
"Healthcare"
],
"content": "Introduction\n\nGlobalization of agrifood has brought about remarkable shifts in diet patterns especially in developing countries which has shown to be a major underlying factor for increasing prevalence of obesity and associated NCDs. With long standing history of infectious diseases, developing countries are now facing a rising tide of non-communicable diseases which is popularly known as the double burden of malnutrition (Coexistence of over- and undernutrition). This dietary transition is basically characterized by a shift from a diet with a higher proportion of carbohydrate based foods such as cereal grains (rice, wheat, maize), vegetables (leaves, roots, legumes) and low animal products (meat, egg, milk) to one which is lower in carbohydrate and higher in animal-based food with high sugar and caloric content and larger amount of processed food (chocolates, soft drinks). Though dietary and epidemiological transition have been two main foci in the study of nutrition transition, more socio-economic and demographic parameters are now being included in the discipline. The term nutrition transition was first coined by Barry M. Popkin who remains one of the most highly cited researchers in the field of human nutrition (Wahdan, 1996). According to Popkin, the scope of nutrition transition encompasses not merely the dietary and physical aspects, but also many other economic, social and environmental factors that is shaping the landscape of modern living as never before. By virtue of increased participation in global trade, South Asian nations have experienced a period of unprecedented economic growth, higher income level, provision of labor-saving technologies, and a significant reduction in the number of people living in extreme poverty. India, the largest economy in the region, rose to global prominence with its trillion dollar economy and is set to be world’s third largest economy by 2050 (World Bank). Despite the global recession, India’s share in global trade increased to 1.28 per cent in 2011 compared to 0.67 per cent a decade earlier. Bangladesh has earned the recognition of being the economic miracle of the decade and is likely to surpass Pakistan to become the second largest economy in the region. Economic transition with improvement in household economic status has shown to be associated with increased consumption of animal products and higher prevalence of overweight, obesity and other NCDs (Salter, 2013), and South Asia appears to be no exception in this regard. Since 1990, South Asian countries are experiencing an increasing trend in the prevalence of overweight and obesity (Popkin et al., 2012). India with around 40% total population living on vegetarian diet, has experienced a doubling in total poultry meat consumption since 2000 while in Pakistan total meat consumption has increased by 130% during the same period. Increased cross-border food trade, advancement in local food technology and in food marketing and processing industry have greatly increased the availability of processed food products even in the remote rural areas where around 70% of the South Asians live. Though chronic dietary (caloric) inadequacy, macronutrient deficiency and infectious diseases are traditionally conceived as the major causes of malnutrition in South Asian countries, the impact of adequate but unhealthy diet along with lifestyle and environmental factors are fast replacing the trend and are becoming the major focus of epidemiologists. Rapid urbanization, access to labour-saving technologies and rise in various non-farm sectors have reduced the need and scope for physical activities to a level a level which is contributing to a sharp rise in the prevalence of overweight and obesity. However, the benefit of economic growth didn’t translate to improved nutritional status for the population at large, a phenomenon which is known as the South Asian enigma (Guha-Khasnobis et al., 2010). More than half of world’s total underweight children live in South Asia [Figure 1]. While malnutrition is the single largest cause of child mortality in the region, childhood obesity is also becoming a public health concern especially in the urban areas (Roux et al., 2008). Thus at one the end of the epidemiological spectrum lies widespread undernutrition, and a rising tide of obesity and associated NCDs on the other. This rising dual burden of disease is posing enormous pressure on the underdeveloped healthcare systems in South Asia and provides an imperative to adopt more mainstreaming and cross-cutting policies in national and regional level.\n\nSource: Global disease burden 2010 (http://goo.gl/Tp7fS1)\n\n\nMethodology\n\nThis study is based on literature published between 1995 and 2014. A systematic literature was conducted in April of 2014 in the following electronic databases: PubMed, Embase, The Cochrane Library and Google Scholar; using the following search terms: ‘nutrition transition’, ‘dietary transition’, ‘epidemiological transition’, ‘obesity’, ‘diabetes’, cardiovascular diseases. Main inclusion criteria was relevance to studies in context of following South Asian countries: India, Pakistan, Bangladesh, Nepal and Sri Lanka. Following the preliminary search, the titles and abstracts of the articles were analysed and selected according to their suitability for this review. Both original and review articles were included. No special exclusion criteria was applied. Additional data were triangulated from various sources mainly from FAO, USDA, WHO, World Bank, and health and nutrition surveys in each countries.\n\n\nDemographic transition in South Asia\n\nSouth Asian demography is characterized by declining fertility and mortality rate [Table 1], high population density (seven times the world average), and a large youth segment. It houses around one fifth of mankind with less than 4% of global land area and contributing to merely 2% of global income (Jacques, 2008). Total population was 1.65 billion in 2012 of which 31% are located in urban settings. Though most people are living in the rural areas, urbanization is occurring at a great speed [Figure 2]. Three of world’s top ten most populous countries (India, Pakistan, Bangladesh) and four of top ten most populous cities are located in this region (Mumbai, Karachi, Delhi, Dhaka). South Asia has registered remarkable progress towards the Millennium Development Goals (MDGs) [Table 1] and has substantially reduced the rate of premature death and disability from infectious diseases such as polio, pneumonia, diarrhea and many malnutrition related deaths thanks strong government commitment and a steady economic growth. In Bangladesh for example, total infant and child mortality rate has decline respectively by around 60 and 50 per cent since 1980. All countries are experiencing increasing trend life expectancy. India accounts for around 75% of total population in South Asia and contributes to around 80% of regional GDP. India’s population surpassed billion mark in 2001 and is likely to exceed China to become the most populated country by 2050 (Doak et al., 2005; World Bank, 2013). Bangladesh, the most densely populated nation in the world, is the third largest country in South Asia and eighth in the world in terms of total population. Population growth rate declined from 2.1% to 1.4% since 1990s which provides a concrete example of how economic growth is linked with decline in population growth, increased life expectancy and reduced child mortality (Bleich et al., 2011; Basu et al., 2013). The country has made good progress in all areas of human development after a long history of huger, food insecurity and human deprivation. Following decades of internal conflicts, Nepal and Sri Lanka have been able to restore peace and have also achieved great success in health and economic terms. Population growth rate is about 0.7% in Sri Lanka and is the least populated country in the region with a population of 20.33 million in 2012. By virtue of this sharp fall in infant mortality rates, the size of the child and youth segment is also expanding. South Asia already has about a quarter of world’s youth and is also the most youthful subregion in Asia (32% population are below 14). Though its a good sign for the economy as more people are being able to join the workforce to supply cost-effective labor for the fledgling economy, the demographic transition is actually serving as a double-edged sword as it has also produced a rapid increase in numbers of older people who are more prone to NCDs and largely account for the rapidly increasing NCD burden (Basnyat & Rajapaksa, 2004). Besides that, this huge youth population is also being exposed to the growing obesogenic environment and a multitude of other risk factors of NCDs while striving to create the conditions for sustainable careers and quality living for themselves. Childhood obesity has shown to be associated with an increased risk of NCDs in later years (Ghose et al., 2013). Since aging population bears increased susceptibility to NCDs, the total burden from these diseases may reach a huge proportion in next two decades, if steps are not taken immediately to avert this situation.\n\nSource: FAO, HDI 2013\n\nTable 1 shows the demographic and economic trends in South Asia. It is evident from this table that income has increased dramatically with a sharp rise in 2011. Infant mortality and net under-five mortality have also declined substantially over past three decades with a significant rise in life expectancy. This demonstrates how economic progress is linked with the improvement in health status of countries. (NA- Data not available)\n\nPakistan has highest urban population in the region while Nepal has the highest rate of urbanization.\n\nSource: UN Population Database, State of world cities (http://goo.gl/6G7S3h)\n\n\nDrivers of nutrition and dietary transition and their impact on NCDs\n\nA dietary transition from high starch and low animal food to higher intake of animal based food, higher proportions of edible oils and sweetener are occurring in almost all developing regions. Nutrition transition is occurring as an inevitable outcome of the changes in global food system triggered mainly by increasing income levels, rapid industrialization and urbanization and globalization of agrifood.\n\nHistorically, nutrition and dietary transition is found to be associated with a shift from a preindustrial agrarian economy to an industrial economy as seen in the context of developed countries in Europe and North America (Barry, 1999). Industrialized countries have experienced a nutritional and epidemiological transition since the industrial revolution. Reform in economic system brings about changes in food system, and when accompanied by modification in lifestyle habits such as reduced degree physical activity, it contributes to changing disease pattern such as rising prevalence of overweight, obesity and associated NCDs (Doak et al., 2005).\n\nToday, people in developing countries can afford more calories than ever before which is largely attributable to increased disposable income and greater availability of food (Josef et al., 2005). As the demand for food is rising in the aggregate, a major shift is also taking place in the type of food demanded. This shift generally occurs to increased demand for fish, egg, meat and dairy based food, and poor households tend to shift to more animal based diet with the rise in income (Misra & Shrivastava, 2013; Singh et al., 2011). Figure 3 shows that total meat consumption has increased significantly in all South Asian countries over past past two decades. Though over 60% of all South Asians still earn less then $1.25 a day, the situation is actually much better compared to three decades back and more and more people are being able to afford these type of food. Per capita GDP growth has seen a dramatic leap since 2001 [Table 2] which has played a catalytic role in increasing the demand for meat, fish, egg, and dairy products (Freedman, 2002). Many researchers attributed the food price inflation of 2007–08 to the rising demand from India and China as the countries are becoming major food importers to meet huge domestic demand.\n\nPer capita meat consumption rate (shown on the Y-axis) has increased in almost all countries with Sri Lanka having the highest rate. Pakistan has the highest per capita meat consumption and is currently world’s tenth largest consumer of beef.\n\nSource: USDA (http://goo.gl/p0dehO), Thepoultrysite (http://goo.gl/Slh83n)\n\nSource: CIA Factbook (http://goo.gl/SqSOTB)\n\nTable 2 illustrates the shift in percent labour force allocation in selected South Asian countries. Despite being agrarian economies, total employment in agriculture has declined in all countries, while that in service sector has increased.\n\nThough around 60% of people are living in the rural areas, all South Asian countries are experiencing a very fast urbanization [Figure 2]. Prevalence of obesity is higher in urban areas as compared to the rural areas, since these are most affected by rapid changes in nutritional pattern and sedentary life style (Shariful Islam et al., 2013). Rapid urbanization has been associated with a shift in labor force from agriculture to service sector which has reduced average physical activity status of the population. Table 3 shows that people are becoming more involved in service jobs in South Asian countries. For example in Bangladesh, prevalence of diabetes in urban areas is twice as high as in the rural areas (Saquib et al., 2012). Dhaka is one of the fastest growing cities in the world which is due mainly to the rapid growth of the garments sector attracting cheap labor from rural areas. Nepal is leading the race of urbanization in South Asia with a rate of 4.9% while that for Sri Lanka is the lowest (0.7%). From a dietary point of view, urbanization is characterized by a marked increase in the intake of energy-dense foods, a decrease in physical activity, and a heightened level of psychosocial stress, all of which promote the risks of developing metabolic syndrome (Barry, 1999). Urbanization affects people’s health and diet in various ways. People living in urban areas consume diets distinctly different from those of their rural counterparts which usually includes an increasing amount of animal and dairy products, sugar rich food, fast foods and less of fruits and vegetables (Barry, 1999). People living in urban areas not only show an increased appetite for processed and convenience foods and diets high in sugar and fat content, but also a more sedentary lifestyle which sets the preconditions for obesity and most other NCDs (Barry, 1999). Urbanization is also accompanying increased female labor force participation. As more and more women are joining the labour force, it greatly influencing the shift from home cooked traditional food towards precooked convenience food and fast food both at home and outside. A substantial increase in childhood as well as adult obesity in the urban population is therefore in line with the radical changes in lifestyle during the last few decades.\n\nSource: Global Health Observatory Data. WHO\n\nTable 3 shows that NCDs account for highest causes of mortality in South Asia. Among all NCDs, contribution of cardiovascular diseases is the highest followed by cancer and diabetes.\n\nThere is a growing evidence that globalization and trade liberalization have played a key role in the dietary and nutrition transition in the developing countries (Ghose et al., 2013; Lim, 2012). With the advent of food biotechnology and the expansion of transnational food corporations (TFCs), global food chain has undergone massive changes in terms of the ways food is grown, processed, stored, transported and consumed. Food mile (The distance that food travels from the place of production) has reached continental scale as a result of improved transportation system and declining trade barriers. Following the Uruguay Round, the average tariff on most goods fell from about 40% in 1947 to 4.7% in 1993 (Paul, 2008). As a consequence, developing countries have enjoyed a huge inflow of processed food products which has remarkably transformed the scenario of food market and people’s food choices. Nutritionists and health researchers around the world are expressing serious concerns as food is being traded the same manner as garments, electronics, and all other products ignoring the long term health and environmental impacts. Historically in South Asia, community food markets have been the main sources of everyday food supply which are mostly locally grown fresh raw foods. The situation has been changing as groceries and super-shops are taking the lead which are usually selling cheap junk foods such as cookies, chips and soft drinks. Even two decades earlier popular processed items were limited to rice cakes, sweetmeats and vegetable/fruit jelly/pickle which are produced using traditional methods mostly by rural households who has no knowledge of artificial food chemicals. However, this culture is on the verge of ruin with the progress of industrialization as these foods are now being manufactured on an industrial scale by food companies which are dependent on a range on food additives. Large supermarkets which are characteristics of affluent societies are now spreading fast across developing regions like South Asia (Reardon et al., 2003). Figure 4 shows the interior of a supermarket in a small town in Nepal teeming with soft drinks, cookies, chips. As a result, children especially in the urban areas are increasingly being exposed a completely new food environment which offers cheap availability of numerous food products which are most often obesogenic and have many potential health hazards.\n\nJanakpurdham is known for many historical and religious sites and has very little traces of industrialization. The super-shops like this in the city however bear the signs of industrialized nations teeming with processed foods such as cookies, chips, and soft drinks with no place for fresh fruits and vegetables.\n\nPhoto credit: Mr. Sudeep Sharma\n\n\nEpidemiological transition in South Asia and the burden of NCDs\n\nEpidemiological transition refers to the shift from acute infectious and deficiency type diseases to chronic non-communicable diseases (NCDs) which reflects changes in the pattern of morbidity and mortality [1, (Doak et al., 2005). In general, this transition occurs through a complex pathway which is influenced by gradual changes in nutritional, demographic and socio-economic parameters (Doak et al., 2005). In industrialized countries, epidemiological transition emerged towards the early 1900s marked by a rising levels of NCDs and a drastic fall in the prevalence infectious disease (Detels, 1997). This rising prevalence NCDs is believed to be an outcome of a complex interplay between a demographic, socio-economic, nutritional, environmental factors along with changes in lifestyle pattern. Once known as the diseases of the wealthy western countries, NCDs are fast becoming more threatening in the developing regions such as Latin America and South Asia. Globally, NCDs have become the leading causes of morbidity and mortality (Accounting for 43% of all disease burden in 1999) and is projected overtake that of infectious diseases within a decade (Wagner et al., 2012).\n\nWhile in the developed countries the battle is chiefly against NCDs, people in developing countries are facing a double burden malnutrition (Paul, 2008), 9]. Like other developing regions, South Asian countries are also facing a double burden of malnutrition [Figure 5]. In 2000, 44% of the burden ofdisease in this region measured in disability adjusted life years (DALYs) was attributed to non-communicable diseases (Basnyat & Rajapaksa, 2004). According to WHO guidelines for Asian populations, a third of the South Asian adult population is classified as obese, and 50% as overweight, by 2030 NCDs (T2DM, CVDs, COPD, cancer) and are going to account for 72% of total mortality by 2030 in South Asia (Shariful Islam et al., 2013; Singh et al., 2011). In India, although under-nutrition and micronutrient deficiencies continue to be major public health problems, over-nutrition and obesity are also emerging as a major problem in many states(Kerala, Tamil Nadu). In Bangladesh, NCDs are responsible for almost half of annual mortality and of 41% total disease burden (Bleich et al., 2011).\n\nThe proportion of stunting and underweight is higher than rest of the world, while that of overweight is about one-third.\n\nSource: WHO, UNICEF (www.childinfo.org)\n\nSouth Asians have a higher prevalence of diabetes mellitus than in any other region in the world (Jafar, 2006) and also have an increased susceptibility to cardiovascular disease (CVD). CVD is the largest single cause of mortality in all South Asian countries [Table 2]. In Bangladesh, total mortality from NCDs increased by 68% between 1986 and 2006 while that from communicable diseases rose by 5% during the same period (Ahsan Karar et al., 2009). Bangladesh alone accounts for 40% of all diabetes patients located in least developed countries and the number is increasing by 5–6 percent a year. An estimated 7 million people are suffering from diabetes in Bangladesh, while the corresponding figure is respective 2 and 1 million for Nepal and Sri Lanka. In Pakistan, about 12.9 million (10% of total population) people are currently living with diabetes which is projected to become 14.5 million in 2025. The scenario of double burden of malnutrition is perhaps most vivid in India than any other place in the world. India has the recognition of housing the highest number of malnourished child in the world yet the number of NCDs is increasing dramatically since 1990. With 30% of all children are born underweight (17% of global total), and 48% of all under-five children are stunted, it also has the second highest prevalence of diabetes (Danaei et al., 2011) and cardiovascular disease in the world (Basu et al., 2013). India diabetic population rose to 63 million (>7% of total adult population) in 2013 which is second only to China (~92 million) and is projected to reach 100 million by 2030. Researchers suggest that demographic, epidemiological and nutrition transition is fueling the NCD epidemic in India particularly in the urban areas (Shetty, 2002).\n\n\nThe economic impacts of NCDs and nutrition transition household and national economy\n\nNCDs are both a determinant and result of poor socio-economic status and exert an important bearing especially on the poor households as they require a long-term treatment. The United Nations High Level Meeting in 2011 declared NCDs a major health, economic and development issue and one of the most significant challenges to poverty eradication in developing countries. World Economic Forum (WEF) has also identified NCDs as the second most severe threat to the global economy. According to the projections of WEF, the burden of NCDs will account for as high as $47 trillion by the year 2030 globally (Bloom et al., 2011). NCDs including diabetes are estimated to reduce GDP by up to 5% in many LDCs. Studies have shown that South Asian nations incur about a 5% loss of their annual GDP due to substandard sanitation and hygiene facilities while infections account for 20% of DALYs (Basnyat & Rajapaksa, 2004). But no such study has yet been conducted focusing on the costs of NCDs. Main direct costs of NCDs include the fees for hospitalization, transportation, drugs and some indirect costs are loss of work days, absenteeism, reduced workplace productivity. Among children, obesity has a detrimental effect not only on health, but also on their psychological wellness and thus can affect lifetime earnings potential (Roux et al., 2008). In 2005, India experienced the highest loss in potentially productive years of life worldwide (Koh-Banerjee et al., 2003). Out of pocket expenditure for NCDs in India increased from 31.6% in 1995–96 to 47.3 percent in 2004, and hospital stays more than doubled during the same time (Jaime & Lock, 2009). Among the NCDs, diabetes is the most costly and is also a major risk factor for a variety of life-threatening and expensive complications such as heart attack, renal disease, neuropathy, visual impairment (Shariful Islam et al., 2013). NCDs are also responsible for unhealthy aging and increases the burden of medical spending in later years which put huge pressure on of savings and household assets.\n\n\nTackling the risk factors of NCDs among children\n\nWhile the burden of NCDs is unquestionably going to multiply in near future, understanding of the risk factors of NCDs is still fairly poor which constitutes a major obstacle for the implementation of effective prevention strategies (Espina et al., 2013). The World Health Organization (WHO) forecasts that in next two decades there will be dramatic changes and transitions in the world's health needs, as a result of epidemiological transition (Ahsan Karar et al., 2009). A multisectoral policy approach must be accompanied by the adoption of national and community level nutrition and health programs to tackle this overwhelming burden of NCDs.\n\nStrategic food policy making to control unhealthy dietary behaviour has been in place in many countries. Bolivia and Montenegro have closed down fast food shops; Hungary and Australia have imposed a surtax on soft drinks, whilst Denmark has increased tax by 25% on ice cream, chocolate and sweets products. Successful implementation of such pricing policies will require strong political will and cooperation from private sectors and civil society. As industrial control on global food systems have become the major driver of nutrition transition, more governmental regulation of food companies must aim to keep the use salt, sugar and other food additives limited according to the WHO guidelines. National food policy must also focus on increasing the access to and affordability of healthy and nutritious food and discourage the consumption of junk food. Food advertisements have a major impact on people’s dietary behaviour especially among school aged children (Barry, 2001; Vineis et al., 2014). Globally, expenditure on food advertisements has almost doubled in real terms since 1980 ($512 billion in 1980 compared to $216 billion in 2004). Advertising junk foods targeted at children must be banned or reduced to a minimum. Regulating school meals is also important reducing obesity among school children. What children eat at school is hard to control but banning the sale of certain types of foods are extremely helpful. In 2005, New Jersey and California banned selling junk food and sodas in public schools, and in Ontario the government banned junk food and pop in elementary schools in 2010. In most cities in south Asia, students buy oily snacks, junk foods from school canteen or nearby shops which are both cheap and delicious. Figure 6 shows the items in a super shop near a school Dhaka city. To prevent this, school authorities must develop healthy eating programs and also ban selling junk foods in canteens and in shops.\n\nThe picture shows that a variety of junk foods are made available to school students. Most of these products are sold at extremely cheap prices (> $0.15).\n\nPhoto credit: Sharmistha Ghosh\n\nSchools have been promoted by policymakers, researchers and media as a strategic setting for implementing nutrition policies as it provides an inviting setting for the promotion of healthy behaviours in children which can ultimately contribute to reducing the prevalence of obesity (Harrison & Jones, 2012). Since children spend more time in schools than in any other environment outside home, it provides an ideal workplace for developing child health intervention programs. A compelling piece of evidence suggests that physical inactivity is a strong determinant of childhood overweight and obesity which increases the risk of many chronic diseases in later life and that modest increases in physical activity have the potential to produce substantial health benefits (Roux et al., 2008). Many countries have adopted physical activity among school children. Studies have shown that apart from controlling weight, physical exercise (PE) is important because it is associated with cardiovascular benefits such as a reduction in low-density lipoproteins, prevention of hypertension, and prevention of chronic disease (Harrison & Jones, 2012). On average, children spend around 6-8 hours per day in school. Which means they get little or no time or interest for physical activities at home after having long days. Spending a long day at school and nights with excessive homeworks not only make children apathetic to school but also have adverse implications for physical and mental status and social relationships. School enrollment has increased considerably in South Asia [Table 1]. South Asia has the highest number of children in primary schools worldwide, accounting for 28% of global primary school enrollments and currently some 200 million children are enrolled in primary education. Given this huge young population, there is a risk of increasing incidence of NCDs in next few decades as obese children bear a greater risk of developing NCDs in later years. However, this risk can be turned to an opportunity for long-term prevention strategies by promoting school-based health and nutrition programs.\n\nPeople in urban areas are not only being deprived of healthy environment and healthy food, but also of adequate levels of physical activity. This is a particular concern for South Asian countries as unbalanced growth of urban areas and poor urban planning continue to aggravate the problem. Understanding the link between environment and the scope for physical activity is important to develop strategies to reduce the prevalence of obesity (Grijalva-Eternod et al., 2012; Trasande et al., 2009). A large volume of literature has been published in the last decade focusing on the importance of physical activity for the effective prevention management obesity and NCDs (Lim, 2012). Most studies have highlighted that the decreasing space for physical activities is an important factor for the higher prevalence of NCDs in urban settings. Arguably, in highly populated cities in South Asia, its hard or impossible for schools to keep sufficient space for physical activities. Besides that, children in slums and poor neighborhoods are almost always deprived of a healthy environmental space where they get little or no scope for outdoor sports and physical exercise. Though heath clubs now days are becoming popular, they are generally expensive and lack facilities for school children. Organizing frequent sports events at schools and community settings may prevent children from getting addicted to computer games. Community-based intervention programs targeting childhood obesity and nutrition-related chronic diseases must be accompanied by improving facilities for physical activities.\n\n\nConclusions and policy recommendations\n\nSouth Asian countries are undergoing various types of transitions. The study sheds some light on the impacts of nutrition transition on the emergence of NCDs in South Asia. Its highly predictable that the economic and social costs associated with nutrition transition will be felt more severely in the upcoming decades. Given the emerging epidemic of NCDs, healthcare system in South Asian countries is at a crossroads. More comprehensive studies are required to gain insight on the disease-specific social, demographic and economic determinants of NCDs in order to design more effective approaches to tackle them in the long run. In conclusion, this study proposes the adoption of following policies to address the challenges of nutrition transition and the emergence of NCDs in South Asia:\n\nNational food policy must focus on improving peoples access to healthy and nutritious food. Better coordination between public and private sectors is required to make the policies function properly. Political will is a most crucial element to regulate and enhance food standards and make food industries abide by the regulations.\n\nLack of experience and inadequate infrastructure are major hindrances to the management of NCDs in South Asia as the healthcare system is originally structured to address the acute type infectious diseases. Addressing obesity and NCDs will therefore require a restructuring of the healthcare system and for that, a well-developed NCD framework must also be put in place.\n\nSince NCD are going to rise, it should be incorporated in the broader development agenda to strengthen healthcare systems for NCD prevention through leveraging nationwide prevention and intervention programs, allocating sufficient resources and improving primary health care.\n\nSchool based nutrition and physical activity programs must be leveraged to reduce childhood obesity. Junk food must be banned in schools and in nearby shops and the advertising of such products must also be controlled.\n\nProviding information about the impacts of unhealthy diets in news papers, national TV channels and radios may help greatly to encourage healthy eating behaviour. People must be made aware of the fact that there is no silver bullets to address NCDs, and prevention remains the best cure. Health policy makers have to take measures to reduce the risky behaviours such as controlling smoking, alcoholism, use of trans-fat in restaurants and food companies, consumption of junk food.\n\nAs the burden of obesity and NCDs is shifting towards the poor, reducing the cost of NCD drugs and ensuring equitable access to NCD care remains a high priority. Governments need to make sure that caregivers don’t profit at the cost of impoverishment of the vulnerable population. Disputes over drug patenting of life saving drugs must be dealt with by prioritizing long term health benefits over momentary economic gain.\n\nSince private sectors are largely responsible for the spread of NCD epidemic, they must also come forward to solve this issue. Building research partnerships among various sectors are essential to measure the magnitude of the problem and design a holistic approach for preventing NCDs.\n\n\nLimitations of the study\n\nThe assessment of the impact of nutrition transition is a rather complex task in the context South Asia due to highly unequal social hierarchies. The impact of recent economic growth has not been even across all societies and is criticized for accelerating income inequality. Evidently, average income is rising and health status is also improving. But the majority of the population is still living below poverty line ($1.25/day) in South Asia and it still remains home to worlds largest proportion of undernourished population. This is a highly paradoxical situation since nutrition transition is expected to have better health outcomes for these historically deprived populations who lack access to sustainable food supply and have no sight of the lifestyle changing technologies. Coexistence of over- and undernutrition is increasing in the same communities which makes it hard to develop strategies for the target population. Surtaxing certain food commodities (Meat and dairy products) might adversely affect food and nutrition security among poor households who spend a bulk of income on food. This situation calls for more comprehensive studies to make more context adjusted food and nutrition policies.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author acknowledges generous help from his colleagues from India, Nepal and Bangladesh for supplying valuable insight, data and the photographs. The author also wishes to extend special thanks to Professor Barry M. Popkin for providing valuable study materials and suggestions which greatly helped to improve the manuscript.\n\n\nReferences\n\nAhsan Karar Z, Alam N, Kim Streatfield P: Epidemiological transition in rural Bangladesh, 1986–2006. Glob Health Action. 2009; 2: 1–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAhsan Karar Z, Alam N, Kim Streatfield P: Epidemiological transition in rural Bangladesh, 1986–2006. Glob Health Action. 2009; 2: 1–9. 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Reference Source\n\nKoh-Banerjee P, Chu NF, Spiegelman D, et al.: Prospective study of the association of changes in dietary intake, physical activity, alcohol consumption, and smoking with 9-y gain in waist circumference in 16 587 US men. Am J Clin Nutr. 2003; 78(4): 719–727. PubMed Abstract\n\nLim SS, Vos T, Flaxman AD, et al.: A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions,1990–2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2224–2260. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMisra A, Shrivastava U: Obesity and dyslipidemia in South Asians. Nutrients. 2013; 5(7): 2708–2733. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaul A, Francis B: Epidemiological and nutrition transition in developing countries: impact on human health and development. Proc Nutr Soc. 2008; 67(1): 82–90. Publisher Full Text\n\nPopkin BM, Adair LS, Ng SW: Global nutrition transition and the pandemic of obesity in developing countries. Nutr Rev. 2012; 70(1): 3–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPopkin BM, Horton SH, Kim S: The Nutrition Transition and Prevention of Diet-related Diseases in Asia and the Pacific. Food Nutr Bull. 2001; 22(4): 3–58. Reference Source\n\nReardon T, Peter Timmer C, Barrett CB, et al.: The Rise of Supermarkets in Africa, Asia, and Latin America. Am J Agr Econ. 2003, 85(5): 1140–1146. Reference Source\n\nRoux L, Pratt M, Tengs TO, et al.: Cost effectiveness of community-based physical activity interventions. Am J Prev Med. 2008; 35(6): 578–88. PubMed Abstract | Publisher Full Text\n\nSalter AM: Impact of consumption of animal products on cardiovascular disease, diabetes, and cancer in developed countries. Animal Frontiers. 2013; 3(1): 20–27. Publisher Full Text\n\nSaquib N, Saquib J, Ahmed T, et al.: Cardiovascular diseases and type 2 diabetes in Bangladesh: a systematic review and meta-analysis of studies between 1995 and 2010. BMC Public Health. 2012; 12: 434. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShariful Islam SM, Lechner A, Ferrari U, et al.: Social and economic impact of diabetics in Bangladesh: protocol for a case-control study. BMC Public Health. 2013; 13: 1217. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShetty PS: Nutrition transition in India. Public Health Nutr. 2002; 5(1A): 175–182. PubMed Abstract | Publisher Full Text\n\nSingh RB, Tomlinson B, Thomas GN, et al.: Coronary artery disease and coronary risk factors: the South Asian paradox. J Nutr Environ Med. 2001; 11(1): 43–51. Publisher Full Text\n\nTrasande L, Cronk C, Durkin M, et al.: Environment and obesity in the National Children’s Study. Environ Health Perspect. 2009; 117(2): 159–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrends and Determinants of Foreign Direct Investment in South Asia. The World Bank Group Office of the Chief Economist South Asia Region. Report No: ACS4862.6/16/2013. Reference Source\n\nVineis P, Stringhini S, Porta M: The environmental roots of non-communicable diseases (NCDs) and the epigenetic impacts of globalization. Environ Res. 2014; 133: 424–30. PubMed Abstract | Publisher Full Text\n\nWagner KH, Brath H: A global view on the development of non communicable diseases. Prev Med. 2012; 54(Suppl): S38–41. PubMed Abstract | Publisher Full Text\n\nWahdan MH: The epidemiological transition. East Mediterr Health J. 1996; 2(1): 8–20. Reference Source"
}
|
[
{
"id": "10887",
"date": "02 Nov 2015",
"name": "Ailiana Santosa",
"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 widespread, well-balanced and comprehensive review of study over the last two decades on the nutrition transition. The manuscript reads well and has important findings. The introduction clearly defines the purpose of the manuscript and the relevance to the policy practice.However, the author does not clearly describe the literature search strategies in the methods section as he mentioned it in the objective of the study. He stated that he used several electronic databases including COCHRANE library. Has he fulfilled the COCHRANE checklist? He also did not clearly state the inclusion and exclusion criteria. Even he stated that there were no special exclusion criteria (why there are no special exclusion criteria?). My concerns are while the method of review paper is unclear, it will dilute the findings and the discussion later. I found that some of findings are diluted and unclear. In revising the manuscript, the author should:Divide the published studies between review studies and empirical studies, and bring to light emerging evidence from existing review studies and clarify what new evidence is learnt from their review. Divide evidence regarding different terms of transition that he focused on and explanatory factors contributing to the nutrition transition in South Asia. In the \"Methods\" section, clarify which are the inclusion and exclusion criteria, the agreement for selecting the published articles used for review, the indicator(s) are used to evaluate the nutrition transition or lack of it, and attempt in the presentation of the findings to interpret the sensitivity of the findings to alternative specification of indicator for measuring the timing and stage of transitions. Summarise in a table the evidences (and number of studies) for the nutrition transition by country coverage (rural, urban, sub-national, national); then address the extent to which the predictive article expected from the nutrition transition has varied across study countries.",
"responses": [
{
"c_id": "1695",
"date": "24 Nov 2015",
"name": "Bishwajit Ghose",
"role": "Author Response",
"response": "Dear Professor,I wish to extend my sincere thanks for your valuable comments and suggestions regarding the paper.The contribution of the present review is that it reaffirms the findings of past studies by cross verifying from multiple studies conducted on the individual countries and proposes a set of policy instruments.As shown in the tables and figures, the findings on the drivers and outcomes of dietary transition were consistent for the countries included in the study. Cochrane Library was searched for this study, but the database provided no directly relevant article itself. Reference lists however were searched manually for articles of relevance and were sourced from the publisher. Authors mentioned that there is no such study in the context of South Asia as yet, and the literature resource is notably meagre. Which is why no special exclusion criteria was applied. Broadening the inclusion criteria facilitated the triangulation and cross-verification of evidences from different sources. Almost of the articles were reviews and no clinical trials were available on the topic.With regard to comment 2, the review summarizes the proximal sociodemographic and economic factors which are driving the dietary and epidemiological transition and have presented concrete evidences on the shifting epidemiological pattern in South Asia. It recognizes the absence of nationally representative studies on exploring more subtle and context specific underlying causes of dietary transition. Knowledge from more in-depth studies is crucial and could be leveraged to develop national nutrition policies targeting the rising burden of NCDs. Previous studies were limited in their scope of showing that nutrition transition is underway in these countries, however missed the opportunity to propose policies based on their findings. To this end, apart from summarizing the past studies, the main aim of this review was to the synthesize a range of policy measures based on studies and policy papers in countries who have long experienced nutrition transition and NCDs prevention policies. The methodology section was rewritten as suggested. Thank you!With regard to part of comment 1 and 4, a new table (4) was included providing the information as suggested. Thank you!"
}
]
},
{
"id": "11199",
"date": "12 Nov 2015",
"name": "Saverio Stranges",
"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\nOverall CommentsThis article discusses the nutrition transition in South Asia, its influence on the nutrition profile of South Asian countries, and its relationship with the increasing burden of non-communicable diseases in the region. The author stipulates that countries in this region are mired by the dual burden of malnutrition and the new emergence of obesity and associated NCDs.While the article and its content are poignant additions to literature on nutrition and NCDs, and the manuscript is in large part quite well-written, it could greatly benefit from a few improvements. Specifically, there are a few claims made by the author which need to be substantiated by more evidence.IntroductionThe section begins with a sentence that is too long, and needs to be either broken up or shortened to improve its coherence.The author mentions several economic statistics in the first paragraph (lines 27 to 30) which need a reference. Similarly, the facts stated between lines 39 and 45 need to be substantiated with a reference.MethodologyLine 2: Please add the word “review” after systematic literature.Demographic transition in South AsiaLine 4: Please replace “mankind” with “global population”.Pages 4 through to 7 present some good evidence with regards to nutrition, NCDs and socioeconomic and demographic factors.However, under “Urbanization” on page 6, the author states that women joining the labour force “greatly influences the shift from home cooked traditional food towards precooked convenience food and fast food”. Currently, this statement is not substantiated by evidence.Epidemiological transition in South Asia and the burden of NCDsThe second paragraph starts as “While in the developed countries the battle is chiefly against NCDs, people in developing countries are facing a double burden [of] malnutrition.” I believe that the first and the second halves of the sentence are not related as the word “while” would suggest. It is possible that the author intends to make a point here which is unclear at the moment, and some paraphrasing or additional information might help clarify the issue.On the same page (7), the caption for Figure 4 ends with “with no place for fresh fruits and vegetables”. I believe that this is a non-factual interpretation of the photograph provided, since all the facts cannot be ascertained from the picture. As such, I believe that this part of the caption should be removed. Also, it would benefit the manuscript if loaded terms such as “teeming” can be removed or substituted with more neutral terms. In this specific example, the word can be removed.In the third paragraph (p. 8, lines 13-15), the statistics on the burden of diabetes in Bangladesh need to be referenced.The economic impacts of NCDsI would like to commend the author on making a very important point about the economic burden of NCDs, especially on vulnerable households; that NCDs are both a socioeconomic determinant of health, as well as a result of poor socioeconomic status.However, in developing countries undergoing economic transition, studies have shown that better socio-economic status puts individuals at greater risk of obesity and associated NCDs. For example:Gouda J, Prusty R (2014) Overweight and obesity among women by economic stratum in urban India. J Health Popul Nutr 32:79–88Pednekar MS, Hakama M, Herbert JR, Gupta PC (2008) Association of body mass index with all-cause and cause-specific mortality: findings from a prospective cohort study in Mumbai. Int J Epidemiol 37:524-535Chhabra P, Chhabra SK (2007) Distribution and determinants of body mass index of non-smoking adults in Delhi, India. J Health Popul Nutr 25:294-301Tackling the risk factors of NCDs among childrenThe suggestions provided at the start of the first paragraph in this section (p. 9) need to be referenced or substantiated with evidence. Have such measures been scientifically confirmed as being effective?Statistics on the global expenditure on food advertisements needs to be referenced.Is banning of “junk food” in schools effective as a health promotion measure for children even beyond school? The author’s suggestion is a very valid point, but it would be even better if studies which have assessed the benefits of such measures are cited.On page 10, under “Community based physical activity campaigns”, the author mentions “most studies” as having highlighted the relationship between lack of open spaces (for physical activity) and NCDs. These studies need to be referenced here.Conclusions and policy recommendationsOn page 11, the final recommendation appears too presumptuous about the private sector’s responsibility in the spread of the NCD epidemic. This might be a fair point, but it can be challenged without any evidence to substantiate it. It is also too broad a claim to be included, and the recommendation to build partnerships for a holistic approach is enough on its own without the preceding sentence.Final commentsI believe that the suggested improvements would advance and improve the manuscript greatly towards acceptance. I would suggest minor language edits, since there appear to be more typographical than syntactic errors, and these can be resolved through proof-reading.",
"responses": [
{
"c_id": "1694",
"date": "24 Nov 2015",
"name": "Bishwajit Ghose",
"role": "Author Response",
"response": "Dear Professors,Thanks indeed for your evaluation and providing valuable insights and suggestion for the article. Below is a step-by-step list of the modifications made in the revised manuscript.1-Introduction: The opening sentences was split into two.New references were added for the suggested sentences. (39-40)2. Methodology: The word 'review' was added after 'systematic literature'.Demographic transition in South Asia. Line 4: “Mankind” was replaced with “global population”.Following references were added regarding demographic, socioeconomic contexts of NCDs: [41-47].3. Epidemiological transition in South Asia and the burden of NCDs The sentence was rephrased to:In contrast with developed countries where the disease burden is dominated by NCDs (with lower proportion of infectious diseases), countries in the developing world are facing a double burden of malnutrition.The following sentence was removed as suggested. Thank you!“The super-shops like this in the city however bear the signs of industrialized nations teeming with processed foods such as cookies, chips, and soft drinks with no place for fresh fruits and vegetables.”New References were added (48-49). 4. The economic impacts of NCDs Completely agree in this respect. Evidences on the impact of changing socioeconomic status on NCDs remain pretty much mixed and unsubstantiated. The author consulted with Prof. Barry regarding this issue and was advised that the confusion lies in the complex geographical and socio-cultural interplay which determines the variation in susceptibility to certain obesogenic factors/environments.Authors understanding is that historically South Asian societies are extremely stratified and each stratum exhibiting varying degrees of proneness to certain diseases and risk factors. The situation is actually very perplexing and requires in-depth epidemiological studies to find an answer to this complexity. However, explaining this issues is not in the scope of the current study and could cause deviation from the central theme. 5. Tackling the risk factors of NCDs among childrenGovernmental policy of surtaxing on tobacco has previously shown to have effectively reduced smoking rates.1] Zhang B. The impact of tobacco tax cuts on smoking initiation among Canadian young adults. Am J Prev Med. 2006 Jun;30(6):474-9.2] D Levy. Increasing taxes to reduce smoking prevalence and smoking attributable mortality in Taiwan: results from a tobacco policy simulation model. Tob Control. 2005 Jun; 14(Suppl 1): i45–i50.Applying such policies on food items is hard compared to tobacco/alcohol. Few countries have only including such policies for selected food items (mentioned in the article) in their national nutrition policy to promote healthy eating habits. Their effectiveness remains to be studied.In the context of South Asia such policies may sound paradoxical since the rate of poverty and undernutrition is still very high. However, given the rising burden of morbidity and mortality from NCDs, such policies deserve due attention in the public health agenda. In theory, raising prices should reduce the consumption of a given product. However, whether or not it will translate to lower prevalence of NCDs in the long term will also require long-term trials. Authors proposed these policies based on the synthesis of policy literature from developing countries, not of epidemiological findings.Reference 51 was added for the comment as suggested.Statistics on the global expenditure on food advertisements was given reference no [50].Majority of the studies have actually focused on the impact of selling of junk food inside schools and have suggested to ban selling junk foods both on campus and in shops nearby. However, evidences from studies focusing exclusively on shops away from school are not yet available.Page 10: References 52-53 were added.ConclusionsThe sentence was removed. Thank you!The manuscript was revised for typographical error was corrected where applicable. Thank you!"
}
]
}
] | 1
|
https://f1000research.com/articles/4-8
|
https://f1000research.com/articles/4-164/v1
|
23 Jun 15
|
{
"type": "Research Article",
"title": "Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus",
"authors": [
"J. Fallon Campbell",
"Sarah J. Swartz",
"Scott E. Wenderfer",
"J. Fallon Campbell",
"Sarah J. Swartz"
],
"abstract": "Hypertension is an important manifestation of systemic lupus erythematosus (SLE) but reports of prevalence vary between 20-70% in published reports of adult and pediatric patients. For both children and adults with SLE, the clinical diagnosis and management of hypertension has traditionally been based on guidelines developed for the general population. In clinical trials, the criteria used for defining participants with hypertension are mostly undefined. As a first step towards formally assessing the blood pressure (BP) patterns of children diagnosed with SLE, 24-hr ambulatory BP monitoring data was analyzed on clinic patients who presented with prehypertension or stage I hypertension. In this pediatric SLE cohort, 20% met daytime criteria for a diagnosis of hypertension. Patterns of BP elevation varied widely with white coat, masked, isolated systolic, and diastolic nocturnal hypertension all identified. Nocturnal hypertension was detected in 60% and attenuated nocturnal BP dipping in 90% of both hypertensive and normotensive SLE patients. In SLE patients, the median nighttime systolic and diastolic loads were 25% and 15.5% compared with median daily loads of 12.5% and 11.5%. Daytime and nighttime systolic and diastolic BP load and nocturnal dipping was compared to a control population consisting of 85 non-SLE patients under 21 years old with prehypertension or stage 1 hypertension presenting to hypertension clinic. Median systolic BP dipped 5.3 mmHg in SLE patients compared to 11.9 mmHg in non-lupus (p-value = 0.001). Median diastolic BP dipped 12.9 mmHg versus 18.5 mmHg in non-lupus (p-value = 0.003). Patterns of BP dysregulation in pediatric SLE merit further exploration. Children with or without SLE displaying prehypertensive or stage 1 casual BP measurements had similar rates of hypertension by ambulatory BP monitoring. However, regardless of BP diagnosis, and independent of kidney involvement, there was an increased proportion with attenuated nocturnal dipping and nocturnal hypertension in SLE patients.",
"keywords": [
"Hypertension",
"Blood pressure",
"SLE",
"Lupus",
"Pediatric",
"ABPM",
"White-coat",
"Prehypertension"
],
"content": "Introduction\n\nAmbulatory blood pressure monitoring (ABPM) is preferred to casual clinic blood pressure (BP) monitoring in the diagnosis of hypertension (HTN). There are many shortfalls of casual BP readings, including the white coat effect, observer bias/measurement error, regression to the mean with repeated measurements, and variability of blood pressure over time (Flynn, 2011). Additionally, the published normative values for casual BP are based on the auscultation method, yet many clinic measurements are taken with oscillometric devices (Woroniecki & Flynn, 2005). Perhaps the most clinically relevant shortfall is the limited outcome data regarding casual BP measurements and end-organ damage or cardiovascular risk.\n\nThere is good evidence supporting the utility of ABPM findings in the prediction of cardiovascular outcomes, both in adults and children (Belsha et al., 1998; Lurbe et al., 2004; Pickering et al., 2006; Singh et al., 2013; Sorof et al., 2002). ABPM can account for the white coat effect as well as measurement and observer errors. Where casual BP measurements account for the magnitude of BP at single points in time, ABPM can define BP loads which measure the proportion of BPs that exceed a defined cutoff, typically the 95th percentile as defined by normative data, over a 24-hr period. Therefore ABPM provides a better appreciation of BP trends which can account for the dynamic nature of BP (such as circadian rhythms, nocturnal BP dipping) (Flynn, 2011).\n\nAlthough ABPM is a valuable piece of the HTN evaluation, there are potential barriers to its widespread utilization related to both financial and clinical considerations. Insurance companies offer limited reimbursement for ABPM placement and interpretation (Swartz et al., 2008). Although there is vast evidence for normative values in adults; there is more limited normative data for ABPM interpretation in children. The current normative values are based on approximately 950 healthy children with limited variability in ethnicity/race (Wühl et al., 2002). Despite these obstacles, ABPM is considered the gold standard for diagnosis of HTN in both adults and pediatrics. It is useful in predicting cardiovascular risk related to HTN but is also helpful in assessing BP in special pediatric populations such as obesity, sickle cell disease, chronic kidney disease, end stage renal disease, and diabetes (Flynn, 2011). There is limited research on the use of ABPM in the pediatric systemic lupus erythematosus (SLE) population (Canpolat et al., 2013), though HTN occurs in 20–70% of these patients (Bogdanovic et al., 2004; Brunner et al., 2002; Lau et al., 2006; Ruggiero et al., 2013). There is also limited data regarding the management of HTN in the SLE population, with management traditionally based on guidelines developed for the general population (Tselios et al., 2014).\n\nCardiovascular disease is a leading cause of mortality in adults with SLE and though there are many non-traditional risk factors including altered renal function, impaired endothelial function, chronic inflammation, and an activated renin-angiotensin system (RAS) (Gustafsson et al., 2012; Kiani et al., 2008; Knight & Kaplan, 2013; Pieretti et al., 2007), HTN is still an important risk factor (Contreras et al., 2005; Ginzler et al., 1993; Petrin et al., 1993; Yang et al., 1994). Therefore the use of standardized definitions to define HTN in SLE patients is crucial in better understanding cardiovascular risk and preventing adverse outcomes. Although isolated nocturnal HTN is not considered sufficient for a diagnosis for systemic hypertension in the general population, it is known to associate with increased risk of cardiac outcomes (Yee, 2015). Additionally, attenuated nocturnal dipping, even in the setting of normal 24-hr BP, was noted to be an independent predictor of cardiovascular mortality in a large prospective cohort study in Japan (Ohkubo et al., 2002). Hence, it is also important to use ABPMs to characterize blood pressure patterns in SLE patients so that specific guidelines for ABPM interpretation can be established for this population.\n\n\nMethods\n\nBP patterns of the 10 SLE study participants (demographics summarised in Table 1) recruited from a single center were retrospectively reviewed using data from 24-hr ambulatory BP monitoring tests performed between February 2012 and April 2013. ABPM was routinely ordered only on SLE patients seen in a multispecialty pediatric lupus clinic when they presented without kidney disease (non-renal lupus or nephritis in remission) and with casual BP measurements in prehypertensive or stage 1 hypertensive range (National High BP Education Program Working Group on High BP in Children and Adolescents, 2004). The inclusion criteria for the SLE cohort were: (1) diagnosis of SLE by American College of Rheumatology (ACR) criteria, (2) age < 21 years, and (3) ABPM performed. There were 85 patients in the non-SLE cohort (43% female, race and ethnicity unknown). The inclusion criteria for the non-SLE cohort included: (1) age < 21 years and (2) ABPM performed. Exclusion criteria for both cohorts: (1) ABPM uninterpretable due to incomplete/missing data, (2) casual BP measurements all < 90th or all > 99th percentile for age, gender, and height, +5 mmHg (3) end stage renal disease, or (4) kidney transplant recipient. The mean age at ABPM for the non-SLE cohort was 12.4 ± 0.4 years, and the mean BMI was 25.1 ± 0.8 kg/m2 using the Mosteller formula. The study protocol was reviewed and approved by the Institutional Review Board for Baylor College of Medicine (H-32061).\n\nParticipants. As this study was a retrospective chart review, there were no dropouts.\n\nSample size. As this was a pilot study, the sample size was not determined by formal power analysis. There were 11 consecutive SLE patients and 100 consecutive non-SLE patients with ABPM data identified, but 1 and 15 patients, respectively did not qualify based on the inclusion and exclusion criteria.\n\nBlinding. All interpretation of ABPM data was performed at the time of clinical testing, prior to inception of the study. There was no formal blinding of ABPM data during the data analysis or sensitivity analysis phases.\n\n24-hr ABPM was performed using SpaceLabs 90217-1Q or 90217A-1 equipment. SpaceLabs Medical Software (version 90219) was used to evaluate the BP patterns. BP measurements were automatically measured every 20 minutes during the daytime and every 30 minutes during the nighttime over a 24-hr period. Mean diastolic and systolic BPs were calculated for both daytime and nighttime periods and compared to normative data for mean BPs based on age and gender (Wühl et al., 2002). BP loads were calculated for both diastolic and systolic BP, reflecting the percentage of BP measurements above the 95th percentile for gender and age. Blood pressure loads >25% were considered abnormal (Urbina et al., 2008). Additionally, nocturnal dipping of BP was defined as the difference between daytime and nighttime BPs. Nocturnal dipping <10% was considered abnormal (Urbina et al., 2008). American Heart Association (AHA) definitions were used to define normal blood pressure, masked, white-coat and sustained HTN (Flynn, 2011).\n\nDemographic and clinical data was collected from medical records for the SLE cohort. Demographic data included race and age at diagnosis. Clinical data (from within 3 months of ABPM date) included height, weight, BMI, laboratory results, eGFR using the Schwartz formula (Schwartz et al., 2009), presence of proteinuria, medications at the time of ABPM, echocardiogram findings including left ventricular hypertrophy (LVH), left ventricular mass index (LVMI), and relative wall thickness (RWT), ACR criteria for SLE, and SLEDAI score (Systemic Lupus Erythematosus Disease Activity Index). Demographic information for the non-SLE cohort was obtained from the SpaceLabs software, including age at the time of ABPM, gender, height, and weight.\n\nStatistical analysis was performed using SigmaPlot software (version 11.0). Patient characteristics and ABPM findings were analyzed using descriptive statistics (medians, and intra-quartile ranges). Fisher exact and Wilcoxon rank sum tests were used to characterize patient demographics and BP patterns. Statistical significance was defined as p-value ≤0.05 (two tailed).\n\n\nResults\n\nThe study population consisted of ten patients, all of whom met ACR diagnostic criteria for SLE (Patient demographics in Table 1). At the time of ABPM, mean age of the SLE cohort was 14.6 years. The mean BMI was 25.1 kg/m2. Nine patients were female. Three SLE patients were Hispanic, three were Caucasian, and four were African American. The non-SLE control population consisted of 85 age- and BMI-matched pediatric patients with casual BP measurements between 90–99th percentile without kidney disease or diabetes.\n\nAll of the SLE patients were antinuclear antibody (ANA) positive, nine were anti-double stranded DNA (anti-dsDNA) antibody positive, and nine tested positive for anti-phospholipid antibodies (aPL). Three SLE patients had echocardiograms and none had sonographic evidence of left ventricular hypertrophy based on adult criteria (LVMI 35.3, 39.2, and 42.9 g/m^2.7, with RWT of 0.40, 0.52, and 0.39). Two patients had proteinuria based on the SLE ACR definition (>0.5 g/day) at the time of ABPM. Among the seven patients with lab values available, the mean ESR was elevated at 58.71 mm/hr (normal <20) and the mean C3 was slightly low at 83.4 mg/dL (normal 90–200).\n\nAll SLE patients were prescribed prednisone at the time of ABPM with a mean dose of 0.31 ± 0.08 mg/kg/day. Three patients also received intravenous (IV) pulse steroids within the 3 months prior to ABPM. Two received weekly doses of IV solumedrol (30mg/kg/dose) and the third received a one-time dose in the week prior to ABPM placement. At the time of ABPM, only one was prescribed an ACE-inhibitor, and none were prescribed diuretics or any other anti-hypertensive. No one was treated with rituximab within the 2 years prior to the ABPM, though one patient did receive Rituximab following ABPM. The SLE patients met a median of six SLE ACR criteria for diagnosis of SLE. Five patients met criteria for malar rash; three met criteria for photosensitivity; five had mouth sores; three experienced serositis; eight had arthritis; six had renal involvement; seven met hematologic criteria; nine met immunologic criteria; and one met neurologic criteria. No patients were noted with a documented discoid rash.\n\nSLE patients tended to have lower daytime systolic blood pressure (SBP) and diastolic blood pressure (DBP) loads and higher nighttime systolic BP loads as compared to the non-SLE patients, and the decreased median SBP load was statistically significant (Table 2). The SLE cohort also showed a significantly higher rate of attenuated nocturnal dipping in both SBP and DBP, when compared to the non-SLE cohort (Figure 1). Ninety percent of SLE patients had attenuated nocturnal dipping compared to only 26% of non-SLE patients. SLE patients also had a higher rate of nocturnal HTN, whether in isolation or in conjunction with daytime HTN.\n\n* based on definition of elevated BP as exceeding the 95th%tile for age and gender (Wuhl, 2002)\n\nBox plots indicate median, 10th, 25th, 75th, and 90th percentile data, based on Wilcoxon Analysis. Dots indicate the outliers. Differences in %SBP dipping (p = 0.001) and %DBP dipping (p = 0.003) and daytime SBP load (p = 0.01) were statistically significant whereas differences in nighttime SBP load (p = 0.36) and DBP load (p = 0.59) failed to reach significance.\n\nSpecifically, only two SLE patients met ABPM criteria for both daytime and nighttime HTN; however four additional patients had isolated nocturnal HTN with normal daytime BPs. Nine of the SLE patients had attenuated nocturnal dipping, regardless of HTN diagnosis. Of the nine SLE patients with attenuated nocturnal dipping, two had proteinuria at the time of ABPM. The one patient with normal dipping had no proteinuria.\n\nThere were no statistically significant associations between most laboratory measures (complement 3 (C3), ANA, anti-dsDNA antibodies, aPL antibodies) and nocturnal HTN or attenuated nocturnal dipping. The two patients without aPL antibodies did not have nocturnal HTN, though they did have attenuated dipping (Table 3). The one patient who had normal nocturnal dipping was African American, had the highest BMI, low C3 levels, a SLEDAI score of 4, received a dose of pulse steroid within 3 weeks of ABPM, and met more than six SLE ACR criteria. She did have nocturnal and masked HTN. There were no obvious associations between ABPM findings and the presence of specific historical ACR criteria for SLE; however, this study is underpowered to perform formal statistical analysis. Of the six SLE patients who historically met diagnostic criteria for kidney disease, five had nocturnal HTN, while only one of the four patients without a history of nephritis had nocturnal HTN. Moreover, five of the six with a history of nephritis and all four of the non-renal SLE patients had attenuated nocturnal dipping.\n\n* based on definition of elevated BP as exceeding the 95th%tile for age and gender (Wuhl, 2002)\n\nThere was also no significant difference in attenuated nocturnal dipping between SLE patients who received pulse corticosteroids within 3 months of ABPM and those who had not. Of the three patients who received pulse steroids, two had nocturnal HTN and one did not. Finally, there were no statistically significant association between use of specific immunosuppressive medication usage (azathioprine, mycophenolate mofetile, hydroxychloroquine, methotrexate) and either nocturnal HTN or attenuated nocturnal dipping. The patient who was on an ACE inhibitor at the time of ABPM did have attenuated nocturnal dipping and nocturnal HTN.\n\nAttenuated dipping was not associated with disease duration. The patient with the longest SLE vintage (9 years from SLE diagnosis to time of ABPM) had both nocturnal HTN and attenuated nocturnal dipping, but the three patients with the shortest disease duration (<1 year from SLE diagnosis to time of ABPM) all had attenuated dipping.\n\nThe two SLE patients with daytime HTN also had nocturnal HTN and attenuated nocturnal dipping. They were both African American and one was the only male in the cohort. One patient’s disease duration was 9 years whereas the other was diagnosed in the past year. The patient with disease duration of < 1 year was on a higher oral steroid dose and had proteinuria and low C3 level. They both met more than six ACR diagnostic criteria.\n\nAll four of the patients with normal nocturnal BPs still had attenuated nocturnal dipping. Three of these patients’ disease duration was ≤ 1 year while the fourth was 6 years. Their SLEDAI scores ranged from 4–8 at the time of ABPM ± 10 days.\n\nTo determine whether disease-specific BP parameters might be influenced by the thresholds used to define hypertension during analysis of the ABPM data, a sensitivity analysis was performed. Since all patients in our pediatric SLE cohort lacked active nephritis and heart disease, a 95% cutoff was used to distinguish normal versus high BP. To test whether using a 90% cutoff would alter the results, all SLE patient ABPM data was re-interpreted. The data was also reanalyzed using BP loads of >30% (per institutional protocol) rather than 25% (Urbina et al., 2008) to define HTN. In addition, to test if the quality of the ABPM data affected the findings, the comparison between cohorts was repeated after ABPM tests were discarded if either <75% of attempted BP measurements were successful, <50 total measurements were successful, or both. Finally, since 90% of the SLE cohort was female, comparisons were made to the non-SLE controls after eliminating ABPM data from males in the non-SLE cohort. Results showed that decrease in prevalence of daytime SBP load in the SLE cohort lost significance using a 90% cutoff, whereas the increase in incidence of nocturnal HTN became significant using BP loads >30% to define hypertension (Figure 2). The attenuation of nighttime BP dipping in the SLE cohort and all other ABPM findings were not significantly altered by any of the changes.\n\nBoxes represent the range of medians (left) or means (right) obtained from sensitivity analysis. Analyses were repeated comparing non-SLE to SLE cohort, using either 90th or 95th percentiles, and 25% or 30% load, in the definition for hypertension, and by restricting dataset to only include ABPM findings when success rates of measurements were >75%, when >50 total successful measurements were recorded, or both. Ranges of p-values are indicated.\n\n\nDiscussion/Conclusions\n\nThis study illustrates the potential benefit for further investigation of ABPM use in characterizing BP patterns in SLE patients. Our results show that pediatric SLE patients have a very high rate of attenuated nocturnal SBP and DBP dipping. This was associated with higher rates of nocturnal HTN (whether isolated or in conjunction with daytime HTN), though with standard ABPM-based definitions, this was not statistically significant. A previous study of subclinical cardiovascular disease in pediatric SLE patients reported similar findings with 14 of 21 patients having attenuated nocturnal dipping and higher nocturnal BPs when compared to daytime BPs (Canpolat et al., 2013). However the prior study’s primary focus was cardiovascular risk. There was no control group, and the relationships between ABPM findings and clinical characteristics tested were limited to echocardiogram findings. The small number of studies using ABPMs to characterize BPs in other pediatric chronic illnesses, such as sickle cell disease, has been revealing. This is the first study to investigate the relationship between BP characteristics on ABPM and clinical characteristics in the pediatric SLE population.\n\nSLE patients are at increased risk for death and cardiovascular disease is a leading cause of mortality in this population. This is related to both traditional and non-traditional risk factors in adult patients (Knight & Kaplan, 2013). In a study of 94 adults with SLE, correlations were noted between intima-medial thickness and clinical disease activity scores (Oryoji et al., 2013). Similarly, in a separate study of 64 adults with SLE and nephritis in complete remission, 53% were hypertensive (Shaharir et al., 2015), and the risk factors identified included disease duration (odds ratio (OR) 1.06), longer duration interval to achieving remission (OR 1.10), and the number of disease relapses (OR 2.5). There were no associations between histological classes of nephritis, body mass index, or waist circumference. A study of 51 children with SLE also demonstrated that functional and morphological cardiovascular changes were independent of traditional risk factors such as daytime HTN, hypertriglyceridemia, diabetes, and chronic kidney disease (Sozeri et al., 2013). In SLE, these changes in arterial stiffness, intima-media thickness, and LV mass (Canpolat et al., 2013; Oryoji et al., 2013; Sozeri et al., 2013) are likely to be almost entirely secondary to non-traditional factors, such as disease-related mechanisms like enhanced apoptosis, aPL antibodies, circulating immune complexes, and vasculitis.\n\nTherefore it is important to understand the BP characteristics of these patients, particularly the nocturnal BP patterns, as our study shows that they differ from the general population. There was no effect on this altered ABPM blood pressure pattern in our cohort attributable to medication usage, complement cascade activation and hypocomplementemia, or titers of ANA, aPL antibodies, or anti-dsDNA antibodies. Future studies of ABPM testing in SLE populations can be designed to further assess clinical parameters such as degrees of systemic inflammation, interferon versus neutrophil signatures, or endothelial cell dysfunction, in order to try to understand the possible mechanisms for elevated nocturnal BPs and attenuated nocturnal dipping in SLE patients.\n\nBased on our study, the duration of disease did not seem to play a role in the attenuated dipping, as this pattern was seen even within the first year after SLE diagnosis. Since 90% of SLE patients had attenuated dipping, compared with only 60% of patients meeting criteria for diagnosis of nocturnal hypertension, one might conclude that attenuated nocturnal BP dipping is an earlier change that progresses to nocturnal HTN in the setting of SLE. However, the SLE patient without attenuated dipping did have nocturnal hypertension. Therefore, it is more likely that the disease process in SLE leads to cardiovascular changes sufficient to cause elevated nighttime BP very early in the disease course. If nocturnal HTN or attenuated BP dipping turn out to be pathogenic in SLE, then monitoring for HTN solely with casual daytime clinic measurements may postpone possible interventions that could potentially reduce the increased cardiovascular risk faced by these patients.\n\nOne limitation of this study is the small number of patients in the SLE group and the resulting low power. Several findings trended toward significance and might become statistically significant with a larger study population. Although there were a limited number of statistically significant findings, strict inclusion of only SLE patients with prehypertension or stage 1 hypertension, without active nephritis, and most without anti-hypertensive medication, provided for a valid comparison between children with SLE and non-lupus controls. Our study does show that nocturnal HTN and attenuated nocturnal dipping do occur more frequently in pediatric SLE patients than in the non-SLE population. Further research is warranted regarding the association of these findings with other clinical characteristics.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw ABPM Data for pSLE Cohort, 10.5256/f1000research.6532.d49239 (Campbell et al., 2015a).\n\nF1000Research: Dataset 2. Raw ABPM Data for non-SLE Cohort, 10.5256/f1000research.6532.d49240 (Campbell et al., 2015b).\n\nF1000Research: Dataset 3. Raw Data: Sensitivity Analysis: non-SLE greater than 50 successful readings, 10.5256/f1000research.6532.d49241 (Campbell et al., 2015c).\n\nF1000Research: Dataset 4. Raw Data: sensitivity analysis: non-SLE, greater than 75 percent successful, 10.5256/f1000research.6532.d49242 (Campbell et al., 2015d).\n\nF1000Research: Dataset 5. Raw Data: Sensitivity Analysis: pSLE greater than 50 successful readings, 10.5256/f1000research.6532.d49257 (Campbell et al., 2015e).\n\nF1000Research: Dataset 6. Raw Data: Sensitivity analysis: pSLE greater than 75 percent successful, 10.5256/f1000research.6532.d49258 (Campbell et al., 2015f).\n\nF1000Research: Dataset 7. Raw Data: Sensitivity analysis: pSLE using 90th percentile, 10.5256/f1000research.6532.d49259 (Campbell et al., 2015g).\n\n\nConsent\n\nA waiver of consent was obtained from the Institutional Review Board for this study.",
"appendix": "Author contributions\n\n\n\nJFC and SEW conceived the study. JFC and SEW designed the experiments. JFC and SEW carried out the research. SJS contributed to the design of experiments and provided expertise in analysis of ambulatory BP monitoring data. JFC and SEW prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded in part by a Pediatric Pilot Award program, granted to SEW by the Department of Pediatrics at Baylor College of Medicine.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank Dr. Michael Braun (BCM) for thoughtful discussions and assistance with reading ambulatory BP monitoring reports, Isenia Medina (BCM) for assistance with performing the ambulatory BP tests, and Debra Canter (BCM) for regulatory support; as well as Dr. Marietta De Guzman and all of the nurses and Pediatric Rheumatologists who see patients in the Texas Children’s Hospital Pediatric Lupus Clinic.\n\n\nReferences\n\nBelsha CW, Wells TG, McNiece KL, et al.: Influence of diurnal blood pressure variations on target organ abnormalities in adolescents with mild essential hypertension. Am J Hypertens. 1998; 11(4 Pt 1): 410–7. PubMed Abstract | Publisher Full Text\n\nBogdanović R, Nikolić V, Pasić S, et al.: Lupus nephritis in childhood: a review of 53 patients followed at a single center. Pediatr Nephrol. 2004; 19(1): 36–44. PubMed Abstract | Publisher Full Text\n\nBrunner HI, Silverman ED, To T, et al.: Risk factors for damage in childhood-onset systemic lupus erythematosus: cumulative disease activity and medication use predict disease damage. Arthritis Rheum. 2002; 46(2): 436–44. PubMed Abstract | Publisher Full Text\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 1 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015a. Data Source\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 2 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015b. Data Source\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 3 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015c. Data Source\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 4 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015d. Data Source\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 5 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015e. Data Source\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 6 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015f. Data Source\n\nCampbell JF, Swartz SJ, Wenderfer SE: Dataset 7 in: Nocturnal Hypertension and Attenuated Nocturnal Blood Pressure Dipping is Common in Pediatric Lupus. F1000Research. 2015g. Data Source\n\nCanpolat N, Kasapcopur O, Caliskan S, et al.: Ambulatory blood pressure and subclinical cardiovascular disease in patients with juvenile-onset systemic lupus erythematosus. Pediatr Nephrol. 2013; 28(2): 305–13. PubMed Abstract | Publisher Full Text\n\nContreras G, Pardo V, Cely C, et al.: Factors associated with poor outcomes in patients with lupus nephritis. Lupus. 2005; 14(11): 890–5. PubMed Abstract | Publisher Full Text\n\nFlynn JT: Ambulatory blood pressure monitoring in children: imperfect yet essential. Pediatr Nephrol. 2011; 26(12): 2089–94. PubMed Abstract | Publisher Full Text\n\nGinzler EM, Felson DT, Anthony JM, et al.: Hypertension increases the risk of renal deterioration in systemic lupus erythematosus. J Rheumatol. 1993; 20(10): 1694–700. PubMed Abstract\n\nGustafsson JT, Simard JF, Gunnarsson I, et al.: Risk factors for cardiovascular mortality in patients with systemic lupus erythematosus, a prospective cohort study. Arthritis Res Ther. 2012; 14(2): R46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKiani AN, Magder L, Petri M: Coronary calcium in systemic lupus erythematosus is associated with traditional cardiovascular risk factors, but not with disease activity. J Rheumatol. 2008; 35(7): 1300–6. PubMed Abstract\n\nKnight JS, Kaplan MJ: Cardiovascular disease in lupus: insights and updates. Curr Opin Rheumatol. 2013; 25(5): 597–605. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLau KK, Jones DP, Hastings MC, et al.: Short-term outcomes of severe lupus nephritis in a cohort of predominantly African-American children. Pediatr Nephrol. 2006; 21(5): 655–62. PubMed Abstract | Publisher Full Text\n\nLurbe E, Sorof JM, Daniels SR: Clinical and research aspects of ambulatory blood pressure monitoring in children. J Pediatr. 2004; 144(1): 7–16. PubMed Abstract | Publisher Full Text\n\nNational High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents. The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics. 2004; 114(2 Suppl 4th Report): 555–76. PubMed Abstract\n\nOhkubo T, Hozawa A, Yamaguchi J, et al.: Prognostic significance of the nocturnal decline in blood pressure in individuals with and without high 24-h blood pressure: the Ohasama study. J Hypertens. 2002; 20(11): 2183–9. PubMed Abstract | Publisher Full Text\n\nOryoji K, Kiyohara C, Horiuchi T, et al.: Reduced carotid intima-media thickness in systemic lupus erythematosus patients treated with cyclosporine A. Mod Rheumatol. 2013; 1–7. Publisher Full Text\n\nPetrin J, Rozman B, Dolenc P, et al.: The dissociation of arterial hypertension and lupus glomerulonephritis in systemic lupus erythematosus. Blood Press. 1993; 2(2): 108–12. PubMed Abstract | Publisher Full Text\n\nPickering TG, Shimbo D, Haas D: Ambulatory blood-pressure monitoring. N Engl J Med. 2006; 354(22): 2368–74. PubMed Abstract | Publisher Full Text\n\nPieretti J, Roman MJ, Devereux RB, et al.: Systemic lupus erythematosus predicts increased left ventricular mass. Circulation. 2007; 116(4): 419–26. PubMed Abstract | Publisher Full Text\n\nRuggiero B, Vivarelli M, Gianviti A, et al.: Lupus nephritis in children and adolescents: results of the Italian Collaborative Study. Nephrol Dial Transplant. 2013; 28(6): 1487–96. PubMed Abstract | Publisher Full Text\n\nSchwartz GJ, Muñoz A, Schneider MF, et al.: New equations to estimate GFR in children with CKD. J Am Soc Nephrol. 2009; 20(3): 629–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShaharir SS, Mustafar R, Mohd R, et al.: Persistent hypertension in lupus nephritis and the associated risk factors. Clin Rheumatol. 2015; 34(1): 93–97. PubMed Abstract | Publisher Full Text\n\nSingh A, Gianos E, Schwartzbard A, et al.: Use of ambulatory blood pressure monitoring to guide hypertensive therapy. Curr Treat Options Cardiovasc Med. 2013; 15(6): 746–60. PubMed Abstract | Publisher Full Text\n\nSorof JM, Cardwell G, Franco K, et al.: Ambulatory blood pressure and left ventricular mass index in hypertensive children. Hypertension. 2002; 39(4): 903–8. PubMed Abstract | Publisher Full Text\n\nSozeri B, Deveci M, Dincel N, et al.: The early cardiovascular changes in pediatric patients with systemic lupus erythematosus. Pediatr Nephrol. 2013; 28(3): 471–6. PubMed Abstract | Publisher Full Text\n\nSwartz SJ, Srivaths PR, Croix B, et al.: Cost-effectiveness of ambulatory blood pressure monitoring in the initial evaluation of hypertension in children. Pediatrics. 2008; 122(6): 1177–81. PubMed Abstract | Publisher Full Text\n\nTselios K, Koumaras C, Urowitz MB, et al.: Do current arterial hypertension treatment guidelines apply to systemic lupus erythematosus patients? a critical appraisal. Semin Arthritis Rheum. 2014; 43(4): 521–5. PubMed Abstract | Publisher Full Text\n\nUrbina E, Alpert B, Flynn J, et al.: Ambulatory blood pressure monitoring in children and adolescents: recommendations for standard assessment: a scientific statement from the American Heart Association Atherosclerosis, Hypertension, and Obesity in Youth Committee of the council on cardiovascular disease in the young and the council for high blood pressure research. Hypertension. 2008; 52(3): 433–51. PubMed Abstract | Publisher Full Text\n\nWoroniecki RP, Flynn JT: How are hypertensive children evaluated and managed? A survey of North American pediatric nephrologists. Pediatr Nephrol. 2005; 20(6): 791–7. PubMed Abstract | Publisher Full Text\n\nWühl E, Witte K, Soergel M, et al.: Distribution of 24-h ambulatory blood pressure in children: normalized reference values and role of body dimensions. J Hypertens. 2002; 20(10): 1995–2007. PubMed Abstract | Publisher Full Text\n\nYang LY, Chen WP, Lin CY: Lupus nephritis in children--a review of 167 patients. Pediatrics. 1994; 94(3): 335–40. PubMed Abstract | Publisher Full Text\n\nYee J: Ambulatory Blood Pressure monitoring: mercury rising. Adv Chronic Kidney Dis. 2015; 22(2): 81–85. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "9811",
"date": "17 Aug 2015",
"name": "Joyce Popoola",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a retrospective study looking at nocturnal blood pressure dips in paediatric patients with lupus. It is descriptive in nature and looks at small numbers as it is a single centre review of a relatively rare condition in the paediatric population. Though the paper shows a statistically significant nocturnal dip in the systolic and diastolic blood pressure compared to the controls it is unable to dissect out demographics or other characteristics that could be used to explain this because of the small numbers. This is an important question as blood pressure will have an impact on long term effect of this condition and potential development of renal disease beyond lupus nephritis. The paper offers no opportunity for mechanistic explanations for the observations because of the retrospective nature and small numbers as recognised by the authors.The statistics are also limited for the same reasons. I feel the paper should be published however to highlight the issue and also to provide a platform for the authors to raise awareness to the issue and enable a multi centre prospective study of the same question dissecting out demographics (age stratification, ethnicity), other co-morbidities (this would be more of an issue in an adult population) and to test out the mechanisms of this process potentially examining vascular resistance and flow mediated dilatation. It would also be of interest to see the effect of treatment on this phenomenon in a longitudinal fashion.",
"responses": []
},
{
"id": "10969",
"date": "03 Nov 2015",
"name": "Joseph Flynn",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present the results of a small retrospective case series of children with systemic lupus studied by Ambulatory Blood Pressure Monitoring (ABPM). Their major finding is that these patients have blunted nocturnal BP dipping, which has been associated with adverse cardiovascular risk. This is a novel finding that while needing to be confirmed in a larger, prospective study, could have major implications for the clinical management of children with SLE.One issue that requires clarification is how the authors characterize the study population. In several places in the paper they state that the SLE patients don't have nephritis, but in reality, about half had a history of nephritis. The authors should restate the description of the SLE patients to clarify that these children were not currently being treated for active nephritis. There are actually some interesting differences in the children with and without a history of nephritis that could be explored in more detail - for example, the ABPM parameters in Table 2 could be further divided into groups of those with and without a history of nephritis. How would that division then affect the results of the other analyses performed?Another major issue that requires greater exploration is the effect of prednisone treatment on BP dipping. All of the SLE patients were on prednisone and it is likely that this affected the dipping patterns. This needs to be explicitly acknowledged in the discussion, and some discussion of prior studies (if they exist) exploring the effect of corticosteroids on the circadian variation of BP needs to be added.Minor suggestions:add the number of subjects to the abstract (it only gives the number of non-SLE controls) shorten the introduction by 50% (first 2 paragraphs and the first 2/3 of the third paragraph could be deleted) I don't think the sensitivity analysis adds anything; this could probably be deleted as well it is unlikely that specific laboratory findings would affect BP outcomes, could some of this information be removed as well?",
"responses": [
{
"c_id": "1698",
"date": "23 Nov 2015",
"name": "Scott Wenderfer",
"role": "Author Response",
"response": "The authors appreciate the detailed and thorough reviews performed by all of the referees, including Dr. Flynn. We have submitted a modified and improved version of the manuscript addressing the comments and suggestions, as summarized in our response here.One issue that requires clarification is the study population. In several places in the paper the authors state that the SLE patients don't have nephritis; but in reality, about half had a history of nephritis. The authors should restate the description of the SLE patients to clarify that these children were not currently being treated for active nephritis.Done (modified the Study Population section of the Methods as well as in the Patient and Clinical Characteristics section of the Results)There are actually some interesting differences in the children with and without a history of nephritis that could be explored in more detail - the ABPM parameters in Table 2 could be further divided into groups of those with and without a history of nephritis.We thank the referee for this suggestion. Table 2 has been modified to sub-divide our pSLE cohort into the 4 non-renal pSLE patients and 6 patients with a history of lupus nephritis which was in remission and not being treated at the time of ABPM. The table now shows that nighttime SBP and DBP loads were higher in SLE patients with a history of nephritis than in non-renal lupus patients, even though the nephritis was in remission at time of ABPM. Moreover, it shows that all SLE patients with nocturnal HTN had a history of nephritis.Another major issue that requires greater exploration is the effect of prednisone treatment on BP dipping.We agree that the effect of pharmacologic therapy in patients with pSLE is a confounding factor in the interpretation of ABPM data in our cohort of patients, and we agree that there may indeed be an effect of the prednisone on the attenuated nocturnal dipping. We have added a paragraph to the discussion describing four studies that support this notion. Unfortunately, our cohort of patients is too small to distinguish between disease-specific and treatment-specific effects. Nonetheless, as long-term prednisone use in patients with SLE is nearly universal, our results emphasize the need to consider nocturnal HTN in the management of these patients.Minor suggestion: add the number of subjects to the abstract.Done (line 9 of the abstract)"
}
]
},
{
"id": "10968",
"date": "16 Nov 2015",
"name": "Coral Hanevold",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIncreased risk for cardiovascular disease is a significant cause of long term morbidity of SLE. In this small retrospective study the authors address one of the potential modifiable risk factors for CV disease in this population, hypertension. In this pilot study the authors focused on children and teens with SLE but no renal disease (or nephritis in remission) with prehypertension or stage 1 HTN who had been evaluated with an ABPM. Although the patient numbers are small, the study is strengthened by the use of the non-SLE control group. With regard to the methods, findings in the SLE patients were compared to a control group comprised of children undergoing ABPM for evaluation of prehypertension or stage 1 hypertension who did not have SLE (though other details regarding this group are not available). This group was reported to be age matched though it is noted that the mean age of this group was 12.4 years as compared to 14.6 for the SLE group. The mean BMI for the 2 groups was well matched. Unfortunately data for the non-SLE group was limited to information on the ABPM report which was limited to age, sex, height, weight (with BMI calculated from these data). Thus it was not possible to compare race or ethnic group of the control group to the SLE group which was 40% African American and 30% Hispanic.The authors found that the SLE patients demonstrated a higher prevalence of reduced dipping than the control group (90% vs 26% respectively). Additionally, 60% of SLE patients had nocturnal HTN as compared to 39% of the control group. Due to the small number of patients the authors were not able to demonstrate an association of findings on ABPM with clinical factors such as steroid exposure, disease activity, proteinuria, echocardiograms, etc. Of note all patients with nocturnal hypertension met historical criteria for nephritis even though not active at time of the study. It is possible that these SLE patients had other risk factors for hypertension not addressed here such as positive family history or elevated BMI. Perhaps the authors could comment on the latter point at least. These factors are important given the high prevalence of African American and Hispanic children in their SLE population. Data also showed that SLE patients had a statistically significant lower daytime systolic load as compared to controls while only 50% had elevated nighttime systolic loads and 60% had nocturnal hypertension. Thus it may be that the low daytime systolic load contributed to the finding of attenuated dipping in some of the SLE cohort. The authors performed various sensitivity analyses that involved using a lower threshold (90th percentile) to define hypertension, use of 30% as cutoff for abnormal load, more stringent requirements for adequacy, and limited non-SLE group to females. These data are somewhat confusing and the authors could consider deleting this section.The study reported here highlights the need for scrutiny of BPs in SLE patients without active nephritis in whom mild elevations in the office might be attributed to white coat hypertension. Additionally the high prevalence of abnormalities of nocturnal pressures, often occurring in isolation, indicates that this method of evaluation (ABPM) is crucial in the evaluation of this population. These findings should be the basis for further study in a larger group of patients. The study has significant limitations as mentioned above but the findings are compelling and thus warrant reporting.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-164
|
https://f1000research.com/articles/4-1314/v1
|
20 Nov 15
|
{
"type": "Method Article",
"title": "A DNA extraction protocol for improved DNA yield from individual mosquitoes",
"authors": [
"Catelyn C. Nieman",
"Youki Yamasaki",
"Travis C. Collier",
"Yoosook Lee",
"Catelyn C. Nieman",
"Youki Yamasaki",
"Travis C. Collier"
],
"abstract": "Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.",
"keywords": [
"DNA extraction",
"mosquito",
"molecular biology"
],
"content": "Introduction\n\nDNA extraction for Anopheles mosquitoes is typically done using commercially available products (Brown et al., 2011; Demirci et al., 2012; Horton et al., 2010; Main et al., 2015; Norris et al., 2015; Weetman et al., 2012). They work well enough to provide a sufficient amount of DNA for PCR-based assays from a single mosquito. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity from a single mosquito using typical extraction protocols (Marsden et al., 2011). Whole genome amplification could alleviate the issue but it also creates bias in genome representation (see Table 1 and results section below).\n\nIncrease or decrease in comparison with sequence from original DNA is marked in up or down arrows in parentheses.\n\nWhile trying to find alternative DNA extraction protocols for improved DNA yield but without compromising the automation option, we found that a combination of tissue lysis protocol from Life Technologies and DNA extraction protocol from Qiagen yielded higher DNA amount than the protocol using Qiagen or Life Technologies kit only.\n\n\nMethod\n\nAdult mosquitoes from a single generation of Pimperena and Mopti-NIH colonies obtained from Malaria Research and Reference Reagent Resources Center (Manassas, VA) were used for this study. Prior to extraction, the whole adult mosquito samples are individually preserved either by freezing at -20°C or storing in 80% ethanol; for the latter case, samples are rehydrated in water for 1 hour prior to DNA extraction.\n\nWe used a protocol adapted from the manufacturer’s mouse tail protocol and Whatman FTA card protocol. For each sample contained in a 1.5mL tube, 8µL or 2µL of proteinase K (Life Technologies, 100mg/mL concentration) were added with 92µL or 98µL PK buffer (Life Technologies), respectively. Three specimens were processed without physical disruption. A 3mm diameter steel bead was added to each of the rest of samples and homogenized using Qiagen Tissulyser (Qiagen, Valencia, CA) for 30sec at 30Hz. Mosquito tissue in PK buffer and proteinase K solution were incubated for 2 hours at 56°C. After incubation, 100µL of DNA lysis buffer was added to each tube. Each tube was vortexed briefly (<10s) and centrifuged at 15,000rpm using an Eppendorf microcentrifuge. This created white precipitate, which was mixed by pipetting up and down several times before transferring to a 2.0mL deep well plate for DNA extraction. 90µL of lysate was used for DNA extraction using the Biosprint 96 instrument. The other 90µL of lysate from the same sample was used for DNA extraction using the MagMAX™ Express-96 Magnetic Particle Processor.\n\nWe used a protocol adapted from the manufacturer’s tissue extraction protocol. For each sample contained in a 1.5mL tube, 10µL or 40 µL of proteinase K (Qiagen, 20mg/mL concentration) were added with 90µL or 60µL ATL buffer (Qiagen, Vallencia, CA), respectively. A 3mm diameter steel bead was added to each of the remaining samples and homogenized using Qiagen Tissulyser (Qiagen, Valencia, CA) for 30sec at 30Hz. Mosquito tissue in ATL buffer and proteinase K solution were incubated for 2 hours at 56°C. Each tube was centrifuged briefly after incubation. 90µL of lysate was used for DNA extraction using the Biosprint 96 instrument. The other 90µL of lysate from the same sample was used for DNA extraction using the MagMAX instrument.\n\nAs noted earlier, we used a protocol adapted from the manufacturer’s mouse tail protocol and Whatman FTA card protocol. 120µL of 100% isopropanol was added to each lysate. The plate containing lysate and isopropanol was gently mixed (220rpm) using a shaker for 3 minutes. 20µL of DNA binding bead mix (16µL binding beads and 4µL of PCR-grade water) was added to each sample and shook for 3 minutes at 220rpm. We used the “4412021 DW Blood” protocol on the MagMAX instrument, which washes lysate once with wash buffer 1 and twice with wash buffer 2 (Life Technologies). Initial heated elution volume was 75µL (elution buffer 1) and then when prompted, 75µL of elution buffer 2 was added to complete the DNA elution step.\n\n100µL of 100% isopropanol, 100µL of AL buffer (Qiagen) and 15µL of MagAttract Suspension (Qiagen) was added to each lysate. We used the “BS96 DNA Tissue” protocol on the BioSprint 96 instrument, which washes lysate twice with AW1 buffer, twice with AW2 buffer (Qiagen), and once with water with added tween 20 (Sigma) at a final concentration of 0.02%. The DNA was eluted in 150µL AE buffer (Qiagen).\n\nDNA yield was measured using a Qubit high sensitivity, double stranded DNA kit (Life Technologies), using 1µL of input DNA. R statistics software version 3.0.0 was used to calculate mean and standard deviation and to perform Wilcoxon rank sum test with α of 0.05 after multiple comparison.\n\n5µL of original input DNA was used to amplify the whole genome using Qiagen Repli-g kit. We followed the manufacturer’s protocol. We followed the library protocol provided in Norris et al. (2015). Genomic DNA libraries were sequenced using Illumina HiSeq2500 platform with paired-end 150 bp reads at the QB3 Vincent J Coates Genomics Sequencing Laboratory at UC Berkeley. Adaptor sequences and poor quality sequences were trimmed from the raw Illumina fastq files using the Trimmomatic software version 0.30 (Bolger et al., 2014) using default options. Reads were aligned to the A. gambiae reference genome AgamP3 (Giraldo-Calderon et al., 2015) using BWA-MEM version 0.7.5 (Li, 2013).\n\n\nResults & Discussion\n\nIn our attempt to sequence whole genomes from field-collected individual mosquitoes, about 50% of specimens failed to pass the DNA quantity required (>30ng) for whole genome sequencing. These DNA samples were extracted using our established DNA extraction protocols using Qiagen kits and instruments (Lee et al., 2009; Main et al., 2015; Marsden et al., 2011; Norris et al., 2015; Slotman et al., 2006). The requirement of high genomic DNA content is not new. In the past, people have circumvented the problem by conducting whole genome amplification (Lee et al., 2013; Marsden et al., 2011; Weetman et al., 2012). We sequenced the whole genomes using original DNA in parallel with whole-genome amplified DNA to test if we can use whole genome amplification to bypass the DNA quantity issue.\n\nWhole genome amplified DNA provided a higher number of reads than the original DNA with less DNA input (Table 1). However, comparison revealed that the particular whole genome amplification kit we used is not suitable for retrieving certain sections of genomes such as mitochondrial genome. This is indicated in the lower depth of coverage in mitochondrial genome in whole genome amplified material while the rest of chromosomes had higher depths relative to the library from original DNA. More importantly, the sequence generated from whole genome amplified samples produced number of inconsistent genotype calls (Table 2). This inconsistency became more apparent in mitochondrial sequences where heterozygous calls were produced where the genome sequence from the same original DNA had no such calls. These biases are likely introduced by the random primers used in the whole genome amplification kit. This result prompted us to pursue developing better DNA extraction protocols to improve DNA yield in an automated setup.\n\nChr stands for chromosome, locus for genomic coordinates, A1 for allele 1, A2 for allele 2, angle for arctangent value from two variable (=atan2(A1,A2)), and GT for genotype calls based on angle value. If arctangent value is less than 0.25, it is considered as A1/A1 homozygote. If value is higher than 1.25, genotypes are called as A2/A2. Otherwise, genotoypes are called as heterozygote (A1/A2).\n\nWe found that the Life Technologies tissue lysis and extraction protocol (Table 3, line 4, in purple) was highly consistent in its DNA yield. A combination of the Life Technologies tissue lysis with the Qiagen BioSprint DNA extraction protocol (Table 3, line 3, in green) gave the highest average DNA yield (Wilcoxon rank sum test P-value=0.0031). The amount of magnetic beads added to tissue lysate had little effect on DNA yield. The amount of proteinase K (2µL vs 8µL) also showed little difference in DNA yield. Chemical lysis alone, without physical disruption, was not sufficient to produce consistency in DNA yield (Table 3, lines 5 and 6).\n\nGroup 1 is the standard protocol used at the Vector Genetics Laboratory over ten years.\n\nFor a typical PCR-based assay, DNA quantity of 0.25–1.8 ng/µL in 200µL volume is sufficient. However, genomic approaches such as whole genome DNA library construction for next generation sequencing demand as little as 30ng of DNA. In our typical Qiagen BioSprint DNA extraction protocol, roughly 50% of DNA samples failed to yield 0.375ng/µL (Table 3, line 1, in blue), which leaves ~50µL of DNA for future study and allows for only a single trial of whole genome library construction. This constraint became a significant hindrance in our research involving whole genome sequencing from an individual mosquito.\n\nThis improved DNA extraction protocol will increase the chance of library construction from a single individual. Our observation is limited to trying out commercially available automated DNA extraction protocols and we do not have sufficient expertise on why certain protocols worked better or worse than others. As genomic approaches are more readily available to researchers, this improved DNA extraction protocol will facilitate such approaches that demand high-quantity DNA input from limited source material.\n\nWe have not rigorously tested all the possible combinations of extraction protocols. We only tested this on mosquito samples, and we only explored high-throughput automated DNA extraction protocols as we typically handle hundreds of mosquito samples at a time for population genetics studies. Therefore, our findings should be noted as suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘A DNA extraction protocol for improved DNA yield from individual mosquitoes’, 10.5256/f1000research.7413.d107517 (Nieman et al., 2015).",
"appendix": "Author contributions\n\n\n\nCCN and YL conceived the study. CCN, YY conducted DNA extractions. CCN conducted library preparations. TCC and YL conducted genome sequence data analysis. CCN and YL conducted data analysis for DNA quantity from different protocols. CCN, YY, TCC and YL wrote manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSignature Research in Genomics (SRG) Program by the UC Davis Office of Research and School of Medicine supported this research.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Miss Allison Weakley for assisting library construction.\n\n\nReferences\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrown JE, McBride CS, Johnson P, et al.: Worldwide patterns of genetic differentiation imply multiple ‘domestications’ of Aedes aegypti, a major vector of human diseases. Proc Biol Sci. 2011; 278(1717): 2446–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDemirci B, Lee Y, Lanzaro GC, et al.: Identification and characterization of single nucleotide polymorphisms (SNPs) in Culex theileri (Diptera: Culicidae). J Med Entomol. 2012; 49(3): 581–8. PubMed Abstract | Publisher Full Text\n\nGiraldo-Calderon GI, Emrich SJ, MacCallum RM, et al.: VectorBase: an updated bioinformatics resource for invertebrate vectors and other organisms related with human diseases. Nucleic Acids Res. 2015; 43(Database issue): D707–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHorton AA, Lee Y, Coulibaly CA, et al.: Identification of three single nucleotide polymorphisms in Anopheles gambiae immune signaling genes that are associated with natural Plasmodium falciparum infection. Malar J. 2010; 9: 160. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee Y, Collier TC, Sanford MR, et al.: Chromosome inversions, genomic differentiation and speciation in the African malaria mosquito Anopheles gambiae. PLoS One. 2013; 8(3): e57887. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee Y, Cornel AJ, Meneses CR, et al.: Ecological and genetic relationships of the Forest-M form among chromosomal and molecular forms of the malaria vector Anopheles gambiae sensu stricto. Malar J. 2009; 8: 75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi H: Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Cornell University Library; 2013; p. arXiv:1303.3997v2. Reference Source\n\nMain BJ, Lee Y, Collier TC, et al.: Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali. Mol Ecol. 2015; 24(20): 5145–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarsden CD, Lee Y, Nieman CC, et al.: Asymmetric introgression between the M and S forms of the malaria vector, Anopheles gambiae, maintains divergence despite extensive hybridization. Mol Ecol. 2011; 20(23): 4983–94. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNieman CC, Yamasaki Y, Collier TC, et al.: Dataset 1 in: A DNA extraction protocol for improved DNA yield from individual mosquitoes. F1000Research. 2015. Data Source\n\nNorris LC, Main BJ, Lee Y, et al.: Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets. Proc Natl Acad Sci U S A. 2015; 112(3): 815–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSlotman MA, Reimer LJ, Thiemann T, et al.: Reduced recombination rate and genetic differentiation between the M and S forms of Anopheles gambiae s.s. Genetics. 2006; 174(4): 2081–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeetman D, Wilding CS, Steen K, et al.: Gene flow-dependent genomic divergence between Anopheles gambiae M and S forms. Mol Biol Evol. 2012; 29(1): 279–91. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "11616",
"date": "17 Dec 2015",
"name": "Beniamino Caputo",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 an interesting update on extraction protocols and highlights the importance of testing different methods when extracting DNA for a wide range of downstream applications. The article’s title is sound and describes well the observations made, the same is true for the abstract which provides an adequate summary of the article. Also the aim of the article and the study design are well described and allow to reproduce the different methods tested. Results and discussions appear to be sound and coherent with the offered data. There are only few minor corrections to make:In Methods: please correct TissuLyser in TissueLyser specify the number of specimens analyzed In Results and Discussion:in the second paragraph it might be noteworthy that genotype call errors do not appear only when considering the mitochondrial DNA, but, at least in A.gambiae 1 listed in Table 2, many errors , such as opposed genotype calls can be observed also for autosomal sequences Table 1:it could be useful to add some information in the caption such as the meaning of ChrM ( I suppose it stands for mitochondrial DNA?) and units of measurements for the given data please correct A.gambia in A.gambiae (1st column)",
"responses": []
},
{
"id": "11972",
"date": "01 Feb 2016",
"name": "Zainulabeuddin Syed",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis m/s by Niema et al. presents some useful information for researchers who routinely struggle with reliable protocols to extract high quality DNA, esp. of great quality needed for sequencing. Library construction for next-generation sequencing commonly requires at least 30 ng of DNA. This can present a challenge for researchers that work with small insects and/or who are very limited based on the tissues they use for library construction. DNA yield is particularly important when studying genetic variation among individuals. The authors assessed several common methods for the extraction of DNA from single Anopheles mosquitoes. The manuscript is well written and the methods are appropriate. Comments:No “non-commercial” methods were included in the analyses, nor were they discussed. For example, a standard CTAB protocol gets excellent yields of good quality DNA. Perhaps CTAB does not produce DNA that is appropriate for library prep or does not work well in an automated system. It would be really helpful if authors can address and elaborate on this issue Were the sizes of the individuals used in the study the same? For example, the mass of males is often much less than females, and rearing conditions can influence size to a great extent.\n\nPlease correct a few spelling errors.",
"responses": []
},
{
"id": "11970",
"date": "10 Feb 2016",
"name": "Karla Saavedra-Rodriguez",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 provides very useful data for researchers aiming to analyze whole mosquito genomes; especially when sequencing is still expensive and having a reliable DNA extraction protocol is essential. The abstract, justification, protocols and results are very well described.Some comments:Did the preservation of individual mosquitoes – 80% alcohol or -20°C– affect the DNA yield? I am not sure if this was an additional variable. Please clarify. Mention the number of specimens you used for each combination of lysis and extraction methods. Table 1: Do ‘X, 2L, 2R, 3L, ..chrM’ columns correspond to mean coverage’s? Please add a brief description of these terms in the table legend. Discussion: the statement ‘the amount of magnetic beads…had little effect on DNA yield’. This variable was not mentioned in the methods section. Please add, mention specific bead amounts tested, sample size and statistical test. Table 2: Please mention in this table (or table 1) what ‘Mt, 2R….X’ stands for. Are ‘Mt and chM’ mitochondrial DNA? Table 2: State the difference between A. gambiae 1 and A. gambiae 2 groups. Table 2: There are significant differences between the genotype-calls in the WGA and original DNA. Would you suggest having a third method to validate your genotypes? Maybe regular DNA sequencing. I think this could add to your discussion. Table 3: Add a column with your sample sizes.",
"responses": [
{
"c_id": "1810",
"date": "11 Feb 2016",
"name": "Yoosook Lee",
"role": "Reader Comment",
"response": "Thank you for your positive feedback. I will try to get the formal revision done in the near future. For your first question, we typically use 80% for storing samples in Africa. Due to hot environmental conditions there we were expecting accelerated evaporation of ethanol which could make ethanol content lower than what we intended (70%). So we put extra to compensate the loss. We have tried this protocol on samples stored dry in silica, dry frozen at -20C or 100% ethanol as well. If samples are stored in high ethanol content, they get harder than 70-80% ethanol. But this doesn't affect DNA extraction because we do hydrate in water for an hour anyway to soften up the tissue except frozen ones.We have not detected any decrease in yield for relatively fresh material (< 2 months). Quality goes down for samples stored without alcohol (either in silica or frozen) resulting in lower yield in target species DNA and lots of microbial content, requiring more sequencing reads to get the same coverage."
}
]
}
] | 1
|
https://f1000research.com/articles/4-1314
|
https://f1000research.com/articles/4-1313/v1
|
20 Nov 15
|
{
"type": "Review",
"title": "Advances in understanding and managing bullous pemphigoid",
"authors": [
"Cathy Y. Zhao",
"Dedee F. Murrell",
"Cathy Y. Zhao"
],
"abstract": "Bullous pemphigoid (BP) is the commonest subtype of autoimmune blistering disease in most countries of the world. It occurs most frequently in elderly patients and is characterised clinically by large, tense blisters in the skin preceded by urticarial plaques and pruritus. Immunopathologically, it is characterised by autoantibodies directed against the 180 kD antigen (BP180) and the 230 kD antigen (BP230). New knowledge regarding BP is being continually uncovered. This article reviews the recent advances in BP, including newer diagnostic tests, standardised outcome measures and emerging therapeutic options, as well as the evidence supporting their use.",
"keywords": [
"Bullous pemphigoid",
"blisters",
"urticarial plaques",
"pruritus"
],
"content": "Introduction\n\nBullous pemphigoid (BP) is the commonest subtype of autoimmune blistering disease (AIBD), a rare but potentially fatal group of skin diseases. BP usually affects the elderly and has an incidence of 12.1 to 66 new cases per million per year in epidemiological studies conducted in Europe1–4. Clinically, it can present heterogeneously but typically manifests with large, tense blisters in the skin preceded by urticarial plaques and intense pruritus. Immunopathologically, it is characterised by subepidermal autoantibodies directed against the 180 kD antigen (BP180) and the 230 kD antigen (BP230), two components of adhesion complexes promoting dermo-epidermal cohesion5.\n\nMedical knowledge regarding BP has progressed considerably in recent years. An important area of progress is newer BP laboratory testing methods, allowing a faster, cheaper and more feasible diagnosis of BP to be established. Another important area is the validation of BP outcome measures, allowing more accurate assessment of disease severity, facilitating the optimal therapy choice and dosage to be administered, and true disease responsiveness to be monitored. As BP usually affects the elderly (over 70 years of age), therapy choice is complicated and needs to be tailored to suit this frail population, balancing between efficacy, practicality and safety. Insights into BP therapies, especially systemic antibody-modulating agents, have increased significantly with regard to both efficacy and safety. This article reviews the recent advances in BP, including diagnostic techniques, outcome measures and therapeutic options, as well as the evidence supporting their use.\n\n\nDiagnosis\n\nThe diagnosis of BP is based on a combination of clinical, histopathological and immunological criteria6. In the setting of tense bullae with dermal-epidermal separation on histology, or of prodromal separations and positive direct immunofluorescence (DIF) for IgG or C3, the diagnosis of BP can be made if three of the following four criteria are present: age of more than 70 years, absence of atrophic scars, absence of mucosal involvement, and absence of predominant bullous lesions on the neck and head7,8. This has a sensitivity of 90%, specificity of 83%, and positive predicative value of 95% when validated using immunoelectron microscopy and a sensitivity of 86%, specificity of 90%, and positive predicative value of over 95% when validated using immunoblotting as the gold standard. Therefore, it is recommended to perform a DIF and serological analysis to exclude a BP in all pruritic skin lesion patients who are at least 65 years old9.\n\nThe diagnosis of BP may be further confirmed by the characterisation of circulating autoantibodies by using methods such as indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), Western blotting and immunoprecipitation. Developed in the 1980s, IIF is the most commonly used method of autoantibody characterisation, which has a sensitivity of 60–80% in detecting IgG autoantibodies that typically bind to the epidermal side of the salt-split human skin10. The substrate may be obtained commercially or alternatively prepared in the laboratory, the latter of which has the disadvantage of being potentially very time-consuming11. On the other hand, introduced in 2002, ELISA for BP using recombinant protein of BP180 NC16 (the extracellular domain harbouring immunodominant epitopes of BP autoantibodies) is a more sophisticated method of autoantibody characterisation12. It has the advantages of allowing multiple-sample testing, is easily reproducible, and provides a quantitative analysis13. Various validation studies have shown the sensitivity of the commercially available BP180 ELISAs to be 70–90%, which increases when using the NC16A domain and other extracellular portions of BP180 or BP230 together6,12,14,15. However, ELISA has two disadvantages: the cost is high and the recombinant proteins used may not contain all of the epitopes present in vivo. In addition, autoantibodies to BP180 and BP230 may be found usually in low levels by ELISA in about 7% of patients with other unrelated diseases or in healthy subjects16. Western blotting and immunoprecipitation are less commonly used given that these methods are often tedious and time-consuming, and have low availability.\n\nDeveloped in Germany in 2012, the BIOCHIP IF mosaic is a new IIF method for the diagnosis of AIBDs and allows both antibody screening and antigen-specific substrate testing in a single miniature incubation field. So far, four validation studies have been performed, showing that for BP180 testing, it has good sensitivities, varying from 77–100%, and even superior specificities of 84–100% (Table 1)17–20. However, for BP230, the sensitivities were much lower, varying from 39–94%, although the specificities were generally good (100% in three studies)18–20. These cumulative findings suggest that the BIOCHIP IF mosaic in detecting BP180 autoantibodies may be comparable to ELISA, and therefore may be used as a faster and cheaper screening test for patients with suspected BP. However, given the low sample size of the studies, the validities of the findings are compromised. A future study with large sample size and inter-rater reliability evaluation may be indicated for the BIOCHIP mosaic IF test.\n\n*Study in progress. ELISA, enzyme-linked immunosorbent assay.\n\n\nOutcome measures\n\nThe standardisation of BP outcome measures is important for the progress of BP treatment development, as it allows the direct comparison and meta-analysis of results from different clinical trials. Formed in 2008, the BP Definitions Group consists of many worldwide AIBD experts21. Over a period of 2 years, the group held seven consensus meetings to establish definitions for the various stages of BP disease activity, therapeutic end-points and the first BP-specific severity outcome measure, called the Bullous Pemphigoid Disease Area Index (BPDAI)22. The BPDAI consists of two components: objective and pruritus. Its objective component has up to 360 points and includes blisters or erosions, urticarial or erythematous lesions, and mucosal involvement, each worth up to 120 points. Its separate subjective pruritus component has up to 30 points and takes into account the subjective severity of pruritus in the last 24 hours, week and month. The BPDAI has been validated in terms of its sensitivity to change, accuracy, and external validity by two separate studies23,24. The studies showed that the BPDAI correlated well with the patient’s erythematous/eczematous/urticarial skin surface, number of daily new blisters, and anti-BP180 titres tested by using ELISA, but not with the anti-BP230 titres. The BPDAI’s inter-rater and intra-rater reliabilities have also been reported to be excellent, and high intra-class correlation coefficients were detailed by a conference abstract summarising their preliminary results25. Future studies to evaluate the interpretability and cross-cultural validity of BPDAI may be indicated in order to complete its validation according to the COSMIN (Consensus-Based Standards for the Selection of Health Status Measurement Instruments) checklist manual26.\n\nOther than disease severity, the BP patient’s quality of life (QOL) may be measured by the Autoimmune Bullous Disease Quality of Life (ABQOL) and Treatment of Autoimmune Bullous Disease Quality of Life (TABQOL) questionnaires. These two questionnaires were developed in 2013, as dermatology disease-specific QOL measures were shown to be more sensitive to changes in disease status than generic QOL measures27. Furthermore, these two questionnaires have been validated as having acceptable levels of construct validity, internal consistency and test-retest reliability, leading to their expansion and further validations in other languages21.\n\n\nTreatment\n\nThe treatment of BP should be aimed at decreasing blistering formation and pruritus, promote the healing of blisters and improve QOL while having a minimally adverse profile28. The therapeutic options for BP have been transformed significantly in the past decade. Topical clobetasol propionate 0.05% (40 grams per day) has been shown to be superior to oral prednisolone (0.5 mg/kg per day) in terms of overall survival, disease control and adverse event profile for patients with extensive BP. It is equally effective for patients with moderate BP, as shown in a randomised controlled trial (RCT)29. For this reason, topical clobetasol has taken over the previous benchmark of BP therapy, oral corticosteroids, as the first-line treatment for BP. This is supported by the consensus statement from the European Dermatology Forum in collaboration with the European Academy of Dermatology and Venereology28,30. However, topical clobetasol has the disadvantages of poor practicality in bedridden patients, higher rates of incompliance, and poor accessibility in certain countries. In these cases, oral prednisolone (0.5–1 mg/kg per day), despite its significantly worse adverse event profile, is recommended as the initial therapy instead29,31.\n\nThe use of immunosuppressive medications for BP has been controversial. Their efficacy is generally inconclusive with significant potential adverse events, given prolonged use31.\n\nThe most well-established immunosuppressive medication is azathioprine, a purine analogue, typically given at 0.5–2.5 mg/kg per day, followed by mycophenolate mofetil, a DNA-synthesis inhibitor, and methotrexate, a folate antagonist28. There have been a few RCTs evaluating immunosuppressive agents32,33. One study found azathioprine to have worse hepatotoxicity than mycophenolate32. However, the evidence for efficacy, measured by disease control and remission, was inconclusive. Furthermore, none of the studies used a placebo, which is a control that may be difficult to justify ethically31. Overall, introducing an immunosuppressive medication depends on several factors, including the efficacy of the first-line topical clobetasol or oral corticosteroids, the patient’s disease extent and co-morbidities, the dermatologist’s experiences, and cost considerations.\n\nIn this era of biological therapies, new antibody modulators, including rituximab and omalizumab, have been suggested for the treatment of BP. It was proposed that they would have a more benign adverse event profile and more selective mechanisms of action. Rituximab is a humanised chimeric monoclonal antibody that targets CD20+ B cells. So far, rituximab has shown promising evidence for other AIBDs such as pemphigus; however, in BP, the evidence for rituximab has been limited to case reports and case series34–36. In a retrospective case series involving five patients with refractory BP, rituximab was administered as 375 mg/m2 given weekly over 4 weeks, resulting in complete remission in three of the five patients, and partial remission in one patient34. However, one other patient died shortly after rituximab therapy. Despite the potential serious adverse events of rituximab, its efficacy for refractory BP was implicated. In a more recent retrospective study, rituximab’s role as first therapy was evaluated37. In this study, a group of 13 patients received 4 weekly infusions of rituximab 500 mg along with oral prednisolone, and a group of 19 patients received oral prednisolone only. The study had a follow-up period of 1 year. It found that the rituximab group had significantly higher rates of complete remission (92% versus 53%) and lower rates of mortality (15% versus 37%). However, there were no significant differences in the patient’s mean BPDAI scores or the cumulative oral corticosteroid dose. The study supported the safety and effectiveness of rituximab but was limited by its retrospective nature and its small sample size. A future RCT with a longer follow-up period, larger sample, and a comparison of various rituximab protocols, may be indicated.\n\nOmalizumab is a humanised monoclonal antibody that inhibits the binding of IgE to its receptors. It has been previously used for asthma and chronic urticaria, and is postulated to be effective for BP as IgE antibodies specific for the BP180 autoantigen have been detected in sera and biopsy samples from the majority of patients with BP38. In an uncontrolled case series of six patients with refractory BP, omalizumab was administered subcutaneously in doses of 300–375 mg from fortnightly to every 6 weeks39. Overall, five of the six patients demonstrated clinical improvements from the omalizumab, and the sixth patient terminated treatment because of inter-current comorbidities. Three of the six patients had their BP180 and BP230 autoantibodies measured by ELISA and showed significant reductions after the use of omalizumab. None of the patients had any significant adverse events. Although the study suggested the efficacy of omalizumab, it was limited by its significantly small sample size, its variation in omalizumab dosage between patients, and its use of non-standardised outcome measures. Future RCTs evaluating omalizumab as a potential treatment for BP may be indicated.\n\n\nConclusions\n\nKnowledge regarding AIBD is rapidly advancing. The diagnosis of BP requires a combination of clinical, histopathological and immunological testing via the detection of tissue-bound and circulating autoantibodies, the latter being the main area of development in recent years. Standardised outcome measures such as the BPDAI have been developed to facilitate the comparison and meta-analysis of clinical trial results. This would likely lead to higher-quality clinical studies evaluating BP treatments, especially immunosuppressives and antibody modulators, with the aim of reducing adverse events associated with oral corticosteroids.",
"appendix": "Competing interests\n\n\n\nThe senior author of the article (Dedee F. Murrell) was involved in the development and validation of the BPDAI mentioned in the article.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMarazza G, Pham HC, Schärer L, et al.: Incidence of bullous pemphigoid and pemphigus in Switzerland: a 2-year prospective study. Br J Dermatol. 2009; 161(4): 861–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLangan SM, Smeeth L, Hubbard R, et al.: Bullous pemphigoid and pemphigus vulgaris--incidence and mortality in the UK: population based cohort study. BMJ. 2008; 337: a180. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGudi VS, White MI, Cruickshank N, et al.: Annual incidence and mortality of bullous pemphigoid in the Grampian Region of North-east Scotland. Br J Dermatol. 2005; 153(2): 424–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBertram F, Bröcker EB, Zillikens D, et al.: Prospective analysis of the incidence of autoimmune bullous disorders in Lower Franconia, Germany. J Dtsch Dermatol Ges. 2009; 7(5): 434–40. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLabib RS, Anhalt GJ, Patel HP, et al.: Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting. J Immunol. 1986; 136(4): 1231–5. PubMed Abstract\n\nDi Zenzo G, Della Torre R, Zambruno G, et al.: Bullous pemphigoid: from the clinic to the bench. Clin Dermatol. 2012; 30(1): 3–16. PubMed Abstract | Publisher Full Text\n\nVaillant L, Bernard P, Joly P, et al.: Evaluation of clinical criteria for diagnosis of bullous pemphigoid. French Bullous Study Group. Arch Dermatol. 1998; 134(9): 1075–80. PubMed Abstract | Publisher Full Text\n\nJoly P, Courville P, Lok C, et al.: Clinical criteria for the diagnosis of bullous pemphigoid: a reevaluation according to immunoblot analysis of patient sera. Dermatology. 2004; 208(1): 16–20. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSchmidt E, Goebeler M, Hertl M, et al.: S2k guideline for the diagnosis of pemphigus vulgaris/foliaceus and bullous pemphigoid. J Dtsch Dermatol Ges. 2015; 13(7): 713–27. PubMed Abstract | Publisher Full Text\n\nGammon WR, Briggaman RA, Inman AO 3rd, et al.: Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin. J Invest Dermatol. 1984; 82(2): 139–44. PubMed Abstract | Publisher Full Text\n\nMascaró JM Jr: Histological and Immunofluorescence Diagnosis of Autoimmune Blistering Diseases. In: Murrell D, ed. Blistering Diseases. Sydney: Springer; 2015; 161–191. Publisher Full Text\n\nKobayashi M, Amagai M, Kuroda-Kinoshita K, et al.: BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid. J Dermatol Sci. 2002; 30(3): 224–32. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLee EH, Kim YH, Kim S, et al.: Usefulness of Enzyme-linked Immunosorbent Assay Using Recombinant BP180 and BP230 for Serodiagnosis and Monitoring Disease Activity of Bullous Pemphigoid. Ann Dermatol. 2012; 24(1): 45–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDi Zenzo G, Thoma-Uszynski S, Fontao L, et al.: Multicenter prospective study of the humoral autoimmune response in bullous pemphigoid. Clin Immunol. 2008; 128(3): 415–26. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSitaru C, Dähnrich C, Probst C, et al.: Enzyme-linked immunosorbent assay using multimers of the 16th non-collagenous domain of the BP180 antigen for sensitive and specific detection of pemphigoid autoantibodies. Exp Dermatol. 2007; 16(9): 770–7. PubMed Abstract | Publisher Full Text\n\nWieland CN, Comfere NI, Gibson LE, et al.: Anti-bullous pemphigoid 180 and 230 antibodies in a sample of unaffected subjects. Arch Dermatol. 2010; 146(1): 21–5. PubMed Abstract | Publisher Full Text\n\nChiang YZ, Zhao CY, Melbourne W, et al.: Biochip immunofluorescence microscopy as a new diagnostic tool for autoimmune blistering skin diseases in Australia. World Congress of Dermatology; Vancouver, Canada; 2015. Reference Source\n\nvan Beek N, Rentzsch K, Probst C, et al.: Serological diagnosis of autoimmune bullous skin diseases: prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy. Orphanet J Rare Dis. 2012; 7: 49. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nZarian H, Saponeri A, Michelotto A, et al.: Biochip technology for the serological diagnosis of bullous pemphigoid. ISRN Dermatol. 2012; 2012: 237802. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTampoia M, Zucano A, Villalta D, et al.: Anti-skin specific autoantibodies detected by a new immunofluorescence multiplex biochip method in patients with autoimmune bullous diseases. Dermatology. 2012; 225(1): 37–44. PubMed Abstract | Publisher Full Text\n\nZhao CY, Murrell DF: Outcome measures for autoimmune blistering diseases. J Dermatol. 2015; 42(1): 31–6. PubMed Abstract | Publisher Full Text\n\nMurrell DF, Daniel BS, Joly P, et al.: Definitions and outcome measures for bullous pemphigoid: recommendations by an international panel of experts. J Am Acad Dermatol. 2012; 66(3): 479–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatsatsi A, Kyriakou A, Giannakou A, et al.: Clinical significance of anti-desmoglein-1 and -3 circulating autoantibodies in Pemphigus Patients Measured by Area Index and Intensity Score. Acta Derm Venereol. 2014; 94(2): 203–6. PubMed Abstract | Publisher Full Text\n\nLévy-Sitbon C, Barbe C, Plee J, et al.: Assessment of bullous pemphigoid disease area index during treatment: a prospective study of 30 patients. Dermatology. 2014; 229(2): 116–22. PubMed Abstract | Publisher Full Text\n\nWijayanti A, Zhao CY, Boettiger D, et al.: The Validity, Reliability and Responsiveness of the Bullous Pemphigoid Disease Area Index (BPDAI). Cutaneous Biologic Meeting; North Stradbroke Island, Australia. 2014. Reference Source\n\nMokkink LB, Terwee CB, Patrick DL, et al.: COSMIN checklist manual. Amsterdam, Netherlands: COSMIN; 2012. Reference Source\n\nFinlay AY, Khan GK, Luscombe DK, et al.: Validation of Sickness Impact Profile and Psoriasis Disability Index in Psoriasis. Br J Dermatol. 1990; 123(6): 751–6. PubMed Abstract | Publisher Full Text\n\nRamirez-Quizon MN, Borradori L, Hall RP III, et al.: Management of Bullous Pemphigoid. In: Murrell DF, ed. Blistering Diseases. Sydney, Australia: Springer; 2015; 543–550. Publisher Full Text\n\nJoly P, Roujeau JC, Benichou J, et al.: A comparison of oral and topical corticosteroids in patients with bullous pemphigoid. N Engl J Med. 2002; 346(5): 321–7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nFeliciani C, Joly P, Jonkman MF, et al.: Management of bullous pemphigoid: the European Dermatology Forum consensus in collaboration with the European Academy of Dermatology and Venereology. Br J Dermatol. 2015; 172(4): 867–77. PubMed Abstract | Publisher Full Text\n\nKirtschig G, Middleton P, Bennett C, et al.: Interventions for bullous pemphigoid. Cochrane Database Syst Rev. 2010; (10): CD002292. PubMed Abstract | Publisher Full Text\n\nBeissert S, Werfel T, Frieling U, et al.: A comparison of oral methylprednisolone plus azathioprine or mycophenolate mofetil for the treatment of bullous pemphigoid. Arch Dermatol. 2007; 143(12): 1536–42. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nGuillaume JC, Vaillant L, Bernard P, et al.: Controlled trial of azathioprine and plasma exchange in addition to prednisolone in the treatment of bullous pemphigoid. Arch Dermatol. 1993; 129(1): 49–53. PubMed Abstract | Publisher Full Text\n\nLourari S, Herve C, Doffoel-Hantz V, et al.: Bullous and mucous membrane pemphigoid show a mixed response to rituximab: experience in seven patients. J Eur Acad Dermatol Venereol. 2011; 25(10): 1238–40. PubMed Abstract | Publisher Full Text\n\nKasperkiewicz M, Shimanovich I, Ludwig RJ, et al.: Rituximab for treatment-refractory pemphigus and pemphigoid: a case series of 17 patients. J Am Acad Dermatol. 2011; 65(3): 552–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nShetty S, Ahmed AR: Treatment of bullous pemphigoid with rituximab: critical analysis of the current literature. J Drugs Dermatol. 2013; 12(6): 672–7. PubMed Abstract\n\nCho YT, Chu CY, Wang LF: First-line combination therapy with rituximab and corticosteroids provides a high complete remission rate in moderate-to-severe bullous pemphigoid. Br J Dermatol. 2015; 173(1): 302–4. PubMed Abstract | Publisher Full Text\n\nMessingham KA, Holahan HM, Fairley JA: Unraveling the significance of IgE autoantibodies in organ-specific autoimmunity: lessons learned from bullous pemphigoid. Immunol Res. 2014; 59(1–3): 273–8. PubMed Abstract | Publisher Full Text\n\nYu KK, Crew AB, Messingham KA, et al.: Omalizumab therapy for bullous pemphigoid. J Am Acad Dermatol. 2014; 71(3): 468–74. PubMed Abstract | Publisher Full Text | F1000 Recommendation"
}
|
[
{
"id": "11297",
"date": "20 Nov 2015",
"name": "Zhi Liu",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11298",
"date": "20 Nov 2015",
"name": "Cassian Sitaru",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1313
|
https://f1000research.com/articles/4-1310/v1
|
20 Nov 15
|
{
"type": "Software Tool Article",
"title": "HiCUP: pipeline for mapping and processing Hi-C data",
"authors": [
"Steven W. Wingett",
"Philip Ewels",
"Mayra Furlan-Magaril",
"Takashi Nagano",
"Stefan Schoenfelder",
"Peter Fraser",
"Simon Andrews",
"Philip Ewels",
"Mayra Furlan-Magaril",
"Takashi Nagano",
"Stefan Schoenfelder",
"Peter Fraser",
"Simon Andrews"
],
"abstract": "HiCUP is a pipeline for processing sequence data generated by Hi-C and Capture Hi-C (CHi-C) experiments, which are techniques used to investigate three-dimensional genomic organisation. The pipeline maps data to a specified reference genome and removes artefacts that would otherwise hinder subsequent analysis. HiCUP also produces an easy-to-interpret yet detailed quality control (QC) report that assists in refining experimental protocols for future studies. The software is freely available and has already been used for processing Hi-C and CHi-C data in several recently published peer-reviewed studies.",
"keywords": [
"Hi-C",
"CHi-C",
"Chromatin",
"Structure",
"Epigenetics",
"Genomics",
"Bioinformatics",
"Pipeline"
],
"content": "Introduction\n\nHi-C is a ligation-based proximity assay utilising the power of massively parallel sequencing to identify three-dimensional genomic interactions1. The method (summarised in Figure 1a) involves fixing chromatin to preserve genomic organisation, followed by restriction enzyme digestion of the DNA. Overhanging single-stranded DNA at the ends of restriction fragments are then filled in with the concomitant incorporation of biotin. Fragments in close spatial proximity are ligated together generating a novel \"modified restriction site\" sequence (see Figure 1b). Following sonication the sheared ligated DNA fragments are enriched by streptavidin pull-down of the biotin residues, and then are ligated between sequencing adapters. The resulting molecule, termed a di-tag, should comprise two different DNA fragments separated by a modified restriction site. Since these two fragments were positioned close to each other during fixation, by analysing the composition of a population of di-tags generated by a Hi-C experiment it is possible to infer genomic three-dimensional organisation.\n\na) Diagram summarising the Hi-C experimental protocol. The red and blue rectangles represent cross-linked restriction fragments while the yellow marker shows the position of biotin incorporation. b) Generation of the Hi-C ligation junction sequence by successive digestion (with HindIII in this example), fill in and blunt-ended ligation steps. The modified restriction site sequence is not found in the original genomic sequence.\n\nA recent variation of the protocol involves enriching Hi-C libraries for di-tags in which one or both reads align to pre-selected regions of a genome2–4. This Capture Hi-C protocol (CHi-C) is advantageous since Hi-C libraries are extremely complex and even with current high-throughput technologies often only a small proportion of a Hi-C library is sequenced. Selecting for di-tags in this way makes it possible to gain a more complete and higher resolution contact profile for loci of interest.\n\nReaching valid conclusions regarding genomic interactions requires Hi-C data to be mapped unconventionally (described below) as compared with most paired-end experiments. Following this, artefacts inherent to the Hi-C protocol should be removed. To meet these demands we developed HiCUP, an easy-to-use Hi-C bioinformatics pipeline which has few dependencies and is coded in Perl. HiCUP produces a detailed quality control (QC) report in an interactive HTML format, enabling the user easily to assess the quality of a library and how the experimental protocol may be improved in the future. HiCUP was designed for mapping Hi-C data and removing artefacts. It does not perform the normalisation and statistical tests needed to interpret Hi-C experiments, rather it is intended as the starting point of processing Hi-C datasets and should be used in conjunction with other Hi-C pipelines.\n\nThere are numerous packages available to perform different steps in the analysis of Hi-C data. These include Hicpipe5, which is used to generate renormalised contact maps by correcting for pre-determined systematic biases, such as the GC content around the Hi-C ligation junction. Similarly, HiC-Lib6 comprises a set of Python scripts for drawing corrected contact heatmaps, only this technique uses an iterative correction algorithm so that potential biases do not need to be identified prior to processing. Homer7 also creates iteratively corrected heatmaps and includes extra features, such as scripts to identify topologically-associated domains (TADs). In addition, Fit-Hi-C8 takes renormalized data to identify mid-range intra-chromosomal interactions; and GOTHiC9, a Bioconductor package, performs a cumulative binomial test to identify contacts between distal genomic loci that have significantly more reads than expected by chance. Finally, CHiCAGO10 is an open-source R package for interaction detection in CHi-C datasets. Many of the existing tools — including CHiCAGO, Hicpipe, Homer and GOTHiC — can take the output of HiCUP as input for the analyses they perform.\n\n\nMethods\n\nHiCUP takes paired-end FASTQ files along with a FASTA reference genome and associated aligner indices and then reports valid di-tags in BAM/SAM format.\n\nMapping: The first stage in the HiCUP pipeline involves truncating reads at the modified restriction site (if present) that separates two DNA fragments. The rationale for this step is similar to that responsible for adapter trimming, namely to remove bases that would otherwise prevent a read mapping to the specified reference genome. After truncation, the resulting trimmed read sent for alignment should, in theory, represent a contiguous genomic sequence derived from a single restriction fragment (i.e. not a hybrid sequence comprising more than one restriction fragment).\n\nHiCUP uses Bowtie11 or Bowtie 212 to map Hi-C di-tags, allowing only unique high-quality alignments. Since valid Hi-C reads do not represent one continuous genomic sequence, the pipeline maps forward and reverse reads independently and then re-pairs sequences in which both ends aligned unambiguously to the genome.\n\nFiltering: HiCUP removes sequences representing experimental Hi-C artefacts and other uninformative di-tags, which is important since even a small number of invalid di-tags could lead to incorrect conclusions being drawn concerning genomic structure. HiCUP identifies and removes such sequences by positioning putative di-tags on an in silico digestion of the reference genome. The processes by which these artefacts are generated experimentally, and how HiCUP identifies them, are described below.\n\nRe-ligation of adjacent restriction fragments. The Hi-C protocol does not prevent entirely two adjacent restriction fragments re-ligating, but HiCUP discards such di-tags since they provide no useful three-dimensional proximity information. Similarly, multiple fragments could re-ligate forming a contig, but here paired reads will not map to adjacent genomic restriction fragments (Figure 2b). Furthermore, it is possible that a genome may not be digested completely, also generating molecules spanning adjacent or multiple restriction fragments. Such species should be selected against during biotin pull-down but, nevertheless, are observed in processed datasets.\n\nThe schematic shows the genome digested into 5 restriction fragments. These fragments may subsequently ligate to each other, or fragments derived from another chromosome, forming valid cis or trans di-tags respectively (a). In contrast, re-ligation or incomplete digestion leads to the generation of invalid contiguous sequences (b). Another common artefact occurs when the sequenced read-pair maps to a single restriction fragment (c), (d) & (e). Further, PCR may result in a fragment being copied multiple times (f). Di-tags are also rejected when the mapped reads are positioned too far away from the putative restriction enzyme cut-site than allowed by the experimental size-selection step (g).\n\nHiCUP takes the expected size range of the sample (which is predetermined by a di-tag length selection step during library construction) and identifies di-tags in which reads, when mapped to the genome, are separated by a distance falling within the size-selection range. Such di-tags are assumed to comprise a contig spanning several restriction cut sites, and so are discarded.\n\nPaired reads mapping to the same restriction fragment. Read pairs mapping to the same restriction fragment do not describe three-dimensional contacts and therefore should be discarded. A mechanism for generating such aberrant species is when fragments circularise by ligating to themselves and then, following sonication, form linear molecules possessing a valid Hi-C junction. Indeed, inadequate cross-linking can make this problem worse, increasing the frequency with which individual restriction fragments become separated and are therefore unable to ligate to other DNA molecules13. These artefacts are identifiable because the read pairs that map to the same genomic restriction fragment are orientated away from each other when aligned to the reference genome (Figure 2c). It is also possible for non-ligated DNA fragments to insert between sequencing adapters, despite the protocol being designed to minimise such events. Consequently, HiCUP identifies and removes these unwanted species by checking if the forward and reverse reads of a di-tag map to the same genomic restriction fragment, but unlike circularised fragments the reads are orientated towards each other. Furthermore, HiCUP divides this category into two sub-categories depending on whether the DNA fragment end overlaps a restriction fragment cut site. If the fragment end does overlap it is termed a \"dangling end\" (Figure 2d), but if it does not it is termed an \"internal fragment\" (Figure 2e). Overlapping a restriction enzyme cut site is important since it is here that biotin is incorporated and consequently a high proportion of dangling ends may be indicative of failure to remove biotin residues from non-ligated DNA fragments during the Hi-C protocol \"chewback\" step13. A high proportion of internal fragments suggests the experimental protocol may be improved in some other way: since internal fragments are derived from DNA positioned away from the restriction enzyme cut-site, these fragments should not have incorporated a biotin tag during the Hi-C ligation step. If, however, the streptavidin pull-down was not efficient, it could be assumed that a large proportion of these internal fragments arose from the genomic background. Alternatively, the presence of these internal fragments may be explained by the aberrant incorporation of biotin, possibly a result of DNA restriction digestion at non-canonical sites.\n\nValidating insert size. HiCUP places aligned di-tags on an in silico digested genome to calculate the theoretical length of the Hi-C insert and removes those not falling within the range set by the size-selection step of the protocol (Figure 2g). Explanations for such discrepancies include a read being incorrectly mapped or a putative di-tag containing multiple internal fragments or dangling ends. It is also possible that sequence variation between the sample DNA and reference genome leads to the loss or creation of restriction sites in the sample material. While such events are not common, the hallmark of restriction enzyme site generation is sometimes observed in Hi-C datasets, manifesting as an aggregation of reads orientated towards the novel restriction site.\n\nHi-C protocol variations. The Hi-C protocol may be modified by substituting sonication for a second digestion step, using a different restriction enzyme. The HiCUP pipeline includes additional filters when following the double-digest protocol, since the start of every read should now correspond to a cut site (in contrast to sonication, which is essentially a random fragmentation process). Reads not beginning exactly at a cut site are removed. Furthermore, to be considered valid a di-tag should have been generated by blunt-ended ligation between fragment ends created by the first restriction enzyme used in the protocol.\n\nPCR duplicates. Considering the huge number of theoretical pairwise interactions, it is likely that observed duplicate di-tags are the result of PCR amplification13 (as demonstrated in the Results section). These duplicates will unduly strengthen the case for a given genomic interaction and therefore HiCUP removes all but one representative copy of the di-tag (Figure 2f).\n\nPipeline output: the final valid dataset is in BAM/SAM format, with read pairs constituting a di-tag placed on adjacent lines within the file. This format is readily amenable for post-pipeline visualisation and analysis.\n\nThe relative abundance of the different classes of di-tag produced by the Hi-C assay are a direct result of the experimental protocol and therefore provide a useful QC diagnostic to identify ways in which the procedure may be improved. To enable users to benefit from these observations, HiCUP provides statistics documenting each step of the pipeline.\n\nHiCUP should be run on a Unix-based operating system (tested using CentOS v6.2) with Perl (tested using v5.10.1) installed. The pipeline requires a functional version of Bowtie (tested using v1.1.0) or Bowtie 2 (tested using v2.2.5) for mapping reads to a specified reference genome. Compression of SAM files to BAM format requires SAMtools (v0.1.18 or later). HiCUP may also compress or decompress files using gzip (tested using v1.3.12). For full functionality HiCUP requires the statistical programming language R (tested using v3.1.2). We recommend each file processed in parallel is allocated at least 5GB RAM.\n\nThe main body of the pipeline consists of four Perl scripts each performing a specific task. The first stage is the truncation step in which the putative Hi-C ligation junction is identified and reads are cut at this point. The second stage involves mapping reads independently and then pairing each read with its counterpart. The third stage filters out di-tags that are Hi-C artefacts, and the final de-duplication step involves removing identical di-tags. There is also a HiCUP master script that regulates data flow and executes each step of the pipeline (Figure 3).\n\nHiCUP takes FASTQ files generated by DNA sequencing and produces cleaned mapped data accompanied with QC reports. The bulk of the pipeline comprises 4 scripts: Truncater, Mapper, Filter and Deduplicator. These are executed in turn by the HiCUP master script which controls data flow through the pipeline. (The diagram uses rectangles with angled or rounded edges to represent data files or HiCUP Perl scripts respectively.)\n\n\nResults\n\nAs discussed previously, the final stage of HiCUP removes — retaining one copy — duplicate di-tags from the dataset. When considering the theoretical number of di-tags that may be generated it is reasonable to assume that exact duplicates are the result of PCR amplification and do not represent independent Hi-C ligation events. We addressed whether this assumption is valid with an experiment in which one of four barcode adapters were ligated randomly to both ends of a di-tag. We followed our standard Capture Hi-C protocol2, except that all four barcode adapters were mixed at equimolar ratios in the ligation reaction. The two technical replicates named here as Sample 1 (European Nucleotide Archive accession SAMEA2421737) and Sample 2 (European Nucleotide Archive accession SAMEA2421733) were CHi-C libraries of foetal liver cells from mouse (strain C57BL/6) embryos at day 14.5 of development.\n\nAssuming equal numbers of each barcode, all 16 barcode-barcode permutations should occur with equal frequency. Crucially, the barcodes are incorporated into the di-tags before PCR amplification, providing a test for the origin of the duplicates: those representing independent Hi-C events are most likely to possess differing barcodes (Figure 4a), whereas di-tags with a single common origin amplified by PCR should have identical barcodes (Figure 4b).\n\nThe two independent libraries were sequenced and the resulting FASTQ reads were classified by barcode i.e. the first four base pairs of the polynucleotide. The reads were mapped and filtered with HiCUP, but duplicates were retained. The barcode sequences were then quantified revealing that 71.3% corresponded to a pre-defined sequence in Sample 1 and 71.5% in Sample 2. Each valid barcode was then quantified. For both samples the barcode CCTT was most prevalent (Sample 1: 9,889,602; Sample 2: 16,368,793), and for both samples the barcode CGCT was the least common (Sample 1: 2,991,820; Sample 2: 5,002,346).\n\nThe diagram shows di-tags (shaded rectangles) delimited by a pair of barcoded sequencing adapters.\n\nDespite the deviation from the ideal and expected result in which all barcode combination frequencies were equal, it was still possible to address the source of duplicate di-tags. To achieve this, the barcode combination of each duplicate was recorded. Duplicates arising from PCR amplification should have had identical barcode combinations, in contrast to bone fide initial Hi-C interaction events. Table 1 shows the result of this quantification, demonstrating that the overwhelming majority of duplicate di-tags are delimited by identical barcode combinations, almost certainly a result of PCR amplification.\n\nThe Copies column refers to how many times a di-tag was observed in the dataset, the Di-tags column refers to the number of different di-tags observed with the specified number of copies, and the One column lists the percentage of di-tags in which all the copies possessed identical barcodes.\n\nTo assess the impact of retaining duplicate di-tags we detected significant interactions in HiCUP-processed CHi-C datasets using the CHiCAGO pipeline (five datasets were processed: Samples 1 and 2 are described previously and Samples 3, 4 and 5 were generated from mouse embryonic stem cells and have the Gene Expression Omnibus accession GSM1888519). The analysis showed that removing duplicate di-tags substantially reduced the number of called significant interactions (see Table 2). This was not surprising owing to the vast number of theoretical interactions, meaning that fragment-fragment contacts repeated only a small number of times were likely to be statistically significant.\n\nThe table shows the number of statistically significant interactions identified in each of the datasets when duplicate di-tags are retained (Dups) or removed (No dups). The percentage of unique di-tags in each dataset is also shown. (*Unique corresponds to the percentage of di-tags retained after removing all but one representative copy of each di-tag.)\n\nConsequently, when considering that PCR results in certain di-tags being amplified disproportionately14, and that only a small number of observed fragment-fragment contacts are needed to qualify an interaction as statistically significant, retaining PCR duplicates will lead to an erroneous interpretation of the data. Furthermore, this over-calling of significant interactions becomes more problematic as the proportion of duplicate di-tags increases.\n\n\nUse cases\n\nHiCUP was used to process several Hi-C datasets available on public repositories. Table 3 summarises the results. The bacterial sample was a species of Caulobacter crescentus (SRR824843)15; human refers to Homo sapiens (SRR027963)1; fruit fly refers to Drosophila melanogaster (SRR389762)16 and yeast was a strain of Schizosaccharomyces pombe (SRR1271321)17.\n\nEach value is represented as a percentage of the reads or read-pairs processed at the given stage of the pipeline. (*Unique corresponds to the percentage of di-tags retained after removing all but one representative copy of each di-tag.)\n\nThe table shows key statistics produced by HiCUP when processing Hi-C datasets. All the samples except Fruit Fly (in which the protocol did not include the fill-in/blunt-ended ligation step) show an appreciable percentage of truncated reads, meaning that the Hi-C ligation junction sequence was identified. Failure to detect the ligation junction sequence after following a standard Hi-C protocol provides a first indication that the Hi-C library is of poor quality. The samples mapped with variable efficiency to their respective reference genomes and the percentage of valid di-tags also varied considerably, but the yeast sample is particularly noticeable in returning a high proportion of internal fragments. This may be the result of additional technical difficulties in generating Hi-C libraries from yeast cells, requiring certain areas of the protocol to be further refined for reasons discussed previously.\n\n\nDiscussion\n\nHiCUP is software tailored for processing Hi-C data and has been publicly available for several years. HiCUP maps sequence data in a manner optimised for the Hi-C protocol and then removes commonly encountered experimental artefacts which could otherwise lead to incorrect inferences being made about the conformation of a genome. Furthermore, HiCUP provides statistics summarising each stage of the pipeline which may help in refining the experimental protocol. For example, a high proportion of circularised di-tags suggests inadequate cross-linking, whereas a high proportion of dangling ends implies the chewback step to remove biotin from unligated restriction fragments was inefficient13. The summary statistics also include the proportion of trans (inter-chromosomal) interactions to cis (intra-chromosomal) interactions. A high trans/cis ratio is indicative of a poor library, since spurious ligation events will tend to be between genomic loci on different chromosomes18.\n\nHiCUP is flexible, allowing the user to specify numerous parameters and the final output is in the commonly used BAM/SAM format: which is compatible with, or may be converted to a format compatible with a myriad of analysis tools. Indeed, HiCUP has already been used to process and analyse Hi-C and CHi-C data in conjunction with GOTHiC2–4, Homer18,19 and Hicpipe18. One recent study used HiCUP-processed Hi-C data to shed light on how GWAS SNPs found in genome deserts may modulate gene activity20. Another publication characterised architectural changes in genome organisation in age-related cellular senescence19. HiCUP was also used in a study to compare the efficiency of two different Hi-C protocols18. Finally, four other research articles pioneered targeted Hi-C capture techniques, enriching for gene promoter-containing fragments to elucidate regulatory networks in mice2,4,21 and humans3. In addition to these studies, HiCUP is being used to process data in numerous ongoing projects and is under active development to meet the demands of the burgeoning fields of Hi-C and CHi-C in furthering our understanding of three-dimensional genomic structure, organisation and regulation.\n\n\nSoftware availability\n\n1. URL link to where the software can be downloaded from or used by a non-coder: www.bioinformatics.babraham.ac.uk/projects/hicup\n\n2. URL link to the author’s version control system repository containing the source code: www.bioinformatics.babraham.ac.uk/projects/hicup\n\n3. Archived source code as at time of publication: http://dx.doi.org/10.5281/zenodo.3338822\n\n4. Software license: GNU GPL v3 or later.",
"appendix": "Author contributions\n\n\n\nSWW wrote the HiCUP code and manuscript. PAE assisted with the HTML HiCUP summary reports. MF and SS devised and performed the random barcode CHi-C experiment. TN outlined the initial HiCUP scope and requirements. PF directed the Hi-C and CHi-C research programmes and SA directed the bioinformatics tool development. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nResearch supported by the Medical Research Council and the Biotechnology and Biological Sciences Research Council of the UK.\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\nMikhail Spivakov, Jonathan Cairns and Paula Freire-Pritchett assisted with identifying significant interactions in HiCUP-processed data using the CHiCAGO pipeline. Felix Krueger and Csilla Várnai gave useful coding advice and Biola-Maria Javierre provided numerous datasets with which to test HiCUP. Sarah Elderkin and Harry Armstrong provided cells for CHi-C libraries. Anne Segonds-Pichon, Cameron Osborne, Bhupinder Virk and Edoardo Petrini provided assistance with the manuscript preparation.\n\n\nReferences\n\nLieberman-Aiden E, van Berkum NL, Williams L, et al.: Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009; 326(5950): 289–293. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchoenfelder S, Furlan-Magaril M, Mifsud B, et al.: The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements. Genome Res. 2015; 25(4): 582–597. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMifsud B, Tavares-Cadete F, Young AN, et al.: Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat Genet. 2015; 47(6): 598–606. PubMed Abstract | Publisher Full Text\n\nSchoenfelder S, Sugar R, Dimond A, et al.: Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome. Nat Genet. 2015; 47(10): 1179–1186. PubMed Abstract | Publisher Full Text\n\nYaffe E, Tanay A: Probabilistic modeling of Hi-C contact maps eliminates systematic biases to characterize global chromosomal architecture. Nat Genet. 2011; 43(11): 1059–1065. PubMed Abstract | Publisher Full Text\n\nImakaev M, Fudenberg G, McCord RP, et al.: Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat Methods. 2012; 9(10): 999–1003. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHeinz S, Benner C, Spann N, et al.: Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B Cell identities. Mol Cell. 2010; 38(4): 576–589. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAy F, Bailey TL, Noble WS: Statistical confidence estimation for Hi-C data reveals regulatory chromatin contacts. Genome Res. 2014; 24(6): 999–1011. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMifsud B, Martincorena I, Darbo E, et al.: GOTHiC, a simple probabilistic model to resolve complex biases and to identify real interactions in Hi-C data. bioRxiv. 2015. Publisher Full Text\n\nCairns J, Freire-Pritchett P, Wingett SW, et al.: CHiCAGO: Robust Detection of DNA Looping Interactions in Capture Hi-C data. bioRxiv. 2015. Publisher Full Text\n\nLangmead B, Trapnell C, Pop M, et al.: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3): R25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLangmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4): 357– 359. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBelton JM, McCord RP, Gibcus JH, et al.: Hi-C: a comprehensive technique to capture the conformation of genomes. Methods. 2012; 58(3): 268–276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAird D, Ross MG, Chen WS, et al.: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol. 2011; 12(2): R18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLe TB, Imakaev MV, Mirny LA, et al.: High-resolution mapping of the spatial organization of a bacterial chromosome. Science. 2013; 342(6159): 731–734. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSexton T, Yaffe E, Kenigsberg E, et al.: Three-dimensional folding and functional organization principles of the Drosophila genome. Cell. 2012; 148(3): 458–472. PubMed Abstract | Publisher Full Text\n\nGullerova M, Proudfoot NJ: Cohesin complex promotes transcriptional termination between convergent genes in S. pombe. Cell. 2008; 132(6): 983–995. PubMed Abstract | Publisher Full Text\n\nNagano T, Várnai C, Schoenfelder S, et al.: Comparison of Hi-C results using in-solution versus in-nucleus ligation. Genome Biol. 2015; 16(1): 175. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChandra T, Ewels PA, Schoenfelder S, et al.: Global reorganization of the nuclear landscape in senescent cells. Cell Rep. 2015; 10(4): 471–483. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDryden NH, Broome LR, Dudbridge F, et al.: Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C. Genome Res. 2014; 24(11): 1854–1868. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSahlén P, Abdullayev I, Ramsköld D, et al.: Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution. Genome Biol. 2015; 16(1): 156. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWingett SW, Ewels PA, Furlan-Magaril M, et al.: HiCUP: pipeline for mapping and processing Hi-C data. Zenodo. 2015. Data Source"
}
|
[
{
"id": "11293",
"date": "02 Dec 2015",
"name": "Ferhat Ay",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes the HiCUP pipeline that has already been used by many for mapping and initial processing of Hi-C data. HiCUP is a useful tool for processing both traditional Hi-C data and more recent Capture Hi-C data. The article clearly describes what HiCUP does and what it does not. It also provides a nice study on the origin of duplicated di-tags showing that nearly all such duplicates are due to PCR amplification. Other than my few comments below, I am happy with the article and want to note that I have successfully downloaded and used HiCUP pipeline to process number of different Hi-C data sets.Even though mentioned as out of scope, it would still be useful to have a simple normalization method implemented with HiCUP. Maybe as simple as \"vanilla coverage normalization\" mentioned in Rao et al. which is a single step of matrix balancing. Another useful feature would be to have HiCUP generate an HTML report that basically links together all different figures created by the pipeline. I wrote such scripts to generate an HTML for my local HiCUP installation. \"Fit-Hi-C takes renormalized data to identify mid-range intra-chromosomal interactions\" --> \"Fit-Hi-C takes normalized data to identify statistically significant intra-chromosomal interactions\". If possible, modify Figure 2F to reflect that one copy of the duplicates is valid and others are discarded. Sequence variation between sample and the reference can be in the form of copy number changes or other aberrations. It should be noted that HiCUP does assume these do not happen. In \"Operation\", put the names of scripts in parentheses where they are described. In Figure 3, indicate in which steps the restriction cut site information is used (e.g. truncater, digester, filter). In \"Results\", mention explicitly that they hold for the analyzed case where sonication is the choice instead of a second digestion with a restriction enzyme. It may be the case that a substantial part of duplicates are not PCR related for the latter case where the theoretical number of di-tags is limited compared to sonication. In Table 3, use horizontal lines to demarcate where one step ends and the other starts. Otherwise, it is difficult to understand what each percentage is out of.",
"responses": []
},
{
"id": "11292",
"date": "07 Dec 2015",
"name": "Juan M. Vaquerizas",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Wingett and collaborators describes HiCUP, a pre-processing pipeline for Hi-C data. The pipeline is designed as an executable command-line tool that includes the most common pre-processing steps for Hi-C datasets, including read truncation and mapping, filtering, assignment of read to restriction fragments, detection and removal of PCR duplicates and generation of quality evaluation plots. The pipeline is solid and well implemented. We have downloaded and tested the current version of the pipeline using one of our locally produced Hi-C datasets, and the filtering results of the pipeline are equivalent to our locally implemented pre-processing pipelines. Therefore, we are happy with the implementation of the software and have no major concerns with the manuscript.We only have a few minor comments that we hope will help the authors to improve the software.Table 1It would be useful to include a further column that would display the % calculated against the total number of di-tags per sample. This would allow the user to quickly determine the overall amount of PCR-duplicates included in the library. HTML reportingGiven that the tool is aimed to provide the user with an easy way to highlight errors with Hi-C libraries, it would be useful to implement the following in the HTML report:- include a clearer description of how the cutoff for reads that are “too short to map” is chosen, and indicate if this can be changed by the user- include labelling to indicate what part of the reporting corresponds to the “read” level and what corresponds to the “pairs” level.- include a plot with the inwards/outwards bias as function of distance from restriction fragment as proposed by Jin et al., (2013)1. This is already calculated by the pipeline, but the representation of the data will help the user to determine whether the library has specific issues with under-digestions or ligation artefacts. ScreenshotsThe manuscript could include some screenshots of the reporting output to guide the user through it and to highlight what are the key indicators of good/bad quality datasets (some of these could even be obtained using simulated datasets with specific biases). This can also be provided as a guided example in the online documentation of the pipeline. Ratio trans/cis interactionsThe manuscript states that “A high trans/cis ratio is indicative of a poor library, since spurious ligation events will tend to be between genomic loci on different chromosomes [ref 18]”.While we agree that this is a possible interpretation of that scenario, it should be noted that the trans/cis ratio depends on the genome’s size and the number of chromosomes that each species has, and it might also be related with specific higher-order chromatin conformations, or depend on the specific Hi-C protocol used in that experiment. Therefore, the authors might not want to generalise the interpretation of the high trans/cis ratio, or, at least, give more specific guidance regarding the interpretation of this ratio. For example, the four datasets included in this manuscript would produce trans/cis ratios of 0, 0.18, 0.52 and 1.18 for bacteria, yeast, fly and human, respectively, which in this case correlate with the genome size and the number of chromosomes in each species, but that are not necessarily indicative of the quality of these datasets. Implementation and code availabilityRun on a sample of one of our locally produced Hi-C data sets (12 million paired reads), the HiCUP pipeline finishes in a reasonable amount of time (~2 hours with sequential processing of two files, a little more than half the time when it is run on two threads [2300 Mhz AMD Opteron Processor, 64Gb RAM]).The configuration file could be very convenient, but required some copy-pasting of file paths. Regarding this, it is unclear why R can be automatically detected, but the full path of bowtie2 has to be specified. In addition, it might be convenient to also add the HiCUP_digester step of the pipeline to the main HiCUP executableThe link to the software package was identical to the version control system link, and it was not straightforward to find the link to the self-hosted bug report system. We think that having the ability to review code online, file bug reports, and contribute to the package using a more generally used online repository (such as GitHub) would make HiCUP even more useful.",
"responses": []
},
{
"id": "11295",
"date": "07 Dec 2015",
"name": "Nicola Neretti",
"expertise": [],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a novel pipeline for mapping and littering of Hi-C data and demonstrate evidence that duplicate di-tags are PCR artifacts. This pipeline has already been successfully used in several studies. Following is a list of questions and comments that should be addressed by the authors to improve the clarity of their manuscript.How does the pipeline handle the case of restriction sites that are very close to the beginning of a read? Is there a minimum size for a truncated read to be included in the analysis? The “Relegation of adjacent restriction fragments” section is not completely clear to me. Fragment nr. 4 in Figure 2b could be quite large and removing fragments which include 2 restriction sizes might remove true events corresponding to local looping of the DNA. Is there any experimental evidence that such fragments are predominantly artifacts of re-ligation or incomplete digestion and should be excluded? Or are the authors using the theoretical length of the Hi-C insert to inform this type of filtering? A more explicit description of size-selection would be beneficial here. The authors present evidence that duplicate di-tags are PCR artifacts. This is in true for the standard Hi-C protocol, which from what I can see, was used to generate the datasets described in the result section. Because the HiCUP pipeline has been developed to handle the double-digestion protocol as well, could the author comment about duplicate di-tags and PCR artifacts in this context? With a double digestion protocol, the probability of truly obtaining the same fragment multiple times is much higher because the DNA is cut at fixed locations (as opposed to randomly via sonication). The pipeline removes fragments with insert size less than expected by size selection. Structural variation (e.g. deletions) could yield di-tags with larger than expected theoretical insert size. Such di-tags would be valid and informative. Either an exploration or discussion of how structural variation affects this filtering step would be be helpful. The percentages reported in Table 3 are difficult to interpret and compare. They clearly do not sum to 100%, so the authors should provide a more detailed description of what each percentage corresponds to (e.g. % of total reads or % of total mapping reads, etc.).",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1310
|
https://f1000research.com/articles/4-1297/v1
|
19 Nov 15
|
{
"type": "Review",
"title": "Post-translational control of RIPK3 and MLKL mediated necroptotic cell death",
"authors": [
"James M. Murphy",
"James E. Vince"
],
"abstract": "Several programmed lytic and necrotic-like cell death mechanisms have now been uncovered, including the recently described receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-dependent necroptosis pathway. Genetic experiments have shown that programmed necrosis, including necroptosis, can play a pivotal role in regulating host-resistance against microbial infections. Alternatively, excess or unwarranted necroptosis may be pathological in autoimmune and autoinflammatory diseases. This review highlights the recent advances in our understanding of the post-translational control of RIPK3-MLKL necroptotic signaling. We discuss the critical function of phosphorylation in the execution of necroptosis, and highlight the emerging regulatory roles for several ubiquitin ligases and deubiquitinating enzymes. Finally, based on current evidence, we discuss the potential mechanisms by which the essential, and possibly terminal, necroptotic effector, MLKL, triggers the disruption of cellular membranes to cause cell lysis.",
"keywords": [
"necroptotic cell death",
"necroptosis",
"RIPK3",
"MLKL",
"mixed lineage kinase domain-like",
"receptor interacting protein kinase-3"
],
"content": "Introduction\n\nNecroptosis is a caspase-independent programmed cell death pathway1. Necroptotic cell lysis and the release of intracellular pro-inflammatory molecules is dependent on phosphorylation of the pseudokinase, MLKL, by the protein kinase, RIPK3. Phosphorylation of MLKL leads to its activation, and subsequent cell death by mechanisms that are currently a matter of debate, although a weight of evidence suggests this involves disruption of cellular membranes, including the plasma membrane. Following tumor necrosis factor (TNF) receptor 1 (TNFR1) ligation, the protein kinase, RIPK1, activates RIPK3 presumably by promoting RIPK3 autophosphorylation, which in turn leads to MLKL activation. However, it is now apparent that RIPK3 can be activated independently of RIPK1 in many circumstances, and that RIPK1 can act to repress RIPK3 activation, both in vitro and in vivo2–8. Consequently, our current thinking is that RIPK3 and MLKL are the core machinery essential for all necroptotic cell death responses.\n\nStudies using RIPK3-deficient mice have implicated pathological RIPK3 signaling, and potentially necroptosis, in many inflammatory diseases, such as atherosclerosis, kidney ischemia reperfusion injury, liver injury, myocardial infarction, and multiple sclerosis (reviewed in 9). However, it has recently been posited that necroptosis may also act in an anti-inflammatory capacity, as cell death abrogates TNF- or toll-like receptor- (TLR) induced transcription of pro-inflammatory cytokines and the ensuing inflammatory response10. Recent research has also revealed that RIPK3 has several non-necroptotic signaling capabilities, both in vitro and in vivo (reviewed in 11). These include the ability to activate caspase-8 dependent apoptosis12–14, trigger interleukin-1β (IL-1β)-dependent inflammation through caspase-8 and/or the Nod-like receptor 3 (NLRP3) inflammasome15–22, and regulate the transcription of cytokines23,24. Hence, the use of MLKL-deficient mice is required to validate necroptosis as a bona fide drug target in many inflammatory disease models where RIPK3 has been implicated. In this regard, murine genetic studies have started to document how unrestrained MLKL-dependent necroptotic signaling can result in embryonic lethality3 and cause liver inflammation25. In addition, the development of phospho-specific MLKL antibodies as markers of activated MLKL have shown that necroptosis is likely to occur in diseases such as toxic epidermal necrolysis26,27, drug-induced liver injury28, and pathogen infection21. Cancer cell lines have also been observed to suppress RIPK3 expression29, which in some circumstances has been attributed to DNA methylation30. As such, chemically induced hypomethylation can restore RIPK3 expression and promote RIPK3-MLKL-induced necroptosis in response to chemotherapeutic treatments. A greater understanding of the mechanisms of necroptosis signaling, and when it occurs, is therefore likely to yield new therapeutic opportunities in a number of different disease states.\n\nSeveral signaling receptors have been documented to activate RIPK3-MLKL dependent necroptosis, including death receptors (i.e., TNFR1), TLRs, the DNA receptor DAI (DNA-dependent activator of interferon [IFN]-regulatory factors or ZBP1/DML-1), and the T-cell antigen receptor. Type I IFN and IFNγ-induced transcriptional responses have also been proposed to cause necroptosis, or to enhance TLR3/4 and TNFR1 necroptosis31–33. While protein kinase R (PKR) was suggested to directly trigger formation of the RIPK1-RIPK3 necrosome downstream of IFNγ signaling33, PKR is not required for type I IFN killing32, and hence the underlying mechanism for IFN-induced necroptosis requires further study. By comparison, necroptotic signaling caused by TNFR1 ligation is better defined (reviewed in 34). In most cases, TNF binding to TNFR1 triggers the formation of a cell surface complex, complex I, that induces the transcription of pro-survival genes and inflammatory cytokines. Mechanistically, the death domain (DD) of TNFR1 interacts with the DD of TNFR1-associated death domain (TRADD) (and potentially the DD of RIPK1) to nucleate the formation of a large multimeric TRADD-RIPK1-TRAF2- inhibitor of apoptosis (IAP) ubiquitylation platform35–38. For example, RIPK1 binding to this complex and its modification with ubiquitin chains by IAP proteins parallels IAP-dependent recruitment of the linear ubiquitin chain assembly complex (LUBAC). Ubiquitylated RIPK1, and LUBAC modification of NEMO (nuclear factor kappa-light-chain-enhancer of activated B cells [NFκB] essential modifier), subsequently activate canonical NFκB signaling. In the absence of optimal RIPK1 ubiquitylation (i.e., when IAPs are lost), RIPK1 dissociates into the cytosol to form a secondary death-inducing complex that can activate caspase-8 (complex II) to cause apoptosis. Caspase-8 represses necroptotic signaling, and hence, when caspase-8 activity is low, RIPK1 can bind RIPK3 to form the necrosome, activate MLKL, and induce necroptotic killing.\n\nBecause under normal cell culture conditions necroptosis is not induced by death receptor or TLR ligation, experimentally, necroptosis is usually studied by deleting or inhibiting key negative regulators of necroptotic signaling, such as caspase-8 or IAP proteins (see below). Physiological settings that trigger necroptosis have been less well defined, although situations where caspase-8 is down-regulated, such as following cutaneous wounding39–41, or IAP protein depletion, such as during TNF-like weak inducer of apoptosis (TWEAK)-FGF-inducible molecule 14 (FN14) TNF superfamily signaling42,43, clearly have the potential to promote a necroptotic response. Along these lines of evidence, biopsies from children suffering from inflammatory bowel disease display decreased caspase-8 expression and elevated RIPK3 and MLKL levels, and may indicate ongoing necroptosis44. More direct experiments have been performed to suggest that bacterial and viral molecules can act to induce or inhibit necroptosis at multiple levels, including direct RIPK1/RIPK3 targeting, or downstream of MLKL phosphorylation21,45–50.\n\nIn this review we summarize recent advances in identifying and understanding positive and negative regulators of necroptotic signaling (Table 1), focusing on findings with strong genetic evidence. We begin by a brief discussion on the best studied necroptotic components; RIPK1, RIPK3 and MLKL.\n\n\nThe core necroptotic machinery\n\nThe defining feature that triggers RIPK3 serine/threonine kinase activity and its phosphorylation of MLKL is RIPK3 oligomerization via RIP homotypic interaction motif (RHIM) containing proteins. For example, RHIM containing signaling components of TNFR1 (RIPK1), TLR3 and TLR4 (TRIF) and DAI itself can all form RHIM-RHIM interactions with RIPK3 to trigger necroptotic death. Similarly, the artificial dimerization/oligomerization of RIPK3 directly suffices to cause its activation and recruitment of MLKL in the absence of any upstream signal4,14,51. The importance of RHIM-RHIM interactions for RIPK3 signaling is highlighted by several viral RHIM-containing proteins, such as cytomegalovirus vIRA and herpes simplex virus ICP-6 and ICP-10, which bind RIPK1 and RIPK3 and can regulate necroptosis to alter host anti-viral responses46–49.\n\nThe interaction of RIPK1 and RIPK3 RHIMs induces the formation of a large amyloid-like necrosome signaling platform52. Recent work has suggested that RIPK1-RIPK3 heterodimers do not suffice to trigger necroptosis, and instead RIPK3 homo-oligomer formation within the necrosome complex is essential for RIPK3 activation, intramolecular RIPK3 phosphorylation, and MLKL recruitment51. This model fits with recent genetic studies showing that the deletion of RIPK1 in vivo can trigger RIPK3-MLKL induced necroptosis3, as the loss of RIPK1 may enhance the propensity for RIPK3 oligomerization4.\n\nActivated RIPK3 phosphorylates MLKL, thereby allowing MLKL association with, and disruption of, phospholipid membranes28,53–55. RIPK3-deficient mice display no overt phenotype, and hence targeting RIPK3 kinase activity with small-molecule inhibitors represents one strategy for the treatment of necroptotic-driven diseases13,56. It is noteworthy, however, that RIPK3 kinase inhibition13, or a D161N mutation in the RIPK3 kinase domain in vivo12, can drive lethal caspase-8-dependent apoptosis by triggering RIPK1 recruitment to RIPK3, and RIPK1-mediated activation of caspase-8. Interestingly, RIPK3 kinase activity per se is not essential for mammalian viability, as an independently generated kinase-dead (K51A) RIPK3 mouse is viable and fertile13. Hence, the development of RIPK3 kinase inhibitors that avoid lethal RIPK3 conformational changes are required. Alternatively, MLKL inhibitors54,57 may be a more attractive strategy for specifically targeting necroptosis, as these would avoid complications that may result from altering non-necroptotic RIPK3 signaling capabilities.\n\nMLKL is an essential necroptosis effector that operates downstream of RIPK357–60. Unlike RIPK3, which is a catalytically active, conventional protein kinase, MLKL belongs to a group of related proteins that are enzymatically-dead, termed pseudokinases61,62. In addition to its C-terminal pseudokinase domain, MLKL contains an N-terminal four-helix bundle (4HB) domain, which is now known to mediate MLKL’s killer function53–55,63. Our current understanding of MLKL activation is that RIPK3 phosphorylates the activation loop in MLKL’s pseudokinase domain57,59,60,64,65, which induces a conformational change that relieves the suppressive function of the pseudokinase domain, allowing the unleashing of the executioner N-terminal 4HB domain54. Several different models for how the MLKL 4HB domain can induce cell death have been proposed, ranging from a direct membrane permeabilization or pore-forming capacity28,55,66, to the implication of downstream effectors53,63 (summarized in Figure 1). Although details are still emerging, recombinant MLKL was found to bind to various lipids in “fat Western” lipid arrays28,55, which was further reflected in a capacity to permeabilize liposomes in vitro28,55,66. Curiously, MLKL most readily permeabilized liposomes containing 15% cardiolipin, a lipid believed to be confined to the mitochondrial inner membrane. The necessity of mitochondria for necroptosis has been challenged67, making the preference for cardiolipin difficult to reconcile. At the molecular level, the mode of 4HB domain engagement of lipids remains of outstanding interest. While initial reports suggested this interaction might be mediated by positively-charged residues in the human MLKL 4HB domain55, these residues are poorly conserved among MLKL orthologs and indeed, curiously, mutation of the acidic counterparts in the mouse 4HB domain similarly compromised the capacity of the domain to induce cell death54. It is therefore possible that, rather than a direct interaction with negatively-charged phospholipids, these charged residues in the MLKL 4HB domain are important to the MLKL homo- and/or hetero-oligomerisation that is necessary for necroptosis.\n\nThe core necroptosis machinery, comprising RIPK3 and MLKL, are activated following RIPK3 interaction with RIPK1, TRIF or DAI via their RHIMs. CYLD-mediated deubiquitylation of RIPK1 is necessary for its participation in cell death pathways, while ubiquitylation of RIPK3 (at Lys5) and MLKL by as-yet-unidentified E3 ligases may promote necroptosis15,97. HSP90 is known to augment the necroptotic functions of RIPK1 and RIPK3. MLKL is believed to induce cell death via a membrane-directed process54, perhaps by directly permeabilizing membranes28,55, with some debate over whether channel activation might be involved28,53,63, or, as one report suggests, there may be a role for Src in promoting MLKL-mediated death downstream of Gβγ. Abbreviations: 4HB, four-helix bundle; CYLD, cylindromatosis; DAI, DNA-dependent activator of interferon [IFN]-regulatory factors; HSP90, heat shock protein 90; IAP, inhibitor of apoptosis proteins; MLKL, mixed lineage kinase domain-like; RHIM, RIP homotypic interaction motif; RIPK, receptor interacting protein kinase; TLR, toll-like receptor; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; TRADD, TNFR1-associated death domain; TRAF2, TNF receptor associated factor 2; TRPM7, transient receptor potential cation channel, subfamily M, member 7.\n\nAnother outstanding question is whether the MLKL 4HB domain forms a transmembrane pore or merely somehow compromises the integrity of the membrane. In concert with a greater understanding of which membrane within a cell is the target of MLKL action, knowledge of the means by which MLKL breaches membranes is essential for a comprehensive understanding of its action. Even though the membrane compromising activity of recombinant MLKL in liposome assays is highly suggestive of MLKL acting as a lone gunman, several lines of evidence have implicated other proteins as either co-effectors that augment or negate the activities of RIPK3 or MLKL, or as effectors that function downstream of MLKL activation (Figure 1 and Figure 2). Of note, the induction of necroptosis is associated with the ubiquitylation of RIPK3 and MLKL15, although the function of these modifications requires further study. We have summarized important necroptotic regulators that promote (Figure 1) or negate (Figure 2) MLKL-mediated death, and elaborate on current knowledge of their activities below.\n\nNegative regulation at the levels of RIPK1, RIPK3 and MLKL have been reported to attenuate necroptotic signalling. Not only is necroptosis by definition a caspase-independent process, but caspase-8 negates cell death, potentially by proteolytically-cleaving CYLD, RIPK1 and RIPK3. Reversible tuning of the pathway is accomplished by introduction or removal of post-translational modifications, including: ubiquitylation of RIPK1 by IAPs; deubiquitylation of RIPK3 on Lys5 by A20; and dephosphorylation of mouse RIPK3 at S231/T232 by Ppm1b. Whether an analogous phosphatase exists to dephosphorylate activated MLKL and inhibit necroptosis is not clear. However, other regulators of MLKL have been proposed, such as TRAF2. It is currently unknown what factors might govern phospho-MLKL assembly into higher order oligomers on the membrane, although a role for additional factors in mediating death is suggested by the lag between MLKL membrane translocation/oligomerisation and cell death65. Abbreviations: 4HB, four-helix bundle; CYLD, cylindromatosis; IAP, inhibitor of apoptosis proteins; MLKL, mixed lineage kinase domain-like; RHIM, RIP homotypic interaction motif; RIPK, receptor interacting protein kinase; TNF, tumour necrosis factor; TRADD, TNFR1-associated death domain; TRAF2, TNF receptor associated factor 2.\n\nAn important role for RIPK1 and its kinase activity in death receptor-induced necroptosis was documented long before RIPK3 was identified as the essential necroptotic RIPK1 binding partner68. The development of the RIPK1 kinase inhibitor necrostatin-169,70 has been widely used to demonstrate the therapeutic potential of targeting RIPK1 kinase activity and necroptosis in disease models. The plausibility of this strategy was recently validated when, in contrast to the embryonic lethality of RIPK1-deficient mice caused by unrestrained apoptotic and necroptotic signaling2,3,5, RIPK1 kinase-dead knockin mice, containing K45A2 or D138N12,71 alleles, were shown to be viable and fertile. Hence, while RIPK1 kinase activity is often a requisite for necroptotic killing, it is not critical for mammalian survival. Instead the kinase-independent scaffolding role of RIPK1 is vital in preventing lethal RIPK3-MLKL and caspase-8 signaling.\n\nMechanistically, RIPK1 phosphorylation of RIPK3 is an attractive hypothesis for why RIPK1 kinase function is required for TNF-induced necroptosis. However, RIPK1 mediated phosphorylation of RIPK3 has not been formally reported to date, and the key target(s) of RIPK1 kinase activity remain unclear. Recently it has been suggested that the inhibition of RIPK1 kinase activity enhances RIPK1’s anti-necroptotic function, and consistent with this, depletion of total RIPK1 in several cell types was shown to sensitize to TNF and TLR-induced necroptotic killing7. Why RIPK1 loss sensitizes some cell lines to TNF-induced necroptosis, while in others it confers protection, may be due to the expression levels of RIPK3 in different cells or tissues, dictating its ability to efficiently engage TNFR1 or TLR complexes and form RIPK3 oligomers. Consistent with this notion, the expression levels of RIPK3 have been shown to largely determine whether TNF can engage RIPK3 killing in the absence of RIPK172. Unlike the direct recruitment of RIPK3 to the RHIM containing receptor DAI46, or the TLR adaptor protein TRIF56,73, the way RIPK3 is recruited to TNF receptor complexes in RIPK1 deficient cells is unknown.\n\n\nPositive regulators of necroptosis\n\nCYLD (Cylindromatosis) is a deubiquitinating enzyme that removes K63-linked and linear ubiquitin chains from its target proteins, which include RIPK1 and TRAFs. This deubiquitinase activity of CYLD is linked with increased TNF-induced death signaling, including RIPK1-RIPK3 necrosome formation and necroptotic cell death74–76. Mechanistically, CYLD has been proposed to deubiquitylate RIPK1 during necrosome complex formation to facilitate RIPK1/3 kinase activity76. In this context, it is interesting to note that the LUBAC component, HOIL-1 interacting protein (HOIP), can ubiquitylate RIPK1 within the necrosome77. Although HOIP loss did not impact necroptotic killing, the functional consequences of necroptotic-associated RIPK1, RIPK3 and MLKL ubiquitylation warrant further investigation.\n\nFollowing TNF stimulation, aberrant CYLD necroptotic activity is held in check by caspase-8 mediated CYLD processing and inactivation74. Consistent with this, colitis induced by deletion of the essential caspase-8 adaptor protein Fas-associated protein with death domain (FADD) in intestinal epithelial cells was ameliorated by loss of either RIPK3 or CYLD activity78. CYLD is thereby important for necroptotic signaling in some situations in vitro and in vivo. In contrast, however, unlike RIPK3-deletion, the in vivo inactivation of CYLD delays, but does not prevent, the inflammation caused by unrestrained necroptotic killing of FADD deficient keratinocytes79. Hence, although CYLD tunes necroptotic activity, it is not essential for it to occur in all cell types.\n\nThe Cdc37-heat shock protein 90 (HSP90) co-chaperone system has been widely implicated as a regulator of protein kinase “clients”. Conventionally, HSP90 interaction is thought to enhance protein stability, often through protection from proteasomal degradation, as is believed to be the case with RIPK180–85. RIPK3 has long been recognized as an HSP90 client protein86,87, although only recently has a less passive regulatory role been suggested88. Inhibition of HSP90 or genetic knockdown of the kinase-interaction adaptor, Cdc37, inhibited RIPK3’s capacity to phosphorylate MLKL to induce necroptosis, but only conferred modest effects on RIPK3 abundance88. The precise underlying mechanism remains a matter of outstanding interest.\n\nMLKL was implicated as a regulator of plasma membrane ion channels in two independent studies: one suggested a role for TRPM7 (transient receptor potential cation channel, subfamily M, member 7) in necroptotic killing53, and another more broadly for Na+ ion channel signaling63. Subsequent studies using ion-free media, however, have challenged whether ion channels play an obligate role in mediating necroptosis28, but instead suggest they contribute to varying extents in different cultured cell lines under some circumstances.\n\nA genetic screen for additional necroptosis effectors identified the transmembrane G-proteins, Gβ and Gγ, as instigators of an alternative pathway that operates in parallel with TNF-induced necroptosis89. Perturbation of Gβγ signaling disrupted MLKL oligomerization and translocation to membranes, a finding attributed to perturbed activation of the protein kinase, Src. The mechanistic underpinnings and universality of this pathway have not yet been fully elucidated, but are illustrative of the many possible signaling effectors that could potentially intersect with and tune the necroptosis signaling pathway. This idea is supported by the recent identification and functional characterization of MLKL phosphorylation sites outside the best-understood phosphorylation sites within the MLKL pseudokinase domain activation loop65. This study is illustrative of a broader potential role of post-translational modifications in modulating the activities of RIPK3 and MLKL.\n\n\nNegative regulators of necroptosis\n\nCaspase-8-deficient embryonic lethality, or inflammatory disease resulting from tissue or cell type specific caspase-8 deletion, is rescued by RIPK3 or MLKL loss (reviewed in 9), demonstrating that caspase-8 is an essential repressor of potentially lethal necroptotic activity. Notably, this pro-survival function for caspase-8 does not require caspase-8 processing90, but appears to be mediated by the catalytic activity of caspase-8/FLICE-inhibitory protein (cFLIP) heterodimers91. The critical caspase-8 targets required to repress necroptosis remain unclear, although caspase-8 cleavage of key necroptotic inducers, including RIPK192, RIPK393, and CYLD74, have been described and are likely to be important.\n\nMammalian IAP proteins are ubiquitin E3 ligases and include cIAP1, cIAP2 and XIAP (reviewed in 94). The cIAPs target RIPK1 for ubiquitylation to propagate TNF-induced NFκB activation and pro-survival responses, and also to prevent RIPK1 from associating with, and activating, caspase-8. XIAP, on the other hand, is a direct inhibitor of apoptotic caspases, although its deletion in mice does not result in an overt phenotype. In contrast, the co-deletion of cIAP1 and cIAP2 causes embryonic lethality, which is rescued in part by loss of RIPK1, RIPK3 or TNFR195. Strikingly, while cIAP1/2 loss in combination with caspase inhibition is sufficient to trigger necroptosis in several cell types, in bone marrow derived macrophages and dendritic cells XIAP is more important for limiting TNF- and TLR-induced killing15,19. For example, XIAP loss alone can confer some sensitivity to TNF- and TLR-induced necroptosis and apoptosis, which is greatly enhanced by cIAP1/2 co-deletion15. Lipopolysaccharide (LPS) treatment of wildtype macrophages triggers IAP-dependent RIPK3 ubiquitylation15. Because LPS stimulation alone does not perturb macrophage viability, this may indicate that, akin to RIPK1 ubiquitylation, RIPK3 ubiquitylation under these conditions regulates transcriptional and/or pro-survival responses. However, LPS-induced necroptotic signaling caused by the loss of IAPs and caspase inhibition is also associated with increased RIPK3 (and MLKL) ubiquitylation15, which in this case may play a pro-necroptotic role96. At this stage the identity of the key XIAP substrate required to limit RIPK3-MLKL signaling remains unclear. It is possible that other ubiquitin E3 ligases also negatively regulate necroptosis signaling. Consistent with this, it has been suggested that the ubiquitin ligase MKRN1 (Makorin RING finger protein-1) represses RIPK1-RIPK3 complex formation, although how it does so is incompletely understood97.\n\nTNF receptor associated factor 2 (TRAF2) binds to cIAP1/2 via a cIAP interaction motif (CIM) and is required for cIAP1/2 recruitment into TNF receptor complexes98. Hence, as one might predict, the loss of TRAF2 may sensitize cells to death receptor mediated necroptotic killing by preventing cIAP1/2 recruitment and ubiquitylation of RIPK199,100. Remarkably, however, TRAF2 was recently reported to also exert anti-necroptotic activity independently of cIAP1/2 by binding directly to MLKL to limit the association of MLKL with RIPK3100. The steady state association of TRAF2 with MLKL was diminished upon TNF-induced necroptosis induction, and this correlated with CYLD dependent deubiquitylation of TRAF2. It will be interesting to define the TRAF2 interacting residues of MLKL, and whether their mutation impacts MLKL binding to RIPK3 and cellular necroptotic activity.\n\nPpm1b was recently identified as a phosphatase that dephosphorylates mouse RIPK3 at T231/S232101, two sites at which phosphorylation is known to enhance RIPK3 catalytic activity102. While deficiency in Ppm1b led to a modest elevation in cell death in the absence of stimulation (5–10% vs 2–5% cell death, depending on cell line studied), the enhancement of cell death observed upon TNF-stimulation of L929 fibrosarcoma cells and TNF+pan-caspase inhibitor (zVAD-fmk) treatment of mouse embryonic fibroblasts was profound103. This is the first illustration that necroptotic signaling can be tuned by dephosphorylation of RIPK3. It remains of outstanding interest whether there are other phosphatases that modulate the activity of RIPK3 and whether dephosphorylation of MLKL might serve as a mechanism to disarm its pro-necroptotic activity.\n\nA20 is a deubiquitinating enzyme that is rapidly induced following TNF- and TLR-stimulation as part of a negative feedback loop to restrict NFκB activation and inflammatory cytokine production. Hence, its mutation or loss in mice and humans results in pronounced inflammatory disease. A20 has been proposed to restrict TNF-induced NFκB by removing K63-linked ubiquitin chains from RIPK1, and at the same time can target RIPK1 for proteasomal degradation through the addition of K48-linked ubiquitin chains103. However, it also exerts its anti-inflammatory properties by targeting a number of TNF receptor and TLR signaling components104. Recently it was found that A20 deletion in T-cells led to RIPK3 dependent T-cell killing, and that the perinatal lethality of A20 deficient mice was significantly rescued by RIPK3 co-deletion96. Mechanistically, A20 loss promoted RIPK1-RIPK3 necrosome formation as a consequence of RIPK3 Lys5 ubiquitylation, while A20 expression and catalytic activity correlated with decreased RIPK3 ubiquitination.\n\n\nOpen questions surrounding MLKL function\n\nBecause MLKL was only recently identified as an effector of the necroptosis cell death pathway, much remains to be garnered about how it functions in this pathway, whether it has additional “moonlighting” functions, and how it might contribute to other death signaling pathways. Should moonlighting functions exist for MLKL, they are likely very subtle, since genetic deletion of MLKL in mice does not noticeably impact viability, fertility or development58,60. The recent observation that, upon phosphorylation, MLKL translocates to the nucleus105 suggests either a second (as-yet-unknown) function for MLKL in the nucleus or that the necroptosis signaling pathway relies on a transition via the nucleus to other membranes. This remains a matter of ongoing interest. The subcellular destination for MLKL that leads to cell death has also been a matter of debate: does MLKL-mediated death rely on translocation to mitochondrial or plasma membranes or another subcellular organelle? Initially, MLKL was implicated as being upstream of the mitochondrial phosphatase, phosphoglycerate mutase family member 5 (PGAM5), whose activation was thought to drive cell death through mitochondrial fragmentation via Dynamin-related protein 1 (Drp1)106. However, several studies indicated that both PGAM5 and Drp1 are dispensable for necroptosis60,107–109 and, as a result, the ubiquity of their involvement in the pathway has been questioned. Indeed, depletion of mitochondria by mitophagy did not prevent necroptotic death67, suggesting that mitochondria are dispensable for necroptosis, at least in a subset of commonly studied laboratory cell lines. Nonetheless, reactive oxygen species (ROS), which emanate from mitochondria, have been widely implicated in necroptotic cell death59,110–112. It remains to be determined whether ROS arise as a consequence of cell death or whether their generation is a driving force or augments necroptotic death and/or contributes to inflammatory responses. As described above, it is of enormous interest to understand the precise mechanism by which MLKL kills cells and whether other factors are involved downstream of MLKL phosphorylation and whether MLKL function can be modulated by ubiquitylation, as suggested by a recent study. Moreover, these modifications may govern whether MLKL can participate in other intersecting signaling pathways, such as inflammatory signaling in the absence of cell death18 or cell death by pyroptosis.\n\n\nAbbreviations\n\n4HB, four-helix bundle; CYLD, cylindromatosis; DAI, DNA-dependent activator of IFN-regulatory factors; DD, death domain; Drp1, Dynamin-related protein 1; FADD, Fas-associated protein with death domain; HOIP, HOIL-1 interacting protein; HSP90, heat shock protein 90; IAP, inhibitor of apoptosis proteins; IFN, interferon; LPS, lipopolysaccharide; LUBAC, linear ubiquitin chain assembly complex; MLKL, mixed lineage kinase domain-like; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; PGAM5, phosphoglycerate mutase family member 5; PKR, protein kinase R; RHIM, RIP homotypic interaction motif; RIPK, receptor interacting protein kinase; ROS, reactive oxygen species; TLR, toll-like receptor; TNF, tumour necrosis factor; TNFR1, TNF receptor 1; TRADD, TNFR1-associated death domain; TRAF2, TNF receptor associated factor 2.",
"appendix": "Competing interests\n\n\n\nJames M. Murphy co-leads a program funded by Catalyst Therapeutics Pty Ltd and the Walter and Eliza Hall Institute to develop necroptosis inhibitors. James E. Vince declares that he has no competing interests.\n\n\nGrant information\n\nThe authors are grateful to the National Health and Medical Research Council of Australia for support via a fellowship to JEV (1052598), project grants (1057905, 1067289, 1051210) and the IRIISS scheme (9000220), and the Australian Research Council for fellowship support to JMM (FT100100100). Additional support was provided by Victorian State Government Operational Infrastructure Support Scheme.\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\nReferences\n\nVandenabeele P, Galluzzi L, Vanden Berghe T, et al.: Molecular mechanisms of necroptosis: an ordered cellular explosion. 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PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLi S, Zhang L, Yao Q, et al.: Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains. Nature. 2013; 501(7466): 242–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nUpton JW, Kaiser WJ, Mocarski ES: DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA. Cell Host Microbe. 2012; 11(3): 290–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nUpton JW, Kaiser WJ, Mocarski ES: Virus inhibition of RIP3-dependent necrosis. Cell Host Microbe. 2010; 7(4): 302–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuo H, Omoto S, Harris PA, et al.: Herpes simplex virus suppresses necroptosis in human cells. Cell Host Microbe. 2015; 17(2): 243–51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang Z, Wu SQ, Liang Y, et al.: RIP1/RIP3 binding to HSV-1 ICP6 initiates necroptosis to restrict virus propagation in mice. Cell Host Microbe. 2015; 17(2): 229–42. PubMed Abstract | Publisher Full Text\n\nOmoto S, Guo H, Talekar GR, et al.: Suppression of RIP3-dependent necroptosis by human cytomegalovirus. J Biol Chem. 2015; 290(18): 11635–48. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu XN, Yang ZH, Wang XK, et al.: Distinct roles of RIP1-RIP3 hetero- and RIP3-RIP3 homo-interaction in mediating necroptosis. Cell Death Differ. 2014; 21(11): 1709–20. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLi J, McQuade T, Siemer AB, et al.: The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012; 150(2): 339–50. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nCai Z, Jitkaew S, Zhao J, et al.: Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol. 2014; 16(1): 55–65. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHildebrand JM, Tanzer MC, Lucet IS, et al.: Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death. Proc Natl Acad Sci U S A. 2014; 111(42): 15072–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDondelinger Y, Declercq W, Montessuit S, et al.: MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep. 2014; 7(4): 971–81. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nKaiser WJ, Sridharan H, Huang C, et al.: Toll-like receptor 3-mediated necrosis via TRIF, RIP3, and MLKL. J Biol Chem. 2013; 288(43): 31268–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSun L, Wang H, Wang Z, et al.: Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 2012; 148(1–2): 213–27. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWu J, Huang Z, Ren J, et al.: Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis. Cell Res. 2013; 23(8): 994–1006. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nZhao J, Jitkaew S, Cai Z, et al.: Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci U S A. 2012; 109(14): 5322–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMurphy JM, Czabotar PE, Hildebrand JM, et al.: The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity. 2013; 39(3): 443–53. PubMed Abstract | Publisher Full Text\n\nMurphy JM, Zhang Q, Young SN, et al.: A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties. Biochem J. 2014; 457(2): 323–34. PubMed Abstract | Publisher Full Text\n\nManning G, Whyte DB, Martinez R, et al.: The protein kinase complement of the human genome. Science. 2002; 298(5600): 1912–34. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChen X, Li W, Ren J, et al.: Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death. Cell Res. 2014; 24(1): 105–21. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nRodriguez DA, Weinlich R, Brown S, et al.: Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ. 2015. PubMed Abstract | Publisher Full Text\n\nTanzer MC, Tripaydonis A, Webb AI, et al.: Necroptosis signalling is tuned by phosphorylation of MLKL residues outside the pseudokinase domain activation loop. Biochem J. 2015; 471(2): 255–65. PubMed Abstract | Publisher Full Text\n\nSu L, Quade B, Wang H, et al.: A plug release mechanism for membrane permeation by MLKL. Structure. 2014; 22(10): 1489–500. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nTait SW, Oberst A, Quarato G, et al.: Widespread mitochondrial depletion via mitophagy does not compromise necroptosis. Cell Rep. 2013; 5(4): 878–85. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHoller N, Zaru R, Micheau O, et al.: Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000; 1(6): 489–95. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDegterev A, Hitomi J, Germscheid M, et al.: Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008; 4(5): 313–21. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDegterev A, Huang Z, Boyce M, et al.: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005; 1(2): 112–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPolykratis A, Hermance N, Zelic M, et al.: Cutting edge: RIPK1 Kinase inactive mice are viable and protected from TNF-induced necroptosis in vivo. J Immunol. 2014; 193(4): 1539–43. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMoujalled DM, Cook WD, Okamoto T, et al.: TNF can activate RIPK3 and cause programmed necrosis in the absence of RIPK1. Cell Death Dis. 2013; 4: e465. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHe S, Liang Y, Shao F, et al.: Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc Natl Acad Sci U S A. 2011; 108(50): 20054–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nO'Donnell MA, Perez-Jimenez E, Oberst A, et al.: Caspase 8 inhibits programmed necrosis by processing CYLD. Nat Cell Biol. 2011; 13(12): 1437–42. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHitomi J, Christofferson DE, Ng A, et al.: Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell. 2008; 135(7): 1311–23. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMoquin DM, McQuade T, Chan FK: CYLD deubiquitinates RIP1 in the TNFα-induced necrosome to facilitate kinase activation and programmed necrosis. PLoS One. 2013; 8(10): e76841. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Almagro MC, Goncharov T, Newton K, et al.: Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination. Cell Death Dis. 2015; 6: e1800. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWelz PS, Wullaert A, Vlantis K, et al.: FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation. Nature. 2011; 477(7364): 330–4. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBonnet MC, Preukschat D, Welz PS, et al.: The adaptor protein FADD protects epidermal keratinocytes from necroptosis in vivo and prevents skin inflammation. Immunity. 2011; 35(4): 572–82. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChen WW, Yu H, Fan HB, et al.: RIP1 mediates the protection of geldanamycin on neuronal injury induced by oxygen-glucose deprivation combined with zVAD in primary cortical neurons. J Neurochem. 2012; 120(1): 70–7. PubMed Abstract | Publisher Full Text\n\nFearns C, Pan Q, Mathison JC, et al.: Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding. J Biol Chem. 2006; 281(45): 34592–600. PubMed Abstract | Publisher Full Text\n\nGentle IE, Wong WW, Evans JM, et al.: In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-kappaB and activation of caspase-8. J Biol Chem. 2011; 286(15): 13282–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLewis J, Devin A, Miller A, et al.: Disruption of hsp90 function results in degradation of the death domain kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factor-induced nuclear factor-kappaB activation. J Biol Chem. 2000; 275(14): 10519–26. PubMed Abstract | Publisher Full Text\n\nPantano C, Shrivastava P, McElhinney B, et al.: Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase. J Biol Chem. 2003; 278(45): 44091–6. PubMed Abstract | Publisher Full Text\n\nVanden Berghe T, Kalai M, van Loo G, et al.: Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis. J Biol Chem. 2003; 278(8): 5622–9. PubMed Abstract | Publisher Full Text\n\nCho YS, Challa S, Moquin D, et al.: Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell. 2009; 137(6): 1112–23. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nPark SY, Shim SH, Cho YS: Distinctive roles of receptor-interacting protein kinases 1 and 3 in caspase-independent cell death of L929. Cell Biochem Funct. 2014; 32(1): 62–9. PubMed Abstract | Publisher Full Text\n\nLi D, Xu T, Cao Y, et al.: A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis. Proc Natl Acad Sci U S A. 2015; 112(16): 5017–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi L, Chen W, Liang Y, et al.: The Gβγ-Src signaling pathway regulates TNF-induced necroptosis via control of necrosome translocation. Cell Res. 2014; 24(4): 417–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang TB, Oh GS, Scandella E, et al.: Mutation of a self-processing site in caspase-8 compromises its apoptotic but not its nonapoptotic functions in bacterial artificial chromosome-transgenic mice. J Immunol. 2008; 181(4): 2522–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nOberst A, Dillon CP, Weinlich R, et al.: Catalytic activity of the caspase-8-FLIPL complex inhibits RIPK3-dependent necrosis. Nature. 2011; 471(7338): 363–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLin Y, Devin A, Rodriguez Y, et al.: Cleavage of the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev. 1999; 13(19): 2514–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng S, Yang Y, Mei Y, et al.: Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell Signal. 2007; 19(10): 2056–67. PubMed Abstract | Publisher Full Text\n\nSilke J, Meier P: Inhibitor of apoptosis (IAP) proteins-modulators of cell death and inflammation. Cold Spring Harb Perspect Biol. 2013; 5(2): pii: a008730. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoulin M, Anderton H, Voss AK, et al.: IAPs limit activation of RIP kinases by TNF receptor 1 during development. EMBO J. 2012; 31(7): 1679–91. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nOnizawa M, Oshima S, Schulze-Topphoff U, et al.: The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis. Nat Immunol. 2015; 16(6): 618–27. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nLee E, Kim J, Ahn Y, et al.: Ubiquitination and degradation of the FADD adaptor protein regulate death receptor-mediated apoptosis and necroptosis. Nat Commun. 2012; 3: 978. PubMed Abstract | Publisher Full Text\n\nVince JE, Pantaki D, Feltham R, et al.: TRAF2 must bind to cellular inhibitors of apoptosis for tumor necrosis factor (TNF) to efficiently activate NF-{kappa}B and to prevent TNF-induced apoptosis. J Biol Chem. 2009; 284(51): 35906–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKarl I, Jossberger-Werner M, Schmidt N, et al.: TRAF2 inhibits TRAIL- and CD95L-induced apoptosis and necroptosis. Cell Death Dis. 2014; 5: e1444. PubMed Abstract | Publisher Full Text\n\nPetersen SL, Chen TT, Lawrence DA, et al.: TRAF2 is a biologically important necroptosis suppressor. Cell Death Differ. 2015; 22(11): 1846–57. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChen W, Wu J, Li L, et al.: Ppm1b negatively regulates necroptosis through dephosphorylating Rip3. Nat Cell Biol. 2015; 17(4): 434–44. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nChen W, Zhou Z, Li L, et al.: Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem. 2013; 288(23): 16247–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWertz IE, O'Rourke KM, Zhou H, et al.: De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling. Nature. 2004; 430(7000): 694–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nWertz I, Dixit V: A20--a bipartite ubiquitin editing enzyme with immunoregulatory potential. Adv Exp Med Biol. 2014; 809: 1–12. PubMed Abstract | Publisher Full Text\n\nYoon S, Bogdanov K, Kovalenko A, et al.: Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it. Cell Death Differ. 2015. PubMed Abstract | Publisher Full Text\n\nWang Z, Jiang H, Chen S, et al.: The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell. 2012; 148(1–2): 228–43. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMoujalled DM, Cook WD, Murphy JM, et al.: Necroptosis induced by RIPK3 requires MLKL but not Drp1. Cell Death Dis. 2014; 5(2): e1086. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRemijsen Q, Goossens V, Grootjans S, et al.: Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. Cell Death Dis. 2014; 5(1): e1004. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSchenk B, Fulda S: Reactive oxygen species regulate Smac mimetic/TNFα-induced necroptotic signaling and cell death. Oncogene. 2015. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLin Y, Choksi S, Shen HM, et al.: Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem. 2004; 279(11): 10822–8. PubMed Abstract | Publisher Full Text\n\nVanlangenakker N, Vanden Berghe T, Bogaert P, et al.: cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production. Cell Death Differ. 2011; 18(4): 656–65. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMarshall KD, Baines CP: Necroptosis: is there a role for mitochondria? Front Physiol. 2014; 5: 323. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcQuade T, Cho Y, Chan FK, et al.: Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis. Biochem J. 2013; 456(3): 409–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBertrand MJ, Milutinovic S, Dickson KM, et al.: cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell. 2008; 30(6): 689–700. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMahoney DJ, Cheung HH, Mrad RL, et al.: Both cIAP1 and cIAP2 regulate TNFalpha-mediated NF-kappaB activation. Proc Natl Acad Sci U S A. 2008; 105(33): 11778–83. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nO'Donnell MA, Legarda-Addison D, Skountzos P, et al.: Ubiquitination of RIP1 regulates an NF-kappaB-independent cell-death switch in TNF signaling. Curr Biol. 2007; 17(5): 418–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVarfolomeev E, Goncharov T, Fedorova AV, et al.: c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. J Biol Chem. 2008; 283(36): 24295–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDynek JN, Goncharov T, Dueber EC, et al.: c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling. EMBO J. 2010; 29(24): 4198–209. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHe S, Wang L, Miao L, et al.: Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009; 137(6): 1100–11. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMiao W, Qu Z, Shi K, et al.: RIP3 S-nitrosylation contributes to cerebral ischemic neuronal injury. Brain Res. 2015; 1627: 165–76. PubMed Abstract | Publisher Full Text\n\nDephoure N, Zhou C, Villén J, et al.: A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 2008; 105(31): 10762–7. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11283",
"date": "19 Nov 2015",
"name": "Pascal Meier",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
},
{
"id": "11284",
"date": "19 Nov 2015",
"name": "Martin Leverkus",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11285",
"date": "19 Nov 2015",
"name": "Peter Vandenabeele",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11287",
"date": "19 Nov 2015",
"name": "Jiahuai Han",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1297
|
https://f1000research.com/articles/4-1296/v1
|
19 Nov 15
|
{
"type": "Review",
"title": "Advances in understanding and managing atopic dermatitis",
"authors": [
"Michael Barton",
"Robert Sidbury",
"Michael Barton"
],
"abstract": "Atopic dermatitis is a chronic, pruritic skin disease characterized by an improperly functioning skin barrier and immune dysregulation. We review proposed atopic dermatitis pathomechanisms, emphasizing how these impact current perspectives on natural history, role of allergic sensitization, and future therapeutic targets.",
"keywords": [
"dermatitis",
"atopic",
"immune"
],
"content": "Introduction\n\nAtopic dermatitis (AD) is a common chronic, pruritic skin disease characterized by an improperly functioning skin barrier and immune dysregulation. Part of the “atopic triad”, it often precedes asthma and seasonal allergies, affecting up to 20% of children and 3% of adults in the United States1,2. The age-old question remains: is AD primarily a skin barrier defect or a dysfunctional immune system? This dichotomy is made explicit in various clinical scenarios. For example, patients with primary immunodeficiency diseases like hyper-IgE (Job’s) syndrome, some with IgE levels of more than 30,000 IU/ml, often have terrible AD, but so do some hypogammaglobulinemia or severe combined immunodeficiency patients who may have little to no IgE at all. Additionally, 80% of patients with AD have a positive atopic history with increased serum IgE levels or immediate skin test reactivity or both, but an identical eczema phenotype is seen in the other 20% of patients with AD, those with so-called intrinsic AD, who have no atopic tendency at all. Furthermore, certain areas of the skin have notoriously been affected (e.g., flexural surfaces of the elbow/knee), whereas other areas appear to be spared (i.e., axilla), which would seem incongruent with constitutive immune dysregulation. This construct extends to the therapeutic realm as immunosuppressive agents like cyclosporin A have demonstrated efficacy, but so too have emollients and barrier repair “devices” that putatively do not affect the immune system.\n\nSo which is the chicken and which the egg? Does barrier dysfunction beget immune dysregulation or vice versa? Improved understanding of the genetic and molecular bases of AD will likely answer this question and, as will be detailed later, lead to better therapies. It may well have broader implications vis-à-vis the “atopic march”: the orderly procession of infantile eczema, to childhood asthma, to adult hay fever that occurs in some patients. A variety of studies, including early application of emollients to high-risk infants, have suggested that not only can eczema be interrupted but other co-morbidities can potentially be prevented3.\n\n\nSkin barrier abnormalities: the “outside-in” theory\n\nThe skin barrier is composed of multiple elements working synchronously to protect the host from various external insults. The physical barrier is composed of the outermost layer of the epidermis, or stratum corneum, and deeper tight junctions. A broad range of proteins, including but not limited to S100 family proteins and filaggrin byproducts, contribute to the skin’s physical integrity. The normal host microbiome plays a pivotal role in the protection of skin from pathogens as well as the determination of immune responses. Immunologically, several barrier components have been shown to be altered in patients with AD. This was elegantly demonstrated by Ong et al., who showed that patients with AD, notorious for being colonized and infected by Staphylococcus aureus, had deficient levels of antimicrobial peptides in biopsy specimens relative to patients with psoriasis, an inflammatory dermatosis with many similarities, except the tendency toward infection4. Both genetic and acquired causes of epithelial skin barrier dysfunction have been identified. Recent advances have focused on the primary importance of this abnormal barrier as a trigger for eczematous inflammation. This “outside-in” construct purports that leakage in the barrier allows the penetration of various allergens, irritants, and microbes into the skin of patients with AD, thereby leading to a cascade of inflammatory events.\n\nAlthough several barrier defects have previously been examined, recent literature seems to be directed toward filaggrin as a major culprit in the development and severity of AD5. Initially demonstrated to have reduced expression in patients with ichthyosis vulgaris, an inherited disorder of keratinization, and a minor diagnostic criterion for AD, filaggrin’s direct association with AD has been elucidated over the last decade6. Filaggrin is the monomeric breakdown product of a polyprotein encoded on the FLG gene. It serves stratum corneum structure and function as well as inhibits transepidermal water loss. Loss-of-function mutations in the FLG gene are a major risk factor for AD, and even variations in gene size have been associated with an increased AD risk7. Within the AD population, these mutations lead to early onset, more severe course, and higher prevalences of asthma, food allergy, and microbial infection8–10.\n\nRegardless of FLG gene status, filaggrin deficiency alone appears to play an important role in the pathogenesis of AD11. Deficiencies in filaggrin-processing enzymes such as caspase-14 have been identified12. Environmental stressors such as low ambient humidity, sunburn, skin irritants, and psychological stress have been proposed to reduce filaggrin levels in the epidermis. Additionally, inflammation itself can downregulate filaggrin expression, revealing the interdependent relationship between skin barrier and immune dysregulation in AD13. Interestingly, current therapies such as topical steroids and calcineurin inhibitors have been shown to restore filaggrin levels back to normal, and new therapies are being studied to directly target the modulation of FLG expression14,15.\n\nSimilar reductions of other structural and tight junction proteins such as loricrin, involucrin, and claudins contribute to the abnormal barrier in AD. Emerging evidence suggests that collectively these defects may contribute to systemic allergic sensitization. A recent study revealed an exposure-response relationship between peanut protein levels found in household dust and peanut skin prick test (SPT) sensitization in patients with AD16. The authors conclude that increased allergen exposure through an impaired skin barrier in inflamed skin may be responsible for sensitization and subsequent allergy. Results from the LEAP (Learning Early about Peanut Allergy) trial, a study designed to assess the development of peanut allergy in infants at high risk for the allergy (which included infants with severe eczema or egg allergy or both), suggest that early introduction of peanuts into an infant’s diet may promote oral tolerance and prevent peanut allergy17. Upon being randomly assigned to either consume or avoid peanuts, 640 infants between the ages of 4 and 11 months were followed until they reached 5 years of age. Among the infants who initially had negative SPTs, the prevalence of peanut allergy was significantly higher among the avoidance group (13.7%) compared with the consumption group (1.9%). Moreover, those infants who initially demonstrated SPT positivity had peanut allergy prevalences of 35.3% in the avoidance group and 10.6% in the consumption group. Collectively, these data support the notion of skin sensitization and oral tolerance among patients with AD, which may impact the approach to high-risk pediatric patients in the future. Early food consumption appears to reduce the risk of developing specific systemic allergy sensitization, but further information is needed to determine whether improved control of skin barrier defects during infancy will have the same effect. These studies raise many additional questions. Should all patients be exposed to peanut early? All eczema patients? Do these data extend to other allergenic foods? If so, when to introduce? In what form? How much? These answers require further study, and the National Institutes of Health has assembled an expert panel to incorporate these important new findings into their previously published food allergy guidelines18.\n\n\nImmune system dysfunction: the “inside-out” theory\n\nImmune dysregulation is critical to the pathogenesis of AD. Several existing therapies target T cell-mediated inflammation. The molecular signature of AD inflammation varies by chronicity and phase of the disease, but TH2, TH22, and TH17 cells predominate, resulting in characteristic inflammatory mediators, including interleukin-4 (IL-4) and IL-13. These cells and their inflammatory cytokines correspondingly have an effect on the epidermal barrier, including suppression of skin cell differentiation, hyperplasia, and apoptosis. Specific cytokines appear to downregulate terminal differentiation genes responsible for protective barrier proteins, including filaggrin, loricrin, and involucrin. Immune activation has a complex and intertwined relationship with the skin barrier defects of patients with AD, and continuing to identify immunologic pathways will have profound effects on understanding and managing AD.\n\n\nAdvances in treatment\n\nOne of the most exciting candidate drugs in this arena is dupilumab. Previously shown to be effective in patients with asthma and elevated eosinophil levels, dupilumab is an injectable, fully human, monoclonal antibody that blocks the cellular messenger signals IL-4 and IL-1319. In a randomized, double-blind, placebo-controlled trial, Beck et al. showed marked improvement in EASI 50 (50% reduction in Eczema Area and Severity Index) score compared with placebo (85% versus 35%, P < 0.001)20. Additionally, patients receiving dupilumab reported a significant decrease in symptomatic itching, and when used in combination, dupilumab served as a topical steroid-sparing agent.\n\nApremilast is an oral phosphodiesterase 4 (PDE-4) inhibitor currently used in the treatment of psoriasis. In an uncontrolled, open-label pilot study of 16 adults with moderate to severe AD receiving either 20 or 30 mg twice daily, clinical responses at 6 months were similar to those seen with systemic immunosuppressants, including cyclosporine, mycophenolate, and methotrexate, based on EASI scores and quality-of-life indices21,22. The ability to improve inflammation, itch, and quality of life while avoiding end-organ damage seen with alternative systemic agents is an appealing characteristic of this drug. Although these results provide a strong foundation to build upon, additional controlled studies with greater sample sizes are necessary to determine efficacy, dosage, and safety.\n\nSeveral topical PDE-4 inhibitors are also being studied but the furthest along is AN272823. In one phase 2 trial, 86 adolescents with mild to moderate AD receiving twice-daily AN2728 treatment revealed a 71% improvement from baseline in their Atopic Dermatitis Severity Index (ADSI) score and total or partial clearance in 66% of the skin lesions24. On the basis of such encouraging phase 2 data, two phase 3 trials with 750 patients with mild to moderate AD have been conducted. AN2728, now named crisaborole, was shown to be superior to vehicle at day 29 in the percentage of patients found clear or almost clear by global assessment: 32.8% versus 25.4% (P < 0.04) in one trial and 31.4% versus 18% (P < 0.001) in the other. Pooled analysis of adverse effects showed that only application site pain was seen at rates above vehicle (4.4% versus 1.2%). Crisaborole appears to be a safe and effective non-steroidal alternative for mild to moderate eczema. Time will tell where it may fit into the therapeutic armamentarium and what current agent it may function most like, but these trial results suggest efficacy akin to pimecrolimus.\n\nOmalizumab is a humanized monoclonal antibody that selectively binds to IgE antibodies. It has been licensed for the treatment of severe allergic asthma and chronic urticaria. Its effectiveness treating severe and treatment-resistant AD has been controversial, and mixed results have derived from several small case studies and uncontrolled trials25,26. One randomized, double-blind, placebo-controlled study of eight patients found comparable improvements in AD between omalizumab and placebo27. A 2014 study assessed the efficacy of omalizumab in patients with and without FLG mutations28. None of the seven FLG mutation carriers responded, whereas the eight patient responders had no FLG mutations, suggesting that patients with a primary skin barrier defect may not respond to anti-IgE therapy. The potential benefit of omalizumab for patients with AD is uncertain at this time and will depend upon the results of additional randomized control trials.\n\nSeveral lines of investigation have suggested a role for vitamin D in AD pathogenesis29–31. There is good evidence to support AD improvement with vitamin D supplementation32–35. An initial study conducted in 2008 revealed that 1,000 IU of vitamin D improved AD in four of the five children studied, compared with only one of the six children in the control group. Subsequent studies have reproduced similar results with larger sample sizes. Despite these encouraging trial results, attempts to correlate AD severity and vitamin D levels have led to mixed results36. Important questions remain, including optimal dosing and target serum levels.\n\nThymic stromal lymphopoietin (TSLP) is another intriguing target for directed therapeutic interventions. TSLP is an epithelial cell-derived cytokine responsible for triggering the differentiation of naïve T cells into TH2 cells and has been shown to have an association with AD and other allergic diseases37–39. Variations within the TSLP-encoding gene have also been associated with a decreased risk of persistent AD and susceptibility to eczema herpeticum40. The first human anti-TSLP drug to be tested, AMG 157, has exhibited promising results in the reduction of allergen-induced asthmatic responses41. Although the effectiveness of AMG 157 for management of AD has not been studied directly, it is reasonable to predict, given the known relationship between AD and TH2 cells, that the drug may show similar results in the skin.\n\nIt would seem prudent to target therapy directly toward pruritus, for a condition known as “the itch that rashes”; however, there is currently no treatment available to single out this neural pathway. CT327 and tradipitant are two medications in phase 2 development aiming to address this aspect of therapy by antagonizing tropomyosin-receptor kinase A (trkA) and neurokinin 1 receptors (NK-1Rs), respectively. A 2010 study of 15 AD patients with moderate pruritus revealed noticeable symptomatic improvements within 8 days with topical CT32742. Itch symptoms improved by up to 59%, and no serious side effects were experienced; CT327 has also shown promising results for the treatment of chronic pruritus in patients with psoriasis43. Similar outcomes are being measured by focusing on NK-1R, which has a high affinity for substance P, a neurotransmitter believed to influence a variety of pathophysiological processes, including itch. Higher blood concentrations of oral tradipitant have been correlated with greater improvements in itch scores compared with control groups44.\n\n\nConclusions\n\nThe future for patients with AD has never looked so promising. Advances in understanding the genetic basis of AD and its associated barrier defects are dovetailing with a better grasp of the molecular basis of eczematous inflammation. The result will hopefully lead to improved therapies in the short term and possible prevention of both AD and atopic co-morbidities down the road.\n\n\nAbbreviations\n\nAD, atopic dermatitis; ADSI, Atopic Dermatitis Severity Index; EASI, eczema area and severity index; IL, interleukin; NK-1R, neurokinin 1 receptor; PDE-4, phosphodiesterase 4; TSLP, thymic stromal lymphopoietin.",
"appendix": "Competing interests\n\n\n\nMB declares that he has no competing interests. RS has been an investigator in a phase 3 trial of the topical phosphodiesterase inhibitor AN2728 sponsored by Anacor. He received no direct payments related to this work.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBoguniewicz M, Leung DY: Recent insights into atopic dermatitis and implications for management of infectious complications. J Allergy Clin Immunol. 2010; 125(1): 4–13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams H, Robertson C, Stewart A, et al.: Worldwide variations in the prevalence of symptoms of atopic eczema in the International Study of Asthma and Allergies in Childhood. J Allergy Clin Immunol. 1999; 103(1 Pt 1): 125–38. PubMed Abstract | Publisher Full Text\n\nSimpson EL, Chalmers JR, Hanifin JM, et al.: Emollient enhancement of the skin barrier from birth offers effective atopic dermatitis prevention. J Allergy Clin Immunol. 2014; 134(4): 818–23. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nOng PY, Ohtake T, Brandt C, et al.: Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med. 2002; 347(15): 1151–60. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nIrvine AD, McLean WH, Leung DY: Filaggrin mutations associated with skin and allergic diseases. N Engl J Med. 2011; 365(14): 1315–27. PubMed Abstract | Publisher Full Text\n\nBrown SJ, Mclean WH: One remarkable molecule: filaggrin. J Invest Dermatol. 2012; 132(3 Pt 2): 751–62. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nRodríguez E, Baurecht H, Herberich E, et al.: Meta-analysis of filaggrin polymorphisms in eczema and asthma: robust risk factors in atopic disease. J Allergy Clin Immunol. 2009; 123(6): 1361–70.e7. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nLeung DY, Guttman-yassky E: Deciphering the complexities of atopic dermatitis: shifting paradigms in treatment approaches. J Allergy Clin Immunol. 2014; 134(4): 769–79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMargolis DV, Apter AJ, Gupta J, et al.: The persistence of atopic dermatitis and filaggrin (FLG) mutations in a US longitudinal cohort. J Allergy Clin Immunol. 2012; 130(4): 912–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBohme M, Söderhäll C, Kull I, et al.: Filaggrin mutations increase the risk for persistent dry skin and eczema independent of sensitization. J Allergy Clin Immunol. 2012; 129(4): 1153–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nThyssen JP, Kezic S: Causes of epidermal filaggrin reduction and their role in the pathogenesis of atopic dermatitis. J Allergy Clin Immunol. 2014; 134(4): 792–9. PubMed Abstract | Publisher Full Text\n\nBroccardo CJ, Mahaffey S, Schwarz J, et al.: Comparative proteomic profiling of patients with atopic dermatitis based on history of eczema herpeticum infection and Staphylococcus aureus colonization. J Allergy Clin Immunol. 2011; 127(1): 186–93, 193.e1–11. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nHowell MD, Kim BE, Gao P, et al.: Cytokine modulation of atopic dermatitis filaggrin skin expression. J Allergy Clin Immunol. 2009; 124(3 Suppl 2): R7–12. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBoguniewicz M, Leung DY: Atopic dermatitis: a disease of altered skin barrier and immune dysregulation. Immunol Rev. 2011; 242(1): 233–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOtsuka A, Doi H, Egawa G, et al.: Possible new therapeutic strategy to regulate atopic dermatitis through upregulating filaggrin expression. J Allergy Clin Immunol. 2014; 133(1): 139–46.e1–10. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBrough HA, Liu AH, Sicherer S, et al.: Atopic dermatitis increases the effect of exposure to peanut antigen in dust on peanut sensitization and likely peanut allergy. J Allergy Clin Immunol. 2015; 135(1): 164–70. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nDu Toit G, Roberts G, Sayre PH, et al.: Randomized trial of peanut consumption in infants at risk for peanut allergy. N Engl J Med. 2015; 372(9): 803–13. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nBoyce JA, Assa'ad A, Burks AW, et al.: Guidelines for the Diagnosis and Management of Food Allergy in the United States: Summary of the NIAID-Sponsored Expert Panel Report. J Allergy Clin Immunol. 2010; 126(6): 1105–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHamilton JD, Suárez-Fariñas M, Dhingra N, et al.: Dupilumab improves the molecular signature in skin of patients with moderate-to-severe atopic dermatitis. J Allergy Clin Immunol. 2014; 134(6): 1293–300. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBeck LA, Thaçi D, Hamilton JD, et al.: Dupilumab treatment in adults with moderate-to-severe atopic dermatitis. N Engl J Med. 2014; 371(2): 130–9. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVolf EM, Au SC, Dumont N, et al.: A phase 2, open-label, investigator-initiated study to evaluate the safety and efficacy of apremilast in subjects with recalcitrant allergic contact or atopic dermatitis. J Drugs Dermatol. 2012; 11(3): 341–346. PubMed Abstract\n\nSamrao A, Berry TM, Goreshi R, et al.: A pilot study of an oral phosphodiesterase inhibitor (apremilast) for atopic dermatitis in adults. Arch Dermatol. 2012; 148(8): 890–7. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nFurue M, Kitahara Y, Akama H, et al.: Safety and efficacy of topical E6005, a phosphodiesterase 4 inhibitor, in Japanese adult patients with atopic dermatitis: results of a randomized, vehicle-controlled, multicenter clinical trial. J Dermatol. 2014; 41(7): 577–85. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMoustafa F, Feldman SR: A review of phosphodiesterase-inhibition and the potential role for phosphodiesterase 4-inhibitors in clinical dermatology. Dermatol Online J. 2014; 20(5): 22608. PubMed Abstract\n\nKim DH, Park KY, Kim BJ, et al.: Anti-immunoglobulin E in the treatment of refractory atopic dermatitis. Clin Exp Dermatol. 2013; 38(5): 496–500. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nEl-Qutob D: Off-Label Uses of Omalizumab. Clin Rev Allergy Immunol. 2015; 1–13. PubMed Abstract | Publisher Full Text\n\nAndreae DA, Wang J: Immunologic effects of omalizumab in children with severe refractory atopic dermatitis: a randomized, placebo-controlled clinical trial. Pediatrics. 2014; 134(Suppl 3): S160. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nHotze M, Baurecht H, Rodriguez E, et al.: Increased efficacy of omalizumab in atopic dermatitis patients with wild-type filaggrin status and higher serum levels of phosphatidylcholines. Allergy. 2014; 69(1): 132–5. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nPeroni DG, Piacentini GL, Cametti E, et al.: Correlation between serum 25-hydroxyvitamin D levels and severity of atopic dermatitis in children. Br J Dermatol. 2011; 164(5): 1078–82. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nBäck O, Blomquist HK, Hernell O, et al.: Does vitamin D intake during infancy promote the development of atopic allergy? Acta Derm Venereol. 2009; 89(1): 28–32. PubMed Abstract | Publisher Full Text\n\nOren E, Banerji A, Camargo CA Jr: Vitamin D and atopic disorders in an obese population screened for vitamin D deficiency. J Allergy Clin Immunol. 2008; 121(2): 533–4. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nSidbury R, Sullivan AF, Thadhani RI, et al.: Randomized controlled trial of vitamin D supplementation for winter-related atopic dermatitis in Boston: a pilot study. Br J Dermatol. 2008; 159(1): 245–7. PubMed Abstract | Publisher Full Text\n\nCamargo CA Jr, Ganmaa D, Sidbury R, et al.: Randomized trial of vitamin D supplementation for winter-related atopic dermatitis in children. J Allergy Clin Immunol. 2014; 134(4): 831–835.e1. PubMed Abstract | Publisher Full Text\n\nAmestejani M, Salehi BS, Vasigh M, et al.: Vitamin D supplementation in the treatment of atopic dermatitis: a clinical trial study. J Drugs Dermatol. 2012; 11(3): 327–30. PubMed Abstract | F1000 Recommendation\n\nJavanbakht MH, Keshavarz SA, Djalali M, et al.: Randomized controlled trial using vitamins E and D supplementation in atopic dermatitis. J Dermatolog Treat. 2011; 22(3): 144–50. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nChiu YE, Havens PL, Siegel DH, et al.: Serum 25-hydroxyvitamin D concentration does not correlate with atopic dermatitis severity. J Am Acad Dermatol. 2013; 69(1): 40–6. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGao PS, Rafaels NM, Mu D, et al.: Genetic variants in thymic stromal lymphopoietin are associated with atopic dermatitis and eczema herpeticum. J Allergy Clin Immunol. 2010; 125(6): 1403–1407.e4. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nNakajima S, Igyártó BZ, Honda T, et al.: Langerhans cells are critical in epicutaneous sensitization with protein antigen via thymic stromal lymphopoietin receptor signaling. J Allergy Clin Immunol. 2012; 129(4): 1048–55.e6. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nLandheer J, Giovannone B, Mattson JD, et al.: Epicutaneous application of house dust mite induces thymic stromal lymphopoietin in non-lesional skin of patients with atopic dermatitis. J Allergy Clin Immunol. 2013; 132(5): 1252–4. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nMargolis DJ, Kim B, Apter AJ, et al.: Thymic stromal lymphopoietin variation, filaggrin loss of function, and the persistence of atopic dermatitis. JAMA Dermatol. 2014; 150(3): 254–9. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation\n\nGauvreau GM, O'Byrne PM, Boulet LP, et al.: Effects of an anti-TSLP antibody on allergen-induced asthmatic responses. N Engl J Med. 2014; 370(22): 2102–10. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nCreabilis SA: Creabilis Announces Positive Phase IIa Results for TrkA Kinase Inhibitor CT327 in Atopic Dermatitis. Luxembourg, 2010. Reference Source\n\nRoblin D, Yosipovitch G, Boyce B, et al.: Topical TrkA Kinase Inhibitor CT327 is an Effective, Novel Therapy for the Treatment of Pruritus due to Psoriasis: Results from Experimental Studies, and Efficacy and Safety of CT327 in a Phase 2b Clinical Trial in Patients with Psoriasis. Acta Derm Venereol. 2015; 95(5): 542–8. PubMed Abstract | Publisher Full Text | F1000 Recommendation\n\nVanda Pharmaceuticals Inc. Vanda Pharmaceuticals Announces Tradipitant Phase II Proof of Concept Study Results for Chronic Pruritus in Atopic Dermatitis. Washington DC, 2015. Reference Source"
}
|
[
{
"id": "11281",
"date": "19 Nov 2015",
"name": "Lawrence F Eichenfield",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11282",
"date": "19 Nov 2015",
"name": "Peter A. Lio",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1296
|
https://f1000research.com/articles/4-134/v1
|
29 May 15
|
{
"type": "Research Article",
"title": "The Resource Identification Initiative: A cultural shift in publishing",
"authors": [
"Anita Bandrowski",
"Matthew Brush",
"Jeffery S. Grethe",
"Melissa A. Haendel",
"David N. Kennedy",
"Sean Hill",
"Patrick R. Hof",
"Maryann E. Martone",
"Maaike Pols",
"Serena Tan",
"Nicole Washington",
"Elena Zudilova-Seinstra",
"Nicole Vasilevsky",
"Resource Identification Initiative Members are listed here: https://www.force11.org/node/4463/members",
"Matthew Brush",
"Jeffery S. Grethe",
"Melissa A. Haendel",
"David N. Kennedy",
"Sean Hill",
"Patrick R. Hof",
"Maryann E. Martone",
"Maaike Pols",
"Serena Tan",
"Nicole Washington",
"Elena Zudilova-Seinstra",
"Nicole Vasilevsky"
],
"abstract": "A central tenet in support of research reproducibility is the ability to uniquely identify research resources, i.e., reagents, tools, and materials that are used to perform experiments. However, current reporting practices for research resources are insufficient to allow humans and algorithms to identify the exact resources that are reported or answer basic questions such as “What other studies used resource X?” To address this issue, the Resource Identification Initiative was launched as a pilot project to improve the reporting standards for research resources in the methods sections of papers and thereby improve identifiability and reproducibility. The pilot engaged over 25 biomedical journal editors from most major publishers, as well as scientists and funding officials. Authors were asked to include Research Resource Identifiers (RRIDs) in their manuscripts prior to publication for three resource types: antibodies, model organisms, and tools (including software and databases). RRIDs represent accession numbers assigned by an authoritative database, e.g., the model organism databases, for each type of resource. To make it easier for authors to obtain RRIDs, resources were aggregated from the appropriate databases and their RRIDs made available in a central web portal (www.scicrunch.org/resources). RRIDs meet three key criteria: they are machine readable, free to generate and access, and are consistent across publishers and journals. The pilot was launched in February of 2014 and over 300 papers have appeared that report RRIDs. The number of journals participating has expanded from the original 25 to more than 40. Here, we present an overview of the pilot project and its outcomes to date. We show that authors are generally accurate in performing the task of identifying resources and supportive of the goals of the project. We also show that identifiability of the resources pre- and post-pilot showed a dramatic improvement for all three resource types, suggesting that the project has had a significant impact on reproducibility relating to research resources.",
"keywords": [
"Resource identifiers",
"Multi-centre initiative",
"Publishing",
"Pre-pilot data",
"Post-pilot data"
],
"content": "Introduction\n\nResearch resources, defined here as the reagents, materials, and tools used to produce the findings of a study, are the cornerstone of biomedical research. However, as has long been bemoaned by database curators and documented recently by Vasilevsky and colleagues, these resources are not very well identified in the scientific literature11. Vasilevsky and colleagues found that researchers did not include sufficient detail for most key research resources to allow someone to identify conclusively an antibody, a genetically modified animal, or cell lines, for example. In most cases, authors would provide insufficient metadata about the resource to conclusively identify a particular resource, e.g., a non-unique set of attributes with no catalog or stock number to specifically identify the resource used. It should be noted that the authors were, generally speaking, following the guidelines offered by the journals. Such guidelines traditionally state that one should include the company name and city in which it was located. Further, even when uniquely identifying information was provided (e.g., a catalog number for a particular antibody), the vendor may have gone out of business or the particular product may no longer be available. Given that in these cases a human cannot find which resources were used, an automated agent, such as a search engine or text mining tools will also not be able to identify the resources.\n\nBecause the current practices for reporting research resources within the literature are inadequate, non-standardized and not optimized for machine-based access, it is currently exceedingly difficult to answer very basic questions about published studies such as “What studies used the transgenic mouse I am interested in?” These types of questions are of interest to the biomedical community, which relies on the published literature to identify appropriate reagents, troubleshoot experiments, and aggregate information about a particular organism or reagent to form hypotheses about mechanism and function. Such information is also critical to funders who paid a research group to generate a particular tool or reagent and the resource providers, both commercial and academic, who would like to be able to track the use of these resources in the literature. Beyond this basic utility, identification of the particular research resource used is an important component of scientific reproducibility or lack thereof.\n\nThe Resource Identification Initiative (RII) is laying the foundation of a system for reporting research resources in the biomedical literature that will support unique identification of research resources used within a particular study. The initiative is jointly led by the Neuroscience Information Framework (NIF; http://neuinfo.org) and the Oregon Health Science University (OHSU) Library, data integration efforts occurring as part of the Monarch Initiative, http://www.monarchinitiative.org), and with numerous community members through FORCE11, the Future of Research Communications and e-Scholarship, a grassroots organization dedicated to transforming scholarly communication through technology. Since 2006, NIF has worked to identify research resources of relevance to neuroscience. The OHSU group has long-standing ties to the model organism community, which maintains databases populated by curating the literature and contacting authors to add links between model organisms, reagents, and other data. In a 2011 workshop (see https://www.force11.org/node/4145) held under the auspices of the Linking Animal Models to Human Diseases (LAMHDI) consortium, various stakeholders from this community drafted recommendations for better reporting standards for animal models, genes, and key reagents.\n\nThe RII initiative was launched as a result of two planning meetings building off of the recommendations of the LAMHDI workshop. The first was held in 2012 at the Society for Neuroscience meeting with over 40 participants comprising editors, publishers and funders (sponsored by INCF; http://incf.org). This meeting outlined the problem of incomplete identification of research resources within papers, and the need for a computational solution for identifying and tracking them in the literature. Recognizing that any solution needed to work for both humans and machines, three broad requirements were identified: 1) the standard should be machine-processable - that is, designed for search algorithms, in addition to human understanding; 2) the information should be available outside the paywall, so that search algorithms and humans have free access to the information across the biomedical literature; and 3) the standard should be uniform across publishers, to make uptake and usage easier for both human and machine.\n\nA follow-up workshop at the NIH (https://www.force11.org/node/4857) was held in June of 2013 to gain agreement from this stakeholder group for the design of a pilot that would explore solutions for this problem. A working group, the Resource Identification Initiative, was established through FORCE11, comprised of publishers, journal editors, antibody manufacturers and distributors, biocurators, software tool developers, and foundations. Based upon agreements garnered at the June 2013 meeting, the RII designed a pilot project to test implementation of a system for authors submitting manuscripts to identify research resources through the use of a unique identifier, termed a Research Resource Identifier or RRID.\n\n\nPilot project overview\n\nThe pilot project has focused on a limited number of resources - antibodies, software tools/databases, and model organisms. These three resource types were chosen because they are a major source of variation across experiments and are used broadly across research communities. For the purposes of this pilot, a critical aspect was that a relatively complete and authoritative central registry existed that could issue an accession number, as Genbank does for gene sequences. To gain broad agreement amongst publishers and editors who were concerned about the potential burden on authors and staff, it was agreed that participation in the pilot project would be voluntary for authors with participation not representing a condition of acceptance for publication. The pilot project was also designed to have minimal requirements for publishers such that modification of manuscript submission systems was not required.\n\nThe pilot project was originally designed to run for 6 months, with each of the participating journals agreeing to participate for at least 3 months. The goal was to ensure a large enough sample to understand author behavior: could they and would they do the task, to test the sufficiency of the infrastructure, and to obtain a sufficiently large dataset to explore and demonstrate the utility of RRIDs. Over the minimum 3-month window, each partner journal would request that authors supply RRIDs in a standard format as a citation to indicate the use of any of these three types of research resources. To be as unambiguous as possible, authors were to include the RRIDs in the text of the materials and methods, but not in the introduction or discussion sections. The RRID syntax comprises an accession number assigned by the authoritative database with the prefix \"RRID:\" prepended (e.g., RRID:AB_2298772 for an antibody). We also requested that closed-source journals include RRIDs in the keyword field as this field is available for indexing in PubMed outside of paywalls. The journals were given flexibility for when and how they wanted to ask authors for these identifiers, namely, at time of submission, during review, or after acceptance. They were not required to modify their instructions to authors or their submission systems. The RII team would be responsible for preparing appropriate materials for requesting RRIDs and for establishing a central portal where these identifiers could be obtained. The RII team also agreed to establish a help desk to assist the authors if they encountered any difficulties.\n\nThe pilot project was designed to address four key questions. A set of evaluation criteria was designed for each question:\n\n1. Compliance: Would authors be willing to add resource identifiers to their publications and register new resources in the system? Compliance was evaluated by examining the number of submissions to the participating journals, the rate of author compliance in providing RRIDs, the number of new resources registered, and direct feedback from authors.\n\n2. Accuracy: Could authors add these identifiers accurately or would additional editorial or staff oversight be necessary? Accuracy was measured by a quantitative analysis of RRID accuracy by RII curators.\n\n3. Identifiability: Would the use of RRIDs improve our ability to identify resources in the literature? Identifiability was measured by assessing the number of catalog numbers pre- and post-pilot in the journals that participated.\n\n4. Utility: Will RRID’s be useful to the scientific community? Can the RRID’s as constructed be used to identify all studies that use a particular research resource? To encourage the development of applications, the data set is being made freely available so that third parties can develop tools to work with RRIDs.\n\nThe pilot began in February 2014, with over 25 journals participating. Journals that sent a letter to authors at some stage of the review process included: Journal of Neuroscience, Brain and Behavior, Journal of Comparative Neurology, Brain Research, Experimental Neurology, F1000Research, PeerJ, Journal of Neuroscience Methods, Neurobiology of Disease, and the Frontiers group of journals. One journal, Neuroinformatics, chose to add the RRIDs to all manuscripts before asking authors to do this. Journals in the Elsevier and BMC groups were participants based upon updates to their instructions to authors. Because of the success of the project, it was subsequently extended and is still active as of this writing. The number of journals participating has expanded, and now includes PLoS Biology and PLoS Genetics as well as multiple immunology journals in the Elsevier family. A list of the participating journals is available on the Force11 website (https://www.force11.org/RII/SignUp).\n\n\nWorkflow\n\nOne of the prime requirements of the pilot project was to make it as easy as possible for authors to obtain the appropriate identifiers and insert them correctly into their manuscripts. As noted above, the three research resources were chosen because each was covered by an authoritative database (Table 1) that assigned unique IDs and a standard set of metadata to each. However, as can be seen by the length of the list in Table 1, authors could potentially be required to visit several databases to obtain the appropriate identifiers.\n\nEach database has a weekly or monthly scheduled frequency of update and all new data is released weekly. If available, data from both model organism authorities is served as well as the list of strains available via particular stock centers. In most cases the stock centers maintain a link between the genotype and the stock center animal identifier. Scheduling and total data count information can be accessed via DISCO (http://disco.neuinfo.org/webportal/dataPipelineViewStatus.do?id=nlx_154697-1).\n\nTo simplify this process, we utilized the data aggregation services of the NIF, provided through a platform known as SciCrunch, to establish a Resource Identification Portal (http://scicrunch.org/resources; Figure 1). The portal provided a unified query across these different databases and displayed the results in a common format. The portal allows search on various facets such as resource name, catalog number, etc. There is a ‘cite this’ link that provides the citation as it should be reported in the paper. The citation generally includes not just the RRID, but a set of appropriate metadata that would identify the vendor and catalog number as well, for example: A polyclonal antibody against tyrosine hydroxylase (TH) (Chemicon, Cat. AB1542, RRID:AB_90755).\n\nThe workflow for authors is to select their resource type (see community resources box), type in search terms (note, the system attempts to expand known synonyms to improve search results) and open the “Cite This” dialog box. The dialog shown here displays the Invitrogen catalog number 80021 antibody with the RRID:AB_86329. The authors are asked to copy and paste this text into their methods sections.\n\nSciCrunch was built based on the extensible Neuroscience Information Framework platform described previously (12,13, RRID:nif-0000-25673), and the portal infrastructure for RII was developed under an award from NIDDK to create a dkNET portal (RRID:nlx_153866), while the customization of the portal was done by Monarch staff. The data is aggregated from the SciCrunch tool registry, the antibody registry, as well as the model organism community databases and stock centers (Table 1). The data infrastructure allows curators to keep indexes synchronized with the source databases by using an automated crawling engine and new data are released on a weekly basis. All open data from each of these databases is available to download from the source sites, where update frequencies are listed.\n\nThe instructions to authors in all cases were consistent, that is, the same set of instructions was provided to each author. For antibodies, we only required authors to identify primary antibodies and not secondary or tertiary complexes. For tools and databases, we focus on freely available tools, generally supported through public funding and not commercial tools. Authors were asked to insert the correct citation for the resource into the text of the materials and methods section and in the keywords. A help desk was established by the RII working group that provided help if an author encountered difficulty. In most cases, requests were handled in less than 24 hours.\n\nIf a resource was not found via the portal, authors were given the option of submitting the resource to obtain an identifier. For antibodies and software/databases, which are found in databases maintained within the NIF, submission was handled through the Resource Identification Portal. For model organisms, the author was referred to the authoritative model organism database. All new submissions were curated by their respective databases and the data was pulled back into the RII portal weekly so that authors could see their newly registered antibodies or software tools in about a week.\n\n\nResults\n\nThe first RRIDs began appearing in the literature in April of 2014. Although the first paper was identified through PubMed, the majority of papers were found via Google Scholar by searching for “RRID”. Google Scholar, unlike PubMed, appears to search the full text of articles, as it returns snippets of text from the materials and methods containing the RRIDs (for example see Figure 2). A search in PubMed returns very few papers, indicating that publishers were not including the RRIDs outside of the pay-wall. As these papers start to appear in PubMed Central where there is full text search, it should be possible to find papers for RRIDs through the National Library of Medicine. Google Scholar possesses the advantage in that it obtains papers without an embargo period and makes them available for search, unlike PubMed Central. We therefore present statistics and analyses in this paper based upon Google Scholar.\n\nA. Google scholar result for the anti-tyrosine hydroxylase antibody RRID (9/2014; http://scholar.google.com/scholar?q=RRID:AB_90755). B. Shows the most used RRIDs in the first 100 papers, by number of papers using the identifier. All data is available in Supplementary Table 1 and all identifiers can be accessed in Google Scholar (see also Supplemental Table 1).\n\nSearch via Google Scholar reveals that the RRID prefix is not a unique string, but is an acronym for several entities, mostly commonly the Renal Risk in Derby clinical study (for example 9). To return examples of RRIDs requires the use of additional filters, e.g., restricting search to the years 2014 and later. The combination of the RRID prefix with the resource accession number is unique however, in that searching for a particular RRID, for example RRID:AB_90755 returns only papers that use this research resource (Figure 2).\n\nTo address the aims of the pilot project, we tracked the use of RRIDs in published papers and journals. We performed an in-depth analysis of the first 100 papers found through Google Scholar that reported RRIDs. For each paper, we examined the methods section to determine whether RRIDs were correctly cited - that is that they pointed to the correct resource (= true positives), and whether all research resources that should have been identified by an RRID were appropriately cited (= true negatives). The total number of research resources reported in the first 100 papers of the Resource Identification Initiative was determined by manual inspection of each paper by two independent people. A Google Scholar alert was used to track all new papers that contained the term RRID. Each paper was downloaded and examined for the snippets of text surrounding research resources (methods or data use sections). Each snippet of text surrounding the RRID was copied and pasted into a shared document (Supplemental table 1). The RRID was then checked against the scicrunch resolving service (for example https://scicrunch.org/resolver/RRID:AB_262044) to determine what the source database lists about the resource. Information was compared in the following way: if the vendor/catalog number was present in the snippet and these matched the resolver data, we considered that the record was marked accurate. If no information about the catalog number was present in the paper, but the antibody target or clone number matched then the record was also marked accurate, but if the vendor/catalog number information was different from the database record or no other information could be found about the reagent, then the record was marked inaccurate. The total accuracy is primarily therefore based on the catalog number match and importantly does not reflect any upstream problems with organism or reagent identification inside of the laboratories themselves.\n\nThe results of the analysis are summarized in Table 2. The spreadsheet containing the full analysis is available as a supplementary data file. The first 100 papers were published in 15 journals and included 630 RRIDs reported by authors. The bulk of the identifiers (572) came from two journals, the Journal of Comparative Neurology and the Journal of Neuroscience, as these two journals were first to participate both starting the pilot in early February.\n\nJournals that added only the instructions to authors are not included in this table (for example BMC). The compliance rate was by far the lowest with only instructions to authors; this hovered between 1 and 15% when authors were asked by blanket mailing in addition to the instructions to authors (A letter to authors from the editor, when this letter included several pages of instructions. Compliance was lowest in Brain Research and Journal of Neuroscience Methods) and very high if the editorial staff asked authors directly or suggested identifiers for their manuscript. For two journals this was further facilitated by a special section in the papers that described tools, Journal of Comparative Neurology and Neuroinformatics both contain sections that normally describe antibodies and software tools, respectively.\n\nAs of March 1, 2015 there were 312 papers published with at least one RRID, from 44 unique journals (Supplementary files show the updated list of journals and a count for each) indicating that hundreds of authors have participated in the pilot project even though it is voluntary. Informal feedback from the editors and authors via help requests and other correspondence indicates that authors who are attempting to find RRIDs are supportive of the aims of the project and readily able to find the correct RRIDs.\n\nAuthors were willing to add resources to the registries if they were not available. Since the project began, over 200,000 antibodies from vendors - both solicited and unsolicited - and at least 200 from individual authors were added to the Antibody Registry (http://antibodyregistry.org). In cases where antibodies are sold by government-led projects such as NeuroMab from UC Davis, antibody identifiers have been included in the antibody manufacturer’s web site. Many of the additions were secondary antibodies, which were not part of the project but authors felt that they should also be identified. In one representative example, Jackson ImmunoResearch was contacted by several authors and submitted their full catalog to the Antibody Registry, allowing authors to identify secondary antibodies. Additionally, there were over 100 software tools and databases registered. Many were for common commercial statistical tools (e.g., SPSS, GraphPad), technically out of scope for the project, but authors did not make the distinction between commercial and non-commercial tools. Figure 2 shows the most common tools identified by RRID in papers from the first 100 papers. Commercial tools such as MATLAB, SAS and GraphPad were cited along with ImageJ and FreeSurfer. The most common antibody was NeuN from Millipore. These same resource identifiers have continued to be very highly cited in subsequent papers, with ImageJ cited in 42 papers and the NeuN antibody cited in 8 papers (Google Scholar March 17, 2015). A comparison of added resources vs those reported in the first 100 papers indicates that the Registries already listed the majority of research resources in each of these categories, as the number of new resources added for this set represented only 10% of the total reported resources.\n\nA major concern of the publishers and editors was whether or not authors could retrieve RRIDs accurately and whether significant editorial oversight would be necessary for quality control (see workshop outcome documents at https://www.force11.org/node/4857). As shown in Table 3, authors of the first 100 papers were 96% accurate when reporting resource identifiers - that is, they used the correct identifier as determined by comparing the metadata supplied in the article with that in the database, accessible via a resolving service (see http://scicrunch.com/resolver/RRID:AB_262044). Authors did not create RRIDs for resources they were either unable to find, or were not in the databases in 34 clear-cut cases. This constitutes a 5.4% false negative rate. In cases where authors tried to identify research resources they tended to include identifiers for over 90% of them.\n\nCounts were ascertained by two curators going through every paper with at least one RRID, and %Error was calculated when the information surrounding the identifier reported in the paper or the identifier did not match the data from the resolving service for each entity. For a complete list of resources, including links to the resolver, see Supplementary table 1.\n\nA total of 24 errors were found in the 630 RRIDs analyzed. For antibodies, 15 antibodies were incorrectly identified (3.8% error rate). Inspection of these errors, showed that: three errors were copy/paste mistakes where authors mixed up the combination of catalog number and identifier for resources actually used in their paper; three errors resulted from identifiers missing a digit at the end of the ID (for example, “Swant, catalog #6B3, RRID: AB_1000032” should have been named RRID: AB_10000320); one error involved reporting a reference PMID instead of the resource identifier. The apparent cause of the other eight antibody errors was not possible to determine. For organisms, seven errors were made (13.2% error rate). All of these errors involved mice for which authors used the appropriate gene or allele identifier from Mouse Genome Informatics, MGI, rather than the stock number or genotype identifying the organism. The allele ID is better than no information, but it is not sufficient for identifying the animal used as the same allele may be inserted into different mice of various backgrounds and with other alleles, and those mice may have different characteristics. It should also be noted that authors using the MGI database (up to October 2014), which maintains the authoritative mouse database, would be given a set of MGI identifiers for genes and alleles, but not genotypes. This shows that authors likely went to MGI to obtain their identifiers, but were not able to find the genotype information and substituted the allele ID. Currently MGI contains a Google site search that now searches the genotype information for all mice suggesting that authors of newer papers can now also find the genotype information more easily at MGI. They have also created a tutorial for how to obtain a genotype identifier that has been posted on the SciCrunch pages. The ideal situation is for MGI, the trusted authority, to point authors to the proper genotype information. The fewest errors were made in identifying software tools and databases, with only one mistake from 129 total (0.8%). The mistake was made as the author apparently used an antibody identifier instead of a tool identifier. From the relatively low error rate, it appears that authors are able to perform the task accurately, at least for software tools, databases, and antibodies.\n\nAn outcome of this study was to determine if the use of RRIDs in the literature increased the identifiability of research resources. As shown in Figure 3, when authors were asked by their editors to provide RRIDs, regardless of their compliance with the RII project, the identifiability of research resources significantly increased. We calculated the percentage of identifiable research resources in the same journals, just before the pilot project and after. The reporting of research resources pre-pilot was consistent with findings from the 2013 study (Vasilevsky et al., 2013), in that roughly 50–60% were found to be identifiable. But when asked by their editors, researchers used identifying information in 80–90% of research resources, showing that they presumably had the data available, but did not put it into their papers unless prompted by communication from the editors.\n\nIdentifiability of the three resource types was determined in journal articles that reported RRIDs (post-pilot), and in articles from the same journals before the pilot started (from approximately January–March 2013, pre-pilot). Resources (primary antibodies, organisms, and tools) were considered identifiable if they contained an accurate RRID or by using the same criteria as described in Vasilevsky et al., 2013 (PMID: 24032093). For software and databases (which were not previously analyzed), these resources were considered identifiable if they contained an RRID or reported the manufacturer and version number. The total number of resources for each type is: primary antibodies pre-pilot, n = 140; primary antibodies post-pilot, n = 433; organisms pre-pilot, n = 58; organisms post-pilot, n = 128; tools pre-pilot, n = 176; tools post-pilot, n = 246. The y-axis is the average for each resource type. Variation from this average is shown by the bars: error bars indicate upper and lower 95% confidence intervals. Asterisks indicate significant difference by a z-score greater than 1.96.\n\nConsistency of syntax and machine-processability: The ability to search all studies that used a particular research resource was a prime motivation for this pilot project. The current project had a loose definition of “machine-processable” because we did not want to impose any requirements on the publishers to modify their journal submission system for a pilot project. Thus, we opted to craft RRIDs as unique, indexable alphanumeric strings that could support use of web search engines to return papers that used a particular research resource. We specifically asked authors to place the RRIDs only in the materials and methods section, where they would normally provide identifying information for a given entity, because we wanted to track actual use of the resource and not just mentions of it.\n\nFor individual RRIDs, the approach was highly successful as is illustrated by the ability to type a particular RRID into three search engines for the biomedical literature: Google Scholar, PubMed and Science Direct and retrieve appropriate papers, e.g., RRID:AB_90755 or AB_2298772 (for Google Scholar see Figure 2). It is important to note that each of these systems will come back with different results because each search tool has different types of data about each paper. For example, ScienceDirect has a good full text search of all Elsevier content, but it does not search other publishers content. Both PubMed and Scopus search only the abstracts and return a subset of articles where authors followed instructions to add RRIDs to the keywords, but not those that are only in the methods section. Google Scholar is the most comprehensive as it appears to search full text and brings back papers that are both published and unpublished (usually these are accepted for publication, but not yet indexed by PubMed). An analysis performed in October, 2014 showed varying results from each search engine: Google Scholar returned 315 results (from 2014, 174 are true RRIDs), and ScienceDirect returned 18 (from 2014, three are RRIDs). PubMed revealed 23 papers that contained RRIDs (from 2014, all identify the resource identification initiative identifiers). Scopus returned 48 documents (from 2014, 18 are RRIDs).\n\nThe use of a unique string to retrieve RRIDs is aided by a common syntax. Thus, in our analysis of RRIDs, we also noted whether or not the RRID was correctly formed. While authors were 96% accurate in supplying identifiers, minor corrections to formatting were needed in 34% of RRIDs (66% accuracy). The most common variant was the addition of extra spaces (RRID:AB_90755 vs RRID: AB_90755), other common variants were failure to include the RRID prefix (RRID as table header), using various symbols or spaces in the identifier, or splitting up the RRID prefix and identifier in a table. These practices make it more difficult to identify the RRID through text search alone, and require some additional natural language processing to identify. We note, however, that the search algorithms are improving. Whereas in June, Google Scholar could not recognize an RRID that included a space after the colon, e.g., RRID:AB_####, by October these types of variants were retrieved. Such minor errors could also be avoided by developing authoring system upgrades that directly identify RRIDs and insert the proper citations into the manuscript.\n\nTo promote the development of 3rd party tools around RRID’s, we created a resolver service for RRIDs using SciCrunch. Typing http://scicrunch.com/resolver/RRID:AB_90755, will resolve to a landing page with meta-data on a particular entity. The resolving service allows applications to make use of RRIDs to, for example, enhance articles with RRIDs by providing additional information about the entity and a link to relevant articles and resources. For instance, Elsevier has released their antibody application, which displays antibody meta-data in the right hand side panel, next to the article (see the screen shot below (Figure 4) for 8: http://www.sciencedirect.com/science/article/pii/S0306452214008458). The reader can browse through antibodies referred to in the article, view complete records in antibodyregistry.org and access additional information via direct links to GenBank, ZFIN and other relevant databases. The application also recommends three most relevant articles published in Elsevier journals that refer to the same antibody. The application is freely available on ScienceDirect.\n\nThe application is available in 206 articles in 19 journals (more information can be found at: http://www.elsevier.com/about/content-innovation/antibodies).\n\nPublication practices: Non-open access journals were asked to add RRIDs to publication keywords, but our initial findings suggest that this practice was not being consistently followed. Only 23 papers out of 41 total (as of Oct 20, 2014) were accessible in PubMed. Additionally it should be noted that in two cases, identifiers were removed at typesetting after the initial online version of the manuscript was published with the RRIDs. These identifiers were removed not only from the manuscript, but also from PubMed keywords. Although this was reversed when noted by the working group, this demonstrates that successful implementation requires knowledge of and agreement by the publishers at all steps.\n\n\nDiscussion\n\nThe pilot RRID project has been highly successful in demonstrating the utility of a system to aid in identification of these three research resources in the literature. We showed that authors were willing to adopt new styles of citation for research resources that promoted more accurate identification of research resources used in a study, and that were more amenable to machine-based identification. To date, RRIDs have appeared in over 300 papers from 40 journals. With one exception (the Journal of Neuroscience), journals have continued their request for RRIDs beyond the initial 3-month pilot project and new journals have signed up beyond the initial set that started the project. We believe that the success of the project was due to the extensive pre-planning that involved the publishers and the editors, the limited scope of the initial request, and the recognized need by researchers for better and more useful reporting standards for research resources.\n\nThe load on curation staff with participating journals has been minimal and the initial portal prototype appears reasonable for the majority of authors to find their resource identifiers. With >10,000 searches in the RII portal, there were approximately 100 help questions. Many of these questions were about scope, i.e., whether a particular research resource should be identified. Others were for assistance in finding a resource or guidance in adding a resource not yet contained in the community authorities. While this is not a large number, it is also not insignificant, particularly as the project expands, and certainly points out the need for specific help functions.\n\nGiven the relative completeness of the registries and the rapid advance of machine-learning based techniques for entity recognition, we can envision a semi-automated system that assists the author in supplying correct IDs. We have already improved our ability to detect digital research resources in the literature using machine learning (Ozyurt et al., submitted). In this system, machine learning is used to identify software tools and databases in text and compare the information to Registry listings. The development of such functions would allow the development of recommender systems for authors and automated fact checkers for journal staffs.\n\nUnique identifiers serve as a primary key or “social security number” for identifying a given entity, and providing the ability for search engines to parse them is paramount. For search engines, unique identifiers are simple methods for disambiguating entities with similar names. For identifiers to function in this mode, they need to be unique - that is, the same ID should not point to two different entities, and they need to be persistent - that is, they need to outlive the entity itself. They also need to be at least minimally machine-processable. While many authors supplied identifying information like the catalog number for an antibody supplied by the vendor, or the official strain nomenclature supplied by the IMSR for a mouse, neither of these served the required functions. A catalog number is not a unique identifier, but rather a useful way for vendors to identify their products. If the same antibody is sold by different vendors, it will have a different catalog number. If the same antibody is sold in different aliquots, it may also have a different catalog number. When the antibody is no longer available, the catalog number may disappear, or in some cases be recycled for use with another antibody. All of these features are undesirable in an identifier system. The Antibody Registry, in contrast, was specifically designed to supply useful and stable identifiers for antibodies and not as a commercial source of antibodies. Similarly, the strain nomenclature developed by the Jackson Laboratory, with its superscripts and special characters, is useful for human curators to identify a particular strain, but causes hiccups in most search engines because of all of the special characters. We believe that a well curated registry is essential to the success of such a system, because of the necessity of these two functions, which currently cannot be replaced with a simple uncurated registration service. For example, we found in the registries we maintain (both software and antibodies) that authors sometimes register an entity that is found by a curator to be a duplicate.\n\nThere are many types and formats of identifiers in use today (e.g., DOIs, URIs, ARCs), each with varying amounts of associated infrastructure and use in different communities. For this project, we elected to use simple alphanumeric strings and a common syntax in the form of accession numbers issued by the authoritative community-based registries. We relied on each registry to impose the uniqueness constraint at the level of the entity, for example ensuring that there was only one mouse genotype per unique ID, and to ensure standard metadata by curating each entry. The reuse of authoritative accessions with the RRID prefix provides maximal flexibility and interoperability and minimal ID churn, whilst also provisioning for resource identification.\n\nA frequent question regarding the RRID is why we did not use a DOI as a unique identifier instead of the Registry Accession number. Part of the reason was cultural: researchers were used to supplying accession numbers for Genbank, Gene Expression Omnibus, Protein Data Bank, etc. and understand this requirement. Part of the reason is practical: unlike DOIs, accession numbers are already available for all of the research resources to be identified in this pilot and did not require special infrastructure to resolve or cost to issue. Part of the reason is also philosophical: DOIs are for digital objects, such as individual articles, that live on the web and need to be resolvable. A DOI resolves to a particular article, which is self-contained - it is the object. In contrast, an antibody does not exist on the web but is an independent entity that has data about it scattered across various articles. There is no single digital record that is the antibody; there are documents and data about the entity. We note that in our community we also do not use DOIs to identify people, but rather an ORCID, which serves the same purpose as the RRID.\n\nA case could be made for using DOIs to identify particular software tools and databases, as they are digital objects. As discussed in the next section, our preference is that DOIs be used to identify the particular instance used, e.g., the version of data or software and any supporting workflows, and that the RRID be used to identify the entity or project referenced. Thus, the RRID would be used to identify the Protein Databank, and a PDB identifier or a DOI used to reference the specific data from the PDB. However, we believe that if the RRID system is adopted, appropriate identifier systems should be set by each community. The RRID syntax is meant to be simple and generic and could, in theory, work with any identifier system.\n\nRRIDs are meant to identify research resources at a fairly high level of granularity. At some of the planning meetings, there was a push for more granular information, like lot and batch numbers, for antibodies. We recognize that this level of granularity is likely an important factor in determining how a given reagent performs10. However, in the analysis by Vasilevsky et al. (2013) and in our experience in resource identification using text-mining, the biggest problem was not that authors were not supplying lot numbers but that they were not even supplying catalog numbers. Given that the catalog numbers themselves do not serve as stable identifiers, because antibodies are bought and sold and redistributed by many vendors, we elected to tackle the problem of identifying the root antibody first, i.e., a particular clone for a monoclonal antibody or a type of polyclonal antibody produced by particular protocol. To illustrate the problem, consider the study by Slotta10 that provided an analysis of the performance of antibodies to NF-ĸβ p65, as a follow up to a similar study by Herkenham4. Both studies performed specificity tests on a variety of antibodies and, as is common, did not produce concordant results on all of them. Slotta had been the original producer of an antibody now commonly known as MAB3026 (AB_2178887) and provided its provenance: “It was transferred to Boehringer-Mannheim as Clone 12H11, resold to Roche and finally bought by Chemicon, and it is now sold as MAB3026.” They then speculate that a mutation may have crept in at some point that altered the specificity of the antibody. However, it may simply be that as the antibody was tested under additional conditions, problems were revealed that had not been apparent during more limited applications. The RRID for this antibody binds these different representations together so that all references to this antibody can be tracked. However, authors are encouraged in the citation format to include details about the particular instance of this antibody, namely, the vendor from which the antibody was purchased and the catalog, batch, and lot numbers. However, we did not want to overload the ID system to require assignment of these different lot numbers different RRIDs and maintain the mappings. We also felt that this would grossly decrease compliance.\n\nSimilarly, for software and databases, we elected to identify just the root entity and not a granular citation of a particular software version or database. Our main goal in the case of software tools and databases was to track broad patterns of utilization of these resources (e.g., how many times NeuroMorpho.org was used, as per mandate of the NIF, and not particular versions). More complete practices for citing software and data sets are emerging from recent efforts like the Joint Declaration of Data Citation principles (https://www.force11.org/datacitation), the W3C HCLS dataset description (http://tiny.cc/hcls-datadesc), the software discovery index (http://softwarediscoveryindex.org/), and many others. In these cases, groups are exploring more complete reporting standards for the individual instances (versions, workflows, virtual machines) that can be used to replicate the findings.\n\nThe RII is an example of a grass-roots type of organization that took advantage of existing investments by the NIH to solve a problem without extensive new infrastructure. The RII is continuing to run and has expanded beyond the initial participants. We believe that the growth of the initiative indicates that it fills a need not currently met by our existing practices and infrastructure.\n\nShould RRIDs be adopted broadly across all of biomedicine? We would argue yes, the RRID syntax should become the standard for reporting on usage of research resources. We have shown that the requirements for this type of broad adoption are: the availability of a comprehensive and authoritative registry for the appropriate entities; a centralized portal or services that aggregate these registries into a single search call, and the willingness of a community including journals and publishers to support this style of reporting. More sophisticated services can be built to improve and automate authoring and editorial oversight, but these are not required. The solution is therefore accessible both to large commercial publishers, and smaller community- or society-based journals.\n\nIf RRIDs were to be broadly adopted tomorrow, what are the outstanding issues regarding implementation and scalability? The first issue is one of scope. The current RII focused on three types of research resources which were broadly used and a known source of variability within experiments. Should all research resources be similarly identified, i.e., every chemical, salt, instrument? We think such an approach would be clumsy and difficult to implement. We can imagine a future where all reagents and tools are bar coded and scanned as they are used in a study. However, as long as humans are responsible for supplying identifiers, we think that the effort should focus on certain types of known problematic entities for which better metadata and ability to query across papers is required. Given the recent problems associated with certain cell lines, for example, these are obvious candidates6. The advantage of the current system is that it allows communities who have taken the steps to aggregate and organize resources that are of use to them to agree to include the RRID syntax and single entry point.\n\nThe second issue is governance. We deliberately designed a decentralized system that gives control of issuing identifiers to multiple authorities. Such a model requires some governance, in the form of willingness of the authorities to maintain the integrity of any identifiers and links and implementation of a policy regarding entities that are no longer available. We would also need some governance to ensure that multiple, uncoordinated authorities are not issuing IDs for the same research resource and that the IDs assigned to each entity are unique. The latter constraint is handled by the centralized aggregation service currently provided by SciCrunch, however it may be handled by Identifiers.org or other services in the future. Further, the RRID project promotes consistent citation of research resources at a first level identifiability. We believe that more granular reporting standards can and should work hand in hand with the RRIDs and could be coordinated with the authoritative communities, for example, versioned software releases in GitHub.\n\nWe believe that the RRID project lays an important foundation for creating a type of “universal product code” (UPC) to help alert the scientific community when issues are raised about key research resources. Reagents and tools are not perfect and problems can arise, as the resources themselves can have issues as they are tested across various paradigms and systems. Even when a resource initially performed well, due to spontaneous mutations in biological resources and interactions between particular software tools and platforms, problems can arise over time. For example, two recent papers have published extensive tests showing that common antibodies for NF-Kb show non-specificity under some circumstances7,10. Many of these antibodies are extensively used in the literature, but readers of a particular article have no way of knowing that concerns have been raised. We have similar examples with software tools3, data sets1,5 and genetically modified animals2. We have an infrastructure in place, CrossMark, to alert readers of a particular article that an addendum or erratum has been posted. The RRID system can serve as the basis for a similar system for research resources.",
"appendix": "Author contributions\n\n\n\nAB, PRH, DNK, NW, EZS NV and members of the Resource Identification Initiative (RII) contributed to data gathering. AB and MB were responsible for portal creation. PRH and MEM acted as advocates for the RII. AB, MEM were responsible for repository management for Scicrunch and the Antibody Registry. JSG was the Scicrunch portal architect. PRH implemented use of RRIDs at the Journal of Comparative Neurology. MEM implemented use of RRIDs for Brain and Behaviour. MP implemented use of RRIDs at F1000Research. ST implemented use of RRIDs at the Journal of Comparative Neurology. DNK implemented the use of RRIDs at NeuroInformatics. SH implemented use of RRIDs at Frontiers and brought together stakeholders at preliminary meetings. NW scripted the model organism data. EZS championed the use of RRIDs at Elsevier, developed the App and created Figure 4. NV and AB were responsible for data analysis. AB, MB, JSG, MAH, PRH, MEM and NV prepared the manuscript. Members of the RINL RII wrote blogs and participated in brainstorming meetings for more than a year. 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\nThis work was supported by: an NIF grant to Martone PI (HHSN271200577531C/PHS HHS/United States); a NIDDK grant to Martone PI (1U24DK097771-01); and a grant from Monarch to Haendel PI (5R24OD011883).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank John Maunsell for his effort in getting this effort going and helpful comments on the paper, as well as the many other editors who wrote letters to their authors and the staff at various publishers and many other community projects that wrote blogs and press releases about this initiative. We would also like to thank NIH and INCF for providing the venues and vision for this pilot project, specifically Dr. Jonathan Pollock, who drove the workshop and saw early the need to bring this particular group together around a central goal of better identification of research resources. We would also like to thank ZFIN, who very early on recognized the need for an RRID system and provided inspiration for this project.\n\n\nSupplementary files\n\nSupplementary data for 'The Resource Identification Initiative: A cultural shift in publishing', including participating papers (RRIDs and papers.csv), Curated RRIDs for antibodies, organisms and software (NV curation Antibodies.csv, NV curation Organisms.csv and NV curation Software.csv), Primary and Secondary antibodies given RRIDs (Primary vs Secondary Abs.csv) and an analysis of the first 100 RRIDs used in published papers (first 100 analysis.csv).\n\nClick here to access the data.\n\n\nReferences\n\nButton KS, Ioannidis JP, Mokrysz C, et al.: Power failure: why small sample size undermines the reliability of neuroscience. Nat Rev Neurosci. 2013; 14(5): 365–76. PubMed Abstract | Publisher Full Text\n\nCone AC, Ambrosi C, Scemes E, et al.: A comparative antibody analysis of pannexin1 expression in four rat brain regions reveals varying subcellular localizations. Front Pharmacol. 2013; 4: 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGronenschild EH, Habets P, Jacobs HI, et al.: The effects of FreeSurfer version, workstation type, and Macintosh operating system version on anatomical volume and cortical thickness measurements. PLoS One. 2012; 7(6): e38234. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHerkenham M, Rathore P, Brown P, et al.: Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies. J Neuroinflammation. 2011; 8: 141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHupé JM: Statistical inferences under the Null hypothesis: common mistakes and pitfalls in neuroimaging studies. Front Neurosci. 2015; 9: 18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInternational Cell Line Authentication Committee (ICLAC): Cell line cross-contamination: WSU-CLL is a known derivative of REH and is unsuitable as a model for chronic lymphocytic leukaemia. Leuk Res. 2014; 38(8): 999–1001. PubMed Abstract | Publisher Full Text\n\nListwak SJ, Rathore P, Herkenham M: Minimal NF-κB activity in neurons. Neuroscience. 2013; 250: 282–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacLaren DA, Markovic T, Daniels D, et al.: Enhanced consumption of salient solutions following pedunculopontine tegmental lesions. Neuroscience. 2015; 284: 381–99. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcIntyre NJ, Fluck R, McIntyre C, et al.: Treatment needs and diagnosis awareness in primary care patients with chronic kidney disease. Br J Gen Pract. 2012; 62(597): e227–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSlotta C, Müller J, Tran L, et al.: An investigation of the specificity of research antibodies against NF-κB-subunit p65. J Histochem Cytochem. 2014; 62(2): 157–61. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVasilevsky NA, Brush MH, Paddock H, et al.: On the reproducibility of science: unique identification of research resources in the biomedical literature. PeerJ. 2013; 1: e148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGardner D, Akil H, Ascoli GA, et al.: The neuroscience information framework: a data and knowledge environment for neuroscience. Neuroinformatics. 2008; 6(3): 149–60. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarenco LN, Wang R, Bandrowski AE, et al.: Extending the NIF DISCO framework to automate complex workflow: coordinating the harvest and integration of data from diverse neuroscience information resources. Front Neuroinform. 2014; 8: 58. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "8814",
"date": "05 Jun 2015",
"name": "Randi Vita",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 is overall well written with an appropriate title. The abstract provides an adequate summary and the design and explanation are sound. I believe the conclusions are justified and the data provided is adequate to support them. The subject is valuable to many researchers and provides an important service to researchers going forward. I have a few minor grammatical issues to report:The sentence \"The pilot project has focused on a limited number of resources - antibodies, software tools/databases, and model organisms\" should be \"The pilot project focused on a limited number of resources...\" in \"accession number, as Genbank does for gene\" should use \"GenBank\" For sentence \"Over the minimum 3-month window, each partner journal would request that authors supply RRIDs in a standard format as a citation to indicate the use of any of these three types of research resources.\"In this context, what the \"three types of research resources\" are is unclear. The sentence \"As these papers start to appear in PubMed Central where there is full text search, it should be possible to find papers for RRIDs through the National Library of Medicine.\" is a little confusing, maybe \"As these papers start to appear in PubMed Central, where full text search is possible, papers utilizing RRIDs will be identifiable through the National Library of Medicine.\" is better? Yes, being overly nit picky, but I had to read it twice to get it. Verb tense should be reviewed throughout the manuscript to ensure consistency. Uses of dashes such as \"...from vendors - both solicited and unsolicited - and at..\" occurs throughout the manuscript. This seems nonstandard? In Figure 3, I could not see the pre-pilot bar. This could be due to something with my browser, but worth checking out. In the Discussion, the sentence \"The pilot RRID project has been highly successful in demonstrating the utility of a system to aid in identification of these three research resources in the literature\" should reiterate what three research resources it is referring to. Review the number of uses of the word \"however\" and how close to each other they occur. For example, \"However, authors are encouraged in the citation format to include details about the particular instance of this antibody, namely, the vendor from which the antibody was purchased and the catalog, batch, and lot numbers. However, we...\"And lastly, the link referred to here \"A list of the participating journals is available on the Force11 website (https://www.force11.org/RII/SignUp).\" did not provide a list of the multiple immunology journals in the Elsevier family that are participating. The page just shows the Elsevier logo.",
"responses": []
},
{
"id": "8811",
"date": "18 Jun 2015",
"name": "William Gunn",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe aim of this study was to determine the feasibility and utility of asking authors to include RRIDs when submitting a manuscript. The process for making these determinations was well-considered and the experience and professional relationships of the authors helped greatly in enabling the study to reach the number of journals and submissions that it reached. The analysis of the data was described well and carried out well, making it possible to replicate the experiment, in theory. As much of this study did depend on professional relationships, it's not clear if it could be replicated in practice, but given the nature of the study, this is expected. The authors are to be commended in particular for supplying the raw data supporting the analysis in a usable form as supplementary data, This improved my understanding of the work.With regard to the 4 outcome measures (Compliance, Accuracy, Identifiability, and Utility) the scoring method is clear and well-described, but I felt like there were some important aspects of the data that were not addressed by the authors. With respect to compliance, it could be said that receiving 312 papers with 572 identifiers shows willingness to contribute, but the data in Table 2 suggest that reaching this level of compliance required a very hands-on approach by the editors. This suggests that compliance may be actually fairly hard to get. It's encouraging that 200 new antibody entries were created by authors, but it would have been good to see more data on the prevalence of other resources.With regard to the accuracy goal, a user study of a group of authors going through the workflow would have a useful addition to the study to help determine where the trouble spots for authors arise. Are they in the lookup on the SciCrunch portal or, as suggested for the case of MGI, at the registry itself?The identifiability pre-pilot results are consistent with the results we reached in our analysis of the Reproducibility Project: Cancer Biology set of papers. The authors assert that authors are able to perform the task accurately for software tools, but it is worth noting that software tools had low identifiability before and improved the least. Some discussion about why that is the case would be useful.Regarding utility, evidence is presented that RRIDs are highly useful because an RRID can be entered into Google Scholar and a link to a paper mentioning that RRID can be retrieved. This is perhaps the weakest part of the study, because the utility criteria were that a query could be constructed to show all the publications in which the resource were used could be found. They report data that only 174 identifiers could be found in Google Scholar, but it's not clear how this data was obtained, not exactly what the numbers refer to. The exact query used for each search index should be supplied & the results themselves should have been stored. Given the lack of detail around these points, it's hard to judge if it's actually possible to conduct these queries in a systematic fashion. The discussion about the failure of researchers to include RRIDs in a consistent form shows the current method to get RRIDs inserted in the literature is not sufficient for widespread use, and the level of support required by the SciCrunch site maintainers (100 queries for 10000 searches) also suggests that this approach would not scale. Given the lack of an API for Google Scholar, the approach used is understandable, and it's noted that the RRID Resolver page at SciCrunch does link to a PubMed query for papers mentioning the resource, but there is a text mining API available for ScienceDirect papers which would have been a better choice for assessing presence in ScienceDirect. Overall, the study would have been improved by the addition of a data scientist & must be judged to have failed on the utility criteria as established. This doesn't mean RRIDs are shown not to be useful; on the contrary, it's clear that RRIDs do allow a researcher to find the actual resource used, so in that respect they're quite useful indeed and the resolver page is very helpful. There's a case for broader utility to be made, as well, with the example of the \"Antibody data for this article\" article enhancement on ScienceDirect.It would also have been useful to see a more extended discussion of the sustainability of this project. For example, are journals to take this on as a means to add value, and if so, who pays for the maintenance of the SciCrunch resolver?Overall, the authors have presented good quality data on the feasibility and usability of asking authors to contribute RRIDs to publications and the publishing community should consider this study when considering ways to enrich the literature for text and data mining purposes.The takeaways for me are:Just asking authors via the information for authors pages or via one-off emails does not work.Letting authors enter RRIDs manually detracts from the utility of the identifiers. There's a big opportunity for machine learning approaches here to identify and suggest RRIDs at submission.Non-open access articles inhibit research by preventing the full-text indexing of their articles by search indexes.",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-134
|
https://f1000research.com/articles/4-1289/v1
|
18 Nov 15
|
{
"type": "Review",
"title": "Pharmacokinetic-pharmacodynamic relationship of anesthetic drugs: from modeling to clinical use",
"authors": [
"Valerie Billard"
],
"abstract": "Anesthesia is a combination of unconsciousness, amnesia, and analgesia, expressed in sleeping patients by limited reaction to noxious stimulations. It is achieved by several classes of drugs, acting mainly on central nervous system. Compared to other therapeutic families, the anesthetic drugs, administered by intravenous or pulmonary route, are quickly distributed in the blood and induce in a few minutes effects that are fully reversible within minutes or hours. These effects change in parallel with the concentration of the drug, and the concentration time course of the drug follows with a reasonable precision mathematical models based on the Fick principle.Therefore, understanding concentration time course allows adjusting the dosing delivery scheme in order to control the effects. The purpose of this short review is to describe the basis of pharmacokinetics and modeling, the concentration-effects relationship, and drug interactions modeling to offer to anesthesiologists and non-anesthesiologists an overview of the rules to follow to optimize anesthetic drug delivery.",
"keywords": [
"pharmacokinetic",
"pharmacodynamic",
"anesthetic",
"Anesthesia",
"anesthetic drugs"
],
"content": "Introduction\n\nAnesthesia is a complex state including several reversible therapeutic effects such as loss of consciousness and recall or lack of response to variable noxious stimulations coming from surgery or anesthetic management. Some effects are quantitative, such as electroencephalogram (EEG) or blood pressure changes, and the intensity of effects increases with the dose. Others are quantal (yes or no), such as being asleep or the absence of movement response to surgical incision. The probability of these quantal effects increases with the dose.\n\nThis is also true for adverse effects (such as hypotension, bradycardia, and respiratory depression), although they usually occur at higher doses than therapeutic effects. Therefore, drug dosages should be chosen to maintain the patient inside a therapeutic window, and dosing should be large enough to achieve therapeutic effects but small enough to avoid late recovery or adverse effects. The wider the therapeutic window, the safer the drug, but even for modern anesthetic drugs, this therapeutic window may be narrow in some patients, depending on age, physiological status, or drug combinations. Anesthetists should first target a window which they consider likely to be adequate for the patient and the procedure and then assess whether the anesthetic effect is at the level expected and titrate if this is not the case.\n\nActing on the central nervous system (CNS), which is a fatty structure, all anesthetic drugs are lipophilic. Consequently, every single dose administered follows a distribution process in the body, and the fraction of dose reaching the CNS competes with the biggest inactive fraction distributed in the blood, muscles, and fat at different rates. At the same time, the last fraction of the dose disappears irreversibly from the body through metabolism or excretion. For anesthetic procedures, lasting from a few minutes to a few hours, steady state is never reached. Consequently, the effects related to a dose will change over time because of this balance but, for most drugs, will be parallel to the concentration at the site of effect.\n\nUnderstanding the pharmacokinetics (PK) allows clinicians to adjust the delivery scheme in order to control the concentration at the site of effect in the present and in the future (control of recovery). Understanding pharmacodynamics (PD) (that is, the relationship between concentration and intensity of effects) helps in titrating anesthesia delivery according to individual needs and to successive surgical end-points.\n\nThe aim of this overview is to present the concepts describing this pharmacokinetic-pharmacodynamic (PKPD) relationship and their clinical implications. Although the PKPD relationship has also been described for volatile or local anesthetics in regional anesthesia, we will focus here on intravenous drugs, except for drug interactions.\n\nMany scientists have helped describe and validate these concepts, as shown in the references. In this short review, I may have forgotten some important names or keystone articles. I hope the authors will forgive me and will recognize their discoveries in the full picture.\n\n\nPharmacokinetics and modeling: compartmental models\n\nAfter giving a dose of an anesthetic drug (bolus or infusion) and drawing blood assays, scientists have observed that the time course of plasma concentration may be modeled by using a two- or three-compartment model with an acceptable precision1. This model can be fully characterized by three volumes of distribution, two distribution clearances, and one elimination clearance (or a bunch of six micro-constants describing exchanges; Figure 1). It is specific to the drug, independent of the dose, and linear: doubling the dose will double the plasma concentration at any time, and if two doses are given (for example, two boluses or bolus + infusion), the plasma concentration will be the sum of the concentrations resulting from each dose. This is called the “superposition principle”.\n\nCL1 = k10*V1, CL2 = k12*V1, CL3 = k13*V1, V2 = CL2/k21, V3 = CL3/k31. CL: clearance; k: micro-constant; V: distribution volume.\n\nPK models describe the time course of plasma concentration, estimated by using the drug doses delivered over time and patient characteristics such as weight, height, age, and gender. But the time course of effects is always delayed and attenuated compared with that of plasma concentration because the site of effect is not plasma but CNS (or muscles for muscle relaxants)2. To model the time course of effect, Sheiner and colleagues added the effect compartment as a fourth compartment to the PK model, having a negligible volume and assumed to be synchronized with effects3. The relationship between plasma and effect-site concentration may be described by a single time constant called ke0, which extends the PK to a PKPD model. At steady state, plasma and effect-site concentration are identical (Figure 1).\n\nWhereas early studies determined PK parameters separately for every patient and averaged them in a second step (which required many samples per patient during and after administration), most of the modeling since the 1990s used population analysis with mixed-effects modeling4. In this approach, samples from all patients are considered together. The modeling provides not only the typical values of the parameters but also an estimation of inter-individual and intra-individual variability of parameters and predicted concentration. Using all samples in the same fit requires a lower number of samples per patient.\n\nMoreover, this type of modeling allows clinicians to add physiological covariates to the model (such as weight, lean body mass [LBM], and age, as shown in Table 15–7), added as additive or scaling factors8, which may improve the fit and thus the prediction of the plasma or effect-site concentration.\n\nCL: clearance; H: height; LBM: lean body mass; V: volume; W: weight.\n\nFor all anesthetic drugs, one or several PKPD compartmental models have been published, most of them using a quantitative effect such as EEG to determine ke0. The models are implemented in simulation software available on the web:\n\n- Stanpump, Stelpump, or Ivasim: http://www.opentci.org\n\n- Rugloop: http://www.demed.be/rugloop.htm\n\n- Pkpdtools: http://www.pkpdtools.com\n\n- Tivatrainer: http://www.eurosiva.org/TivaTrainer/tivatrainer_main.htm\n\nAfter the drug, the patient characteristics, and the PK model are chosen, these software packages simulate the time course of plasma and effect-site concentration for any drug-delivery scheme. Some of them, such as Stanpump or Rugloop, can drive a syringe pump, combining the delivery data setup by the user and the events coming from the patient (such as venous line occlusion and empty syringe) to display past, present, and future predicted concentration. Their clinical use should be limited to research study with institutional review board approval and insurance, since none of the software packages mentioned above has a CE (“European Conformity”) mark or US Food and Drug Administration (FDA) approval for clinical use.\n\nBy displaying the time course of effect-site and plasma concentration, these software packages are wonderful teaching tools, showing, for example, how long a drug takes to achieve its maximal concentration after a bolus. Knowing this delay, called time to peak effect, which is specific for the anesthetic drug and independent of the dose, can help to anticipate the delivery toward the end-point requiring the effect or to synchronize drugs having different time to peak9. From the measured time to peak effect, it is also possible to estimate ke0 for a drug when only a PK but not a full PKPD model is available10.\n\nSoftware computes the context-sensitive half-time: that is, the delay to decrease by 50% from the current concentration11 or the decrement time (delay to decrease to a chosen concentration such as the concentration expected at recovery)9. When an infusion is terminated, both will depend upon the ratio of the clearance and the rate at which it re-equilibrates between the vascular space and peripheral sites. Because the distribution of anesthetic drugs to peripheral sites almost never reaches steady state and redistribution from the CNS remains a relevant phenomenon during recovery, these decrement times are much faster than the elimination half-life but increase with duration of delivery, especially in drugs with big volumes of distribution and accumulation. When two drugs with different accumulations (for example, propofol + opioid) are administered, decrement time displays may help to adjust the balance to shorten recovery delay12.\n\nBut the main clinical use of PK models in anesthesia is undoubtedly target-controlled infusion (TCI). In this delivery mode, the user does not choose the dose to give but directly selects the target plasma or effect-site concentration he or she wants to achieve and maintain13. The software computes by iterations the dose to achieve this “target” as fast as possible without overshoot, drives the pump, gets back the dose which has been given, and updates the infusion rate at regular intervals (1 to 10 sec). If the anesthetist decides to decrease the target concentration, the software stops the pump, continues predicting the concentration at regular intervals, and resumes the infusion as soon as the lower target concentration is reached. In case of interrupted drug delivery (because of an empty syringe or occlusion on the venous line), the software calculates the bolus dose necessary to regain the selected target concentration.\n\nSeveral manufacturers have implemented TCI software inside high-infusion-rate syringe pumps. They obtained CE mark approval and released a family of devices which today are clinically used worldwide except in North America, where it is still waiting for FDA approval. Released in 1996 for propofol14, TCI devices were later extended to sufentanil, remifentanil, and then alfentanil from 2003.\n\nA faster control of response to incision15 and a better hemodynamic stability have been described with propofol16 or remifentanil17 TCI as well as a good control of spontaneous ventilation18,19, and use in patient-controlled analgesia mode for postoperative analgesia is possible20. The benefits induced by TCI on the amount of drugs given or on recovery times were heterogeneous and depended widely on the decision criteria chosen in each study to adjust the dosing in both TCI and manual control groups21. Optimizing the combination between bolus and infusion helped reduce the variability of the concentration between patients22. Finally, the most undisputable benefits of TCI were a reduction of workload for the same quality of control21 and better comfort and understanding for anesthetists23.\n\nDespite the clinical usefulness of compartmental models, some limitations have been pointed out over the years.\n\nFirst, these models do not accurately describe the concentrations during the early phase of administration. The compartmental model assumes that any dose given is instantaneously and homogeneously diluted in the whole blood volume, which is unlikely24. Early distribution needs time and may depend on the location of the venous line and also on cardiac output. This influence may be displayed in models called front-end kinetics, where the volume of the central compartment is expressed by a time-dependent process rather than a constant25. Rarely implemented in drug-delivery software today, these models would provide a better control of the concentration in the first minutes after each delivered dose.\n\nSecond, most compartmental models assume that drugs are eliminated only from the central compartment24. If they are also eliminated from tissues such as cisatracurium or remifentanil, this assumption may underestimate elimination clearance and overestimate concentration. Third, the PKPD approach assumes that only the parent drug concentration is responsible for expected effects. When drugs have active metabolite at relevant concentrations, having different PK and different potency, more sophisticated models should be used, as shown with morphine26.\n\nFinally, there is a remaining debate about modeling and TCI in obese patients. Some models used LBM as a covariate6,27. This LBM is estimated from weight and height by using a historical formula established on moderately obese patients: body mass index (BMI) of less than 42 kg/m2 in men and less than 35 kg/m2 in women. Extrapolating this LBM formula to morbidly obese patients markedly underestimates LBM and results in a relevant underdosage for remifentanil or overdosage for propofol. To avoid this risk, TCI manufacturers have limited BMI to 42 and 35 kg/m2 and recommend compensating for this error by titration. In the future, solutions might use a more extended formula for LBM or choose a model with a scaling value of the real weight for the covariate rather than LBM.\n\n\nPharmacodynamic modeling and drug interactions\n\nControlling a predicted concentration, as allowed by PKPD modeling and TCI, is not a clinical goal per se. The goal is to control the effects in order to maintain the therapeutic effects while avoiding or minimizing the adverse overdosage effects. Given that the effects are related to the effect-site concentration, which is the fundamental rule for PKPD analysis, the next issue is to understand which concentration induces which effects.\n\nAs stated in the Introduction, the most-often considered effects in PKPD modeling are quantal effects (yes/no) such as loss of consciousness or absence of response to incision. They are not quantitative but their probability of occurring is. Other therapeutic effects, such as the neuromuscular blockade, are quantitative and measurable. Some adverse effects, such as the degree of respiratory depression, are also quantitative. Finally, some effects are not clinically relevant end-points but surrogate measurable end-points. They are highly correlated with therapeutic effects, i.e. the same concentration of a drug will always induce the same level of both clinical and surrogate effect. Thus, titration of delivery to maintain a “target” chosen value of the surrogate measure will also induce the linked value of the clinical effect. Examples are EEG-related parameters such as Bispectral Index (BIS™), which are often used to titrate anesthesia depth or to express anesthesia depth in concentration-effect modeling28. In all cases, the intensity (or the probability) of the effect increases with the concentration and achieves a maximal value for high concentrations29. Therefore, the relationship between effect-site concentration and each effect can be modeled in most cases by a sigmoidal model described by Hill more than 100 years ago for other drugs (Figure 2)30.\n\nGeneral formula: E=E0+Emax∗CγC50γ+Cγ, inhibitory effect: E=E0-Emax∗CγC50γ+Cγ, probability (as a percentage): P=0+100∗CγC50γ+Cγ.\n\nEach effect will have different Hill curve parameters (C50 and γ). For example, propofol C50 values have been estimated at 4 µg/ml for loss of consciousness, 10 µg/ml for skin incision, and 17 µg/ml for intubation31.\n\nIn clinical practice, the goal is usually not the C50 for therapeutic effects because half of the patients would react at that level; instead, the goal is around C95 (and the probability of no response is around 95%). A higher probability would require a much higher concentration in this very flat part of the Hill curve and would induce a risk of adverse effects and recovery delay. Conversely, the aim is around C50 for EEG effects because the depth of anesthesia inducing maximal EEG effects (flat EEG) requires much higher concentrations than those necessary in clinical practice and the EEG-derived parameters have been designed to offer the maximal sensitivity (steeper part of the Hill curve) in the clinical utility range of concentrations32.\n\nTo titrate anesthesia to individual needs, the main components of general anesthesia may be split between hypnosis (amnesia and loss of verbal contact), analgesia (limited response to noxious stimulations by movement or autonomic nervous system activation), and muscle relaxation.\n\nMuscle relaxation is fully ensured by neuromuscular blocking agents, and modeling using Hill’s model is simple.\n\nFor both other components, no anesthetic drug induces specifically one or the other, but all have a predominant effect on one component and some interaction with other drugs on the other. In other words, opioids (fentanyl, alfentanil, sufentanil, and remifentanil) are mainly analgesics, and huge concentrations induce only a weak sedative effect and moderately enhance the effect of hypnotic drugs on loss of consciousness. Intravenous hypnotics such as propofol, thiopentone, etomidate, or midazolam are mainly hypnotics with almost no analgesic effect when given alone but offer a synergistic interaction with opioids on the analgesia component (that is, the resulting effect is much more intense than the sum of the effects of each drug given alone).\n\nMany studies looked at propofol and opioids33,34 or volatile anesthetics and opioids35 or three drugs including midazolam36 and described their interactions on hypnosis or analgesia components. They focused on C50 and graphically showed the synergism between hypnotic and opioid on a two-dimensional isobole. This interaction may also be modeled by expressing the effect as a linear combination of drug A concentration, drug B concentration, and the product of drug A and drug B concentrations (effect = α.[A] + β.[B]+ γ.[A].[B]), also called the Greco model. The synergism or antagonism is given by the value of γ37.\n\nIn 2000, Minto and colleagues proposed a response surface approach38. They suggested that any drug combination can be considered a virtual “new” drug of which the effect follows a sigmoidal Emax model. The new drug concentration is defined by the ratio of the concentrations of each single drug, each of them weighted by its potency (C50).\n\nThe whole model can be displayed on a three-dimensional graph having drug A and B concentrations as x- and y-axis and effect as z-axis (Figure 3, top). From drug A alone (ratio B/(A+B) = 0) to drug B alone (B/(A+B) = 1), every ratio corresponds to a radial sigmoidal curve. Therefore, the two-dimensional C50 isobole described earlier is a particular horizontal slice of the response surface.\n\n(Top) Minto response surface. (Bottom) Bouillon indirect model.\n\nBoth the Greco and the Minto models assume that the effect is achieved by each single drug. However, in clinical practice, suppression of movement response to surgical stimulation requires high concentrations of propofol whereas loss of consciousness is achieved only at extremely high concentrations of opioid.\n\nTherefore, Bouillon and colleagues proposed another model based on the fact that opioids act not only on the brain but also on ascending neuropathways39. The authors assumed that the control of pain at that level decreases the ascending afferent stimuli going up to the brain and enhances the effect of hypnotics on the brain (Figure 3, bottom).\n\nThis hierarchical model focuses on the reduction of the C50 of propofol or volatile anesthetics by opioids to achieve unconsciousness and to suppress response to stimulation but does not assume that opioids on one hand and propofol or volatile anesthetics on the other hand have the same effect. In fact, the hierarchical model by Bouillon and colleagues is a simplification of the Greco model40.\n\nBoth models, the empirical one by Minto and hierarchical one by Bouillon express the predicted effect for the whole range of concentrations (not only the concentrations studied), based on a limited number of measures per patient using population analysis29. The hierarchical model seemed to describe the reality more closely, possibly because it is closer to anatomical sites and the mechanism of actions41.\n\nFor clinical use, the whole drug interaction model is too difficult to read as a response surface but may be restricted to a two-dimensional family of isoboles showing 50% and 90% probabilities of response to standardized noxious and non-noxious stimuli. The patient’s pharmacological status, based on estimated effect-site opioid and hypnotic drug concentrations, can be displayed in relation to these isoboles in real time as well as in the past and the near future (Figure 4). This display, designed to help physicians to adjust drug delivery, may be useful to predict the response to noxious stimulations, whereas EEG monitoring predicted better the degree of sedation42.\n\nExample of the SmartPilot View™ device (Dräger Medical, Lübeck, Germany). Dark grey area: 50%–90% probability of no motor response to incision. Light grey area: 50%–90% probability of no response to verbal command. White dot: current position of the patient. White arrow: future position in the next 10 minutes without changes in the dosing. White line: past pathway of the patient status since induction. During surgery, this display should complete clinical evaluation: if a patient is responding, it suggests that he or she should be shifted to a deeper anesthesia isobole until the noxious stimulation decreases.\n\n\nFuture developments of pharmacokinetic-pharmacodynamic relationship\n\nOne criticism of TCI is that it is based on the typical population values of the parameters, whereas every patient may differ from this typical value regarding the PK or the PD of the drug, even with models including physiological covariates, or may vary over time from his or her own average PKPD model. If the plasma concentration (or the effect resulting from this concentration) can be measured and compared with the population value, a Bayesian algorithm can adjust the model to make it closer to the patient studied. Initially described with alfentanil and measured concentrations43, this customized PKPD modeling has been applied to rocuronium with repeated measures of effect44. The authors showed that, after two measures, only minor changes in the PK model were observed, suggesting that the intra-individual variability is low compared with inter-individual variability.\n\nIn the future, this kind of algorithm may be used in many contexts, such as to adapt propofol PK to online measured concentrations or to real-time EEG effect, especially in patients who are likely very far from the population model (such as those with cardiac failure, obesity, or various organ failures in intensive care).\n\nClosed loop is often compared to autopilot in airplanes. After a target value for a quantitative effect of anesthetic drugs is defined, a dedicated algorithm repeatedly compares the measured value of this effect to the target and adjusts the drug delivery to achieve and maintain the target. Outside of anesthetic drug delivery, closed loop has been proposed to adjust antihypertensive drugs or controlled ventilation.\n\nIn anesthesia, several systems have been developed in the last 30 years, and all of them are still prototypes without a CE mark or FDA approval for clinical use. They drive either muscle relaxants based on neuromuscular blocking agent monitoring or hypnotic delivery based on EEG-derived parameters which have shown a reasonable performance to estimate depth of anesthesia, whereas quantitative monitoring of analgesia and its specificity are still debated.\n\nThe simplest algorithm is probably the industry proportional-integral-derivative controller, in which the rate of infusion is adjusted on the basis of the effect error (measured–predicted effect), its time course over time, and its trend. It requires no PKPD model but may oscillate around the target value, is delayed in case of abrupt variations, and may become erratic if no measured value is available because of artifacts45.\n\nProbably safer is the model-based controller, in which the drug is delivered according to a full PKPD model as described above. In this case, the error on the effect does not change the rate of infusion but instead changes the model parameters45, achieving a particular case of Bayesian process. With this approach, if the measure of effect is disturbed after a few measures, the model will be already adapted to minimize inter-individual variability and the accuracy of control will remain sufficient.\n\nClinically, closed loop has demonstrated an improvement in the duration of control (increasing the duration with desired BIS™ of EEG values from 70% to 90% of the anesthesia time)46 and in the quality of control (also improving blood pressure stability)47. All these clinical results were combined with a marked reduction of the workload48. Many applications can be imagined in both general anesthesia and sedation in the operating room, intensive care, and transport of patients.\n\n\nConclusions\n\nPKPD analysis offers an irreplaceable tool to understand and model the pharmacology of drugs and fits especially well in intravenous anesthetics. From theoretical properties, described using sophisticated mathematical techniques, it has been converted to safe and user-friendly clinical applications in order to provide a more stable but adjustable level of anesthesia. Displaying prediction, it does not replace but rather completes the clinical assessment of the patient. Future perspective will likely pursue this customization of drug delivery.",
"appendix": "Competing interests\n\n\n\nIn the last 5 years, the author has served as a consultant for Dräger Medical (Lübeck, Germany) and Fresenius Medical Care (Bad Homburg, Germany) and as a speaker at symposia for Covidien (Dublin, Ireland), AbbVie (North Chicago, IL, USA), Dräger Medical, Fresenius Medical Care, and Baxter (Deerfield, IL, USA).\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHull CJ: Models with more than one compartment. In: Hull CJ, ed. Pharmacokinetics for anaesthesia, Oxford: Butterworth-Heinemann, 1991; 170–86.\n\nFuseau E, Sheiner LB: Simultaneous modeling of pharmacokinetics and pharmacodynamics with a nonparametric pharmacodynamic model. Clin Pharmacol Ther. 1984; 35(6): 733–41. PubMed Abstract | Publisher Full Text\n\nSheiner LB, Stanski DR, Vozeh S, et al.: Simultaneous modeling of pharmacokinetics and pharmacodynamics: application to d-tubocurarine. Clin Pharmacol Ther. 1979; 25(3): 358–71. PubMed Abstract\n\nMould DR, Upton RN: Basic concepts in population modeling, simulation, and model-based drug development-part 2: introduction to pharmacokinetic modeling methods. CPT Pharmacometrics Syst Pharmacol. 2013; 2(4): e38. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nMarsh B, White M, Morton N, et al.: Pharmacokinetic model driven infusion of propofol in children. Br J Anaesth. 1991; 67(1): 41–8. PubMed Abstract | Publisher Full Text\n\nSchnider TW, Minto CF, Gambus PL, et al.: The influence of method of administration and covariates on the pharmacokinetics of propofol in adult volunteers. Anesthesiology. 1998; 88(5): 1170–82. PubMed Abstract | Publisher Full Text\n\nCoppens MJ, Eleveld DJ, Proost JH, et al.: An evaluation of using population pharmacokinetic models to estimate pharmacodynamic parameters for propofol and bispectral index in children. Anesthesiology. 2011; 115(1): 83–93. 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PubMed Abstract | Publisher Full Text\n\nVuyk J, Mertens MJ, Olofsen E, et al.: Propofol anesthesia and rational opioid selection: determination of optimal EC50-EC95 propofol-opioid concentrations that assure adequate anesthesia and a rapid return of consciousness. Anesthesiology. 1997; 87(6): 1549–62. PubMed Abstract | Publisher Full Text\n\nEgan TD: Target-controlled drug delivery: progress toward an intravenous \"vaporizer\" and automated anesthetic administration. Anesthesiology. 2003; 99(5): 1214–9. PubMed Abstract | Faculty Opinions Recommendation\n\nGlen JB: The development of 'Diprifusor': a TCI system for propofol. Anaesthesia. 1998; 53(Suppl 1): 13–21. PubMed Abstract | Publisher Full Text\n\nServin FS: TCI compared with manually controlled infusion of propofol: a multicentre study. Anaesthesia. 1998; 53(Suppl 1): 82–6. PubMed Abstract | Publisher Full Text\n\nPassot S, Servin F, Pascal J, et al.: A comparison of target- and manually controlled infusion propofol and etomidate/desflurane anesthesia in elderly patients undergoing hip fracture surgery. Anesth Analg. 2005; 100(5): 1338–42. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDe Castro V, Godet G, Mencia G, et al.: Target-controlled infusion for remifentanil in vascular patients improves hemodynamics and decreases remifentanil requirement. Anesth Analg. 2003; 96(1): 33–8. PubMed Abstract | Publisher Full Text\n\nLallo A, Billard V, Bourgain JL: A comparison of propofol and remifentanil target-controlled infusions to facilitate fiberoptic nasotracheal intubation. Anesth Analg. 2009; 108(3): 852–7. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMoerman AT, Herregods LL, De Vos MM, et al.: Manual versus target-controlled infusion remifentanil administration in spontaneously breathing patients. Anesth Analg. 2009; 108(3): 828–34. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSchraag S, Kenny GN, Mohl U, et al.: Patient-maintained remifentanil target-controlled infusion for the transition to early postoperative analgesia. Br J Anaesth. 1998; 81(3): 365–8. PubMed Abstract | Publisher Full Text\n\nLeslie K, Clavisi O, Hargrove J: Target-controlled infusion versus manually-controlled infusion of propofol for general anaesthesia or sedation in adults. Cochrane Database Syst Rev. 2008; (3): CD006059. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHu C, Horstman DJ, Shafer SL: Variability of target-controlled infusion is less than the variability after bolus injection. Anesthesiology. 2005; 102(3): 639–45. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nEcoffey C, Viviand X, Billard V, et al.: [Target controlled infusion (TCI) anesthesia using propofol. Assessment of training and practice in the operating room]. Ann Fr Anesth Reanim. 2001; 20(3): 228–45. PubMed Abstract | Publisher Full Text\n\nFisher DM: (Almost) everything you learned about pharmacokinetics was (somewhat) wrong! Anesth Analg. 1996; 83(5): 901–3. PubMed Abstract | Publisher Full Text\n\nKrejcie TC, Avram MJ: What determines anesthetic induction dose? It's the front-end kinetics, doctor! Anesth Analg. 1999; 89(3): 541–4. PubMed Abstract | Publisher Full Text\n\nMazoit JX, Butscher K, Samii K: Morphine in postoperative patients: pharmacokinetics and pharmacodynamics of metabolites. Anesth Analg. 2007; 105(1): 70–8. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMinto CF, Schnider TW, Egan TD, et al.: Influence of age and gender on the pharmacokinetics and pharmacodynamics of remifentanil. I. Model development. Anesthesiology. 1997; 86(1): 10–23. PubMed Abstract | Publisher Full Text\n\nStanski DR, Shafer SL: Measuring depth of anesthesia. In: Miller RD, ed. Miller's Anesthesia, 6 Edition. Churchill Livingstone, 2004; 1227–64.\n\nUpton RN, Mould DR: Basic concepts in population modeling, simulation, and model-based drug development: part 3-introduction to pharmacodynamic modeling methods. CPT Pharmacometrics Syst Pharmacol. 2014; 3(1): e88. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolford NH, Sheiner LB: Understanding the dose-effect relationship: clinical application of pharmacokinetic-pharmacodynamic models. Clin Pharmacokinet. 1981; 6(6): 429–53. PubMed Abstract | Publisher Full Text\n\nKazama T, Ikeda K, Morita K: Reduction by fentanyl of the Cp50 values of propofol and hemodynamic responses to various noxious stimuli. Anesthesiology. 1997; 87(2): 213–27. PubMed Abstract | Publisher Full Text\n\nRampil IJ: A primer for EEG signal processing in anesthesia. Anesthesiology. 1998; 89(4): 980–1002. PubMed Abstract | Publisher Full Text\n\nVuyk J, Lim T, Engbers FH, et al.: The pharmacodynamic interaction of propofol and alfentanil during lower abdominal surgery in women. Anesthesiology. 1995; 83(1): 8–22. PubMed Abstract | Publisher Full Text\n\nKazama T, Ikeda K, Morita K: Reduction by fentanyl of the Cp50 values of propofol and hemodynamic responses to various noxious stimuli. Anesthesiology. 1997; 87(2): 213–27. PubMed Abstract | Publisher Full Text\n\nKatoh T, Ikeda K: The effects of fentanyl on sevoflurane requirements for loss of consciousness and skin incision. Anesthesiology. 1998; 88(1): 18–24. PubMed Abstract\n\nShort TG, Plummer JL, Chui PT: Hypnotic and anaesthetic interactions between midazolam, propofol and alfentanil. Br J Anaesth. 1992; 69(2): 162–7. PubMed Abstract | Publisher Full Text\n\nBillard V, Moulla F, Bourgain JL, et al.: Hemodynamic response to induction and intubation. Propofol/fentanyl interaction. Anesthesiology. 1994; 81(6): 1384–93. PubMed Abstract | Publisher Full Text\n\nMinto CF, Schnider TW, Short TG, et al.: Response surface model for anesthetic drug interactions. Anesthesiology. 2000; 92(6): 1603–16. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBouillon TW, Bruhn J, Radulescu L, et al.: Pharmacodynamic interaction between propofol and remifentanil regarding hypnosis, tolerance of laryngoscopy, bispectral index, and electroencephalographic approximate entropy. Anesthesiology. 2004; 100(6): 1353–72. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBouillon TW: Hypnotic and opioid anesthetic drug interactions on the CNS, focus on response surface modeling. Handb Exp Pharmacol. 2008; (182): 471–87. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nHeyse B, Proost JH, Schumacher PM, et al.: Sevoflurane remifentanil interaction: comparison of different response surface models. Anesthesiology. 2012; 116(2): 311–23. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLuginbühl M, Schumacher PM, Vuilleumier P, et al.: Noxious stimulation response index: a novel anesthetic state index based on hypnotic-opioid interaction. Anesthesiology. 2010; 112(4): 872–80. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nMaitre PO, Stanski DR: Bayesian forecasting improves the prediction of intraoperative plasma concentrations of alfentanil. Anesthesiology. 1988; 69(5): 652–9. PubMed Abstract | Publisher Full Text\n\nMotamed C, Devys JM, Debaene B, et al.: Influence of real-time Bayesian forecasting of pharmacokinetic parameters on the precision of a rocuronium target-controlled infusion. Eur J Clin Pharmacol. 2012; 68(7): 1025–31. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nDumont GA, Ansermino JM: Closed-loop control of anesthesia: a primer for anesthesiologists. Anesth Analg. 2013; 117(5): 1130–8. PubMed Abstract | Publisher Full Text\n\nDussaussoy C, Peres M, Jaoul V, et al.: Automated titration of propofol and remifentanil decreases the anesthesiologist's workload during vascular or thoracic surgery: a randomized prospective study. J Clin Monit Comput. 2014; 28(1): 35–40. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nStruys MM, De Smet T, Versichelen LF, et al.: Comparison of closed-loop controlled administration of propofol using Bispectral Index as the controlled variable versus “standard practice” controlled administration. Anesthesiology. 2001; 95(1): 6–17. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLiu N, Chazot T, Genty A, et al.: Titration of propofol for anesthetic induction and maintenance guided by the bispectral index: closed-loop versus manual control: a prospective, randomized, multicenter study. Anesthesiology. 2006; 104(4): 686–95. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation"
}
|
[
{
"id": "11273",
"date": "18 Nov 2015",
"name": "Thomas Schnider",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11274",
"date": "18 Nov 2015",
"name": "Harald Ihmsen",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11275",
"date": "18 Nov 2015",
"name": "Martin Luginbuhl",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11276",
"date": "18 Nov 2015",
"name": "Carl Rosow",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1289
|
https://f1000research.com/articles/4-1288/v1
|
18 Nov 15
|
{
"type": "Review",
"title": "A dynamic architecture of life",
"authors": [
"Beatrix P. Rubin",
"Jeremy Brockes",
"Brigitte Galliot",
"Ueli Grossniklaus",
"Daniel Lobo",
"Marco Mainardi",
"Marie Mirouze",
"Alain Prochiantz",
"Angelika Steger",
"Jeremy Brockes",
"Brigitte Galliot",
"Ueli Grossniklaus",
"Daniel Lobo",
"Marco Mainardi",
"Marie Mirouze",
"Alain Prochiantz",
"Angelika Steger"
],
"abstract": "In recent decades, a profound conceptual transformation has occurred comprising different areas of biological research, leading to a novel understanding of life processes as much more dynamic and changeable. Discoveries in plants and animals, as well as novel experimental approaches, have prompted the research community to reconsider established concepts and paradigms. This development was taken as an incentive to organise a workshop in May 2014 at the Academia Nazionale dei Lincei in Rome. There, experts on epigenetics, regeneration, neuroplasticity, and computational biology, using different animal and plant models, presented their insights on important aspects of a dynamic architecture of life, which comprises all organisational levels of the organism. Their work demonstrates that a dynamic nature of life persists during the entire existence of the organism and permits animals and plants not only to fine-tune their response to particular environmental demands during development, but underlies their continuous capacity to do so. Here, a synthesis of the different findings and their relevance for biological thinking is presented.",
"keywords": [
"computational biology",
"development",
"limb regeneration",
"neuroplasticity",
"plant epigenetics",
"tissue homeostasis",
"repair regeneration"
],
"content": "Introduction\n\nLife from its unicellular form to the most complex organism is increasingly understood as being composed of molecular and cellular components, which are inherently dynamic in nature and undergo specific changes in response to alterations in the internal or external environment of the organism. This insight has forced the re-evaluation of established concepts, such as cellular differentiation, as certain processes, formerly considered to occur only during development, continue life-long or can be re-induced to different degrees in the adult organism. It has also drawn anew the attention to the heritability of acquired changes and adaptations and their possible contribution to the evolution of biological systems (Grossniklaus et al., 2013; Jablonka & Lamb, 2014). From a historic perspective, one might consider the turn of the 20th to the 21st century as a period of profound conceptual transformation occurring in parallel in different fields of biological research and converging towards an increasingly dynamic conception of life (Rubin, 2009; Sánchez Alvarado & Yamanaka, 2014).\n\nThe perception that an understanding of the basic organisation of life is emerging, which incorporates novel insights about changeability, adaptation, and heritability, has been taken as an incentive to organise an interdisciplinary workshop addressing these themes. The event took place at the Accademia dei Lincei in Rome in May 2014 and was hosted by the president of the academy, Lamberto Maffei. It is the result of a collaboration between the Collège de France, Paris and the Collegium Helveticum of the University and ETH of Zurich. The title of the event - “A dynamic architecture of life?” - was chosen to reflect the ambitions of the meeting. The juxtaposition of a wide spectrum of different scientific perspectives was sought in order to pinpoint important commonalities, but also decisive differences between the different models and empirical approaches, which are being used to investigate the dynamic nature of life and its evolution.\n\nWith the understanding in mind that it is in relation to a particular empirical context that concepts in biological research acquire a specific meaning, the aim of this workshop has not been to work towards an unambiguous and comprehensive definition of the terms in use. Instead, the interest has been in the knowledge gained about important characteristics of a dynamic architecture of life that become apparent, if the empirically substantiated articulations of the relevant concepts are presented and are discussed by experts from different fields, who usually do not interact in the framework of a workshop. The represented fields included plant genetics, evolutionary biology, developmental biology, regeneration of simple and complex organisms, the development of the vertebrate nervous system, and computational biology. The presentations on biological research were complemented by a commentary from a historian of science. The discussions produced a rich and very differentiated picture on the multiple ways the dynamic complexity of life has arisen during evolution. In reflection of the theme of the conference, one can state that all contributions to this review ascertain that an understanding of plant and animal physiology as largely static, determined by fixed set points and unchangeable structures, is no longer considered valid. In contrast, ample evidence is provided for a persisting potential of organisms to adapt to novel functional requirements life-long. This potential is tightly regulated and differentially activated by a wide range of external and internal signals and is being discussed as subject to selection during evolution. In the following, you will find short introductions to the different topics, followed by reviews of the different fields, and a general discussion.\n\n\nEpigenetics\n\nDuring the past decades, DNA, which might be considered the central element of a dynamic architecture of life, has been documented as a site of both targeted and stochastic changes. These changes, as presented by Ueli Grossniklaus and Marie Mirouze, include genetic and epigenetic alterations that can also be interrelated, including the reorganization of the genome by transposable elements and a wide spectrum of epigenetic modifications, such as the methylation of cytosine in DNA or various modifications of the proteins associated with DNA. The epigenome is considered an important intermediate step between the genetic information and its expression, permitting differential interpretation of the primary sequence in different cells during development. The frequency and reversibility of epigenetic changes make them attractive candidates to mediate relatively rapid adaptation of plants to changes in their environment. However, in which ways these epigenetic changes are transmitted and influence the evolutionary trajectory of a species, be it in plants or in animals, is still a matter of debate and will require further empirical work.\n\n\nRegeneration\n\nThe capacity for regeneration is present amongst species belonging to a broad range of phyla. This widespread occurrence implies that - over evolutionary time – natural selection has favoured species capable of partial or complete regeneration in response to various circumstances. Jeremy Brockes presents the salamander as an instructive system, which permits to study the conditions and characteristics of limb regeneration and their correlation to particular developmental features. In respect to a dynamic architecture of life it is noteworthy that no senescent cells persist after repeated limb ablation, due to a specific mechanism of surveillance, permitting the repetitive reconstitution of a range of tissues. This relates the phenomenon of limb regeneration to the homeostatic and developmental plasticity of the fresh water polyp hydra, whose different contributions to the maintenance of the body architecture are being discussed by Brigitte Galliot.\n\n\nPlasticity\n\nMarco Mainardi presents the regulation of energy metabolism in mice as much more dynamic than previously understood, reflecting the plasticity of the developing and the adult nervous system. His analysis underscores the interplay of physiological, social, and cognitive demands in shaping the functionality of the hypothalamus, as a key regulator of lipid metabolism. Accordingly, possible strategies to alter the plasticity of specific brain areas for therapeutic purposes are being discussed. Importantly, a critical period of heightened plasticity and sensitivity to external signals during development seems one amongst several features of a dynamic neuronal architecture common to both cortical and subcortical structures of the mammalian brain. The critical period of visual development has been very instructive in dissecting the interplay “between nature and nurture” in shaping the visual cortex. Alain Prochiantz points out that the homeodomain transcription factor Otx2 is both necessary and sufficient for initiating and terminating the critical period of increased malleability of the visual cortex. The possibility to modify the onset and termination of the critical period by Otx2 and other factors intrinsic and extrinsic to the nervous system reflects the fact that plasticity constitutes the tightly controlled default state of the adult cortex.\n\n\nComputational biology\n\nThe opportunities, but also the demanding tasks to realistically model the different aspects of a dynamic architecture of life based on a range of empirical data, become apparent in the contributions by Daniel Lobo and Angelika Steger. Lobo uses the possibility of reverse engineering to group and order a large quantity of existing empirical data in an encompassing model on regenerative events in the planarian flatworm. This model is automatically inferred from experiments described in an unambiguous mathematical language. It can be used to predict the results of novel interventions in the modelled organism, which can then be validated by further empirical work. Steger applies modelling to analyse information storage in the brain. Her work leads her to propose that variability of parameters representing neuronal functions has important ramifications both in terms of increased speed of signal transmission, but also heightened stability of the signalling processes within recurrent networks, as well as lowering energy demands. Such models complement data gained from empirical work by addressing the relevance of specific factors and parameters in processes of regeneration and plasticity.\n\n\nEpigenetic variation within and across generations (Ueli Grossniklaus)\n\nOver the last decade the term “epigenetics” has appeared more and more often in the popular press and has been linked to effects on health or behaviour that have been claimed to depend on particular circumstances that our parents or grandparents had experienced. Such reports of “non-genetic” inheritance have refuelled the debate on the role of “nurture versus nature”. In this contribution it will be discussed in which ways epigenetic regulation might contribute to a dynamic architecture of life by rendering the nature of DNA and its functions more variable.\n\nSo what is epigenetics? In fact, it is an old concept in developmental biology dating back to the 1940’s that has gained great momentum over the last 15 years, as many of the underlying mechanisms have been unravelled. Originally, Waddington defined epigenetics as ‘‘the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being’’ (Waddington, 1942; Waddington, 1968). In the era of molecular biology, the focus has shifted to the hereditary material and a commonly used definition states that epigenetics concerns the “study of mitotically and/or meiotically heritable changes in gene expression that occur without a change in DNA sequence” (Riggs et al., 1996). Thus, in principle, epigenetic regulation plays a role at two levels. First, it is involved in development – within a generation – leading to the specification of cells and assuring the faithful inheritance of their differentiated state through a series of mitotic cell divisions. Second, epigenetic states can be inherited meiotically – across generations – endowing the progeny with information gathered during the lifetime of the parents. While the former is widely accepted and contributes – due to the metastable nature of epigenetic variation – to both developmental stability and plasticity, the relevance of the latter is being debated. Most evolutionary biologists do not attribute a role to epigenetic variation in evolutionary processes. However, the more rapidly changing nature of epigenetic variation makes it particularly attractive for adaptations to a changing environment.\n\nDespite the great attention that transgenerational epigenetic inheritance has gained in the popular press, there have been only a few – often controversially discussed – cases documented in mammals. Environmental influences that can be traced back to parents or grandparents are widespread, but they might be mediated by a wide variety of mechanisms and the underlying nature may or may not necessarily be epigenetic. Controversies arise because many authors do not distinguish between (grand)parental effects and true transgenerational inheritance, which must be demonstrated to occur in several generations after the exposure to a particular environmental condition. For instance, if a pregnant mouse is exposed to a particular treatment, e.g. a specific nutrient regime, not only the mother but also the fetus (F1 generation) and the germ line cells set aside in this foetus that will give rise to future offspring (F2 generation) will be affected by the same treatment. Thus, effects in the children and grandchildren of the treated mouse are not considered as evidence for transgenerational epigenetic inheritance and are now often referred to as “intergenerational”. In cases where effects on subsequent generations were studied in detail, the effects disappeared in the great-grandchildren (F3 generation), leading to the conclusion that they were not transgenerational in nature (Radford et al., 2014; Waterland et al., 2007). Nevertheless, there are some intriguing cases of potential transgenerational effects in humans (Pembrey, 2010) that were identified in epidemiological studies, whose underlying nature has not yet been investigated further.\n\nIn contrast to the scarce data in mammals, there are many well-studied examples of stably inherited epialleles in round worms (Guerin et al., 2014) and plants (Hirsch et al., 2012; Paszkowski & Grossniklaus, 2011). While the (meta)stable inheritance of epiallelic variants is firmly established in plants, it is not clear whether specific treatments (e.g. heat stress) cause epigenetic changes that can be passed on to the next generation and lead to offspring that is better adapted to the specific conditions the parental generation has been exposed to. While there are several publications reporting adaptation to specific biotic and abiotic stresses in the progeny of treated plants, these experiments have been difficult to reproduce and such an adaptive memory does not seem to be a general response to stress in plants (Hirsch et al., 2012; Pecinka & Mittelsten Scheid, 2012). However, in plants new epigenetic variation is generated in each generation, e.g. by spontaneous changes in cytosine methylation (Becker et al., 2011; Schmitz et al., 2011), and it seems that – just like genetic variation – epigenetic variation can also be selected upon (Heichinger and Grossniklaus, unpublished). As epigenetic variation in a population is much higher than genetic variation, epialleles may indeed contribute to the rapid adaptation of plants to novel environmental conditions, provided the epigenetic change is stable enough. Indeed, there are yet unpublished observations that plants of the genus Diplacus can change the colour, morphology, and pollinator under certain environmental conditions and that this change is heritable over several generations (Baumberger and Grossniklaus, unpublished). Since this epigenetic switch leads to a change in pollinator, and thus establishes a reproductive barrier, it will affect the evolutionary trajectory of this species, suggesting a role of epigenetics in ecology and evolution (Hirsch et al., 2012).\n\nAlthough many of the mechanisms underlying epigenetic regulation have been unravelled over the last 15 years, the role and importance of epigenetics in ecology and evolution still remains unclear. In the future, it will be interesting to see whether non-genetic information influenced by the environment indeed plays a role in adaptation and how important and wide-spread such processes are in comparison to genetic ones.\n\n\nGenome plasticity and the underestimated role of mobile genetic elements (Marie Mirouze)\n\nThe genome has long been seen as a static entity, transmitted throughout generations with high fidelity, and only rearranged in sexual organisms through the mechanisms of crossing-over occurring during meiotic recombination. The discovery made by Barbara Mc Clintock that small pieces of the genome (called Transposable Elements, TEs) could be mobilised and, upon stress, induce chromosomal rearrangements in the absence of meiotic recombination had broken the dogma in the early fifties (McClintock, 1984). Later the extensive genome sequencing projects have revealed that TEs make up most of sequenced eukaryotic genomes, from 45% in the human to up to 85% in the maize genome, revealing that big genomes reflect a high amount of TEs and not a difference in gene content.\n\nThe studies of genome expression have revealed another layer of complexity in the last decades, as a code on top of the genomic code, hence called the epigenome (epi=above). Conrad Waddington had been the first one to coin the term epigenetics to describe how the action of genes could give rise to a landscape of different developmental possibilities that would determine the cell fate during embryo development (Slack, 2002). Although his concept was visionary, he was not aware of the molecular details of what would become the focus of research of a large scientific community decades later. The numerous studies on epigenetic marks, chemical modifications of the DNA sequence itself, or on the histone proteins around which the DNA is wrapped, have shown that the epigenome is at the interface between the code (the genome) and its function (expression). Mammalian X chromosome inactivation (Chaligné & Heard, 2014) or flowering time control in plants (Song et al., 2012) are famous examples in which expression of genes are prevented by epigenetic mechanisms during development. Because of their plurality and versatility these epigenetic marks can vary in different cells and tissues within one organism, hence allowing different interpretations of the genomic code during development but also upon stress. Recent studies have further contributed to demonstrate that the manipulation of the epigenome in wild-type organisms (plants in this case) leads to the release of the epigenetic silencing of TEs and to their active mobility in the genome across generations (Mirouze et al., 2009). As expected for a biological phenomenon, there is not only one, but several different and complementary mechanisms that contribute to TE silencing under normal conditions. Hence the epigenome can be seen as a guardian of the genome, preventing uncontrolled TE mobility that would affect the host fitness. Nevertheless, TEs have been successful in proliferating in most eukaryotic genomes, and their role, if not yet clear at the organismal level, has to be envisioned at the evolutionary scale. Indeed owing to their inherent ability of movement, TEs represent a molecular force in evolution. Three specific features of TEs have been shown to contribute to genome evolution. Firstly some essential functions (DNA sequence recognition through transposase, insertion in the genome through integrase) have been co-opted or domesticated by different host genomes and have led to the evolution of important molecular functions such as the telomerase in eukaryotes or the apparition of adaptive immunity (CRISPR in prokaryotes and V(D)J recombination in mammals are good examples, see Koonin & Krupovic, 2015). Secondly, as transcribed entities, TEs possess regulatory cis elements that can be amplified in the genome and have an impact on neighbouring genes leading to the rewiring of regulatory networks (Feschotte, 2008). This is best exemplified in the apparition of the placental function in mammals (Lynch et al., 2011). Finally, the mobility of TEs themselves has been shown to contribute to genome plasticity in the brains of mammals (Baillie et al., 2011). The causes and consequences of the mobility of some TEs in the human brain are not yet clear. However, these studies illustrate another break in the dogma of genomic stability: multicellular organisms are not composed of one but of a myriad of slightly different genomes, the germline genome being most likely the most stable and best protected against TE mobility. Cancer cells with uncontrolled genome plasticity represent an extreme, and the role of TEs in their proliferation is just being investigated (Lee et al., 2012). In normal cells, genome plasticity might have a role in development but this is yet to be discovered. Single cell sequencing together with targeted sequencing of active TEs should allow us to better appreciate the extent of genome plasticity and the role played by TEs.\n\n\nMechanism and evolution in vertebrate limb regeneration (Jeremy P. Brockes)\n\nThe ability to regenerate complex structures in an adult animal is indicative of an underlying dynamic architecture of life. In vertebrates, the most extensive repertoire of regeneration is found in the urodele amphibians or salamanders. This includes most notably the limbs, and salamanders are the only adult tetrapods able to regenerate these appendages. Studies on the mechanism of limb regeneration have considered aspects such as the reprogramming of differentiated cells, and the requirement for concomitant regeneration of peripheral nerves (Brockes & Gates, 2014). These features of a dynamic architecture have been of considerable mechanistic interest, as well as providing valuable pointers for extending the possibilities of mammalian regeneration. The central, and most difficult, questions are concerned with limb regeneration as an evolutionary variable. For example, should limb regeneration be regarded as an ancestral property of tetrapods, that was lost in anurans and amniotes, or did it evolve as a new property in the salamander lineage? The answers to the evolutionary questions encompass other noteworthy issues, such as the relationship between the regeneration of the adult limb, and its embryonic and larval development. In my contribution to the workshop I considered first a rather understudied aspect of the mechanism, which provides interesting pointers for mammals, and second recent evidence on limb regeneration as a salamander novelty (Garza-Garcia et al., 2010).\n\nOne characteristic of regeneration in animals such as salamanders is the ability to undergo multiple sequential episodes with minimal change in outcome, even in some cases in ageing animals. This has recently been shown in relation to newt lens regeneration, in which a cohort of animals was followed over eighteen annual episodes (Eguchi et al., 2011). There have also been several studies of fin regeneration in the zebrafish that have explored this property and concluded that the outcome of regeneration cannot be limited by replicative senescence. The state of cell senescence was originally identified in relation to the arrest of proliferation that occurred after a certain number of divisions in culture, and it has also been implicated as an anti-tumour mechanism. More recently, senescent cells have been found in certain contexts in mouse development, for example the kidney and the limb, suggesting that we do not fully understand the significance of this state. We have established a culture system where newt limb cells are induced to enter senescence, and display a variety of markers found in mammalian senescence. This allowed us to show that a cohort of senescent cells are induced early after transection of the salamander limb, but they are removed effectively during the course of one cycle of regeneration (Yun et al., 2015). The ability to implant normal and senescent cells from culture into the limb has allowed us to analyse this process, and to determine that it is dependent on macrophage-mediated removal. Senescent cells accumulate in mammals with age, and there is some evidence that genetic ablation of senescent cells may ameliorate certain age-related disorders in mice. The salamander is apparently an animal with an active and efficient mechanism of surveillance that removes senescent cells and prevents their accumulation, even after multiple cycles of limb regeneration. Nonetheless, it is possible that the transient cohort may play an important role in regeneration. We think that this context provides a valuable perspective for efforts to target senescent cells in mammals, as well as underlining a new element of the dynamic architecture of the limb that supports regeneration.\n\nIf limb regeneration is an ancestral property in vertebrates, it might have persisted in salamanders from fin regeneration following the fin-limb transition. It would then have been lost in anurans and amniotes. Alternatively, the regeneration of the limb may have presented a barrier that required additional evolutionary novelty over the background level of regeneration present in salamanders. Note that both views of limb regeneration imply selective pressure for this property in salamanders, either to maintain it, or to make the transition from a non-regenerative state. One line of evidence that supports some contribution of 'local' evolution is the identification of salamander-specific proteins in regenerating limbs. This includes the salamander orphan gene Prod 1, a member of the Three Finger Protein (TFP) superfamily, and previously implicated in nerve dependence and positional identity (Brockes & Gates, 2014; Garza-Garcia et al., 2010). Prod 1 is probably present in all salamanders, as it has recently been identified in one of the basal families, the Hynobiidae, which diverged in the early Jurassic. Other putative taxon-specific genes expressed during regeneration have been identified in proteomic and transcriptomic databases from salamanders (Looso et al., 2013). While this microevolutionary evidence is a valuable inroad for our understanding of regeneration, more functional studies will be necessary to determine whether these genes are embedded in the mechanism.\n\nThere are certain established aspects of limb development in salamanders that are different from other tetrapods (Fröbisch & Shubin, 2011). These relate to the order of appearance of the digits, and the order of their subsequent ossification. It is often pointed out that limb development occurs after hatching in many aquatic salamander larvae, and the developing limb is subject to selective pressures for anchorage, balance, and propulsion. These pressures might have extended to engendering its regeneration after loss by injury or predation, and illustrate the interaction between the organism and its environment. The precocious extension of digits 2 and 1 may be critical to allow rapid elongation of the developing limb in this context. In contrast to regeneration, there is a clear consensus that these differences in timing, referred to as preaxial dominance, represent a salamander novelty imposed on the mechanism of limb development found in other tetrapods, termed postaxial dominance. It has been suggested that there is a connection between the evolution of this property, and that of limb regeneration (Brockes, 2015; Fröbisch & Shubin, 2011). The relevance of preaxial dominance to regeneration, and whether it occurs during regeneration as well as development, are both unclear. An intriguing recent contribution comes from the analysis of the fossil record of Paleozoic amphibians. Preaxial dominance is readily visible in the developing bones of the limbs, and is clearly detectable in fossil larvae of the temnospondyl species Apateon in the early Permian (about 300 MYA). It has recently been reported that fossils of Micromelerpeton, a close relative of Apateon and inhabiting the same mountain lakes, show evidence for limb regeneration in the presence of certain characteristic abnormalities in the limbs (Fröbisch et al., 2014). It is possible that regeneration and preaxial dominance originated in the same species at a time close to the divergence of the salamander lineage (Brockes, 2015). Our ability to promote mammalian regeneration will be aided by further understanding of the evolution of regeneration, and the factors underlying the dynamic architecture of the salamander limb.\n\n\nPlasticity of homeostasis and regeneration: what can we learn from Hydra? (Brigitte Galliot)\n\nHow do we maintain the shape and functionalities of our body over years? As adult organisms, we continuously replace - in the absence of any stress or injury - tissues thanks to genetic programs that strictly control the size, the anatomical organization, and the function of these tissues. Such replacement processes that characterize the maintenance of homeostasis occur at paces specific to each organ, typically slow for brain, lungs, kidneys, and rapid for intestine, blood cells, and skin. Genetic programs are constrained cascades of events orchestrated in nuclei by transcription factors, RNA-binding proteins, microRNAs, long noncoding RNAs, which all together modulate the activity of target genes whose activities modify the behaviours of cells and tissues. An excellent example of such a genetic cascade is the Hippo pathway, initially identified in Drosophila, which similarly regulates the homeostasis of numerous organs in planarians and mammals (Piccolo et al., 2014).\n\nIn conditions of stress or injury, exogenous cues play a key role to trigger immediate responses involved in wound healing, and to activate evolutionarily conserved genetic programs that repair damaged tissues or injured organs by restoring size, anatomy, and function but also prevent degeneration and uncontrolled growth (Cordeiro & Jacinto, 2013; Vriz et al., 2014). However, these repair mechanisms reach some limits as the genetic programs we used to develop during our embryonic life progressively close to become, in most cases, irreversibly inaccessible in adulthood. This implies that repaired tissues are often not fully restored, as skin or hearts that become fibrotic after repair and poorly functional. This also implies that we are not able to reconstruct complex structures such as limbs or even complex organs as kidneys, heart, and lungs. When I say “we” I mean the large mammalian family, as most mammals including humans exhibit limited regenerative abilities. By contrast a cohort of non-mammalian species spread over a wide phylogenetic spectrum, including sponges, cnidarian polyps, planarians, annelids, crustaceans, echinoderms, urochordates, fish, salamanders, and lizards, is able to regenerate missing body parts, amputated appendages, and destroyed organs.\n\nOne possible hypothesis is that most metazoans are highly plastic as adult organisms, a feature subsequently lost in mammals. Here, I will consider two forms of plasticity, homeostatic and adult developmental, both taking place in fully developed sexually mature organisms. The term “homeostatic plasticity” refers to the adaptation of an intact organism to external or internal changes, whereas “adult developmental plasticity” refers to the possibility for adult organisms to trigger developmental programs upon injury or damage (Galliot & Ghila, 2010). These programs usually share similarities with embryonic developmental programs, but the main difference is that they are used in a fixed and highly constrained manner during embryonic development, but with much plasticity during adulthood.\n\nHydra is a small freshwater cnidarian polyp that provides a unique model for investigating homeostatic and developmental plasticity: these animals remain fit over weeks of starvation, and do not display signs of senescence when regularly fed. Moreover they reproduce asexually through budding, they regenerate any missing part when bisected along the body axis, and they can even reaggregate and reshape to form the original polyp shape when dissociated as a cell suspension (Galliot, 2012). They also survive the elimination of their nervous system becoming “epithelial” but are still able to bud and regenerate if force-fed. All this is possible due to three large pools of stem cells that continuously divide and provide differentiated cells. So Hydra, which can easily be propagated as mass culture, provides a model system for monitoring a highly dynamic homeostasis, the response to change in environmental conditions (feeding, temperature), the response to injury, and the reactivation of two distinct developmental programs, head and foot regeneration (Figure 1). Interestingly, a large number of genes are conserved from Hydra to humans (Wenger & Galliot, 2013) and studies over the past 25 years convincingly showed that the genetic programs active in Hydra often play similar functions in Drosophila and mammals, as for example neurogenic genes (Galliot et al., 2009; Miljkovic-Licina et al., 2007).\n\nThe scheme represents the typical epithelial bilayered anatomy of Hydra polyps with interstitial stem cells (green dots) distributed along the central body column (framed box). By contrast cells at the apical and basal extremities are terminally differentiated. A highly dynamic homeostasis with continuous renewal of stem cells supports the fitness and the low senescence of Hydra. Animals survive long periods of starvation as well as the elimination of the nervous system if force-fed. After bisection at any level along the body column, the stump is able to reestablish an organizer centre and regenerate the missing part, either basal or apical. However, some Hydra oligactis strains show a very low level of plasticity: they do not adapt to the loss of neuronal progenitors induced by the transfer to cold temperature, they rapidly lose the ability to regenerate, undergo aging, and finally die in a couple of months.\n\nHowever, some Hydra oligactis strains undergo a rapid aging process when transferred to cold temperature (Tomczyk et al., 2015; Yoshida et al., 2006). After few weeks these animals lose (i) their highly dynamic homeostasis as evidenced by the decrease in sustained proliferation of epithelial stem cells, (ii) the ability to adapt to a stressful environment as shown by the dramatic impact of the loss of neurogenesis, and (iii) the ability to regenerate missing body parts (Tomczyk et al. in preparation). As a consequence, these animals lose their fitness in a month and survive no longer than two months. Interestingly, the dissection of the molecular and cellular mechanisms underlying aging in such Hydra points to processes highly reminiscent of those active in humans.\n\nIn summary, Hydra provides a potent model system to understand how a dynamic homeostasis can support a robust adaptability to environmental changes, and how developmental plasticity can promote regeneration. Each of these properties has biomedical implications of utmost interest, as the characterization of the mechanisms underlying plasticity in Hydra should help identify candidate regulators of regeneration and aging in humans.\n\n\nPlasticity in hypothalamic circuits controlling leptin sensitivity and food intake (Marco Mainardi)\n\nMetabolic homeostasis has been traditionally thought to be a static process, characterized by a relatively narrow range of physiological and behavioural responses to the abundance or scarcity of nutrients, e.g, regulation of glycaemia, food seeking, and food intake. However, neurons have been shown to change their activity in response to changes in the levels of metabolic hormones (Mainardi et al., 2013; Mainardi et al., 2015). In addition, the basal hypothalamus, a key area for energy homeostasis, has recently been shown to host a new neurogenetic niche (Kokoeva et al., 2005), along with the subventricular and subgranular zones (see also Prof. Prochiantz’s contribution). Thus, regulation of metabolism can definitely be considered as a plastic and dynamic process, leading to a substantial revision of the concept of a “set-point” for metabolism, towards a more dynamic view.\n\nThe plastic potential of the hypothalamus is an essential feature for the adaptation of neuronal output to variations in environmental stimuli, in close similarity with the role of sensory experience in shaping the cortex. Indeed, brain areas taking part in metabolic homeostasis have to adjust their response not only to drive food-seeking behaviour, but also to maintain a proper amount of energy stores. The net result of this process regulates the balance between energy accumulation and expenditure, thus giving an important contribution in determining body weight and metabolic activity.\n\nOur previous work (Mainardi et al., 2010) has shown that the external environment can dynamically affect the development of hypothalamic neurons’ sensitivity to leptin, the hormone secreted by adipose tissue to negatively regulate food intake and stimulate energy expenditure (Friedman, 2014). In these experiments, mice were exposed to environmental enrichment since birth to enhance the motor, cognitive, and sensory stimulation. Under these conditions, a reduction in circulating leptin levels was observed, which, surprisingly, did not produce the expected increase in food intake. This finding was suggestive of enhanced leptin sensitivity and, consistently, neurons located in the hypothalamus were more responsive to this hormone. In addition, food intake repression by leptin was accentuated in comparison to mice reared under standard conditions. Taken together, these results indicate that the quantity and quality of external stimuli are able to modulate the development of the set-point for lipid homeostasis.\n\nAt the synaptic level, we found rearrangements in the excitation/inhibition ratio of hypothalamic synapses, which indicated a shift towards activity patterns aimed at repressing food intake. This structural observation was put in correlation with a higher expression of the gene encoding for BDNF, a neurotrophin and a master regulator of neural plasticity.\n\nOne of the most interesting aspects is that synaptic plasticity was not observed if mice were exposed to physical exercise alone (although leptin sensitivity was enhanced at a comparable level), indicating that cognitive stimulation and social interaction also have an important role in determining the metabolic set-point.\n\nA striking aspect is that programming of leptin sensitivity by environmental stimuli was shown to be possible only during early postnatal development, suggesting the existence of a critical period, as it has been described for sensory cortices. The analogy with the cortex also extends to the main mediators of plasticity: modulation of the excitation/inhibition ratio and BDNF abundance.\n\nThis parallel between cortical and sub-cortical areas leads to the speculation that the plasticity of functionally distinct brain districts is actually regulated according to the same general, unifying principles. According to this view, the same array of molecules are organized into a common framework that provides the substrate for generating the dynamic architecture of the brain.\n\nFurther experimental efforts are now aimed at finding strategies to artificially manipulate sensitivity to metabolic hormones and plasticity of their target brain areas. The need of tools to harness and control the dynamic potential of the brain-metabolism interplay has a great therapeutic potential. Indeed, severe obesity is usually accompanied by loss of response to leptin and insulin.\n\nThe basic idea is based on restoring a juvenile-like status of heightened plasticity, which would help in restoring an optimal metabolic set-point, ultimately leading to body weight normalization.\n\n\nThe non-cell autonomous regulation of cerebral cortex plasticity by homeoprotein Otx2 (Alain Prochiantz)\n\nThe conception that development brings individuals to a state of perfection from which they will progressively depart in the course of ageing is anchored into a vision of living organisms as thermodynamic machines obeying the second law of thermodynamics. Best illustrated by Bichat in the 19th century, this conception is also present in Schrödinger’s philosophy of life or in the work of the cyberneticists. Several extremely productive consequences have emerged from the latter views, among which the spectacular post-war ascent of molecular biology. However, the 19th century has also been at the origin of a completely opposite view theorized by Claude Bernard, who proposed that life is a permanent movement of construction and deconstruction, taking place at all levels, and that adult organisms are renewed in permanence through what he poetically named \"silent embryogenesis\".\n\nThe recent development of stem cell research and the discovery that we regain our own weight in cells every year has lent weight to the Bernardian view of a very plastic state for all living objects. A possible physiological justification of such plasticity is that it is easier, in many instances, to replace bad cells than to repair them, and also that new elements - if in excess - could favour somatic Darwinian selection, that is adaptation through individuation.\n\nIn the case of the nervous system, in particular the mammalian one, things are slightly more complicated because, with the exception of the two main adult neurogenic regions, the dentate gyrus zone in the hippocampus and the subventricular zone, most neurons are not renewed (Alvarez-Buylla & Lim, 2004; Deng et al., 2010; Ernst & Frisen, 2015). This is possibly because they are engaged in neural networks of high physiological value that have to be maintained throughout life. However, this opens a very important issue: how is learning, and thus adaptation to the environment, made possible not only at early post-natal stages when neural networks are built, but also in the adult when they are completed.\n\nOur laboratory has tackled this issue by working on critical periods (CPs) in the development of the mouse cerebral cortex. Without going into detail, CPs correspond to periods of time during which the cortex can easily learn from the environment and respond by adapting its neuronal circuits to the sensory inputs it receives (Bavelier et al., 2010). For example, the competition between the two eyes for binocular vision in the visual cortex 1 (V1) takes place between post-natal day 20 (P20) and P40 in the mouse. It means that closing an eye between P20 and P40 reduces its visual acuity (amblyopia), but that this functional “disconnection” does not take place if the eye is closed before P20 (plasticity onset) or after P40 (plasticity closure) (Maffei et al., 2010).\n\nThe passage from no plasticity (P20) to maximal plasticity (P30) and no plasticity again (P40) is due to the progressive maturation of inhibitory Fast-Spiking Parvalbulmine neurons (FSPV-cells) and to an ensuing shift in the in Excitatory/Inhibitory (E/I) balance (Hensch & Fagiolini, 2005). We discovered, in collaboration with the group of TaKao Hensch, that the homeodomain transcription factor Otx2 is necessary and sufficient to open plasticity at P20 and close it at P40. We proposed that Otx2 progressively accumulates within FSPV-cells, opens plasticity at a first concentration threshold and closes it at a second one (Figure 2), (Beurdeley et al., 2012; Spatazza et al., 2013; Sugiyama et al., 2008). Throughout adulthood, Otx2 concentration normally remains above the second threshold maintaining the system in a non-plastic state (or a poorly plastic one).\n\nSensory signals, for example eye opening at P14 in the mouse, triggers Perineuronal net (PNN) assembly at the surface of Parvalbumin neurons in layers III/IV of the visual cortex. PNN complex sugars specifically recognize Otx2 en route from extra-cortical sources and enhance its internalization by Parvalbumin neurons, thus triggering the maturation of this class of interneurons. Otx2 reaches a first intracellular concentration threshold that opens plasticity and accumulates until a second threshold closes plasticity. The maintenance of an adult non-plastic state requires the continuous import of Otx2 from extra-cortical sources and blocking the latter import reopens a window of plasticity in the adult.\n\nImportantly, Otx2 is not produced by FSPV-cells and is thus imported from external sources. Among the possible sources is the choroid plexus, which synthesizes Otx2 and secretes it into the cerebro-spinal fluid. It was demonstrated that indeed Otx2 is imported from this source and that its levels within FSPV-cells is reduced upon recombination of the Otx2 locus, specifically in the choroid plexus (Spatazza et al., 2013). This reduction in the adult brings Otx2 concentration back below the second threshold and reopens plasticity allowing one to restore normal binocular vision to previously amblyopic mice, or (and less nicely) to induce amblyopia in the adult.\n\nWithout elaborating further, this finding, which, in fact, might be generalized to other regions of the cerebral cortex, demonstrates that plasticity is the default state of the adult cortex and that the continuous arrival of Otx2 from the choroid plexus (and possibly other sources) into FSPV-cells maintains the cortex in a non-plastic state. It also suggests that transient local changes in Otx2 levels could reopen windows of plasticity in the adult.\n\nIn other words a tightly-controlled potential for adaptive change continues to persist in the adult nervous system of higher vertebrates, implying both the need for stability, but also the possibility for local renewal.\n\n\nThe dynamic encoding and processing of morphological information during development and regeneration (Daniel Lobo)\n\nDuring development and after injuries, organisms orchestrate a highly dynamic process to converge into predetermined forms and shapes – a target morphology whose specification must reside within the organism. Understanding how this target morphology is encoded, stored, and retrieved is essential to explaining the mechanisms controlling these multicellular processes. Moreover, the characteristic feedback loops and nonlinear dynamics typical of biological regulation prevent us from easily inferring the complex processes controlling these systems. Computer science theory and practice can readily assist us with the mathematical formalisms necessary to rigorously describe and analyse these processes and with the computational tools to automate the discovery of such complex dynamic regulatory networks. These computational approaches may represent an inflection point in our goal to explain the formation and repair of biological form and shape, and facilitate the much sought after biomedical and synthetic biology applications.\n\nThe regeneration of planarian worms is one of the most extraordinary examples of dynamic development and restoration of form and shape. Planarians are freshwater flatworms with a complex morphology, possessing a nervous system with a true brain, a diverse set of sensory organs including eyes, a complex musculature, and a branched digestive system. Despite their complex morphology, planarians can regenerate a complete body from almost any amputated piece, growing anew missing organs and resizing existing ones (Lobo et al., 2012). These remarkable abilities, whose principles are not yet well understood, makes the flatworm an extraordinary model system for studying the dynamics of morphological formation and repair.\n\nInterestingly, although the target morphology of the wild-type planaria is a head-trunk-tail phenotype, a transient blockage of gap junction communication with octanol after specific surgical amputations can cause the regeneration of permanent multi-headed morphologies (Figure 3): subsequent amputations produce the same multi-head morphology, even without the application of any drug (Lobo et al., 2014b; Oviedo et al., 2010). Remarkably, neither the octanol treatment nor the surgical manipulation directly cause any genetic change in the worm, suggesting that the target morphology to be regenerated is not encoded in the genome, but possibly by chemical morphogens, mechanical stresses, or electrical signals maintained within the worm’s body. Moreover, the ability to alter these morphological encodings with localized interventions (each surgical amputation produces a head in that location) points towards a linear encoding of the target morphology, similarly to one-to-one expression patterns, such as the homeotic gene expression patterns in the Drosophila embryo, which can be altered with localized interventions. In contrast, small alterations in nonlinear encodings, characteristic of gene regulatory networks such as the Drosophila gap gene network, produce large defects in the resultant morphology.\n\nThe regenerating phenotype from an amputated planarian trunk piece can be locally and permanently altered by non-genetic perturbations, suggesting a linear encoding of the target morphology outside the genome. Experiments extracted from Planform (Lobo et al., 2013a).\n\nMoreover, the nonlinear interactions and feedback loops in nonlinear encodings represent an extraordinary barrier to understand, and possibly to control, the dynamics of development and regeneration. Reverse-engineering dynamical systems with feedback loops is indeed very hard, and no analytical solution exists to solve this inverse problem. Fortunately, novel heuristic computational methods together with the increase of available computational power are showing promising results in aiding in the discovery of such regulatory mechanisms (Villaverde & Banga, 2013).\n\nIn order to apply computational methods to the discovery of dynamic networks from morphological experiments, the manipulations and resultant phenotypes need to be encoded in a mathematical format comprehensible for the computer, a non-trivial task for developmental and regenerative datasets. To this end, mathematical ontologies for morphological and experimental data have been proposed for planarian and limb regeneration experiments, and, based on these formalizations, centralized databases have been curated containing thousands of experiments published in the literature (Lobo et al., 2014a; Lobo et al., 2013b). These freely available resources allow any scientist to instantaneously search for the resultant morphologies from specific genetic knock-downs or pharmacological interventions among thousands of experiments in the literature. More importantly, the mathematical nature of these resources makes the application of computational approaches amenable to reverse-engineer the underlying mechanisms explaining the experimental results in the datasets.\n\nRecent advances in machine learning and artificial intelligence have facilitated the application of automated inferring methods for the automatic discovery of dynamic regulatory networks directly from the morphological outcomes of surgical, genetic, and pharmacological experiments (Lobo & Levin, 2015; Lobikin et al., 2015). These methods are based on the in silico evolution of dynamic networks that can recapitulate, in a virtual environment, the precise resultant phenotypes after applying the same surgical and genetic perturbations contained in the in vivo dataset of experiments. This approach has been successfully applied to the main head versus tail planarian regeneration experiments published in the literature, resulting in the reverse-engineering of a comprehensive dynamic regulatory network of planarian regeneration and demonstrating the power of these automated computational methods.\n\nIn summary, the emergent dynamics of development and regeneration represent a current challenge to understand these processes. To this end, it is important to differentiate between two types of morphological encodings present in biological systems: nonlinear encodings typically stored in the genome, and linear encodings (possibly originally formed during development from the nonlinear encoding) typically stored in chemical, mechanic, or bioelectric patterns. A linear encoding is easy to alter or repair and amenable for direct medical interventions. In contrast, a nonlinear encoding is hard to understand and control. For this reason, novel automatic computational tools are being proposed to aid us in the reverse engineering of these nonlinear encodings, such as the dynamic regulatory networks controlling planarian regeneration. This approach will pave the way for the understanding and control of the outstanding capacity of living organisms to self-construct and self-repair.\n\n\nThe importance of variance in the nervous system (Angelika Steger)\n\nIn the digital world we have a very precise understanding of how information is stored: a single bit stores a value of either 0 or 1, and the combination of bits can be used to store more interesting facts. On the other hand, even after decades of intensive research, our understanding of how information is stored in the brain is still embarrassingly poor. Naturally, we do know basics facts: in the brain a neuron conveys information to its neighbours in form of spike trains, that is, sequences of electric pulses. Pulses per se seem to not differ from each other, so the information must lie either in the number or the timing of the pulses. Both options do not exclude each other, and it is non-trivial to distinguish between the two (Stein et al., 2005). There are also many indications that a single bit of information is not stored by a single neuron, but instead by a large group of neurons. This is known as population coding. Unfortunately, again, so far we have no idea of how exactly this is done.\n\nThere is no obvious way of how to resolve this riddle. Two approaches seem natural: (i) collect as many neurophysiological data as possible, and (ii) develop (mathematical) models that can explain phenomena observed in the brain. A challenging part, however, remains: bridge the gap between these approaches. A major hindrance here is that biological reality comes with a broad range of varieties: many different neuron classes and even within the same class varying properties (threshold potential, membrane capacity, etc.), synaptic connections that come with a wide range of synaptic strengths and failure rates of up to 80%, structural properties of the underlying brain tissue that are similar but not identical throughout neo cortex, and so on.\n\nA standard approach in mathematics and many other disciplines is to first understand how a system works in a ‘pure’ setting and then generalize it step by step in order to transfer it to the noisy ‘real world‘ scenario. In brain research, however, a challenging and still not well resolved first step is: what is an appropriate ‘pure’ setting? Before we know how the brain functions it is hard to come up with classifications that differentiate between ‘meaning’ and unintended variations and imperfections that can be summarized as ‘noise’. But without such a classification it seems impossible to come up with a model that can subsequently be matched successfully to experimental observations. This circular argument nicely explains the problems we are facing. A common approach to overcome this is to start with very simple models -- and to then learn from failures.\n\nIn our work we study the effect of adding variance to neuronal processes (Lengler et al., 2013). Similarly, as was observed in other contexts (Wiesenfeld & Moss, 1995) we show that variance within defining properties of neurons and their synapses is not a handicap of neural systems but that, instead, predictable and reliable functional behaviour of neural systems depends crucially on this variability. In particular, we show that higher variance allows a recurrently connected neural population to react more sensitively to incoming signals, and processes them faster and more energy-efficiently. This challenges a widely spread assumption that variability of neurons in the brain is a defect that has to be overcome by synaptic plasticity in the process of learning. Instead, it might well be that the variability is actually a feature and not a bug!\n\nAnother and perhaps even more surprising result is that variability in the neuronal parameters guarantees stability. We illustrate this at one of the basic processes that occur in neural processing: an external input activates some brain area and this activation spreads due to local recurrent connections. While an initial spread of activity may well be a desired feature, it certainly needs to be stopped before an epileptic seizure occurs. As it turns out, it is not at all easy (neither in simulations nor in theory) to come up with models that exhibit such behaviour, as either inhibition kicks in too early (and in turn nips all activity in the bud) or inhibition is too weak to ever have a sizeable effect and systems thus do not have a decorrelating effect as observed in nature (Ranganathan & Koester, 2011). We could overcome this well-known phenomenon by adding a lot of variance to the system: variance in the definition of the neuron parameters as well as variance in the reliability of the synaptic connections. In fact, our simulations show that an increased level of variance actually leads to more and not less stability in the dynamics of the considered network, and that it is most helpful to have variance in many parameters and not just one. It remains a challenging question to understand whether neuronal plasticity actually enhances variance purposefully in order to strengthen these effects.\n\n\nGeneral discussion\n\nThe format of this conference enabled the juxtaposition and comparison of different model systems, which are being used to uncover the physiological underpinnings of a dynamic architecture of life. One conclusion that can be drawn is that very different balances between change on one side and constancy on the other have evolved in animals and plants: On one end of the spectrum, one might locate animals, showing a capacity for life-long regeneration of their body or appendages. On the other end of the spectrum animals and plants can be found, which possess a much more limited regenerative faculty, but harbour a potential for life-long individual development through adaptive change.\n\nThe reader might comment that this distribution has been known and has been well accepted for most of the 20th century. However, the increasing body of evidence provided for the continuous capacity for plasticity in higher order species puts novel demands to understand the dynamics inherent in living systems, whose high degree of structural and functional complexity has been understood to exclude most change in the mature organism. How is the level of control achieved that permits adaptive change, but bars excessive alterations that would threaten the functionality of complex organs, such as the mammalian nervous system? How are the adaptive responses organized? Do they follow a pre-established hierarchy or is their sequence a reflection of the particular demands to the organism? The latter implies that changes can be elicited at any functional level, for example genes, proteins, cells, tissues or, in the case of the brain, also different parts of the organ, and are then transmitted to the other levels to achieve a coordinated adaptation (for discussion see Morange, 2009). This is also relevant in the context of therapeutic applications addressed below.\n\nEpigenetics seems to provide an important link transforming individual adaptations garnered during the existence of the organism into a property that might be selected for and thus contribute to advances at the species level. The extensive knowledge of epigenetics in plants makes them particularly attractive to discuss these questions. Interestingly, the caution that still prevails in the plant field in accrediting epigenetic change a role in evolution seems to be contrasted by an overly eager reception of evidence for epigenetic changes in animals, which does not stand up to the application of more rigorous criteria. As Ueli Grossniklaus discusses, the establishment of unambiguous parameters for the intergenerational passage of epigenetic modifications is an important pre-requisite for clarifying the importance of epigenetics in animal studies. This example also demonstrates that direct encounters like this conference might be helpful in getting essential know-how across disciplinary divides.\n\nMarie Mirouze states in her contribution that epigenetically induced genetic variation (e.g. the activation of transposons) amongst cellular constituents of an organism can be advantageous and underscores the importance of epigenetic inheritance in rendering differentiation transferable from mother to daughter cells. As Alain Prochiantz discusses, variability amongst newly generated cells might also convey a selective advantage to specifically adapted individual cells within the nervous system. Interestingly, the efforts to model the enormous complexity of information storage in the brain lead Angelika Steger to conclude that variance of neuronal parameters is not something to be necessarily eliminated, but rather might be favourable for improving the overall efficiency of signalling. This provokes the question to which degree variance amongst cellular constituents of organisms, regardless of its origins, is productive and desired and how it might be controlled and selected for.\n\nWhat do we make of the fact that the regenerative capacity is much more limited in mammals? Are the claims made about the application of the insights gained about regeneration in different animal models for therapeutic interventions in humans very ambitious or justified in view of the fact that genetic pathways underlying regenerative processes are widely conserved, as Brigitte Galliot emphasizes? Do therapeutic measures have to address entire physiological processes or might it be sufficient to tweak one or a few particular parameters, implying a plastic capacity of the system that can be induced in different ways to respond in a therapeutically desirable fashion?\n\nIn simple as well as complex organisms, adaptive change and an environment guiding and shaping that change seem to go hand in hand. While in the case of Hydra and plants modelling the environment in an ecologically relevant fashion is relatively easily to achieve, it is more demanding to do so in the case of rodents. In the neurosciences the recourse to a so-called “enriched environment” has become the method of choice to provide an experimental environment that is demanding to the animals on different levels. As the work by Marco Mainardi and others shows, specific changes are elicited in the nervous system in response to a range of physiological stimuli. One might wonder which changes might become possible if the complexity of the environment was raised beyond that of an enriched environment to a more natural environment and in which ways this might promote differences in individual animals (e.g. Freund et al., 2013).\n\nAs the contributions to this review show, the use of central terms - epigenetics, plasticity, and regeneration - is partially overlapping, as different authors emphasize different aspects of the processes of adaptive change they are studying. In reflection of a variable use of concepts in biology, in the history and philosophy of science, the argument has been put forward that it is the differential applicability of biological concepts, which renders them useful in propelling forward a largely empirical science like biology, which advances by posing ever more refined questions about the character and content of its concepts (see the analysis of the concept of the gene during the twentieth century by Rheinberger et al., 2015). However, the variable and widespread use of biological concepts has also been criticized, as implying a loss in precision, ultimately rendering them too vague for proper scientific exchange. In the case of plasticity, suggestions have been made to specify the use of the concept in the neurosciences in order to provide an appropriate framework for further empirical studies (e.g. Will et al., 2008). In view of the fact that the knowledge of the inherent dynamics of biological systems is steadily increasing, it might be necessary to develop a novel terminology, which takes into consideration the recent advances and introduces unambiguous criteria for the use of specific concepts in different fields. Modelling approaches like the one presented by Daniel Lobo could contribute to a discussion on universally applicable criteria, which are sufficiently stringent to guide researchers in the common use of defined terms.",
"appendix": "Author contributions\n\n\n\nB.P.R. wrote the introduction and the discussion, the other authors contributed the reviews on their fields, as indicated in the text. All authors have read and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work of B.P.Rubin was supported by the Collegium Helveticum and the Stiftung für Humanwissenschaftliche Grundlagenforschung (SHG).\n\n\nReferences\n\nAlvarez-Buylla A, Lim DA: For the long run: maintaining germinal niches in the adult brain. Neuron. 2004; 41(5): 683–686. PubMed Abstract | Publisher Full Text\n\nBaillie JK, Barnett MW, Upton KR, et al.: Somatic retrotransposition alters the genetic landscape of the human brain. Nature. 2011; 479(7374): 534–537. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBavelier D, Levi DM, Li RW, et al.: Removing brakes on adult brain plasticity: from molecular to behavioral interventions. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nMainardi M, Pizzorusso T, Maffei M: Environment, leptin sensitivity, and hypothalamic plasticity. Neural Plast. 2013; 2013: 438072. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMainardi M, Scabia G, Vottari T, et al.: A sensitive period for environmental regulation of eating behavior and leptin sensitivity. Proc Natl Acad Sci U S A. 2010; 107(38): 16673–16678. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcClintock B: The significance of responses of the genome to challenge. Science. 1984; 226(4676): 792–801. PubMed Abstract | Publisher Full Text\n\nMiljkovic-Licina M, Chera S, Ghila L, et al.: Head regeneration in wild-type hydra requires de novo neurogenesis. Development. 2007; 134(6): 1191–1201. PubMed Abstract | Publisher Full Text\n\nMirouze M, Reinders J, Bucher E, et al.: Selective epigenetic control of retrotransposition in Arabidopsis. Nature. 2009; 461(7262): 427–430. 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}
|
[
{
"id": "11799",
"date": "29 Jan 2016",
"name": "Richard Festenstein",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 summarises the key points of a recent meeting bringing together scientists working on diverse biological systems linked mainly by their approach to understanding the dynamic aspects of biological states. The report highlights the importance of reversibility of differentiation states and cellular responses to external stimuli as well as the importance of this in the context of adaptive evolution. Clearly there are many unanswered questions and the historian’s perspective pointed out that there is much more experimental work required to formulate general principles linking the described systems. Meetings such as these are of great value in allow abstraction of such principles and are to be encouraged. The inclusion of an historian in such meetings is an innovative approach which could be developed further to allow a real in-depth analysis of how ideas evolve over time.",
"responses": []
},
{
"id": "12521",
"date": "18 Feb 2016",
"name": "Pierre J. Magistretti",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 group of experts met at Accademia dei Lincei to discuss new concepts emerging from experimental data on the dynamic process of living organisms particularly as they relate to differentiation, adaptation, plasticity and memory. The report provides snapshots from selected fields of biology illustrating the paradigm shift that has occurred over the last two decades concerning the dynamics of a variety of molecular and cellular processes. The view that emerges is one of permanent instability that allows for dynamic adaptations to environmental cues. The set of process underlying this instability most likely provides the key to species survival and adaptation, but also, and just as importantly, the emergence of individuality. The paragraphs reporting on the various presentations are sharp and informative and the introductory and concluding ones provide a faithful synthesis of the questions at stake and their elaboration. The only aspect that could have been treated more directly would have been that of synaptic plasticity in the adult which represents undoubtedly an important element of the dynamic architecture of life. This review article represents a remarkable scholarly effort to provide novel insights on a paradigm shift about our understanding of the dynamics of life.",
"responses": []
}
] | 1
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https://f1000research.com/articles/4-1288
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https://f1000research.com/articles/4-1287/v1
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18 Nov 15
|
{
"type": "Review",
"title": "Current controversies in infective endocarditis",
"authors": [
"Thomas J. Cahill",
"Bernard D. Prendergast",
"Thomas J. Cahill"
],
"abstract": "Infective endocarditis is a life-threatening disease caused by a focus of infection within the heart. For clinicians and scientists, it has been a moving target that has an evolving microbiology and a changing patient demographic. In the absence of an extensive evidence base to guide clinical practice, controversies abound. Here, we review three main areas of uncertainty: first, in prevention of infective endocarditis, including the role of antibiotic prophylaxis and strategies to reduce health care-associated bacteraemia; second, in diagnosis, specifically the use of multimodality imaging; third, we discuss the optimal timing of surgical intervention and the challenges posed by increasing rates of cardiac device infection.",
"keywords": [
"Infective endocarditis",
"Bacteraemia",
"Antibiotic prophylaxis",
"Transcatheter valves",
"Multimodality imaging",
"Cardiac device infection"
],
"content": "Introduction\n\nIn 1885, William Osler described ‘malignant’ endocarditis as an infection on the scarred heart valves of young adults with rheumatic heart disease1. At the time, infective endocarditis (IE) was caused mainly by microorganisms originating in the oral cavity (oral streptococci). In 2015 in the developed world, IE looks dramatically different2. Rheumatic fever is now rare, and in 25% of cases the causative infection is health care-acquired3,4. With the exception of intravenous drug users and those with congenital heart disease, patients typically are elderly. The focus of infection is frequently on prosthetic material within the heart: for example, cardiac devices (pacemakers, implantable cardioverter defibrillators and cardiac resynchronisation therapy) or prosthetic heart valves. In parallel, the bacteria underlying IE have changed: highly destructive staphylococci have now overtaken oral streptococci as the most common cause4,5. With an incidence of 3 to 10 per 100,000, IE is rare but carries an in-hospital mortality of 20%2. Improving outcomes for patients with this complex and heterogeneous disease is challenging. In this review, we provide a focused update on current controversies in disease prevention, diagnosis and management.\n\n\nPrevention of infective endocarditis\n\nBacteria enter the bloodstream from the mouth in the course of daily life, and poor dental hygiene has long been recognised as a risk factor for IE6. The significance of bacteraemia that occurs with dental extraction is still debated, as is the role of oral antibiotic prophylaxis to prevent it. In hospital, bacteraemia occurs as a complication of invasive procedures and indwelling venous catheters. Bacterial adherence, inflammation of the cardiac endothelium and thrombus deposition lead to formation of an infected ‘vegetation’7.\n\nPoor oral hygiene is a risk factor for bacteraemia and subsequent IE8. In addition to maintaining good hygiene, oral antibiotic prophylaxis has traditionally been prescribed for those at risk of IE prior to dental and surgical procedures. In 2008, the UK National Institute for Health and Care Excellence recommended against further use of prophylaxis, considering the potential hazards of widespread antibiotic use to outweigh the individual risks9. The European Society of Cardiology (ESC) and American College of Cardiology/American Heart Association continued to recommend prophylaxis but only for patients at highest risk: those with previous IE, prosthetic valves and cyanotic congenital heart disease10.\n\nThe effect of restricting antibiotic prophylaxis has now been examined in several studies11–13. Recently, a UK study found that over a 5-year period from 2008 there had been a small but significant increase in the (already-rising) number of IE cases alongside a fall in prescriptions for prophylaxis12. These findings have been highly controversial. A causal link between IE cases and the withdrawal of systematic prophylaxis cannot be established by observational data, and the findings may be confounded by increased rates of bacteraemia or a growing at-risk population. Importantly, microbiological data were not available, so it is unclear whether the additional cases were caused by oral streptococci (the target of prophylaxis). In the absence of a randomised controlled trial (RCT)—which faces logistical, funding and ethical barriers—this study may tip the balance back toward prophylaxis for high-risk groups.\n\nAt least a quarter of cases of IE are now acquired in the health-care setting14. Mortality for health care-acquired IE exceeds 40% and this is due to the susceptible population, often with multiple comorbidities, and higher rates of Staphylococcus aureus infection15. Health care-associated bacteraemia, the upstream cause of IE, is the target of several preventative strategies. Basic hand hygiene, aseptic technique, sterile barrier clothing and avoidance of the femoral route for venous catheters are all effective at reducing bacteraemia, but getting staff to adhere to best practice can be challenging16,17. ‘Practice-changing’ approaches, such as the use of a checklist to improve adherence to sterile technique, are powerful interventions which have been shown to reduce catheter-related infections18,19.\n\nInfection during the implantation of cardiac devices is an important and preventable cause of IE and is reduced by use of perioperative antibiotics20. Vaccination against S. aureus, which directly damages the cardiac endothelium to cause IE, is an attractive theoretical strategy21. Unfortunately, a number of phase II and III trials have shown negative results, and the vaccine was associated with increased mortality in a 2012 study in patients undergoing cardiothoracic surgery22. Other approaches in preclinical studies include novel materials designed to prevent bacterial adherence and agents that target bacterial biofilm production23,24.\n\n\nDiagnosis of infective endocarditis\n\nDiagnosis of IE requires evaluation of clinical presentation, microbiology results and cardiac imaging2. Reaching a definitive diagnosis can be challenging, particularly in suspected prosthetic valve IE and cardiac device infection (CDI), in which findings can be non-specific. The modified Duke criteria are useful for defining a positive diagnosis but were not intended as a clinical tool and cannot be rigidly applied to the individual patient25. Strategies that facilitate earlier diagnosis, risk stratification and therapy are key to improving survival.\n\nTransthoracic and transoesophageal echocardiography (TOE) are the mainstay of cardiac imaging for IE—for diagnosis, detection of complications and follow-up (Figure 1A). Even after TOE, however, diagnosis is inconclusive in 10% to 20% of patients because of limited resolution or image artefacts26, and adjunctive imaging techniques are showing promise in this group.\n\n(A) Echocardiography remains the core imaging modality in IE. Here, a vegetation (arrow) is visualised on the aortic valve by transoesophageal echocardiography. (B) Computed tomography (CT) is excellent at defining the anatomical extent of complex endocarditis. A large paravalvular abscess (asterisk) can be seen complicating a case of prosthetic valve IE. (C) Three-dimensional transoesophageal echocardiography provides a reconstructed view of the valve and here demonstrates dehiscence of a prosthetic mitral valve (arrow), an indication for surgical intervention. (D) Positron emission tomography/CT has shown value for diagnosis of prosthetic valve IE and cardiac device infection. A focus of fludeoxyglucose-18F (18F-FDG) uptake (arrow) can be seen at the site of a prosthetic aortic valve, separate from the myocardium, consistent with prosthetic valve IE. Adapted from Teoh et al.34.\n\nCardiac computed tomography (CT) (Figure 1B) is emerging as a valuable modality for detecting and defining IE complications in the aortic root (for example, formation of abscess/pseudoaneurysm)27,28. Identification of a paravalvular lesion (abscess, pseudoaneurysm or fistula) on cardiac CT is now a major criterion for the diagnosis of IE in the newly updated 2015 ESC IE guidelines29. CT is less sensitive than transoesophageal echocardiography at detecting valvular perforations and may miss small vegetations, and interpretation can be challenging in the early postoperative period. Three-dimensional TOE can assist with detection of leaflet perforation and define the anatomy of valvular dehiscence (Figure 1C)30,31.\n\n18F-FDG-PET/CT (fludeoxyglucose-18F/positron emission tomography/CT)—which shows uptake of radiolabelled glucose by metabolically active tissues—can visualise the infected vegetations of IE and has been successfully used in patients with suspected prosthetic valve IE or CDI, in which interpretation of TOE can be challenging (Figure 1D)32–34. In a recently published study of 92 patients with suspected prosthetic valve IE or CDI, hybrid PET-CT enabled reclassification of 90% of patients (45 out of 50) with possible IE by Duke criteria and provided a conclusive diagnosis (definite/rejected) in 95% of cases35. Addition of PET-CT to the Duke criteria raised the diagnostic sensitivity from 52% to 91%. As such, positive PET-CT (or single-photon emission computed tomography-CT) signal at the site of a prosthetic valve (at least 3 months after implant) has been included as a new major criterion for diagnosis of prosthetic valve IE in the 2015 ESC guidelines29.\n\nBeyond the heart, CT, magnetic resonance imaging (MRI) and PET imaging are facilitating early detection of embolism36. Systematic MRI of the brain detects abnormalities in up to 80%, and imaging evidence of embolism is now a minor diagnostic criterion in the 2015 ESC guidelines37. PET-CT imaging is useful for detection of peripheral vascular complications such as abscess, mycotic aneurysm and emboli35. Given the range of imaging options now available, the next challenge is to define the optimal imaging strategy for specific patient subsets.\n\n\nManagement of infective endocarditis\n\nClinical care of patients with IE is complex and is reviewed in detail elsewhere2. Management revolves around prolonged parenteral antibiotic therapy (typically 4 to 6 weeks in duration), surgical intervention for those at high risk or not responding to antibiotics, and surveillance for complications. Input is required from a wide range of specialists, including cardiologists, cardiothoracic surgeons, microbiologists and specialists in cardiovascular imaging and infectious diseases. Coordinating care can be a logistical challenge leading to delayed definitive management. Formation of a dedicated IE multidisciplinary team (MDT) is a simple strategy to improve clinical care. In a 2013 study from Italy, introduction of an IE MDT reduced in-hospital mortality from 28% to 13%38. Recent UK guidelines advocate the formation of an IE team in every major centre39.\n\nSurgery is currently performed in 40% to 50% of patients with IE40. There are three main indications: valve dysfunction causing heart failure, uncontrolled infection, and prevention of embolism. Although seemingly clear-cut, the appropriateness and timing of surgery for an individual patient sometimes carry considerable uncertainty. In 2012, a landmark RCT compared early surgery (within 48 hours) with conventional treatment in stable patients with native valve IE, echocardiographically severe valvular regurgitation and large vegetations41. The cohort was young (mean age of 47 years), was infected mainly by oral streptococci and had little comorbidity. The ‘early surgery’ group had a significant reduction in a composite endpoint of in-hospital death or embolism, driven by a reduction in the rate of embolism. This trial has led to a trend toward earlier surgery, but enthusiasm has been dampened by concerns that early surgery does not have the same benefits in the older, frailer IE population in much of the industrialized world. In prosthetic valve IE, retrospective studies looking at early surgery in patients have failed to find a benefit42,43.\n\nSurgery to prevent embolism remains the most controversial indication, as the embolic risk for a specific individual is often difficult to predict. In general, surgery should be considered for those in the highest-risk groups: those with very large vegetations (>30 mm) or persistent large vegetations (>10 mm) after an embolic event and despite antibiotics29. Mobile vegetations, staphylococcal infection and mitral valve location are also associated with increased embolic risk44. The risk is highest in the first 2 weeks of antibiotic therapy and so intervention for prevention of embolism must be performed early to optimise the risk-to-benefit ratio of major surgery.\n\nThe indications and implantation rates for cardiac devices have expanded dramatically in the last decade. Cardiac resynchronisation therapy is used widely for patients with advanced heart failure, and implantable (intracardiac) cardioverter defibrillators are commonly inserted for prevention of ventricular arrhythmias45. Similarly, in the last 15 years, transcatheter aortic valve implantation (TAVI) has gone from concept to clinical reality, and over 100,000 valves have now been implanted worldwide46. Cardiac device infection (CDI) currently accounts for roughly 10% of IE, and as use of cardiac devices and valve prostheses increases further, cardiologists should expect increasing rates of IE3.\n\nCDI affects 1% to 2% of patients in the first 5 years following device implantation and may involve the pocket housing the generator box, the leads or the endocardial surface20. There are limited studies to direct clinical management of CDI, but UK guidelines outlining current best practice and recommendations for research have recently been published20. Unless infection is superficial and limited to the healing wound, device extraction is required, and this can usually be achieved percutaneously but carries a small risk of mortality. The optimal duration of antibiotic therapy and risks of further infection after device re-implantation are unknown.\n\nReports of small cohorts of patients with IE following TAVI are starting to emerge47–49. A recently published multicentre registry identified a cohort of 53 patients with IE post-TAVI, representing a frequency of 0.5% at 1 year48. The mean time to IE from TAVI procedure was 6 months. Interestingly, use of the self-expanding CoreValve system (Medtronic, Dublin, Ireland) was independently associated with increased risk of IE (hazard ratio 3.12, 95% confidence interval 1.37 to 7.14, P = 0.007). In-hospital mortality for this group was 47%, reflecting the high rates of staphylococcal infection and frailty of the study population. Further studies are required to better define the incidence, risk factors and clinical outcomes of IE following TAVI.\n\n\nConclusions\n\nIE is a rare and multifaceted disease whose heterogeneity is a barrier to well-powered research trials. As a consequence, much of the evidence base is derived from observational studies. Despite ongoing uncertainty, some conclusions can be drawn. Given the increasing use of intracardiac prostheses and devices, an ongoing focus on IE prevention strategies is warranted. Reducing time to diagnosis and definitive management requires set-up of IE clinical teams and full use of multimodality imaging alongside echocardiography. The importance of abnormal CT and hybrid PET-CT imaging is now reflected in the 2015 ESC guidelines on IE, in which abnormal imaging findings are novel diagnostic criteria. Finally, research networks should move toward a focus on multicentre trials, for example to address uncertainties in the timing of surgery and management of CDI.\n\n\nAbbreviations\n\nCDI, cardiac device infection; CT, computed tomography; ESC, European Society of Cardiology; IE, infective endocarditis; MDT, multidisciplinary team; MRI, magnetic resonance imaging; PET, positron emission tomography; RCT, randomised controlled trial; TAVI, transcatheter aortic valve implantation; TOE, transoesophageal echocardiography.",
"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\nAcknowledgements\n\nThe authors thank Jim Newton, Andrew Kelion and Nik Sabharwal for assistance with Figure 1.\n\n\nReferences\n\nOsler W: The Gulstonian Lectures, on Malignant Endocarditis. Br Med J. 1885; 1(1264): 577–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCahill TJ, Prendergast BD: Infective endocarditis. Lancet. 2015; pii: S0140-6736(15)00067-7. PubMed Abstract | Publisher Full Text\n\nMurdoch DR, Corey GR, Hoen B, et al.: Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis-Prospective Cohort Study. Arch Intern Med. 2009; 169(5): 463–73. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSelton-Suty C, Célard M, Le Moing V, et al.: Preeminence of Staphylococcus aureus in infective endocarditis: a 1-year population-based survey. Clin Infect Dis. 2012; 54(9): 1230–9. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFowler VG, Miro JM, Hoen B, et al.: Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA. 2005; 293(24): 3012–21. PubMed Abstract | Publisher Full Text\n\nLockhart PB, Brennan MT, Sasser HC, et al.: Bacteremia associated with toothbrushing and dental extraction. Circulation. 2008; 117(24): 3118–25. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nWerdan K, Dietz S, Löffler B, et al.: Mechanisms of infective endocarditis: pathogen-host interaction and risk states. Nat Rev Cardiol. 2014; 11(1): 35–50. PubMed Abstract | Publisher Full Text\n\nLockhart PB, Brennan MT, Thornhill M, et al.: Poor oral hygiene as a risk factor for infective endocarditis-related bacteremia. J Am Dent Assoc. 2009; 140(10): 1238–44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRichey R, Wray D, Stokes T: Prophylaxis against infective endocarditis: summary of NICE guidance. BMJ. 2008; 336(7647): 770–1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHabib G, Hoen B, Tornos P, et al.: Guidelines on the prevention, diagnosis, and treatment of infective endocarditis (new version 2009): the Task Force on the Prevention, Diagnosis, and Treatment of Infective Endocarditis of the European Society of Cardiology (ESC). Endorsed by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the International Society of Chemotherapy (ISC) for Infection and Cancer. Eur Heart J. 2009; 30(19): 2369–413. PubMed Abstract | Publisher Full Text\n\nPasquali SK, He X, Mohamad Z, et al.: Trends in endocarditis hospitalizations at US children's hospitals: impact of the 2007 American Heart Association Antibiotic Prophylaxis Guidelines. Am Heart J. 2012; 163(5): 894–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nDayer MJ, Jones S, Prendergast B, et al.: Incidence of infective endocarditis in England, 2000-13: a secular trend, interrupted time-series analysis. Lancet. 2015; 385(9974): 1219–28. PubMed Abstract | Publisher Full Text\n\nPant S, Patel NJ, Deshmukh A, et al.: Trends in infective endocarditis incidence, microbiology, and valve replacement in the United States from 2000 to 2011. J Am Coll Cardiol. 2015; 65(19): 2070–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFernández-Hidalgo N, Almirante B, Tornos P, et al.: Contemporary epidemiology and prognosis of health care-associated infective endocarditis. Clin Infect Dis. 2008; 47(10): 1287–97. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLomas JM, Martínez-Marcos FJ, Plata A, et al.: Healthcare-associated infective endocarditis: an undesirable effect of healthcare universalization. Clin Microbiol Infect. 2010; 16(11): 1683–90. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nO'Grady NP, Alexander M, Burns LA, et al.: Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis. 2011; 52(9): e162–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPratt RJ, Pellowe CM, Wilson JA, et al.: epic2: National evidence-based guidelines for preventing healthcare-associated infections in NHS hospitals in England. J Hosp Infect. 2007; 65(Suppl 1): S1–64. PubMed Abstract | Publisher Full Text\n\nPronovost P, Needham D, Berenholtz S, et al.: An intervention to decrease catheter-related bloodstream infections in the ICU. N Engl J Med. 2006; 355(26): 2725–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBlot K, Bergs J, Vogelaers D, et al.: Prevention of central line-associated bloodstream infections through quality improvement interventions: a systematic review and meta-analysis. Clin Infect Dis. 2014; 59(1): 96–105. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nSandoe JA, Barlow G, Chambers JB, et al.: Guidelines for the diagnosis, prevention and management of implantable cardiac electronic device infection. Report of a joint Working Party project on behalf of the British Society for Antimicrobial Chemotherapy (BSAC, host organization), British Heart Rhythm Society (BHRS), British Cardiovascular Society (BCS), British Heart Valve Society (BHVS) and British Society for Echocardiography (BSE). J Antimicrob Chemother. 2015; 70(2): 325–59. PubMed Abstract | Publisher Full Text\n\nFowler VG Jr, Proctor RA: Where does a Staphylococcus aureus vaccine stand? Clin Microbiol Infect. 2014; 20 Suppl 5: 66–75. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nFowler VG, Allen KB, Moreira ED, et al.: Effect of an investigational vaccine for preventing Staphylococcus aureus infections after cardiothoracic surgery: a randomized trial. JAMA. 2013; 309(13): 1368–78. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nBeloin C, Renard S, Ghigo JM, et al.: Novel approaches to combat bacterial biofilms. Curr Opin Pharmacol. 2014; 18: 61–8. PubMed Abstract | Publisher Full Text\n\nDesrousseaux C, Sautou V, Descamps S, et al.: Modification of the surfaces of medical devices to prevent microbial adhesion and biofilm formation. J Hosp Infect. 2013; 85(2): 87–93. PubMed Abstract | Publisher Full Text\n\nPrendergast BD: Diagnostic criteria and problems in infective endocarditis. Heart. 2004; 90(6): 611–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBruun NE, Habib G, Thuny F, et al.: Cardiac imaging in infectious endocarditis. Eur Heart J. 2014; 35(10): 624–32. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFeuchtner GM, Stolzmann P, Dichtl W, et al.: Multislice computed tomography in infective endocarditis: comparison with transesophageal echocardiography and intraoperative findings. J Am Coll Cardiol. 2009; 53(5): 436–44. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nFagman E, Perrotta S, Bech-Hanssen O, et al.: ECG-gated computed tomography: a new role for patients with suspected aortic prosthetic valve endocarditis. Eur Radiol. 2012; 22(11): 2407–14. PubMed Abstract | Publisher Full Text\n\nAuthors/Task Force Members, Habib G, Lancellotti P, et al.: 2015 ESC Guidelines for the management of infective endocarditis: The Task Force for the Management of Infective Endocarditis of the European Society of Cardiology (ESC)Endorsed by: European Association for Cardio-Thoracic Surgery (EACTS), the European Association of Nuclear Medicine (EANM). Eur Heart J. 2015; pii: ehv319. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nThompson KA, Shiota T, Tolstrup K, et al.: Utility of three-dimensional transesophageal echocardiography in the diagnosis of valvular perforations. Am J Cardiol. 2011; 107(1): 100–2. PubMed Abstract | Publisher Full Text\n\nKronzon I, Sugeng L, Perk G, et al.: Real-time 3-dimensional transesophageal echocardiography in the evaluation of post-operative mitral annuloplasty ring and prosthetic valve dehiscence. J Am Coll Cardiol. 2009; 53(17): 1543–7. PubMed Abstract | Publisher Full Text\n\nSarrazin JF, Philippon F, Tessier M, et al.: Usefulness of fluorine-18 positron emission tomography/computed tomography for identification of cardiovascular implantable electronic device infections. J Am Coll Cardiol. 2012; 59(18): 1616–25. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nSaby L, Laas O, Habib G, et al.: Positron emission tomography/computed tomography for diagnosis of prosthetic valve endocarditis: increased valvular 18F-fluorodeoxyglucose uptake as a novel major criterion. J Am Coll Cardiol. 2013; 61(23): 2374–82. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nTeoh EJ, Backhouse L, Chandrasekaran B, et al.: Mycotic aneurysm of the superior mesenteric artery and other sequelae of prosthetic valve endocarditis on 18F-FDG PET/CT. Eur J Nucl Med Mol Imaging. 2014; 41(10): 1993–4. PubMed Abstract | Publisher Full Text\n\nPizzi MN, Roque A, Fernández-Hidalgo N, et al.: Improving the Diagnosis of Infective Endocarditis in Prosthetic Valves and Intracardiac Devices With 18F-Fluordeoxyglucose Positron Emission Tomography/Computed Tomography Angiography: Initial Results at an Infective Endocarditis Referral Center. Circulation. 2015; 132(12): 1113–26. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nVilacosta I, Graupner C, San Román JA, et al.: Risk of embolization after institution of antibiotic therapy for infective endocarditis. J Am Coll Cardiol. 2002; 39(9): 1489–95. PubMed Abstract | Publisher Full Text\n\nDuval X, Iung B, Klein I, et al.: Effect of early cerebral magnetic resonance imaging on clinical decisions in infective endocarditis: a prospective study. Ann Intern Med. 2010; 152(8): 497–504, W175. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChirillo F, Scotton P, Rocco F, et al.: Impact of a multidisciplinary management strategy on the outcome of patients with native valve infective endocarditis. Am J Cardiol. 2013; 112(8): 1171–6. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChambers J, Sandoe J, Ray S, et al.: The infective endocarditis team: recommendations from an international working group. Heart. 2014; 100(7): 524–7. PubMed Abstract | Publisher Full Text\n\nPrendergast BD, Tornos P: Surgery for infective endocarditis: who and when? Circulation. 2010; 121(9): 1141–52. PubMed Abstract | Publisher Full Text\n\nKang DH, Kim YJ, Kim SH, et al.: Early surgery versus conventional treatment for infective endocarditis. N Engl J Med. 2012; 366(26): 2466–73. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nLalani T, Chu VH, Park LP, et al.: In-hospital and 1-year mortality in patients undergoing early surgery for prosthetic valve endocarditis. JAMA Intern Med. 2013; 173(16): 1495–504. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nChirouze C, Alla F, Fowler VG Jr, et al.: Impact of early valve surgery on outcome of Staphylococcus aureus prosthetic valve infective endocarditis: analysis in the International Collaboration of Endocarditis-Prospective Cohort Study. Clin Infect Dis. 2015; 60(5): 741–9. PubMed Abstract | Publisher Full Text | Free Full Text | Faculty Opinions Recommendation\n\nHubert S, Thuny F, Resseguier N, et al.: Prediction of symptomatic embolism in infective endocarditis: construction and validation of a risk calculator in a multicenter cohort. J Am Coll Cardiol. 2013; 62(15): 1384–92. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nThe Task Force on cardiac pacing and resynchronization therapy of the European Society of Cardiology (ESC). Developed in collaboration with the European Heart Rhythm Association (EHRA), Brignole M, Esquivias G, et al.: 2013 ESC Guidelines on cardiac pacing and cardiac resynchronization therapy. Rev Esp Cardiol (Engl Ed). 2014; 67(1): 58. PubMed Abstract | Publisher Full Text\n\nRuparelia N, Prendergast BD: TAVI in 2015: who, where and how? Heart. 2015; 101(17): 1422–31. PubMed Abstract | Publisher Full Text\n\nOlsen NT, De Backer O, Thyregod HG, et al.: Prosthetic valve endocarditis after transcatheter aortic valve implantation. Circ Cardiovasc Interv. 2015; 8(4): pii: e001939. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nAmat-Santos IJ, Messika-Zeitoun D, Eltchaninoff H, et al.: Infective endocarditis after transcatheter aortic valve implantation: results from a large multicenter registry. Circulation. 2015; 131(18): 1566–74. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation\n\nPuls M, Eiffert H, Hünlich M, et al.: Prosthetic valve endocarditis after transcatheter aortic valve implantation: the incidence in a single-centre cohort and reflections on clinical, echocardiographic and prognostic features. EuroIntervention. 2013; 8(12): 1407–18. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "11263",
"date": "18 Nov 2015",
"name": "Ralph Corey",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11264",
"date": "18 Nov 2015",
"name": "Xavier Duval",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required 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": "11265",
"date": "18 Nov 2015",
"name": "Ron Blankstein",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions",
"responses": []
}
] | 1
|
https://f1000research.com/articles/4-1287
|
https://f1000research.com/articles/3-285/v1
|
19 Nov 14
|
{
"type": "Software Tool Article",
"title": "G-Links: a gene-centric link acquisition service",
"authors": [
"Kazuki Oshita",
"Masaru Tomita",
"Kazuharu Arakawa",
"Kazuki Oshita",
"Masaru Tomita"
],
"abstract": "With the availability of numerous curated databases, researchers are now able to efficiently use the multitude of biological data by integrating these resources via hyperlinks and cross-references. A large proportion of bioinformatics research tasks, however, may include labor-intensive tasks such as fetching, parsing, and merging datasets and functional annotations from distributed multi-domain databases. This data integration issue is one of the key challenges in bioinformatics. We aim to solve this problem with a service named G-Links, 1) by gathering resource URI information from 130 databases and 30 web services in a gene-centric manner so that users can retrieve all available links about a given gene, 2) by providing RESTful API for easy retrieval of links including facet searching based on keywords and/or predicate types, and 3) by producing a variety of outputs as visual HTML page, tab-delimited text, and in Semantic Web formats such as Notation3 and RDF. G-Links as well as other relevant documentation are available at http://link.g-language.org/",
"keywords": [
"databases",
"bioinformatics",
"data integration",
"molecular biology"
],
"content": "Introduction\n\nThe use of large-scale data or multi-domain information is becoming a prerequisite in all fields of molecular biology, in light of the advent of high-throughput measurement technologies exemplified by the new generation DNA sequencers, and further driven by the conceptual progress in integrative systems biology approaches. Typical analysis encompasses multiple genes in a pathway or in a regulatory network, uses orthologous gene sets in related organisms, and merges information from multiple-omics layers such as genome, transcriptome, proteome, and metabolome (Arakawa & Tomita, 2013). Bioinformatics researchers therefore need to collect and integrate data from a variety of sources, each with diverse syntax, semantics, protocols, identifiers and naming conventions (Bhagat et al., 2010; Brazas et al., 2012; Katayama et al., 2010). This data integration issue is one of the key challenges in the field of bioinformatics (Stein, 2002; Stein, 2008). While the integration of web services under standardized protocols has seen a sound progress over the last few years (Katayama et al., 2011), data integration with efficient cross-domain queries still requires the use of large-scale data warehouses such as BioMart (Smedley et al., 2009) and InterMine (Smith et al., 2012).\n\nSince the majority of biological databases are well curated with cross-references, related information can be retrieved ad hoc from dispersed databases using hyperlinks. In order to facilitate such processes, web services that collect and provide the cross-reference information from different databases (Diehn et al., 2003; Wu et al., 2013) as well as ID conversion services that assist cross-referencing have been developed (Cote et al., 2007). MyGene.info, for example, provides rapid programmatic access through a RESTful interface for gene-centric queries to retrieve cross-reference information from numerous databases. Gene-centric aggregation, which integrates databases using genes as the primary key, is a highly efficient approach in molecular biology, since the majority of databases have some sort of connection to genes or proteins, due to the success of the “central dogma” of molecular biology. Ideally, a database should be free from predefined schema or primary keys, and should have controlled syntax and semantics. Semantic Web initiatives are therefore collaboratively aiming to provide such framework through HyperText Transfer Protocol (HTTP) with Resource Description Framework (RDF) and Web Ontology Language (OWL) (Katayama et al., 2013). At the current state of Semantic Web technologies, however, cross-domain queries require extensive reasoning or manual curation of ontologies, and the cross-reference-based approach still has an advantage in terms of user experience with lower latency.\n\nCross-reference services usually provide database name and identifiers that do not explicitly define the actual location of the data. Moreover, gene-centric data aggregation services usually do not allow querying of gene sets. To this end, here we describe a new RESTful service named G-Links, which gathers Uniform Resource Identifiers (URI) from more than 100 databases in a gene-centric manner, and provide querying interface based on gene sets for hundreds of species. G-Links can be used programmatically as text data, from Semantic Web services, or from graphical HTML pages.\n\n\nImplementation\n\nG-Links is implemented with Perl programming language and MySQL 5.0, and has a straight-forward RESTful user interface. It internally resolves cross-references in four steps: ID conversion, retrieval of cross-references, filtering and extraction, and formatting of output. G-Links stores all cross-reference information in a gene-centric manner, and for this purpose, it utilizes UniProt IDs as the primary key. Therefore, G-Links first converts the user input to UniProt ID by ID conversion, based on 80 databases supported by UniProt ID Mapping Service (Huang et al., 2011). When a nucleotide or amino acid sequence is given as the query, G-Links searches the corresponding UniProt IDs by sequence similarity search using BLAT (Kent, 2002) against Swiss-Prot database (Bairoch et al., 2004), and when RefSeq ID for bacterial genomes or taxonomy ID is used as the input, G-Links collects all UniProt IDs of genes within the given species based on UniProt taxonomy (http://www.uniprot.org/taxonomy/). In the second step, G-Links collects all text annotations and database cross-references about the gene of interest, gathered from over 130 databases. Here the mapping to Gene Ontology slim (Harris et al., 2004) is pre-computed using map2slim (http://search.cpan.org/~cmungall/go-perl/scripts/map2slim), and resulting URLs for over 30 RESTful bioinformatics analysis web services supported by the G-language Web Services (Arakawa et al., 2010) and Keio Bioinformatics Web Service (KBWS) (Oshita et al., 2011) are generated on-the-fly. KBWS is an European Molecular Biology Open Software Suite (EMBOSS) (Rice et al., 2000) associated software package for accessing popular bioinformatics web services such as BLAST. All cross-references include the URI of the actual location of data, often expressed as Persistent Uniform Resource Locators (PURLs). Retrieved gene set and annotations are optionally filtered in the third step according to user input, and are formatted in the specified output format in the last step.\n\n\nResults\n\nG-Links is available at http://link.g-language.org/ as a RESTful web service, which is suited for resource-centric access and highly accessible via HTTP. Users can rapidly retrieve annotations and cross-references related to a given gene ID, taxonomy ID, or raw sequence data by simply accessing a certain URL. An overview of the URL syntax is presented in Figure 1. For example, the URL to retrieve all annotations and cross references related to the human BRCA1 gene (UniProt ID: BRCA1_HUMAN) is simply http://link.g-language.org/BRCA1_HUMAN (Figure 2). The ID of gene used in this query can be any of the identifiers used in 80 databases supported by G-Links. Programmatic access to this URL can retrieve all 653 annotations and cross-references within 0.2 seconds (tested on dual Xeon X5470 server). G-Links automatically adjusts the output format according to the user context, and outputs the results in human-readable interactive HTML format when accessed from modern HTML browsers, or in Tabular Separated Values (TSV) text format for programmatic access. The HTML format displays a gallery of image resources on the top, such as the pathway maps from KEGG database (Kanehisa et al., 2012), co-expressed gene network from COXPRESdb (Obayashi et al., 2013), and protein 3D structure from Protein Data Bank (Rose et al., 2013), followed by a long table of text annotations and cross-references including database name, ID, and resource URL. Table 1 shows an overview of the categories of databases and web services supported by G-Links output (see http://link.g-language.org/input_list and http://link.g-language.org/output_list for complete listings). In addition to the human-friendly HTML format and computer readable TSV as well as JavaScript Object Notation (JSON) output, G-Links supports RDF/XML and Notation3 (http://www.w3.org/TeamSubmission/n3/) formats, so that the query results can be readily integrated with Semantic Web technologies. For RDF and Notation3 predicate information is given by EMBRACE Data and Methods (EDAM) ontology.\n\nG-Links is implemented as a RESTful service that can be queried by altering the URL. Full documentation and example queries are available at http://www.g-language.org/wiki/glinks.\n\nBy default, access to G-Links with web browsers displays the results in interactive HTML, with related image gallery implemented with CoverFlow (http://imageflow.finnrudolph.de/) on the top, followed by a large table of annotations and cross-references.\n\nDetailed list of Input/Output databases are available at http://link.g-language.org/input_list and http://link.g-language.org/output_list.\n\nOne of the advantages of G-Links over existing cross-referencing services is its ability to retrieve information related to gene sets or all genes of organisms, and to filter out non-related genes by keyword search (filter option) or to extract necessary fields (extract option). Using the filtering option, users can retrieve only the subset of genes related to the given keyword. For example, retrieval of all human (taxonomy ID: 9606) genes having GO slim function including the word “transport” is as simple as http://link.g-language.org/9606/filter=GOslim_function:transport/format=tsv/. Similarly, extraction of only the “DISEASE” annotation of BRCA1 gene is simply http://link.g-language.org/BRCA1_HUMAN/extract=DISEASE. Multiple filtering and extraction conditions can be specified using “|” (vertical bar) as the separator, in order to formulate complex queries. For example, retrieval of SNP information from dbSNP and SNPedia for human genes with known polymorphisms related to breast and ovarian cancer in tabular format is queried as http://link.g-language.org/9606/format=tsv/filter=DISEASE:cancer/filter=:breast|:ovarian|:snps|:polymorphisms/extract=dbSNP|SNPedia.\n\n\nConclusions\n\nBy serving as a data hub of linked open biological data, G-Links can be a starting point in retrieval of gene-centric information. Users can quickly obtain related links and annotations of a gene of interest either graphically via HTML or programmatically via REST interface, such as the orthologs, Gene Ontology terms, protein structure, pathways, SNPs, and publications.\n\n\nSoftware availability\n\nG-Links is a RESTful service with base URL http://link.g-language.org/. Detailed documentation is available at http://www.g-language.org/wiki/glinks including service description, syntax, list of all available options, example queries (URLs) and sample scripts for programmatic access in Perl, Ruby, Python, and Java. Comprehensive lists of supported input/output databases and web services are available at http://link.g-language.org/input_list and http://link.g-language.org/output_list. Internal database of G-Links is regularly updated every month, and the source code is freely available from GitHub repository (http://github.com/cory-ko/G-Links).\n\nhttp://github.com/cory-ko/G-Links\n\nhttps://github.com/F1000Research/G-Links/releases/tag/v1.0\n\nhttp://dx.doi.org/10.5072/zenodo.12701 (Oshita & Arakawa, 2014)\n\nMIT License",
"appendix": "Author contributions\n\n\n\nKO and KA conceived and designed the software, and KO implemented the software. MT provided supervision for the study. KO and KA drafted the manuscript, and 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 KAKENHI Grant Number 222681029 from the Japan Society for the Promotion of Science (JSPS), and by funds from the Yamagata Prefectural Government and Tsuruoka City.\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 development of web services described in this work was significantly facilitated by the DBCLS BioHackathon 2012 hosted by the National Bioscience Database Center/Database Center for Life Science (NBDC/DBCLS). We thank the members of G-language Project at the Institute for Advanced Biosciences, Keio University, and elsewhere, for their extremely valuable feedback and support.\n\n\nReferences\n\nArakawa K, Kido N, Oshita K, et al.: G-language genome analysis environment with REST and SOAP web service interfaces. Nucleic Acids Res. 2010; 38(Web Server issue): W700–705. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArakawa K, Tomita M: Merging multiple omics datasets in silico: statistical analyses and data interpretation. Methods Mol Biol. 2013; 985: 459–470. PubMed Abstract | Publisher Full Text\n\nBairoch A, Boeckmann B, Ferro S, et al.: Swiss-Prot: juggling between evolution and stability. Brief Bioinform. 2004; 5(1): 39–55. PubMed Abstract | Publisher Full Text\n\nBhagat J, Tanoh F, Nzuobontane E, et al.: BioCatalogue: a universal catalogue of web services for the life sciences. Nucleic Acids Res. 2010; 38(Web Server issue): W689–694. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrazas MD, Yim D, Yeung W, et al.: A decade of Web Server updates at the Bioinformatics Links Directory: 2003–2012. Nucleic Acids Res. 2012; 40(Web Server issue): W3–W12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCote RG, Jones P, Martens L, et al.: The Protein Identifier Cross-Referencing (PICR) service: reconciling protein identifiers across multiple source databases. BMC Bioinformatics. 2007; 8: 401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDiehn M, Sherlock G, Binkley G, et al.: SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data. Nucleic Acids Res. 2003; 31(1): 219–223. 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): D258–261. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang H, McGarvey PB, Suzek BE, et al.: A comprehensive protein-centric ID mapping service for molecular data integration. Bioinformatics. 2011; 27(8): 1190–1191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKanehisa M, Goto S, Sato Y, et al.: KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012; 40(Database issue): D109–114. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatayama T, Arakawa K, Nakao M, et al.: The DBCLS BioHackathon: standardization and interoperability for bioinformatics web services and workflows. The DBCLS BioHackathon Consortium*. J Biomed Semantics. 2010; 1(1): 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatayama T, Wilkinson MD, Micklem G, et al.: The 3rd DBCLS BioHackathon: improving life science data integration with Semantic Web technologies. J Biomed Semantics. 2013; 4(1): 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatayama T, Wilkinson MD, Vos R, et al.: The 2nd DBCLS BioHackathon: interoperable bioinformatics Web services for integrated applications. J Biomed Semantics. 2011; 2: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKent WJ: BLAT--the BLAST-like alignment tool. Genome Res. 2002; 12(4): 656–664. PubMed Abstract | Publisher Full Text | Free Full Text\n\nObayashi T, Okamura Y, Ito S, et al.: COXPRESdb: a database of comparative gene coexpression networks of eleven species for mammals. Nucleic Acids Res. 2013; 41(Database issue): D1014–1020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOshita K, Arakawa K, Tomita M: KBWS: an EMBOSS associated package for accessing bioinformatics web services. Source Code Biol Med. 2011; 6: 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOshita K, Arakawa K: G-Links: F1000Research/G-Links. ZENODO. 2014. Data Source\n\nRice P, Longden I, Bleasby A: EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 2000; 16(6): 276–277. PubMed Abstract | Publisher Full Text\n\nRose PW, Bi C, Bluhm WF, et al.: The RCSB Protein Data Bank: new resources for research and education. Nucleic Acids Res. 2013; 41(Database issue): D475–482. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmedley D, Haider S, Ballester B, et al.: BioMart--biological queries made easy. BMC Genomics. 2009; 10: 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmith RN, Aleksic J, Butano D, et al.: InterMine: a flexible data warehouse system for the integration and analysis of heterogeneous biological data. Bioinformatics. 2012; 28(23): 3163–3165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStein L: Creating a bioinformatics nation. Nature. 2002; 417(6885): 119–120. PubMed Abstract | Publisher Full Text\n\nStein LD: Towards a cyberinfrastructure for the biological sciences: progress, visions and challenges. Nature Rev Genet. 2008; 9(9): 678–688. PubMed Abstract | Publisher Full Text\n\nWu C, Macleod I, Su AI: BioGPS and MyGene.info: organizing online, gene-centric information. Nucleic Acids Res. 2013; 41(Database issue): D561–565. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "6750",
"date": "27 Nov 2014",
"name": "Mark A. Ragan",
"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 introduces a simple approach to data integration that can assist bioinformatics researchers. The RESTful API is easy-to-use and allows gene-centric linking of information from a very large numbers of data sources. We recommend the following revisions: ScopeThe authors rightly present data integration as a key challenge in bioinformatics (Abstract), and present their work as aiming to solve this problem. However, data integration is a much deeper problem than just data aggregation. Literature reviewThe authors might mention earlier link-based aggregators, e.g. BioMOBY. Gene centricityA gene-centric approach is appropriate for some organisms (notably bacteria and archaea) and problems, but many questions in eukaryote systems biology require a transcript-centric approach. The authors might mention this (significant) limitation. Aliases and catching errorsAOF2 is an alias for KDM1A. A search for KDM1A works fine. A search for AOF2 using the search box on the website or using http://link.g-language.org/AOF2 returns a blank page with no error message. A search using http://link.g-language.org/AOF2_HUMAN gives an inappropriate error message (contact root@localhost). The article should describe whether and how the system tries to deal with gene aliases, and appropriate feedback should be given to the user when no results are found. WebsiteG-Links appears to be an extension of the G-language Project. Having the G-Links homepage within the G-language Project wiki is confusing, as menu-items on the left are relevant to the latter. We also suggest that the ‘Quick Star’ should be near the top of the page to facilitate ease of use. Comments on current Semantic Web technologiesThe last sentence of the second paragraph of the Introduction states that with current Semantic Web technologies, cross-domain queries require extensive reasoning or manual curation of ontologies. This is not an accurate description of the current state of Semantic Web technologies. The authors might alternatively make reference to the latest version (July 2014) of the Bio2RDF resource, which enables integration and federated queries across 35 datasets, and suggest that a limitation (in comparison to their RESTful API approach) is that the user is required to be familiar with the SPARQL search language. Example use casesThe use cases provided in the manuscript are quite simple and don’t really demonstrate why the G-Links approach can be more powerful than using (for instance) GeneCards to get an overview of a gene such as BRCA1, or just going to the KEGG website and running a search on ‘cancer’. A more comprehensive use case that shows, for example, how easily results from a gene list search in (for instance) TSV format can be programmatically searched to identify common elements for the genes of interest, might better demonstrate the utility of G-Links. Data integration as a current challengeThe references given to support the claim that data integration is currently a major challenge for the bioinformatics field are 12 and 6 years old. Several features claimed as advantages for G-Links (linking to multiple databases, retrieving information on genes from multiple organisms, filtering by keywords, extracting by fields) are reasonably common, e.g. in BioGrid or Cytoscape. LicensingIf any linked resources have license rights that need to be adhered to, this information should be brought to the user’s attention, perhaps using a note on the G-Links website. ConstraintsFor best practice, the system should meet the constraints for RESTful – stateless, cacheable and so on; these might be described briefly in the paper. The authors might mention that multiple URIs can point to the same resource. A resource can exist in different versions; does the system capture and display the version from which information has been captured? Finally, we recommend that the authors present a Semantic Web-specific example for their statement that ‘G-links can be used programmatically as text data, from Semantic Web services..’.",
"responses": [
{
"c_id": "1685",
"date": "18 Nov 2015",
"name": "Kazuharu Arakawa",
"role": "Author Response",
"response": "We would like to thank the reviewer for thorough review, and apologize for the extreme delay in our revision. Following are point-by-point comments for our revision.ScopeThe authors rightly present data integration as a key challenge in bioinformatics (Abstract), and present their work as aiming to solve this problem. However, data integration is a much deeper problem than just data aggregation. We have toned down this claim as follows: “We aim to provide an identifier conversion and data aggregation system as a part of solution to solve this problem”. Literature reviewThe authors might mention earlier link-based aggregators, e.g. BioMOBY. BioMOBY is a registry of bioinformatics web services, and is not a link-based aggregators. It allow the suggestion and discovery of web services based on the data type (ex. to discover BLAST services from a FASTA file – discovery of a service for a given input, or NCBI data retrieval services that produces a FASTA file – discovery of a service for a given output.). As an example of earlier link-based aggregators, we have mentioned MyGene.info service. Gene centricityA gene-centric approach is appropriate for some organisms (notably bacteria and archaea) and problems, but many questions in eukaryote systems biology require a transcript-centric approach. The authors might mention this (significant) limitation. We thank the Reviewer for this important comment. We have added the following paragraph in the manuscript to clarify this limitation.“The gene-centric approach is effective for data aggregation from a variety of databases, especially for prokaryotes, where the genes, transcripts, and proteins are mostly synonymous. On the other hand, this approach can be a limitation for many questions in eukaryote systems biology that require a transcript-centric approach due to the large complexity and diversity of transcriptome regulated by alternative splicing {Nilsen, 2010 #178}. Currently G-Links lists information of all transcript isoforms, their structures and other annotations, and therefore the gene-centric information can be queried from the identifiers related to the isoforms, but not necessarily the other way around.“ Aliases and catching errorsAOF2 is an alias for KDM1A. A search for KDM1A works fine. A search for AOF2 using the search box on the website or using http://link.g-language.org/AOF2 returns a blank page with no error message. A search using http://link.g-language.org/AOF2_HUMAN gives an inappropriate error message (contact root@localhost). The system is modified to provide 404 and 500 HTML errors with corresponding error messages when ID cannot be resolved. Redirection for aliases are likewise revised, and now http://link.g-language.org/AOF2_HUMAN shows a list of redirections. The article should describe whether and how the system tries to deal with gene aliases, and appropriate feedback should be given to the user when no results are found. Error handling is updated as described above. We do not specifically implement support for gene symbols, but UniProt aliases are now correctly handled. For gene symbols, ChiTaRS database contains the information for model eukaryotes, so all information about KDM1A can be retrieved from http://link.g-language.org/KDM1A, although it takes a little while to load all information. WebsiteG-Links appears to be an extension of the G-language Project. Having the G-Links homepage within the G-language Project wiki is confusing, as menu-items on the left are relevant to the latter. We also suggest that the ‘Quick Star’ should be near the top of the page to facilitate ease of use. The website (http://link.g-language.org/) is now not redirected and is given a dedicated page, and the ‘Quick Start’ menu is moved to the top as suggested. Comments on current Semantic Web technologiesThe last sentence of the second paragraph of the Introduction states that with current Semantic Web technologies, cross-domain queries require extensive reasoning or manual curation of ontologies. This is not an accurate description of the current state of Semantic Web technologies. The authors might alternatively make reference to the latest version (July 2014) of the Bio2RDF resource, which enables integration and federated queries across 35 datasets, and suggest that a limitation (in comparison to their RESTful API approach) is that the user is required to be familiar with the SPARQL search language. Revised accordingly. Example use casesThe use cases provided in the manuscript are quite simple and don’t really demonstrate why the G-Links approach can be more powerful than using (for instance) GeneCards to get an overview of a gene such as BRCA1, or just going to the KEGG website and running a search on ‘cancer’. A more comprehensive use case that shows, for example, how easily results from a gene list search in (for instance) TSV format can be programmatically searched to identify common elements for the genes of interest, might better demonstrate the utility of G-Links. We now provide illustrative examples of programmatic access from the UNIX commandline for Gene Ontology classification of all genes in E.coli, as well as for specific set of genes of interest for possible Gene Ontology enrichment analysis, or KEGG BRITE enrichment analysis in the website, and it is also mentioned in the text. Data integration as a current challengeThe references given to support the claim that data integration is currently a major challenge for the bioinformatics field are 12 and 6 years old. Two more latest reviews are added. Several features claimed as advantages for G-Links (linking to multiple databases, retrieving information on genes from multiple organisms, filtering by keywords, extracting by fields) are reasonably common, e.g. in BioGrid or Cytoscape. We have removed the mention that these are advantages, and mentioned that these features are common as in BioGrid and Cytoscape. LicensingIf any linked resources have license rights that need to be adhered to, this information should be brought to the user’s attention, perhaps using a note on the G-Links website. The information is added to the website. ConstraintsFor best practice, the system should meet the constraints for RESTful – stateless, cacheable and so on; these might be described briefly in the paper. Brief description is added to the manuscript: “The server provides a uniform interface based on URL and HTTP in a client-server model, which is stateless and therefore the server does not store any client context information, and the clients and intermediates can cache responses between server update cycles, duration of which is specified by HTML META tag.” The authors might mention that multiple URIs can point to the same resource. Revised accordingly. A resource can exist in different versions; does the system capture and display the version from which information has been captured? G-Links only provides the latest resource, and the manuscript is revised to include this information. Finally, we recommend that the authors present a Semantic Web-specific example for their statement that ‘G-links can be used programmatically as text data, from Semantic Web services..’. Since the use of RDF data requires storage of data obtained from G-Links in a triple store with other semantic web resources, we have removed the claim “from Semantic Web services”."
}
]
},
{
"id": "7340",
"date": "22 Jan 2015",
"name": "Kenji Satou",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAs described in this paper, G-Links system provides a sophisticated way of accessing gene-related information scattered in various databases. The revisions recommended by the first reviewer are still helpful. I think this paper is worth indexing after following the recommended revisions as much as possible. Minor comment: Isn't the number 22681029?",
"responses": [
{
"c_id": "1684",
"date": "18 Nov 2015",
"name": "Kazuharu Arakawa",
"role": "Author Response",
"response": "Minor comment: Isn't the number 22681029? The KAKENHI Grant Number is revised accordingly."
}
]
}
] | 1
|
https://f1000research.com/articles/3-285
|
https://f1000research.com/articles/4-432/v1
|
30 Jul 15
|
{
"type": "Software Tool Article",
"title": "Discovering, Indexing and Interlinking Information Resources",
"authors": [
"Fabrizio Celli",
"Johannes Keizer",
"Yves Jaques",
"Stasinos Konstantopoulos",
"Dušan Vudragović",
"Johannes Keizer",
"Yves Jaques",
"Stasinos Konstantopoulos",
"Dušan Vudragović"
],
"abstract": "The social media revolution is having a dramatic effect on the world of scientific publication. Scientists now publish their research interests, theories and outcomes across numerous channels, including personal blogs and other thematic web spaces where ideas, activities and partial results are discussed. Accordingly, information systems that facilitate access to scientific literature must learn to cope with this valuable and varied data, evolving to make this research easily discoverable and available to end users. In this paper we describe the incremental process of discovering web resources in the domain of agricultural science and technology. Making use of Linked Open Data methodologies, we interlink a wide array of custom-crawled resources with the AGRIS bibliographic database in order to enrich the user experience of the AGRIS website. We also discuss the SemaGrow Stack, a query federation and data integration infrastructure used to estimate the semantic distance between crawled web resources and AGRIS.",
"keywords": [
"Linked Data",
"Text Categorization",
"Recommender Systems",
"Web Crawling",
"AGRIS",
"SemaGrow"
],
"content": "Introduction\n\nAGRIS (http://agris.fao.org/) is the International System for Agricultural Science and Technology, a collection of nearly 8 million multilingual bibliographic resources spanning the last forty years and produced by a network of more than 150 institutions from 65 countries. AGRIS is currently part of the CIARD initiative (http://www.ciard.net/), a self-described “global movement dedicated to open agricultural knowledge”. Some AGRIS data sources are unique (http://aims.fao.org/activity/blog/agris-enriched-data-fao) to the system and AGRIS is the only way in which they can be accessed. The system’s goal is to make agricultural research globally discoverable, and as evidenced by Google Analytics it supports both developed and developing countries. Indeed, AGRIS is accessed from more than 200 countries and territories, reaching peaks of 250,000 visits per month. AGRIS users belong to two very different categories: the general public and agriculture professionals. In particular, a survey conducted at the end of 2014 helped to better describe the AGRIS audience [Celli et al., 2015]: researchers, professors, and graduate students looking for bibliographies, librarians, cataloguers, and people responsible for managing and disseminating research outcomes to the community and the rest of the world (including small and big journal publishers), and government officers asking for reports on specific topics. Since December 2013, AGRIS adopted a LOD (Linked Open Data) infrastructure [Anibaldi et al., 2015], which allowed the creation of mashup pages, where users looking for specific topics (e.g. impacts of climate change in a country) can access a publication from the AGRIS database, together with other related resources extracted from other preselected datasets. External resources available in AGRIS mashup pages are not only bibliographic metadata, but also distribution maps, statistics, germplasm accessions, and so on. In this paper we explore a new data source available in AGRIS mashup pages: the web itself.\n\nNowadays, scientists and researchers publish their results not only in journals or at conferences, but also via web 2.0 tools and other media [Kouper, 2010; Shema et al., 2012] in order to efficiently and broadly communicate their outcomes, a technique that also helps scientific research reach the general public, since newspapers, magazines and science blogs are often the quickest way to reach people informally. Blogs and other websites may also contain a corpus of ongoing research activities, unpublished material, grey literature, quick discussions, and experiments with negative results and ideas. The problem is that this information is usually not exposed using web services that can be consumed by machines, and the only way to access this rich amount of data is to use web search engines that typically return thousands of results, largely meaningless. In addition, most blogs and websites are not well categorized and so it is difficult for users and machines to discover what is actually relevant to the topic of interest.\n\nIn this context, we believe that it is important for AGRIS users – especially for researchers – to have access to those valuable pieces of information that are neither exposed in a database nor accessible via web service. Our goal is to crawl the web, starting from a list of manually preselected websites and then apply a set of machine learning algorithms to categorize discovered web resources. In recent years, much research has been done to crawl and mine the web. Numerous solutions have been proposed [Liakos et al., 2015; Soulemane et al., 2012; Suganya et al., 2015] to cope with the size of both the publicly indexable and the hidden web employing added semantics to discovered resources and to reuse them in fact sheets and mashups. In fact, it is not only important to discover web links, but also to process them in a way that allows reuse in multiple scenarios. The adoption of ontologies and LOD methodologies helps the analysis and enrichment of discovered web resources [Berendt et al., 2004]. Our work shows how it is possible to apply semantic enrichment to crawled web resources and to use this semantic knowledge to enhance the AGRIS web portal. More specifically, our work leverages Semantic Web technologies and the knowledge encoded in the AGROVOC (http://aims.fao.org/standards/agrovoc/about) thesaurus in order to recommend web resources that are relevant to a given AGRIS bibliographic item. AGROVOC is a multilingual vocabulary containing more than 32,000 agricultural concepts in 22 languages, aligned with 16 other multilingual knowledge organization systems related to agriculture, and developed by FAO over the course of thirty years [Caracciolo et al., 2011]. Adopting Semantic Web and LOD best practices and technologies, AGROVOC vocabulary items have been assigned URIs, organized into a SKOS-XL concept scheme (http://www.w3.org/TR/skos-reference/skos-xl.html), and served both as Linked Open data and via SPARQL endpoint (http://www.w3.org/TR/sparql11-overview).\n\nIn this article we discuss crawling and analyzing web resources to populate our “Crawler Database”; a SPARQL endpoint with AGROVOC annotations of web resources identified by the URL from which they were crawled. By providing web resources with semantics we can use the AGROVOC descriptions of AGRIS bibliographic entries to interlink AGRIS and the Crawler Database. This linking is then exploited by a recommender that identifies web resources that are relevant to AGRIS entries. Furthermore, we also discuss the preliminary testing of the SemaGrow Stack (http://www.semagrow.eu/) as the computational infrastructure for interlinking the AGRIS bibliographic database with the Crawler Database. The query federation and data integration functionalities of the SemaGrow Stack facilitate setting up experiments aiming at estimating semantic similarity between AGRIS entries and other resources. Although the core example that we discuss in this paper is based on the entities described in the Crawler Database, the power of the SemaGrow Stack is that it allows the re-use of this software in the context of different mashup pages combining AGRIS with a variety of LOD sources.\n\nThe entire process we discuss in this paper has already been implemented and integrated in the AGRIS website. While the tuning of the recommender system to compute accurate similarities is still an ongoing process, AGRIS mashup pages are enriched with the content of the Crawler Database and statistics are being collected in order to train the recommender system using user behavior. In addition, the workflow and the components described in this paper can be used in any domain, so they are not restricted to agriculture; one can simply use another thesaurus to annotate web resources and populate the Crawler Database.\n\n\nCrawling and indexing the web\n\nThe process of discovering and tagging web resources to display new content in the AGRIS website is based on two backend components: a customized Apache Nutch web crawler and AgroTagger. Figure 1 provides an overview of the entire process. As a starting point, in order to display relevant content in AGRIS, we manually preselect related websites to be used as input for the web crawler. Using these URLs the web crawler discovers other related web URLs, while AgroTagger assigns AGROVOC URIs to web URLs and creates the Crawler Database. In sections 2.1 and 2.2 we describe the two backend components.\n\nA web crawler is a piece of software that methodically and automatically analyzes web pages provided as input. Each input web page is a ROOT of the crawling process. During the analysis of a web page, the web crawler discovers all the hyperlinks available in that page, adding them to the list of web pages to be visited. The process stops at a specific depth, i.e. the number of hops a discovered link is from the ROOT. The depth parameter is defined by the user of the web crawler, with the idea that links decrease in relevance as their distance from the ROOT grows. At the end of the crawling process, a list with discovered web URLs is produced. Figure 2 shows an example execution of the web crawler.\n\nIn order to implement the process of enriching the AGRIS website with relevant web resources, we used a customized version of Apache Nutch (http://nutch.apache.org/), a highly extensible, scalable and configurable open-source web crawler. Web URLs provided as input were manually selected by experts in the domain of agriculture, in order to start the process from “trusted” and valuable websites. Currently, the depth parameter is set to 5, since this value seems to be a good compromise between discovering a good quantity of relevant resources and completing the task within an acceptable response time. The application, including the customized Apache Nutch web crawler and some Bash scripts to run it is available on GitHub (https://github.com/fcproj/agrotagger/tree/master/crawler). The application requires three input parameters:\n\n- The depth of the crawling process\n\n- The path to the directory that stores the output of the process\n\n- The path to the text file that contains the list of web URLs used as ROOTs by the crawler\n\nThe output of the application is a text file containing a structured list of discovered web URLs. Here is a snapshot of the output file:\n\n\n\nAgroTagger engine (https://github.com/fcproj/agrotagger) is a toolkit that assigns semantic terms to textual content. At a high level of abstraction, it can be considered as a keyword extractor that uses the AGROVOC thesaurus to extract keywords from a set of web URLs. It is based on MAUI (https://code.google.com/p/maui-indexer), a tool that combines a keyphrase extraction algorithm and a machine learning toolkit for the identification of topics in textual documents. AgroTagger has been trained to assign terms from the AGROVOC thesaurus, even if it currently works only with English documents. Training MAUI with AGROVOC in other languages will allow the applicability of AgroTagger in a multilingual environment, even if further tests need to be performed to understand how the tool behaves with non-Latin characters. Regarding accuracy, a recent test in which AgroTagger results were analyzed by professional indexers showed an accuracy of approximately 80%; in 20% of the cases the results were too broad. In brief, the accuracy measurement was carried out by domain experts actively involved in the development of the AGROVOC vocabulary based on manual annotations of a test sample from the AGRIS database.\n\nIn the process described in this paper, we apply AgroTagger to web URLs discovered by the web crawler and we annotate such URLs with AGROVOC URIs. Annotations are stored in a triple store (the Crawler Database) after which a recommender system defines some relevant combinations between AGRIS bibliographic resources and web documents, making use of AGROVOC as the backbone of the entire process. Considering AgroTagger as a black box, we can describe its I/O as:\n\n- Input: web URLs discovered by the web crawler\n\n- Output: a set of triples that annotate web URLs\n\nAgroTagger is a multi-threaded application, guaranteeing better performance while manipulating web URLs. For each web URL available in the input file, AgroTagger:\n\n- Downloads the resource available at the given web URL and converts it to a text file\n\n- Runs the MAUI indexer trained with AGROVOC\n\n- Produces a set of annotations as RDF triples\n\nCurrently, AgroTagger produces an RDF NTRIPLE file that describes semantic annotations for web URLs. Here is an example of an annotated web URL:\n\n\n\nTraining AgroTagger with a different thesaurus allows one to reuse the entire workflow and components described in this paper in completely different research domains.\n\n\nThe SemaGrow Stack\n\nSemaGrow is an FP7 European Project that seeks to develop scalable, efficient, and robust data services needed to take full advantage of the data-intensive and inter-disciplinary Science of 2020, re-shaping the way that data analysis techniques are applied to the heterogeneous data cloud. The core technical outcome of the project is the SemaGrow Stack. The SemaGrow Stack implements a SPARQL endpoint that federates SPARQL endpoints, transparently optimizing federated queries and dynamically integrating heterogeneous data models by applying the appropriate vocabulary transformations to queries and results [Konstantopoulos et al., 2013].\n\nThere are several key features of the SemaGrow Stack that address AGRIS use cases: it provides a querying interface that uses the result of ontology alignment to completely hide schema heterogeneity and also applies methods from database research and artificial intelligence that take into account data contents and optimize federated querying plans. The ontology alignment and dynamic vocabulary transformation facilities allow us to take advantage of multiple agricultural knowledge organization systems that have been aligned to AGROVOC. In this manner, one can develop AGROVOC-aware applications and use them over non-AGROVOC (but aligned) datasets without modification. The query optimizer is based on methods that automatically extract detailed metadata about the content of the federated endpoints, overcoming the lack of detail in manually provided annotations [Zoulis et al., 2015]. As an added benefit, the SemaGrow Stack implements fail-over mechanisms that fall back to alternatives in the face endpoint unavailability. It furthermore does not require any modification of the federated endpoints or any other obtrusion of current workflows.\n\nThe SemaGrow Stack is developed and distributed as open-source software (https://github.com/semagrow) and requires an Apache Tomcat environment (http://tomcat.apache.org/) in order to be deployed and executed.\n\n\nThe recommender system\n\nThe Crawler Database is composed of triples generated by AgroTagger. At this stage, the biggest problem is to compute a meaningful intersection with the AGRIS bibliographic database in order to display relevant information in an AGRIS mashup page. First of all we need to define the concept of “meaningful combinations”. A naïve approach is based on counting the number of AGROVOC URIs in common between resources from the Crawler Database and resources from the AGRIS bibliographic database. Thus, for an AGRIS mashup page, we can state that we want to display those resources from the Crawler Database having as many AGROVOC URIs as possible in common with the AGRIS bibliographic entry. To implement this naïve algorithm we developed a recommender system (https://github.com/fcproj/recommender), a JAVA component that computes meaningful combinations between the Crawler Database and the AGRIS database, and generates a new triplestore: the “Recommender Database”. The recommender system runs as required as new data is periodically generated by the web crawler.\n\nThe recommender system can make use of the SemaGrow Stack as the backbone of the process: in this way, the recommender system is able to compute meaningful combinations between all datasets federated by SemaGrow. Combinations are computed by counting the number of common “dcterms:subject” URIs (http://dublincore.org/documents/dcmi-terms/) between entities of the federated datasets (in the case of the AGRIS dataset, “dcterms:subject” URIs are AGROVOC URIs). The SemaGrow Stack is not strictly necessary, since the recommender system can also work querying two target SPARQL endpoints one by one. In any case, the SemaGrow Stack allows code reuse for all datasets; it is sufficient to configure the SemaGrow Stack by defining the URLs of the SPARQL endpoints included in the federation, and the recommender system can work using a single endpoint. Without the usage of the SemaGrow Stack as an intermediate layer, there is the need to modify the code to add further SPARQL endpoints to the process. Let’s explain this statement with an example. Currently, the recommender system has two ways of working (the user can configure it to use either mode): “federated”, which requires a single SPARQL endpoint, and “individual”, which accepts two SPARQL endpoints. The “federated” mode makes use of the SemaGrow Stack: if the user configures the Stack defining three or four endpoints in the federation, the recommender system can combine all of them. On the contrary, the “individual” mode works with a maximum of two SPARQL endpoints and would require additional software code if one wanted to add further endpoints to the process.\n\nOur experiments focus on the usage of the recommender system to interlink the AGRIS database and the Crawler Database, storing computed combinations in the Recommender Database. At this stage, the algorithm is very easy: we simply need to count the number of common AGROVOC URIs (expressed as objects of a “dct:subject” predicate) between entities of the AGRIS dataset and the Crawler Database. Using the SemaGrow Stack and the single query “federated” mode, the SPARQL query to implement the algorithm is:\n\n\n\nThis SPARQL query sorts resources from the Crawler Database by the number of AGROVOC URIs in common with a given AGRIS entity <$AGRIS_URI> after which it takes the 20 most relevant web resources and stores them in the Recommender Database as a set of recommendations for an AGRIS URI. In the query, the statement including the rdf:type predicate is only needed in order to get results from the Crawler Database; since the recommender system can work with any dataset federated by the SemaGrow Stack, the type needs to be an application-dependent parameter so that it can be configured to query different datasets.\n\nUnfortunately, there are still few limitations with the SemaGrow Stack. First of all, SPARQL queries must be optimized and they are affected by their content. For instance, using a FILTER statement makes for a high response time. Thus, in order to identify entities from the different datasets federated by the SemaGrow Stack, it is necessary that such datasets define an “rdf:type” (http://www.w3.org/TR/rdf-schema/) for their entities in order to avoid the usage of the FILTER statement. Moreover, given that at the time of these experiments the SemaGrow Stack distribution was limited and allowed only SPARQL queries with dct:subject and/or rdf:type predicates, we ran our experiments using the “individual” mode during processing and testing. At the time of publication, our final experiments using the “federated” mode are very promising; SPARQL queries that combine triples from federated datasets show a response time comparable to the execution time of a process that runs individual SPARQL queries to different endpoints and programmatically combines their results.\n\nThe “individual” mode that makes use of two different SPARQL queries to two different SPARQL endpoints: AGRIS and the Crawler Database. The first SPARQL query provides the list of AGROVOC URIs (“dct:subject” predicate) for a given <$AGRIS_URI>:\n\n\n\nFor all AGROVOC URIs provided by the previous query, a second SPARQL query computes the crawled web URLs which contain that AGROVOC URI:\n\n\n\nThen, a custom algorithm implemented in JAVA is used to count the number of common AGROVOC URIs between the AGRIS entity and web URLs returned by the previous query, storing the top 20 most relevant web resources in the Recommender Database. The overall custom algorithm can be defined as:\n\n\n\nThe algorithm was executed on 134,847 AGRIS URIs. The recommender system ran in a CentOS 5 environment with the following configuration:\n\n- Processor: Intel(R) Core(TM) i7 2.80GHz\n\n- Recommender system: 2GB RAM on an 8GB RAM machine, hosted in Rome\n\n- AGRIS SPARQL endpoint: hosted in Malaysia, at MIMOS (http://www.mimos.my/), configuration unknown\n\n- Crawler Database SPARQL endpoint: hosted in Serbia, at IPB (http://www.ipb.ac.rs/index.php/en/), configuration unknown\n\nDuring the execution of the experiment, the “IPTraf” (http://iptraf.seul.org/) tool computed a TCP flow rate of between 30 and 45 Kbits/s.\n\nFor each AGRIS URI, the computation of combinations required 3.65 seconds on average. The execution time range was between 2.34 and 5.33 seconds for each AGRIS URI. Differences depended on three aspects:\n\n- the number of AGROVOC URIs for a given AGRIS URI, which affects the response time of the AGRIS SPARQL endpoint;\n\n- the specificity of an AGROVOC concept; broader concepts are associated to many web URLs, so they affect the response time of the Crawler Database SPARQL endpoint and\n\n- network speed and delays.\n\nIt is important to remember that this is a background, offline process that runs periodically to keep the dataset up to date. End users querying AGRIS resources will never have to wait for these processes as they have already been performed. Thus, the aforementioned response times are perfectly acceptable.\n\nIn the Recommender Database, recommendations for the same AGRIS URI are sorted by relevance (currently, the relevance is given by the custom algorithm calculated using the number of AGROVOC URIs in common with the AGRIS resource). The recommender system provides a Similarity Score for each recommended web URL, i.e. the percentage of similarity between an AGRIS resource and a recommended web URL. There are three variables to take into account in order to determine the Similarity Score:\n\n- the number of AGROVOC URIs associated with an AGRIS record (we will refer to this variable as #AGRIS);\n\n- the number of AGROVOC URIs associated with a recommended web URL (#WEB) and\n\n- the number of common AGROVOC URIs between the web resource and the AGRIS record (#COMMON).\n\nA naïve approach would consider the Similarity Score as the ratio given by the division between #COMMON and #AGRIS:\n\n\n\nUnfortunately, this approach has some problems when the AGRIS record is associated with few AGROVOC URIs. For instance, consider the scenario described in Table 1.\n\nThe final two scores have an obvious problem of overestimation: the AGRIS record is associated with only 3 AGROVOC URIs and, even if the web URL has 3 common URIs with the AGRIS record, a score of 1.0 (100%) is too much to predict the similarity. Thus, we need an index to adjust the score when the AGRIS record has few AGROVOC URIs associated. This Similarity Index must be 1 in the case of maximum similarity, and 0 in the case of no common AGROVOC URIs. A naïve Similarity Index would be:\n\n\n\nThe problem with this index is that #WEB is equal to 10 in most cases, since AgroTagger assigns 10 AGROVOC URIs to web resources crawled by the web crawler. Thus, S = 1 only if #AGRIS is 10 and #COMMON is 10, but this situation is highly improbable. If we define a threshold τ to determine when a number of common AGROVOC URIs is relevant to determine a good Similarity Index, we can improve the quality of the Similarity Score. Currently, we have defined τ equal to 6, which means that all cases where #COMMON is equal or bigger than 6, the Similarity Index must be 1.0. In this way, defining the correction factor K:\n\nk = min(τ, #COMMON), where τ = 6\n\nThe Similarity Index σ can be computed as:\n\n\n\nThis index has all the properties we are looking for: if #COMMON is 0, σ is 0; if #COMMON is equals or bigger than the threshold τ (6), σ is 1. At this point, we can redefine the Similarity Score as:\n\n\n\nThis approach further improves the Similarity Score. Since #WEB is never bigger than 10, while #AGRIS could be ideally any positive integer, we can modify the denominator of the first factor introducing the upper limit T, which is the maximum number of AGROVOC URIs associated to an AGRIS resource that is meaningful to the computation of the Similarity Score (we define T = 10):\n\n\n\nWhere: T = 10 and τ = 6.\n\nTable 2 revises the scenario described in Table 1, computing the similarity score with the last formula.\n\nCurrently, the recommender system provides RDF in the RDF/XML serialization. As an example:\n\n\n\nThe custom algorithm to compute meaningful combinations is an area for further research and improvement. In particular, other parameters may play a key role to define the relevance of a web resource. For instance, the system might check if some AGROVOC terms used by an AGRIS record appear in the title of the crawled web resource, or in its description. Then, AgroTagger could also be improved to return the ranking score of each AGROVOC URI assigned to a web resource, in order to use such a score in the algorithm that computes combinations. In addition to that, we can observe users' behavior in an AGRIS mashup page in order to assign more relevance to web resources more frequently clicked by AGRIS users. Furthermore, interviews of users will be conducted to evaluate the relevance of resources displayed in AGRIS mashup pages. Finally, there may be other more exotic algorithms to calculate similarity that can give greater relevance to end users, but such experiments were outside the scope of this project.\n\n\nThe AGRIS front-end\n\nThe creation of an AGRIS mashup page is a dynamic and sensitive process, which is made possible by the usage of AGROVOC as the backbone of the system. In fact, AGRIS records come with AGROVOC URIs and, relying on AGROVOC formal alignments to many thesauri, it is possible to query publicly accessible web services or SRARQL endpoints to provide access to resources indexed with various thesauri. Thus, when the user selects a publication from the AGRIS database, the system can display related information on the same topic. External data sources are identified based on the content, the relevancy to the AGRIS domain, and the information provider [Celli et al., 2015].\n\nThis paper presented a set of components that implement a process of discovering web resources related to AGRIS records in order to enrich the user experience in AGRIS mashup pages. We also ran experiments with the SemaGrow Stack as a backend component that can easily extend the data sources federated by the AGRIS mashup pages. These experiments led directly to the addition of a new data source to the AGRIS mashup pages: the dataset of related resources crawled from the web.\n\nThe creation of this new data source required setting up a process that used numerous components. We implemented an automatic process that makes use of a web crawler to discover web resources, and relies on AgroTagger to annotate discovered URLs with AGROVOC URIs. Then, in order to compute meaningful combinations between the AGRIS database and the Crawler Database, we implemented a recommender system to define the relevance of web resources. The output of this process is a widget in the AGRIS mashup pages that, reading the content of the Recommender Database (that is continuously updated by an offline process), can display relevant resources from the web. In this way, we believe that AGRIS users may find relevant data that assists them in working with agricultural issues and food security.\n\nIn this section we show a recommendation example in order to start a brief discussion about the relevance of resources contained in the widget of Figure 7. Our starting point is the AGRIS article “Water Conservation of Qanat, using Optimum use of Water in Unused Seasons (Case study: Dehraz Qanat of Sabzevar)” (http://agris.fao.org/agris-search/search.do?recordID=IR2015000018). This article exposes some criteria to optimize the use of water carried by qanats (a qanat is an underground channel to transport water from an aquifer under a hill, especially for irrigation of hot and arid environments). Thus, as it is highlighted by the title, the article is mainly about “water conservation”. It has been indexed with eleven AGROVOC terms, including water management, soil sciences, soil conservation, and water conservation. The recommender system suggests five web resources related to this article, with a similarity score ranging between 42% and 60%. Such web resources are available in the widget “Activities from the web”.\n\nA manual analysis of results across a sample of AGRIS records by FAO consultants indicated that a Similarity Score bigger than 50% for results showed a good level of precision. In fact, in the example we are analyzing, the first suggestion is a web page of the USDATA.GOV website (http://afsic.nal.usda.gov/soil-and-water-management/water-conservation); the score is 60% and the resource is completely related with the article, since it contains other resources about “water conservation” coming from USA universities and other agencies. Matching AGROVOC terms of the AGRIS article with the title of this resource would have further increased the Similarity Score, since the title itself is about “water conservation”. Regarding the remaining four suggestions, the example shows how a similarity score of 42% does not always predict good results. For instance, the web page of Cornell University (http://conservationagriculture.mannlib.cornell.edu/pages/resources/photosvideos.html) is relevant to the article, since it contains – among other things – some Power Point presentations about “water management” and “soil management”. Conversely, the other resources with the same similarity score of 42% are irrelevant. As previously discussed, improving the recommender system by matching AGROVOC terms of the AGRIS article with the titles and descriptions of crawled web resources should greatly improve the quality of results: in this example, the improved algorithm would have suggested the XML feeds from EPRINTS about “agricultural water management”, or the PDF document entitled “Agricultural Perspectives on Water Resource Management in the Americas”, both of which are quite relevant and yet missing from the current list.\n\n\nConclusions\n\nThe web contains much latent knowledge, especially when that knowledge is expressed as unstructured and poorly categorized full-text content. This paper describes a proposed solution to discover such knowledge making use of modified open source software (Nutch and Maui) together with the SemaGrow Stack and a custom recommender in order to enrich the relevance of AGRIS bibliographic resources and hence the AGRIS web portal mashup.\n\nThe adoption of the SemaGrow Stack as a backend facilitated the development of a recommender engine as it was possible to implement the system without requiring any prior knowledge of the specifics of the datasets that are combined with the AGRIS database. In this manner, the system can be re-used with any dataset using AGROVOC (or any terminology aligned to AGROVOC) to describe websites, experiments, software, or any resources relevant to agriculture. We are now able to show to AGRIS users any relevant content extracted from the web, something possible thanks to the adoption of semantic web technologies. The entire process will continue to be extended and improved as experiments continue: AgroTagger and the recommender system will be tuned to guarantee better precision in the computation of recommendations (for instance, trying to match AGROVOC keywords with titles of target resources and experimenting with alternative algorithms), while the SemaGrow Stack will be optimized and used to federate additional data sources.\n\nFurthermore, as previously mentioned, training AgroTagger with a different thesaurus allows to apply the entire process to different research domains, and not only to the AGRIS website.\n\n\nSoftware availability\n\nLatest source code (AgroTagger): https://github.com/fcproj/agrotagger\n\nLatest source code (recommender system): https://github.com/fcproj/recommender\n\nArchived source code at the time of publication (AgroTagger): http://dx.doi.org/10.5281/zenodo.20777 (Celli, 2015a).\n\nArchived source code at the time of publication (recommender system): http://dx.doi.org/10.5281/zenodo.20775 (Celli, 2015b).\n\nLicense: CC BY 4.0 http://creativecommons.org/licenses/by/4.0/",
"appendix": "Author contributions\n\n\n\nFC conceived conceptual ideas, implemented all the software components (except for the SemaGrow Stack), designed the experiments, and prepared the manuscript.\n\nJK made provided guidance to the development of the AGRIS website, as well as conceptual suggestions to the implementation of the crawling and tagging processes.\n\nYJ participated in AGRIS modelling, study design and selection of technology solutions. He also prepared and revised the language of the manuscript.\n\nSK contributed sections 3 and part of section 1. He was also responsible for the conceptual and technical development of the SemaGrow Stack.\n\nDV supported the testing of the crawling and recommendation processes, providing access to computing resources of PARADOX supercomputing facility at the Scientific Computing Laboratory of the Institute of Physics Belgrade.\n\nAll 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 European Commission under EU FP7 project SemaGrow (Grant No. 318497), and in part by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (under project ON171017).\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 acknowledge use of computing resources of PARADOX supercomputing facility at the Scientific Computing Laboratory of the Institute of Physics Belgrade.\n\n\nReferences\n\nAnibaldi S, Jaques Y, Celli F, et al.: Migrating bibliographic datasets to the Semantic web: The AGRIS case. Semantic Web. 2015; 6(2): 113–120. Publisher Full Text\n\nBerendt B, Hotho A, Mladenic D, et al.: A roadmap for web mining: from web to semantic web. Web Mining: From Web to Semantic Web. vol. 3209 of Lecture Notes in Computer Science, Springer, Berlin, Germany. 2004; 3209: 1–22. Publisher Full Text\n\nCaracciolo C, Morshed A, Stellato A, et al.: Thesaurus Maintenance, Alignment and Publication as Linked Data: the AGROOVOC use case. In Proceedings of the 5th Intl Conference on Metadata and Semantic Research Proceedings, Izmir, Turkey. 2011; 240: 489–499. Publisher Full Text\n\nCelli F, Malapela T, Wegner K, et al.: AGRIS: providing access to agricultural research data exploiting open data on the web [v1; ref status: approved 1 http://f1000r.es/599]. F1000Res. 2015; 4: 110. Publisher Full Text\n\nCelli F: agrotagger: crawler_agrotagger_1_2_5_DOI. Zenodo. 2015a. Data Source\n\nCelli F: recommender: agris_recommender_system_1_3_2_DOI. Zenodo. 2015b. Data Source\n\nKonstantopoulos S, Koukourikos A, Karampiperis P: Improving the Real-time Performance of Heterogeneous Extremely Large Datasets. In Proc. of the 17th Panhellenic Conference on Informatics, Thessaloniki, Greece. 2013; 287–293. Publisher Full Text\n\nKouper I: Science blogs and public engagement with science: Practices, challenges and opportunities. Journal of Science Communication. 2010. Reference Source\n\nLiakos P, Ntoulas A, Labrinidis A, et al.: Focused crawling for the hidden web. In Proceedings of the World Wide Web 2015 Conference Proceedings, Florence, Italy. 2015. Publisher Full Text\n\nShema H, Bar-Ilan J, Thelwall M: Research Blogs and the Discussion of Scholarly Information. PLoS ONE. 2012; 7(5): e35869. Publisher Full Text\n\nSoulemane M, Rafiuzzaman M, Mahmud H: Crawling the Hidden web Approach to Dynamic web Indexing. International Journal of Computer Applications. 2012; 55(1): 7–15. Publisher Full Text\n\nSuganya Devi R, Manjula D, Siddharth RK: An Efficient Approach for Web Indexing of Big Data through Hyperlinks in Web Crawling. ScientificWorldJournal. 2015; 2015: 739286. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZoulis N, Mavroudi E, Lykoura A, et al.: Workload-Aware Self-Tuning Histograms of String Data. In Proceedings of the 26th DEXA Conference (DEXA 2015), 1–4 September, Valencia, Spain. 2015."
}
|
[
{
"id": "9730",
"date": "24 Aug 2015",
"name": "Paolo Missier",
"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\nOverall, I found the article interesting and easy to read. In line with the F1000 reviewers' guidelines, I am going to comment \"on the quality of the research and whether the article is scientifically sound (i.e. whether the work has been well designed, executed and discussed)\", rather than on novelty, also making suggestions for improvements in the presentation.The work partly builds on prior established tools designed for the AGRIS system, mainly the AGROVOC thesaurus, as well as methods for federating SPARQL queries over heterogeneous resources (the SemaGRow stack).The original research contribution consists mainly of a focused crawler that looks for web resources that are relevant to the AGRIS community, the AgroTagger annotation tool, which annotates those web resources with semantic terms from the AGROVOC thesaurus, and a recommender tool that uses the annotated database to suggest web resources that are likely to be relevant to a given AGRIS bibliographic item. The annotation model is fine, however the evaluation part of the paper is weak, especially with regards to recommendation. This is where I think the authors have an obligation to report on the accuracy (ie F-scores) of their method on a benchmark of test cases, especially since substantial effort is spent in the paper to explain the similarity score used by the recommender. This is completely missing, however, the only evidence of the method at work being an example towards the end. For an experimental paper, this is hardly acceptable. So my main substantial recommendation is to strengthen the evaluation section (this may appear in other articles cited in the references, however that was not immediately clear to me).On matters of form and presentation, I found that the methods, including the recommender algorithm, are very simple (by the authors' own admission) and thus in my opinion the exposition can be greatly abbreviated. For example, fig. 2 and 3 are not very informative and could be removed. Also the lengthy RDF fragments are not very significant (but if you need them, please switch to n3 notation!), the annotation model of fig 4 is very simple (essentially, just a dcterms:subject), the 6-points algorithm is a straightforward intersection operation and has already been described in the text, the queries on pg 7 are fairly simple, etc.Further minor presentation suggestions:You may not need to build up the similarity score from naive to final, possibly just describe the final version?There is a mix of design and implementation / performance considerations, these may be best kept separate.A new section header may be missing after the first paragaph of \"agris front end\"?",
"responses": [
{
"c_id": "1682",
"date": "17 Nov 2015",
"name": "Fabrizio Celli",
"role": "Author Response",
"response": "We would really like to thank Dr. Paolo Missier for his useful comments.We ran an analysis of relevance on a test set of resources in the domain of fisheries. We were able to compute precision, but not recall. In fact, recall is mainly related to the web crawling, and it is not easy to estimate it. In addition to that, in the context of the AGRIS website it is not strongly important: we are more interested in a good precision, especially because we display only 5 recommendations per AGRIS resource.We separated the performance experiments from the deployment section, adding further details. Performance experiments were performed again using a bigger test set and we compared execution time in the “individual” mode with the execution time in the “federated” mode.We abbreviated a bit the exposition (for instance, we removed the six-points algorithm and the RDF fragment related to the output of the recommender system), but we maintained the discussion about the flow of thoughts leading from the naïve similarity score to the final one because we thought it helps in justifying some parameters available in the final formula.We hope that, based on these modifications, the referee can now fully approve our article."
}
]
},
{
"id": "10291",
"date": "28 Sep 2015",
"name": "Kei Kurakawa",
"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 proposed a functional enhancement for the agricultural journal article bibliographic database search, AGRIS, whose bibliographic page has additional web resources information relevant to its content. The web resources are automatically crawled by a customized Nutch web crawler, and labeled with some agricultural index terms, i.e. AGROVOC by a kind of machine learning module, the AgroTagger classifier. Since AGRIS bibliographies are already labeled with AGROVOC, the paper proposed a similarity measure between AGRIS bibliographies and web resources based on the number of common and originally labeled AGROVOC terms on them. AGRIS bibliographies and web resources are stored in SPARQL servers, so that the SemaGrow stack is used to easily send queries to them to calculate the similarity scores. The recommender system provides at most top 20 relevant web resources for an AGRIS bibliography by the calculation, which is serialized on RDF/XML. I recognize that the whole framework to construct the new functionality is valuable, but the paper is not sufficient with computational performance evaluation on the framework and quality evaluation for the linkages between bibliographies and web resources in an approved scientific method. Below are the suggestions for minor improvements. In page 3:In the “Crawling and indexing the web” section, the author describes “In the section 2.1 and 2.2” in the leading paragraph. The section numbers might not be forbidden to point to sections of this article. In page 4:In the left column of the “web crawler” sub-section, the author shows a snap shot of the output file of a Nutch web clawer. Please describe the structure of the file. I would like to know what some tags mean to construct the structure. Why is a blank URL listed in the example? In the left column of the “AggroTagger classifier” sub-section, the author mentions the quality of tagging by AgroTagger such as “AgroTagger results were analyzed by professional indexers showed an accuracy of approximately 80%; in 20% of the cases the results were too broad.” I would like to know more precise descriptions of the quality of tagging. How large is the training set? How did the author construct the training set? Please describe the training set. In addition, the test set for tagging and examining its accuracy should be described. In the “AggroTagger classifier” sub-section, the figure 3 and 4 should be explicitly referenced. In the current article, those figures are not referenced in any sentences. In the first phrase of “The SemaGrow Stack” section, the author refers “the data-intensive and inter-disciplinary Science of 2020”. Does “2020” means “Horizon 2020”? Or, does it refer to another funding framework? Science of 2020? If it is another funding framework, please cite a relevant web site for it. Please make sure what it is. In page 6:In the “recommender system” section, the second paragraph of “The recommender system can make use of … add further endpoints to the process”, the figure 5 should be explicitly referenced. In addition, for good understanding of reconfigurable structures consisting of the “federated” and the “individual”, the author had better depict both structures in the figure 5. In page 7:In the right column, the author describes “the overall custom algorithm” as a list form. For better understanding, this algorithm should be represented in a pseudo-programming code form. In the right column, the author mentions some experimental conditions and results of the number of AGRIS URIs and the average execution time. In addition, how many AGROVOC URIs are prepared for AGRIS URIs or Web URLs in this experiment? How long does it take for all processing? Please figure out the time of processing in the “individual” mode and the “federated” mode. Which is better from the viewpoint of computation? In page 8:In the right column, line 13, “factor K” should be changed to the lower case “factor k”. In the last similarity formula, “braces ( )” should be written in the proper place. In table 2, “Real Score” might be “ Revised Score”? In page 9:In the right column, the section “AGRIS front-end”, please indicate explicitly figure 6 in a sentence.",
"responses": [
{
"c_id": "1681",
"date": "17 Nov 2015",
"name": "Fabrizio Celli",
"role": "Author Response",
"response": "We would really like to thank Dr. Kei Kurakawa for this useful review. The paper was improved following his comments. In particular:We separated the performance experiments from the deployment section. Performance experiments were performed again using a bigger test set and we compared execution time in the “individual” mode with the execution time in the “federated” mode.We ran an analysis of relevance on a test set of resources in the domain of fisheries. We were able to compute precision of linkages, adding some additional details.The “web crawler” section was revised: we described the structure of the output file, which is generated using the “readseg –dump” command line tool provided by Apache Nutch. The blank URL listed in the example was meaningless, so we removed it.The “AgroTagger” section was revised too, with the addition of a more precise description of the test sample used to measure accuracy. Then, the paper including the work of training MAUI was cited.In the “SemaGrow Stack” section, the reference to the “Science of 2020” was removed, since it was misleading. In fact, it is part of SemaGrow proposal description, so it does not explicitly refer to Horizon 2020 or to any other framework, but to a general view of the world in 2020.All minor improvements suggested by the reviewer were implemented.We hope that, based on these modifications, the referee can now fully approve our article."
}
]
},
{
"id": "10769",
"date": "12 Oct 2015",
"name": "Leonidas Papachristopoulos",
"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\nCurrent study entitled \"Discovering, Indexing and Interlinking Information Resources\" consists of a technical evaluation of an enriching system attached to an agricultural bibliographic database called AGRIS. I am not sure about the novelty of the system that is presented but I can admit that is an interesting work which does not pose any reading difficulties to the reader, even to those who are not so familiar. Lately, recommending systems have been set on the center of the research activity and the specific work is following the research trends of the area. In my opinion there are some gaps in the study. For example on the description of the Agrotagger crawler researchers say: “Regarding accuracy, a recent test in which AgroTagger results were analyzed by professional indexers showed an accuracy of approximately 80%; in 20% of the cases the results were too broad. In brief, the accuracy measurement was carried out by domain experts actively involved in the development of the AGROVOC vocabulary based on manual annotations of a test sample from the AGRIS database”It is not clear to me if the aforementioned test has been held in the context of another previous study or it covers the specific study. If it has been held in the past a reference is necessary. Otherwise researchers need to provide further details. Additionally I think that authors should provide more information about accuracy tests. In my opinion the paper describes adequately the design and implementation but needs further enrichment regarding evaluation data. Authors should consider papers restructuring as in many cases I get lost between implementation and evaluation points. Some minor syntax comments are: In Page 1: “AGRIS database, together with other related resources extracted from other preselected datasets”… “together with” should be changed to “combined with”. In Page 1: \"Nowadays, scientists and researchers publish their results not only in journals or at conferences, but also via web 2.0 tools and other media [Kouper, 2010; Shema et al., 2012] in order to efficiently and broadly communicate their outcomes, [a technique that also helps scientific research reach the general public, since newspapers, magazines and science blogs are often the quickest way to reach people informally].\" For better understanding the sentence should be split into two. From the point “a technique’ should start another sentence. In Page 1: \"Blogs and other websites may also contain a corpus of ongoing …\" “Corpus” should be changed into “corpora” In Page 2: \"Adopting Semantic Web and LOD best practices and technologies, AGROVOC vocabulary items have been assigned URIs, organized into a SKOS-XL concept scheme (http://www.w3.org/TR/skos-reference/skos-xl.html), and served both as Linked Open data and via SPARQL endpoint (http://www.w3.org/TR/sparql11-overview).\" Re-write the sentence for better understanding.",
"responses": [
{
"c_id": "1680",
"date": "17 Nov 2015",
"name": "Fabrizio Celli",
"role": "Author Response",
"response": "Really thanks for your comments. As you suggested, the “AgroTagger” section was improved. We added a reference to a paper including details about the MAUI training set. We also added a more precise description of the test sample used to measure accuracy of AgroTagger. In addition to that, evaluation of performance was moved to a separate section, together with further details as the comparison between the execution time in the individual and federated modes. Moreover, we improved the evaluation of relevance of recommendations: we ran an analysis of relevance on a test set of resources in the domain of fisheries, computing precision and providing other comments. We hope that, based on these modifications, the referee can now fully approve our article."
}
]
}
] | 1
|
https://f1000research.com/articles/4-432
|
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