Assessing Social – Ecological Trade-Offs to Advance Ecosystem-Based Fisheries Management

Modern resource management faces trade-offs in the provision of various ecosystem goods and services to humanity. For fisheries management to develop into an ecosystem-based approach, the goal is not only to maximize economic profits, but to consider equally important conservation and social equity goals. We introduce such a triple-bottom line approach to the management of multi-species fisheries using the Baltic Sea as a case study. We apply a coupled ecological-economic optimization model to address the actual fisheries management challenge of trading-off the recovery of collapsed cod stocks versus the health of ecologically important forage fish populations. Management strategies based on profit maximization would rebuild the cod stock to high levels but may cause the risk of stock collapse for forage species with low market value, such as Baltic sprat (Fig. 1A). Economically efficient conservation efforts to protect sprat would be borne almost exclusively by the forage fishery as sprat fishing effort and profits would strongly be reduced. Unless compensation is paid, this would challenge equity between fishing sectors (Fig. 1B). Optimizing equity while respecting sprat biomass precautionary levels would reduce potential profits of the overall Baltic fishery, but may offer an acceptable balance between overall profits, species conservation and social equity (Fig. 1C). Our case study shows a practical example of how an ecosystem-based fisheries management will be able to offer society options to solve common conflicts between different resource uses. Adding equity considerations to the traditional trade-off between economy and ecology will greatly enhance credibility and hence compliance to management decisions, a further footstep towards healthy fish stocks and sustainable fisheries in the world ocean.Open in a separate windowFigure 1Summary of multispecies management options in the Baltic.(A) Profit maximum. (B) Economic optimum while respecting sprat B_PA. (C) Equitable optimum while respecting sprat B_PA. Central numbers indicate total profits (million €/year) as well as an equity measure (in brackets). Area of each pie slice is relative to status quo values 2008-2010 (black circle), with error bars from sensitivity analysis.     

What Can Article-Level Metrics Do for You?

Article-level metrics (ALMs) provide a wide range of metrics about the uptake of an individual journal article by the scientific community after publication. They include citations, usage statistics, discussions in online comments and social media, social bookmarking, and recommendations. In this essay, we describe why article-level metrics are an important extension of traditional citation-based journal metrics and provide a number of example from ALM data collected for PLOS Biology.The scientific impact of a particular piece of research is reflected in how this work is taken up by the scientific community. The first systematic approach that was used to assess impact, based on the technology available at the time, was to track citations and aggregate them by journal. This strategy is not only no longer necessary—since now we can easily track citations for individual articles—but also, and more importantly, journal-based metrics are now considered a poor performance measure for individual articles [1],[2]. One major problem with journal-based metrics is the variation in citations per article, which means that a small percentage of articles can skew, and are responsible for, the majority of the journal-based citation impact factor, as shown by Campbell [1] for the 2004 Nature Journal Impact Factor. Figure 1 further illustrates this point, showing the wide distribution of citation counts between PLOS Biology research articles published in 2010. PLOS Biology research articles published in 2010 have been cited a median 19 times to date in Scopus, but 10% of them have been cited 50 or more times, and two articles [3],[4] more than 300 times. PLOS Biology metrics are used as examples throughout this essay, and the dataset is available in the supporting information (Data S1). Similar data are available for an increasing number of other publications and organizations.Open in a separate windowFigure 1Citation counts for PLOS Biology articles published in 2010.Scopus citation counts plotted as a probability distribution for all 197 PLOS Biology research articles published in 2010. Data collected May 20, 2013. Median 19 citations; 10% of papers have at least 50 citations.Scientific impact is a multi-dimensional construct that can not be adequately measured by any single indicator [2],[5],[6]. To this end, PLOS has collected and displayed a variety of metrics for all its articles since 2009. The array of different categorised article-level metrics (ALMs) used and provided by PLOS as of August 2013 are shown in Figure 2. In addition to citations and usage statistics, i.e., how often an article has been viewed and downloaded, PLOS also collects metrics about: how often an article has been saved in online reference managers, such as Mendeley; how often an article has been discussed in its comments section online, and also in science blogs or in social media; and how often an article has been recommended by other scientists. These additional metrics provide valuable information that we would miss if we only consider citations. Two important shortcomings of citation-based metrics are that (1) they take years to accumulate and (2) citation analysis is not always the best indicator of impact in more practical fields, such as clinical medicine [7]. Usage statistics often better reflect the impact of work in more practical fields, and they also sometimes better highlight articles of general interest (for example, the 2006 PLOS Biology article on the citation advantage of Open Access articles [8], one of the 10 most-viewed articles published in PLOS Biology).Open in a separate windowFigure 2Article-level metrics used by PLOS in August 2013 and their categories.Taken from [10] with permission by the authors.A bubble chart showing all 2010 PLOS Biology articles (Figure 3) gives a good overview of the year''s views and citations, plus it shows the influence that the article type (as indicated by dot color) has on an article''s performance as measured by these metrics. The weekly PLOS Biology publication schedule is reflected in this figure, with articles published on the same day present in a vertical line. Figure 3 also shows that the two most highly cited 2010 PLOS Biology research articles are also among the most viewed (indicated by the red arrows), but overall there isn''t a strong correlation between citations and views. The most-viewed article published in 2010 in PLOS Biology is an essay on Darwinian selection in robots [9]. Detailed usage statistics also allow speculatulation about the different ways that readers access and make use of published literature; some articles are browsed or read online due to general interest while others that are downloaded (and perhaps also printed) may reflect the reader''s intention to look at the data and results in detail and to return to the article more than once.Open in a separate windowFigure 3Views vs. citations for PLOS Biology articles published in 2010.All 304 PLOS Biology articles published in 2010. Bubble size correlates with number of Scopus citations. Research articles are labeled green; all other articles are grey. Red arrows indicate the two most highly cited papers. Data collected May 20, 2013.When readers first see an interesting article, their response is often to view or download it. By contrast, a citation may be one of the last outcomes of their interest, occuring only about 1 in 300 times a PLOS paper is viewed online. A lot of things happen in between these potential responses, ranging from discussions in comments, social media, and blogs, to bookmarking, to linking from websites. These activities are usually subsumed under the term “altmetrics,” and their variety can be overwhelming. Therefore, it helps to group them together into categories, and several organizations, including PLOS, are using the category labels of Viewed, Cited, Saved, Discussed, and Recommended (Figures 2 and ​and4,4, see also [10]).Open in a separate windowFigure 4Article-level metrics for PLOS Biology.Proportion of all 1,706 PLOS Biology research articles published up to May 20, 2013 mentioned by particular article-level metrics source. Colors indicate categories (Viewed, Cited, Saved, Discussed, Recommended), as used on the PLOS website.All PLOS Biology articles are viewed and downloaded, and almost all of them (all research articles and nearly all front matter) will be cited sooner or later. Almost all of them will also be bookmarked in online reference managers, such as Mendeley, but the percentage of articles that are discussed online is much smaller. Some of these percentages are time dependent; the use of social media discussion platforms, such as Twitter and Facebook for example, has increased in recent years (93% of PLOS Biology research articles published since June 2012 have been discussed on Twitter, and 63% mentioned on Facebook). These are the locations where most of the online discussion around published articles currently seems to take place; the percentage of papers with comments on the PLOS website or that have science blog posts written about them is much smaller. Not all of this online discussion is about research articles, and perhaps, not surprisingly, the most-tweeted PLOS article overall (with more than 1,100 tweets) is a PLOS Biology perspective on the use of social media for scientists [11].Some metrics are not so much indicators of a broad online discussion, but rather focus on highlighting articles of particular interest. For example, science blogs allow a more detailed discussion of an article as compared to comments or tweets, and journals themselves sometimes choose to highlight a paper on their own blogs, allowing for a more digestible explanation of the science for the non-expert reader [12]. Coverage by other bloggers also serves the same purpose; a good example of this is one recent post on the OpenHelix Blog [13] that contains video footage of the second author of a 2010 PLOS Biology article [14] discussing the turkey genome.F1000Prime, a commercial service of recommendations by expert scientists, was added to the PLOS Article-Level Metrics in August 2013. We now highlight on the PLOS website when any articles have received at least one recommendation within F1000Prime. We also monitor when an article has been cited within the widely used modern-day online encyclopedia, Wikipedia. A good example of the latter is the Tasmanian devil Wikipedia page [15] that links to a PLOS Biology research article published in 2010 [16]. While a F1000Prime recommendation is a strong endorsement from peer(s) in the scientific community, being included in a Wikipedia page is akin to making it into a textbook about the subject area and being read by a much wider audience that goes beyond the scientific community. PLOS Biology is the PLOS journal with the highest percentage of articles recommended in F1000Prime and mentioned in Wikipedia, but there is only partial overlap between the two groups of articles because they focus on different audiences (Figure 5). These recommendations and mentions in turn show correlations with other metrics, but not simple ones; you can''t assume, for example, that highly cited articles are more likely to be recommended by F1000Prime, so it will be interesting to monitor these trends now that we include this information.Open in a separate windowFigure 5 PLOS Biology articles: sites of recommendation and discussion.Number of PLOS Biology research articles published up to May 20, 2013 that have been recommended by F1000Prime (red) or mentioned in Wikipedia (blue).With the increasing availability of ALM data, there comes a growing need to provide tools that will allow the community to interrogate them. A good first step for researchers, research administrators, and others interested in looking at the metrics of a larger set of PLOS articles is the recently launched ALM Reports tool [17]. There are also a growing number of service providers, including Altmetric.com [18], ImpactStory [19], and Plum Analytics [20] that provide similar services for articles from other publishers.As article-level metrics become increasingly used by publishers, funders, universities, and researchers, one of the major challenges to overcome is ensuring that standards and best practices are widely adopted and understood. The National Information Standards Organization (NISO) was recently awarded a grant by the Alfred P. Sloan Foundation to work on this [21], and PLOS is actively involved in this project. We look forward to further developing our article-level metrics and to having them adopted by other publishers, which hopefully will pave the way to their wide incorporation into research and researcher assessments.     

Rapid Assessment Procedure for Loiasis and Mapping Lymphatic Filariasis: Two Perfect Illustrations of “To Be in English or Not to Be”

Interest in filariasis has found a new impetus now that neglected tropical diseases have their own journal. However, some of the advances published in renowned international journals have completely ignored previous publications on the subject, particularly those in languages other than English. The rapid assessment procedure for loiasis and the mapping of lymphatic filariasis provide two perfect illustrations of this. This problem may seem a bit outdated, given that all “good authors” now publish exclusively in English. It certainly is outdated for most areas of medicine. But, surely, this should not be the case for neglected tropical diseases, for which certain long-standing findings are every bit as important as what may be presented as new discoveries. One possibility would be for certain journals, such as PLOS Neglected Tropical Diseases, to include a specific heading permitting the publication in English of older studies that initially appeared in a language other than English. The texts would be English versions respecting the entirety of the original text. Submission should be accompanied by a presentation of the problem, with details and explanatory comments, with submission at the initiative of the authors of the former article in question or their students or sympathizers.Interest in filariasis has found a new impetus now that neglected tropical diseases (NTDs) have their own journal. However, some of the advances published in renowned international journals have completely ignored previous publications on the subject, particularly those in languages other than English. This Viewpoint article is intended to make us ponder the issue of a language gap or discrimination existing in publishing outcomes and reference citations. This is also the question of deleterious effects of the obligation “to be in English or not to be”.The rapid assessment procedure for loiasis (RAPLOA) and the geographical distribution of lymphatic filariasis provide two perfect illustrations of this.The RAPLOA has recently been widely used to determine the regional endemicity of loiasis and to update existing endemicity data for this disease over its global distribution range. This important work has been recently published in PLOS NTDs [1]. The determination, within a population of the prevalence or, preferably, the annual incidence of episodes, of conjunctival migration by adult worms is a simple, non-invasive, relatively sensitive and specific method for evaluating the endemicity of Loa loa. This approach has proved particularly useful in areas in which both loiasis and onchocerciasis are observed: the mass treatment program to control onchocerciasis is based on the use of ivermectin and there is a risk of adverse treatment outcomes in patients carrying large numbers of L. loa worms [2]. In regions of high endemicity, the correlation between the conjunctival migration index and the microfilarial index is strong overall, both for villages and for age groups. Its use as an epidemiological index was clearly proposed in a publication in 1994 [3]. However, as this article was published in French, in Médecine Tropicale (Marseille), it has never been cited, despite being listed in international databases, including PubMed. A poster communication concerning the same issue had no real impact either, despite being presented at an international congress [4]. The origin of this new epidemiological index (RAPLOA) is systematically attributed to two World Health Organization (WHO) publications in 2001 [5] and 2002 [6]. It is true that the studies reported in these publications validated the concept at a large scale and in different endemic foci.The usefulness of specific clinical manifestations (eye worm and Calabar swelling) to assess L. loa had been recommended by different authors as early as 1950 [7]. But the correlation between the microfilarial index and the frequency of ocular migration has not been studied, and even less attention was paid to the notion of an epidemiological index until the epidemiological studies carried out in Congo Republic (former People''s Republic of the Congo) during the 1980s [8]. However, the index as such was clearly defined in 1994 [3]. Here is a direct translation of an excerpt of the French text published in 1994: “For loiasis, the usual parasitological indices (microfilarial index and mean microfilarial density) are the only measures recognized as providing information about the level of endemicity in humans. In addition to requiring blood samples standardized in terms of both volume and sampling time, these indices do not reflect the real level of parasitism, given the high frequency of infected subjects without microfilaria in the blood. Subjects infested with mature, fertile adult worms, as demonstrated by the removal of a subconjunctival female containing microfilaria from a patient with no detectable microfilaria in the blood, are frequently observed. Two symptoms are both specific and frequent in infected subjects both with and without microfilaria in the blood: subconjunctival migration of an adult worm and elusive, migrating edemas of the hands, wrists and lower part of the forearm” [9]. “The index of subconjunctival filarial migration over the preceding year is particularly useful, because it correlates well with the microfilarial index but is more sensitive” (Figure 1). “Its determination involves precise questioning of the patient, which can be facilitated by the use of a demonstration chart, with diagrams and photographs” (see Figure 2).Open in a separate windowFigure 1Index of the subconjunctival migration of Loa loa adult worms and microfilarial index.Reproduced from Medicine Tropicale [3], released under CC BY 2.0 by Medicine Tropicale. IMSC = Indice de Migration Sous-Conjonctivale in French and Index of the SubConjunctival Migration in English. IM = Indice Microfilarien in French and Microfilarial Index in English.Open in a separate windowFigure 2Illustration of the passage of an adult worm (Loa loa) across the eye.This illustration (diagram and photograph) was made for presentation to patients questioned in endemic regions.The conclusion of this article was formulated as follows: “Screening for foci of filarial endemicity could be improved by the use of a simplified method and the validation of simple, inexpensive indices. Once these foci have been identified, a more precise evaluation can be carried out.”What is most astounding about the two WHO publications cited as the origin of this “new epidemiological index” [5], [6] is that the principal authors come from French-speaking African countries and/or work in or with this institution. They would therefore have been able to understand articles written in French. Furthermore, the WHO has a long-standing culture of multilingualism, particularly in English and French.Against this background, the rejection by the Bulletin of the World Health Organization and by other international journals published in English of an opinion article dealing with this issue and using the example of lymphatic filariasis does not seem to be justified, and is another illustration of “to be in English or not to be.”Indeed, filariasis due to Wuchereria bancrofti is systematically described as endemic in Congo and Gabon, two French-speaking countries, in non-specialist works on tropical medicine and in more specialist publications (WHO) despite a total absence of epidemiologic studies and/or confirmed case report over the last 30 years to prove it. What is certain is that no case was found when the last studies were conducted in these countries at the end of the 1970s and during the 1980s but unfortunately published in French. The studies that we carried out in the Congo as part of the National Project on Onchocerciasis and Other Filariases (between 1982 and 1987) confirmed the presence of four types of human filariasis: onchocerciasis, loaiasis, and the filariases caused by Mansonella perstans and M. streptocerca. There was a total absence of confirmed cases of lymphatic filariasis (bancroftosis). In this case, it is not a question of the attribution of merit for a particular “discovery”, but of basic knowledge of the geographic distribution of a scarcely studied disease, lymphatic filiariasis, in French Central Africa. Taking into account only publications in English, even older and poorly structured data, have been, in our opinion, a source of confusion and has led to false conclusions being drawn about the distribution range of this disease. This undoubtedly highlights the need to update knowledge by carrying out prospective studies (which seem to be underway), but these studies do not seem to be considered a matter of priority given the low levels of resources available and current health priorities.This has drawn us to publish this article in a French-language journal, but together with an entire translation into English [10]. Despite the bilingual nature of this publication, the international PubMed database identifies this article as being published in French, effectively ensuring that it will never be consulted, a classic “catch 22” situation! Indeed, this reference has never yet been cited by another author in a journal published in English. It may be that publication of an article in another language than English makes it more likely that it will not be cited, even if the authors of a subsequent article have access to the journal in which it was published and can understand the language used. Here, we begin to encroach on ethical problems and it is probably best not to delve too deeply. However, suffice it to say that the limited dissemination of publications in a language other than English may account for such equivocal attitudes.The problem is not a rivalry between French and English, but the confrontation between English and all other languages of the world. Moreover, the problem is undoubtedly worse for works published in non–Western European languages such as Chinese, Russian, and Japanese, which are arguably even less accessible.All things considered, this problem may seem a bit outdated, given that all “good authors” now publish exclusively in English. It certainly is outdated for most areas of medicine, where everything that is old is assigned to being nothing more than the history of medicine. But, surely, this should not be the case for NTDs, for which certain long-standing findings are every bit as important as what may be presented as new discoveries.One possibility would be for certain journals, such as PLOS NTDs, to include a specific heading permitting the publication of older studies that initially appeared in a language other than English (and are therefore currently little known). The texts included in this heading would essentially be English versions of these articles previously published in other languages, respecting the entirety of the original text.This would concern studies considered of importance because they highlight a point that remains unclear or describe an aspect considered innovative in a review but for which the originality of the article is due more to an incomplete reference list than to a true advance in knowledge. These articles should be judged in light of the knowledge and technical and methodological means available at the time at which they were initially published. Submission should be accompanied by a presentation of the problem, with details and explanatory comments, with submission at the initiative of the authors of the article in question or their students or sympathizers.     

Triple X Egyptian woman and a Down's syndrome offspring

The 47, XXX karyotype (triple X) has a frequency of 1 in 1000 female newborns. However, this karyotype is not usually suspected at birth or childhood. Female patients with a sex chromosome abnormality may be fertile. In patients with a 47, XXX cell line there appears to be an increased risk of a cytogenetically abnormal child but the extent of this risk cannot yet be determined; it is probably lower in the non-mosaic 47, XXX patient than the mosaic 46, XX/47, XXX one. We describe a new rare case of triple X woman and a Down''s syndrome offspring. The patient is 26 years of age. She is a housewife, her height is 160 cm and weight is 68 kg and her physical features and mentality are normal. She has had one pregnancy at the age of 25 years resulted in a girl with Down''s syndrome. The child had 47 chromosomes with trisomy 21 (47, XX, +21) Figure 1. The patient also has 47 chromosomes with a triple X karyotype (47, XX, +X) Figure 2. The patient''s husband (27 years old) is physically and mentally normal. He has 46 chromosomes with a normal XY karyotype (46, XY). There are neither Consanguinity between her parent''s nor she and her husband.Open in a separate windowFigure 1Karyotype 47, XX + 21 of the daughter of Triple X syndromeOpen in a separate windowFigure 2Karyptype 47, XX + X of the Down syndrome''s mother     

Inside the Mucosal Immune System

An intricate network of innate and immune cells and their derived mediators function in unison to protect us from toxic elements and infectious microbial diseases that are encountered in our environment. This vast network operates efficiently by use of a single cell epithelium in, for example, the gastrointestinal (GI) and upper respiratory (UR) tracts, fortified by adjoining cells and lymphoid tissues that protect its integrity. Perturbations certainly occur, sometimes resulting in inflammatory diseases or infections that can be debilitating and life threatening. For example, allergies in the eyes, skin, nose, and the UR or digestive tracts are common. Likewise, genetic background and environmental microbial encounters can lead to inflammatory bowel diseases (IBDs). This mucosal immune system (MIS) in both health and disease is currently under intense investigation worldwide by scientists with diverse expertise and interests. Despite this activity, there are numerous questions remaining that will require detailed answers in order to use the MIS to our advantage. In this issue of PLOS Biology, a research article describes a multi-scale in vivo systems approach to determine precisely how the gut epithelium responds to an inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), given by the intravenous route. This article reveals a previously unknown pathway in which several cell types and their secreted mediators work in unison to prevent epithelial cell death in the mouse small intestine. The results of this interesting study illustrate how in vivo systems biology approaches can be used to unravel the complex mechanisms used to protect the host from its environment.Higher mammals have evolved a unique mucosal immune system (MIS) in order to protect the vast surfaces bathed by external secretions (which may exceed 300 m² in humans) that are exposed to a rather harsh environment. The first view of the MIS is a single-layer epithelium covered by mucus and antimicrobial products and fortified by both innate and adaptive components of host defense (Figure 1). To this, we can add a natural microbiota that lives in different niches, i.e., the distal small intestine and colon, the skin, the nasal and oral cavities, and the female reproductive tract. The largest microbial population can reach ∼10¹² bacteria/cm³ and occurs in the human large intestine [1]–[3]. This large intestinal microbiota includes over 1,000 bacterial species and the individual composition varies from person-to-person. Other epithelial sites harbor a separate type of microbiota, including the mouth, nose, skin, and other wet mucosal surfaces, that contributes to the host; in turn, the host benefits its microbial co-inhabitants. Gut bacteria grow by digesting complex carbohydrates, proteins, vitamins, and other components for absorption by the host, which in return rewards the microbiota by developing a natural immunity and tolerance (reviewed in [4]–[7]). Finally, the host microbiota influences the development and maturation of cells within lymphoid tissues of the MIS [8],[9].Open in a separate windowFigure 1The gut, nasal, upper respiratory and salivary, mammary, lacrimal, and other glands consist of a single layered epithelium.Projections of villi in the GI tract consist mainly of columnar epithelial cells (ECs), with other types including goblet and Paneth cells. Goblet cells exhibit several functions including secretion of mucins, which form a thick mucus covering. Paneth cells secrete chemokines, cytokines, and anti-microbial peptides (AMPs) termed α-defensins.Mucosal epithelial cells (ECs) are of central importance in host defense by providing both a physical barrier and innate immunity. For example, goblet cells secrete mucus, which forms a dense, protective covering for the entire epithelium (Figure 1). Peristalsis initiated by the brush border of gastrointestinal (GI) tract ECs allows food contents to be continuously digested and absorbed as it passes through the gut. In the upper respiratory (UR) tract, ciliated ECs capture inhaled, potentially toxic particles, and their beating moves them upward to expel them, thereby protecting the lungs. Damaged, infected, or apoptotic ECs in the GI tract move to the tips of villi and are excreted; newly formed ECs arise in the crypt region and continuously migrate upward. Paneth cells in crypt regions of the GI tract produce anti-microbial peptides (AMPs), or α-defensins, while ECs produce β-defensins [10],[11] for host protection (Figure 1). A major resident cell component of the mucosal epithelium are intraepithelial lymphocytes (IELs). The IELs consist of various T cell subsets that interact with ECs in order to maintain normal homeostasis [12]. Regulation is bi-directional, since ECs can also influence IEL T cell development and function [12]–[14].The MIS, simply speaking, can be separated into inductive and effector sites based upon their anatomical and functional properties. The migration of immune cells from mucosal inductive to effector tissues via the lymphatic system is the cellular basis for the immune response in the GI, the UR, and female reproductive tracts (Figure 2). Mucosal inductive sites include the gut-associated lymphoid tissues (GALT) and nasopharyngeal-associated lymphoid tissues (NALT), as well as less well characterized lymphoid sites (Box 1). Collectively, these comprise a mucosa-associated lymphoid tissue (MALT) network for the provision of a continuous source of memory B and T cells that then move to mucosal effector sites 13,14. The MALT contains T cell regions, B cell–enriched areas harboring a high frequency of surface IgA-positive (sIgA⁺) B cells, and a subepithelial area with antigen-presenting cells (APCs), including dendritic cells (DCs) for the initiation of specific immune responses (Figure 2). The MALT is covered by a subset of differentiated microfold (M) cells, ECs, but not goblet cells, and underlying lymphoid cells that play central roles in the initiation of mucosal immune responses. M cells take up antigens (Ags) from the lumen of the intestinal and nasal mucosa and transport them to the underlying DCs (Figure 2). The DCs carry Ags into the inductive sites of the Peyer''s patch or via draining lymphatics into the mesenteric lymph nodes (MLNs) for initiation of mucosal T and B cell responses (Figure 2). Retinoic acid (RA) producing DCs enhance the expression of mucosal homing receptors (α₄β₇ and CCR9) on activated T cells for subsequent migration through the lymphatics, the bloodstream, and into the GI tract lamina propria [15],[16]. Regulation within the MIS is critical; several T cell subsets including Th1, Th2, Th17, and Tregs serve this purpose [13],[14],[17] (Figure 2).Open in a separate windowFigure 2The mucosal immune system (MIS) is interconnected, enabling it to protect vast surface areas.This is accomplished by inductive sites of organized lymphoid tissues, e.g., in the gut the Peyer''s patches (PPs) and mesenteric lymph nodes (MLNs) comprise the GALT. Lumenal Ags can be easily sampled via M cells or by epithelial DCs since this surface is not covered by mucus due to an absence of goblet cells. Engested Ags in DCs trigger specific T and B cell responses in Peyer''s patches and MLNs. Homing of lymphocytes expressing specific receptors helps guide their eventual entry into major effector tissues, e.g., the lamina propria of the gut, the upper respiratory (UR) tract, the female reproductive tract, or acinar regions of exocrine glands. Terminal differentiation of plasma cells producing polymeric (mainly dimeric) IgA is then transported across ECs via the pIgR for subsequent release as S-IgA Abs.

Box 1. Major Inductive Sites for Mucosal Immune Responses

1. GALT (gut-associated lymphoid tissues)
   • Peyer''s patches (PPs)
   • Mesenteric lymph nodes (MLNs)
   • Isolated lymphoid follicles (ILFs)
2. NALT (nasopharyngeal-associated lymphoid tissues)
   • Tonsils/adenoids
   • Inducible bronchus-associated lymphoid tissue (iBALT)
   • Cervical lymph nodes (CLNs)
   • Hilar lymph nodes (HLNs)

Mucosal effector sites, including the lamina propria regions of the GI, the UR and female reproductive tracts as well as secretory glandular tissues (i.e., mammary, lacrimal, salivary, etc.) contain Ag-specific mucosal effector cells such as IgA-producing plasma cells, and memory B and T cells [18]. Adaptive mucosal immune responses result from CD4⁺ T cell help (provided by both CD4⁺ Th2 or CD4⁺ Th1 cells), which supports the development of IgA-producing plasma cells (Figure 2). Again, the ECs become a central player in the MIS by producing the polymeric Ig receptor (pIgR) (which binds both polymeric IgA and IgM) [19]. Lamina propria pIgA binds the pIgR on the basal surface of ECs, the bound pIgA is internalized, and then transported apically across the ECs (Figure 2). Release of pIgA bound to a portion of pIgR gives rise to secretory IgA (S-IgA) antibodies (Abs) with specificities for various Ags encountered in mucosal inductive sites [13],[14],[19]. In addition, commensal bacteria are ingested by epithelial DCs, which subsequently migrate to MLNs for induction of T cell–independent, IgA B cell responses [20]. In summary, two broad types of S-IgA Abs reach our external secretions by transport across ECs and protect the epithelial surfaces from environmental insults, including infectious diseases.It should be emphasized that several unique vaccine strategies are being developed to induce protective mucosal immunity. In this regard, delivery of mucosal vaccines by oral, nasal, or other mucosal routes requires specific adjuvants or delivery systems to initiate an immune response in MALT [21],[22]. However, a major benefit of mucosal vaccine delivery is the simultaneous induction of systemic immunity, including CD4⁺ Th1 and Th2, CD8⁺ cytotoxic T lymphocytes (CTLs), and Ab responses in the bloodstream, which are predominantly of the IgG isotype [21]. This, of course, provides a double layer of immunity in order to protect the host from microbial pathogens encountered by mucosal routes. This is especially promising for development of vaccines for developing countries, as well as those to protect our aging population [23],[24].In this issue of PLOS Biology, Lau et al. used a multi-scale in vivo systems approach to assess how cells of the intestinal MIS communicate with intestinal ECs in response to an inflammatory signal [25]. The present study centered on the use of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) given intravenously (i.v.) to assess its effects on the gut epithelium in the presence (wild-type [WT] mice) or absence (Rag1 knockout mice) of adaptive T and B lymphocytes. It is well known that TNF-α regulates many EC effects, including programmed cell death (apoptosis), survival, proliferation, cell cycle arrest, and terminal differentiation [26]. The authors had previously shown that TNF-α given i.v. to WT mice resulted in two different response patterns in the small intestine [27]. In the duodenum, which adjoins the stomach, TNF-α enhanced EC apoptosis, while in the ileum, the part next to the colon, an enhancement of EC division was seen [27]. In the present study, i.v. injection of TNF-α induced apoptosis in the duodenum (but not ileum) of WT, with heightened cell death in Rag1 mice [25]. Loss of either T or B lymphocytes also led to increased EC apoptosis, suggesting that both cell types are required to protect the epithelium from cell death. Also intriguing was the finding that eliminating the gut microbiota by antibiotic treatment did not affect the degree of EC apoptosis seen. Mathematical modeling allowed the group to show that TNF-α-induced apoptosis involved several steps in mice lacking functional T and B cells. Analysis of potential cytokines involved revealed that only a single chemokine, monocyte chemotactic protein-1 (MCP-1, C-C motif ligand 2 [CCL2]), protected ECs from apoptosis [25]. This new finding complements recent studies showing that IL-22, which is produced by several immune cells in the gut, plays a major role in protecting ECs from inflammation, infection, and tissue damage (Figure 3) [28].Open in a separate windowFigure 3The gut epithelium exhibits several pathways that protect the integrity of this organ.Intestinal epithelial cells (ECs) produce stem cell factor (SCF), which induces proliferation and resistance to bacterial invasion. In addition, neighboring γδ intraepithelial lymphocytes (IELs) produce keratinocyte growth factor (KGF), which also stabilizes ECs. IL-22 produced by Th17, Th22, and γδ T cells as well as natural killer (NK) and lymphoid tissue inducer (LTi) cells plays a key role in both early and late phases of innate immunity in order to maintain the EC barrier. In addition, monocyte chemotactic protein (MCP-1) produced by Paneth cells and goblet cells down-regulates migration of plasmacytoid DCs (pDCs) into the intestinal lamina propria in order to decrease TNF-α-induced EC apoptosis.Several unexpected discoveries followed. First, both goblet and Paneth cells were the major sources of MCP-1, and not the lymphoid cell populations that normally produce this chemokine (Figure 3). Second, the MCP-1 produced did not directly protect ECs, but instead acted via downregulation of plasmacytoid DCs (pDCs), a lymphocyte-like DC that produces various cytokines [29]. Finally, the study established that loss of adaptive (immune) lymphocytes resulted in decreased MCP-1 production, leading to increased pDC numbers and enhanced EC apoptosis. In the final experiment, the authors again showed that pDCs in the duodenum of Rag-1 mice produced increased levels of interferon-gamma that directly induced EC apoptosis. The message given by this intricate study is that systems biology approaches are quite useful in unraveling the complexities posed by the MIS in both health and disease.The model developed by Lau et al. [25] could be useful to study several major problem areas. For example, a paucity of murine models exist to study food or milk allergies that usually affect the duodenum of the small intestine [30]. It is known that chemokine receptors control trafficking of Th2-type cells to the small intestine for IgE-dependent allergic diarrhea [31],[32]. The multi-scale systems approach could be used to assess much earlier responses to food or milk allergies in TNF-α-treated mice. A second avenue could well include the cell and molecular interactions that lead to intestinal EC damage resulting in IBD [33]. Clearly, progress is being made to study genetic aspects, regulatory T cells, and the contributions of the host microbiota to IBD development [17],[34]. Nevertheless, current mouse models have their “readout” as weight loss and chronic inflammation of the colon [17],[33],[34]. The Lau et al. approach could reveal cell-to-cell linkages that ultimately resulted in EC damage [25]. Further, this approach could reveal the earliest stages of pathogenesis of IBD before the influx of inflammatory cells causes the macroscopic changes characteristic of these diseases. Since the duodenum is normally sterile, one could have predicted their finding that antibiotic treatment to remove the gut microbiota would indeed be without effect. However, one wonders what effects would be seen in the stomach or in the colon, both of which can harbor a natural microbiota. Does TNF-α and antibiotic treatment alter the EC program in these mucosal tissue sites?Finally, the intriguing question arising from the Lau et al. study [25] involves the finding that a full-blown adaptive immune system was required to maintain homeostasis and thus reduce EC apoptosis in the GI tract. Note that the response to in vivo TNF-α was assessed after only 4 hours, well before T and B cell responses could be manifested. How are early T and B cell signals transmitted to ECs? What are the mediators involved between the innate and adaptive components in the MIS for communication with the epithelium? As always, insightful studies raise many more questions than are answered. Nevertheless, the multi-scale in vivo systems analysis identified effects on the epithelium in a manner not appreciated up to now. The advantages of using an in vivo perturbation system is far superior to cell culture studies where only a few cell types are present.     

Promoting Effects of the Adipokine,Apelin, on Diabetic Nephropathy

Angiogenesis, increased glomerular permeability, and albuminuria are thought to contribute to the progression of diabetic nephropathy (DN). Apelin receptor (APLNR) and the endogenous ligand of APLNR, apelin, induce the sprouting of endothelial cells in an autocrine or paracrine manner, which may be one of the mechanisms of DN. The aim of this study was to investigate the role of apelin in the pathogenesis of DN. Therefore, we observed apelin/APLNR expression in kidneys from patients with type 2 diabetes as well as the correlation between albuminuria and serum apelin in patients with type 2 diabetes. We also measured the proliferating, migrating, and chemotactic effects of apelin on glomerular endothelial cells. To measure the permeability of apelin in glomerular endothelial cells, we used transwells to detect FITC-BSA penetration through monolayered glomerular endothelial cells. The results showed that serum apelin was significantly higher in the patients with type 2 diabetes compared to healthy people (p<0.05, Fig. 1B) and that urinary albumin was positively correlated with serum apelin (R = 0.78, p<0.05). Apelin enhanced the migration, proliferation, and chemotaxis of glomerular endothelial cells in a dose-dependent manner (p<0.05). Apelin also promoted the permeability of glomerular endothelial cells (p<0.05) and upregulated the expression of VEGFR2 and Tie2 in glomerular endothelial cells (p<0.05). These results indicated that upregulated apelin in type 2 diabetes, which may be attributed to increased fat mass, promotes angiogenesis in glomeruli to form abnormal vessels and that enhanced apelin increases permeability via upregulating the expression of VEGFR2 and Tie2 in glomerular endothelial cells.Open in a separate windowFigure 1Correlation between apelin and albuminuria.A: The apelin concentration in serum was positively correlated with albuminuria (R = 0.78, *p<0.05). B: The apelin concentration in serum was significantly increased in patients with type 2 diabetes (2DM, n = 60) compared to healthy people (control, n = 32). The data are expressed as the means±SD (*p<0.05 vs. control grou ). C: The graphs show the promoting effect of apelin on FITC-BSA passing through the glomerular endothelial cell monolayers at the indicated time point. The data are expressed as the means±SD (n = 6, *p<0.01 vs. control group).     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

A photoreceptor going nowhere but the nucleus

Cryptochrome 2 (CRY2) is a blue/UV-A light receptor that regulates light inhibition of cell elongation and photoperiodic promotion of floral initiation in Arabidopsis. We and others have previously shown that CRY2 is a nuclear protein that regulates gene expression to affect plant development. We also showed that CRY2 is phosphorylated in response to blue light and the phosphorylated CRY2 is most likely active and degraded in blue light. Given that protein translation (and probably chromophore attachment) takes place in the cytosol and that a photoreceptor would absorb photon instantaneously, it would be interesting to know where those inter-connected events occur in the cell. Our results showed that freshly synthesized CRY2 photoreceptor is inactive in the cytosol although it may be photon-excited, it is imported into the nucleus where the photoreceptor is phosphorylated, performs its function, becomes ubiquitinated, and eventually gets degraded (Fig. 1).¹ To our knowledge, this is the first example in any organism that a photoreceptor is shown to complete its post-translational life cycle in a single subcellular compartment.Open in a separate windowFigure 1A model depicting the post-translational life cycle of CRY2. Pi, phosphate group; Ubq, ubiquitin.Key words: blue light, cryptochrome, ubiquitination, phosphorylation, Arabidopsis     

Sequencing the Connectome

Connectivity determines the function of neural circuits. Historically, circuit mapping has usually been viewed as a problem of microscopy, but no current method can achieve high-throughput mapping of entire circuits with single neuron precision. Here we describe a novel approach to determining connectivity. We propose BOINC (“barcoding of individual neuronal connections”), a method for converting the problem of connectivity into a form that can be read out by high-throughput DNA sequencing. The appeal of using sequencing is that its scale—sequencing billions of nucleotides per day is now routine—is a natural match to the complexity of neural circuits. An inexpensive high-throughput technique for establishing circuit connectivity at single neuron resolution could transform neuroscience research.Neuroscientists seek neural explanations of perception, thought, and behavior. What does such an explanation look like? One of the earliest examples is Descartes'' account [1] of the reflex withdrawal of a foot from a fire (Figure 1A). Descartes hypothesized that small particles of the fire displace the skin of the foot, which pulls on a tiny thread and thereby opens a pore in the pineal gland, releasing animal spirits, which flow back via a hollow tube into the foot to cause retraction. Although more modern accounts of the spinal reflex arc differ in important mechanistic and anatomical details, the kernel of Descartes'' explanation is both correct and intellectually satisfying: the neural circuit he describes immediately implies the causal relationship between the stimulus and the resultant action. Circuit-level explanations of computation and behavior represent the gold standard.Open in a separate windowFigure 1The wiring of neural circuits is highly structured.(A) Descartes'' model of the foot withdrawal reflex. (B) Two similar circuits in which the computation is readily deduced from the wiring. The circuit on the top is directionally selective, whereas the one on the bottom performs a center-surround computation. (C) The costs of DNA sequencing are falling exponentially. From 2001 to 2007, the costs of sequencing dropped exponentially, in pace with Moore''s law [18] for computation. Since the introduction of “next generation” sequencing technologies in 2008, the cost of sequencing has fallen more than 10-fold every year, compared with the steady 1.4-fold yearly drop for computing power. Data from http://www.genome.gov/sequencingcosts/.     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

A Large Number of Reviews on Physician Rating Websites May Reflect Reputation Management

BackgroundPhysicians with a large number of reviews and a high rating may be employing reputation management strategies. Specialists may be more likely than non-specialists to employ such strategies. This should be apparent in a study of online physician reviews on physician rating websites (PRW).MethodsUsing one physician rating website, we gathered orthopedic surgeon and family physician reviews. We measured Spearman correlations between the number of reviews and average numerical rating and used chi-squared to test threshold relationships.ResultsThere were very small negative Spear-man correlations between the number of online reviews and the average numerical rating for orthopedic surgeons (p= -0.097, p-value=<0.001) family medicine physicians (p= -0.170, p-value=<0.001; Figure 2). Physicians with more than 100 reviews had a greater average numerical rating than physicians with fewer than 50 reviews. Orthopedic surgeons are more likely than family medicine physicians to have a large number of reviews and average numerical rating greater than 3.Open in a separate windowFigure 2.Family medicine physicians average rating plotted against number of reviews.ConclusionThe small fraction of physician with a high number of reviews may be utilizing reputation management strategies, and this seems relatively specific to specialists rather than non-specialists. Level of Evidence: III     

How Insect Flight Steering Muscles Work

Insights into how exactly a fly powers and controls flight have been hindered by the need to unpick the dynamic complexity of the muscles involved. The wingbeats of insects are driven by two antagonistic groups of power muscles and the force is funneled to the wing via a very complex hinge mechanism. The hinge consists of several hardened and articulated cuticle elements called sclerites. This articulation is controlled by a great number of small steering muscles, whose function has been studied by means of kinematics and muscle activity. The details and partly novel function of some of these steering muscles and their tendons have now been revealed in research published in this issue of PLOS Biology. The new study from Graham Taylor and colleagues applies time-resolved X-ray microtomography to obtain a three-dimensional view of the blowfly wingbeat. Asymmetric power output is achieved by differential wingbeat amplitude on the left and right wing, which is mediated by muscular control of the hinge elements to mechanically block the wing stroke and by absorption of work by steering muscles on one of the sides. This new approach permits visualization of the motion of the thorax, wing muscles, and the hinge mechanism. This very promising line of work will help to reveal the complete picture of the flight motor of a fly. It also holds great potential for novel bio-inspired designs of fly-like micro air vehicles.The ability for powered flight has evolved four times in the animal kingdom and, thanks to their ability to fly, insects have diversified and moved into new regions and habitats with enormous success [1]. Powered flight requires an integrated system consisting of wings to generate aerodynamic force, muscles to move the wings, and a control system to modulate power output from the muscles. Insects are bewilderingly diverse with respect to flight morphology and behaviors, which in turn provides a real challenge to researchers wishing to understand how insects fly. In particular, the impressive flight maneuvers in flies, such as blowflies and fruit flies, have inspired scientists for many years [2]. The ability of a fly to accelerate, make tight turns, rolls, and loops that allow the creature to land upside down on a ceiling is unparalleled in any other organisms, as well as any manmade aircraft. Everybody knows how difficult it is to swat a fly with bare hands—the fly''s capacity for rapid take-off and accurate movement away from a perceived approaching threat is exquisite [3].The flight muscles of many insects, including flies, bees, and mosquitoes, are divided into a few large power muscles that simply contract cyclically to generate sheer power output and a greater number of smaller steering muscles that control the force transmission from the power muscles to the wing [4]–[6]. The power muscles of a fly consist of two sets of antagonistic muscles attached to the inside of the thorax (exoskeleton) (Figure 1). In many insects, including flies, these muscles are asynchronous, which means their contractions are uncoupled to the firing rate of the associated motor neuron [6],[7], i.e., the muscles continue to contract as long as the nerve tickles them. Another characteristic feature of the power muscles is that they are stretch-activated and contract as a response to being lengthened. Both sets of power muscles deform the thorax when contracted such that when the dorso-ventral muscles contract, the thorax is squeezed together dorso-ventrally while expanding longitudinally, and vice versa when the dorsal-longitudinal muscles contract as a response to prior lengthening. The result is an alternate contraction and lengthening of these perpendicular muscle groups and a resonance of the entire thorax that drives the wingbeat. Typical wingbeat frequencies are in the range from 100 Hz and even up to 1,000 Hz in the smallest species [5],[8].Open in a separate windowFigure 1The thorax with and dorsal longitudinal (upper left) and dorso-ventral (upper right) power flight muscles of a fly.The cartoon (bottom) shows a transverse section through the thorax with dorso-ventral muscles (DVM) and dorsal longitudinal muscles (DLM) indicated. The two upper illustrations are redrawn from [6].The forces from the flight muscles are transmitted to the wing through an intricate hinge mechanism (Figure 2). The hardened plates of cuticle between the thorax and wing (sclerites) are mobile and their positions relative to the thoracic outgrowths and wing determine the extent of the wing motion, i.e., the angular amplitude of the wingbeat [6].Open in a separate windowFigure 2Cartoon illustration of a transverse section of the thorax of a fly in rear view, showing some elements of the complex wing hinge of a fly, consisting of ridges and protrusions on the thorax and a number of hardened plates of cuticle (sclerites) between the body (thorax) and the wing root.The basalare sclerite (not shown) is positioned anterior of the first axillary sclerite (Ax1). The indicated structures are dorso-ventral power muscle (DVM), pleural wing process (PWP), post-medial notal process (PMNP), parascutal shelf (PSS), axial wing sclerites (Ax1, Ax2, Ax3), and radial stop (RS). Redrawn and modified from [18].Flight maneuvers arise owing to asymmetric force generation between the left and right wing. Aerodynamic force is proportional to the angle of attack (the angle between the wing surface and the airflow) and the speed squared relative to the air [9],[10]. Except from the turning points of each half-stroke, when the wings rotate about their span wise axes, the angle of attack is usually quite constant during the translational phases of the wingbeat [10], while asymmetric forces are mainly created by changing the wingbeat amplitude in flies [11]–[14]. With wingbeat frequency kept constant, changed amplitude changes the speed and hence force generated.The control of the elements forming the hinge mechanism of the wing is achieved by the steering muscles, which are tiny in terms of mass (<3% of the power muscle mass), but mean everything when it comes to making flight maneuvers. In contrast to the power muscles the steering muscles are synchronous, i.e., there is a 1∶1 correspondence between neural spikes and muscle contraction. No less than some 22 pairs of steering muscles are involved in the force transmission; a few of these indirectly modulate the output by affecting the resonating properties of the thorax, while others are directly attached to the sclerite elements of the hinge mechanism [6],[15]. Three small muscles (b1–b3) are attached to the basalare plate that is directly involved in wing articulation (Figure 3). The actual wing sclerites (Figure 2) are also controlled by specific steering muscles, also with the function of moving the sclerites in relation to required wing motion. The main control function of the hinge mechanism appears to be of the downward movement of the wing, i.e., the angle at the turning point at end of downstroke. For a detailed review about the steering muscles and their function see Dickinson and Tu [6].Open in a separate windowFigure 3The position of the three steering flight muscles b1–b3 inserted to the nail-shaped basalare sclerite.Contraction by the b1 and b2 muscles move the basalare forward and their antagonist b3 moves it backwards when contracted. Redrawn from [6].To date, the function of the steering muscles has been revealed mainly by electrophysiological studies on tethered subjects. Tethering means that the animal is glued to the end of a thin rod, often with force sensors attached to it, and then stimulated to “fly.” In many insects this can be achieved by simply blowing at them or placing them in a wind tunnel. On the tether the insect can either be presented with a visual stimulus or be rotated, which flies can sense via their halteres (hind wings modified to sensory gyroscopic sensory organs) [16]. By inserting electrode wires into the steering muscles, the neural impulses are measured at the same time as the wingbeat kinematics is recorded [13],[17]. What we know about the function of the steering muscles comes from the meticulous studies of correlations between muscle activity and the associated wing movement, including how the hinge mechanism works [6],[18]. Needless to say, such experiments are extremely difficult to achieve in small insects like blowflies and fruit flies that flap their wings at high frequencies. Recent studies of the wing and hinge kinematics provide some support for the hypothesis that the hinge may have a gear function that affects stroke amplitude, as well [18]. However, there are still many open questions regarding the exact function of the steering muscles and how they help in generating laterally asymmetric forces during a fly''s flight maneuver [6].In an article published in this issue of PLOS Biology, Walker and colleagues take a new approach for studying how steering muscles regulate the power output from power muscles [19], using time-resolved x-ray microtomography [20]. By rotating tethered blowflies (Calliphora vicina) in the X-ray beam, a 3D-movie was captured that shows how the steering muscles move. This by itself is a grand achievement at a wingbeat frequency of 145 Hz. As the flies could sense being rotated the steering muscles acted accordingly to achieve an asymmetric power output as a response to a perceived turn. The movies that accompany the article show how several of the key steering muscles and their sclerites operate in concert during the course of a wingbeat, and the visual results are supported by advanced statistical analyses of muscle strain rates and their phase offset. For example, the b1 and b3 muscles (Figure 3) work antagonistically, as was known before, but on the low-amplitude wing the oscillations are delayed by about a quarter of a wingbeat. The strain amplitudes of b1 and b3 were different between the two wings, which were found to be due to dorso-ventral movement of the basalare sclerite on the high-amplitude side and rotation on the low-amplitude side. This shows even higher complexity of the wing hinge than was previously envisaged.The measurements of strain rate in the muscle confirmed the results of a previous study, which showed that asymmetric power output is partially achieved by negative work [21], i.e., absorption of work, by the b1 muscle on the low-amplitude wing. As with other muscles, the steering muscles insert on the skeletal parts and sclerites by tendons. The tendon of the muscle (I1) associated with the first axillary sclerite was observed to buckle when the wing was elevated above the wing hinge, indicative of compressive force acting on it near the top of the wing stroke. This buckling of the tendon forces a reinterpretation of the function of this muscle: it is involved in reducing stroke amplitude at the bottom of the downstroke rather than exerting stress near the opposite end of the stroke. Tendon buckling was seen in some other muscles as well, and although this is its first observation, it may be a more general mechanism involved in control of insect wingbeat kinematics.What are the wider implications of this new study? First, it demonstrates the utility of a new approach to examine the in vivo operation of several insect flight muscles. This alone signals a methodological breakthrough that promises more. So far the flies were tethered and studied during one behavioral treatment (rotation about the yaw axis). Real flight maneuvers, however, also involve angular rotation about pitch and roll axes, acceleration, and braking. Thus, it remains to be seen how the steering muscles operate to control more subtle changes in wing kinematics during the turning saccades and advanced flight maneuvers that take place during free flight. The method involved exposure to lethal X-ray doses, which of course limits how long the experiments can be. Second, tethering is the prevailing paradigm for studying insect flight, but because it interrupts the sensory feedback loop [22], it would be useful for future studies to compare tethered and free flight in some commonly studied species. Furthermore, a more complete understanding of the flight muscle-hinge mechanism may help bio-inspired design of wing articulation systems for fly-like micro air vehicles. Until then, we can enjoy the stunning videos of the oscillating thorax and flight muscle system of the blowfly [19]. See the video from the related research article here (http://youtu.be/P6lBkK3J9wg) or [19].     

Gamete attachment process revealed in flowering plant fertilization

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule (Fig. 1). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants.Open in a separate windowFigure 1.Illustration of the fertilization process in flowering plants. First, each pollen tube accesses an ovule containing egg and central cells. Next, the 2 sperm cells face the female gametes in the ovule after the pollen tube bursts. Finally, each sperm cell simultaneously fuses with either egg or central cell.     

Centre of the Cell: Science Comes to Life

Centre of the Cell is a unique biomedical science education centre, a widening participation and outreach project in London’s East End. This article describes Centre of the Cell’s first five years of operation, the evolution of the project in response to audience demand, and the impact of siting a major public engagement project within a research laboratory.Centre of the Cell is a unique cell-shaped science centre suspended above a real biomedical research laboratory in the heart of London’s East End. It is one of the few, perhaps the only, science education centres in the world to be situated inside a research lab—in the Blizard Institute at the Whitechapel medical and dental campus of Queen Mary University of London (QMUL). Since its opening in September 2009, over 100,000 people have participated in Centre of the Cell activities (Fig 1) with approximately one million visits to the interactive website www.centreofthecell.org. With visitor numbers and activities increasing year on year, we believe this is an example of an innovative and successful public engagement project that may serve as an example for similar initiatives in biomedical research institutes.Open in a separate windowFig 1Visitor statistics for Centre of the Cell years 3–6 (2011–2015).These monthly statistics include all visitors on site as well as visitors to our workshops and shows in schools and other locations. Year 1 and 2 figures (not shown here) were 15,387 and 19,585, respectively. These data show a consistent pattern from month to month. 2013–2014 numbers were lower, due to planned maintenance shutdown of the Pod and introduction of charging for Pod shows that led to a dip in visitor numbers that largely recovered during 2014.     

Interactions between Silicon Oxide Nanoparticles (SONPs) and U(VI) Contaminations: Effects of pH,Temperature and Natural Organic Matters

The interactions between contaminations of U(VI) and silicon oxide nanoparticles (SONPs), both of which have been widely used in modern industry and induced serious environmental challenge due to their high mobility, bioavailability, and toxicity, were studied under different environmental conditions such as pH, temperature, and natural organic matters (NOMs) by using both batch and spectroscopic approaches. The results showed that the accumulation process, i.e., sorption, of U(VI) on SONPs was strongly dependent on pH and ionic strength, demonstrating that possible outer- and/or inner-sphere complexes were controlling the sorption process of U(VI) on SONPs in the observed pH range. Humic acid (HA), one dominated component of NOMs, bounded SONPs can enhance U(VI) sorption below pH~4.5, whereas restrain at high pH range. The reversible sorption of U(VI) on SONPs possibly indicated that the outer-sphere complexes were prevalent at pH 5. However, an irreversible interaction of U(VI) was observed in the presence of HA (Fig 1). It was mainly due to the ternary SONPs-HA-U(VI) complexes (Type A Complexes). After SONPs adsorbed U(VI), the particle size in suspension was apparently increased from ~240 nm to ~350 nm. These results showed that toxicity of both SONPs and U(VI) will decrease to some extent after the interaction in the environment. These findings are key for providing useful information on the possible mutual interactions among different contaminants in the environment.Open in a separate windowFig 1Binding modes and reversibility of interactions among SONPs, U(VI), and HA.The interactions of different systems occur in the aqueous phase. Blue particle presents SONPs; Gray particle presents U(VI); Brown part presents HA.     

Conserved residues in the ankyrin domain of VAPYRIN indicate potential protein-protein interaction surfaces

Plant VAPYRINs are required for the establishment of arbuscular mycorrhiza (AM) and root nodule symbiosis (RNS). In vapyrin mutants, the intracellular accommodation of AM fungi and rhizobia is blocked, and in the case of AM, the fungal endosymbiont cannot develop arbuscules which serve for nutrient exchange. VAPYRINs are plant-specific proteins that consists of a major sperm protein (MSP) domain and an ankyrin domain. Comparison of VAPYRINs of dicots, monocots and the moss Physcomitrella patens reveals a highly conserved domain structure. We focused our attention on the ankyrin domain, which closely resembles the D34 domain of human ankyrin R. Conserved residues within the petunia VAPYRIN cluster to a surface patch on the concave side of the crescent-shaped ankyrin domain, suggesting that this region may represent a conserved binding site involved in the formation of a protein complex with an essential function in intracellular accommodation of microbial endosymbionts.Key words: VAPYRIN, arbuscular mycorrhiza, petunia, symbiosis, glomus, ankyrin, major sperm protein, VAPPlants engage in mutualistic interactions such as root nodule symbiosis (RNS) with rhizobia and arbuscular mycorrhiza (AM) with Glomeromycotan fungi. These associations are referred to as endosymbioses because they involve transcellular passage through the epidermis and intracellular accommodation of the microbial partner within root cortical cells of the host.^1,2 Infection by AM fungi and rhizobia is actively promoted by the plant and requires the establishment of infection structures namely the prepenetration apparatus (PPA) in AM and a preinfection thread in RNS, respectively.^3–5 In both symbioses the intracellular microbial accommodation in epidermal and root cortical cells involves rebuilding of the cytoskeleton and of the entire membrane system.^6–8 Recently, intracellular accommodation of rhizobia and AM fungi, and in particular morphogenesis of the AM fungal feeding structures, the arbuscules, was shown to depend on the novel VAPYRIN protein.^9–11VAPYRINs are plant-specific proteins consisting of two protein-protein interaction domains, an N-terminal major sperm protein (MSP) domain and a C-terminal ankyrin (ANK) domain. MSP of C. elegans forms a cytoskeletal network required for the motility of the ameboidal sperm.¹² MSP domains also occur in VAP proteins that are involved in membrane fusion processes in various eukaryotes.¹³ The ANK domain, on the other hand, closely resembles animal ankyrins which serve to connect integral membrane proteins to elements of the spectrin cytoskeleton,¹⁴ thereby facilitating the assembly of functional membrane microdomains in diverse animal cells.¹⁵ Ankyrin repeats exhibit features of nano-springs, opening the possibility that ankyrin domains may be involved in mechanosensing.¹⁶ Based on these structural similarities, VAPYRIN may promote intracellular accommodation of endosymbionts by interacting with membranes and/or with the cytoskeleton. Indeed, VAPYRIN protein associates with small subcellular compartments in petunia and in Medicago truncatula.^9,10Ankyrin repeats typically consist of 33 amino acids, of which 30–40% are highly conserved across most taxa. These residues confer to the repeats their basic helix-turn-helix structure.¹⁷ Ankyrin domains often consist of arrays of several repeats that form a solenoid with a characteristic crescent shape.¹⁷ Besides the ankyrin-specific motiv-associated amino acids there is little conservation between the ankyrin domains of different proteins, or between the individual repeats of a given ankyrin domain,¹⁷ a feature that was also observed in petunia VAPYRIN (Fig. 1A).⁹ However, sequence comparison of VAPYRINs from eight dicots, three monocots and the moss Physcomitrella patens revealed a high degree of sequence conservation beyond the ankyrin-specific residues (Fig. 1B and Sup. Fig. S1). When the degree of conservation was determined for the individual ankyrin repeats among all the 12 species, it appeared that repeats 7, 9 and 10 exhibited particularly high conservation (Fig. 1C).Open in a separate windowFigure 1Sequence analysis and phylogeny of VAPYRIN from diverse plants. (A) Predicted amino acid sequence of the petunia VAPYRIN protein PAM1. The 11 repeats of the ankyrin domain are aligned, and the ankyrin consensus sequence is shown below the eleventh ankyrin repeat (line c). Conserved residues that are characteristic for ankyrin repeats (Mosavi et al. 2004)¹⁷ are depicted in bold face. (B) Unrooted phylogenetic tree representing the VAPYRINs of eight dicot species (Petunia hybrida, Solanum lycopersicon, Solanum tuberosum, Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula and Glycine max) three monocot species (Sorghum bicolor, Zea mays and Oryza sativa), and the moss Physcomitrella patens. (C) Degree of conservation of the individual ankyrin repeats of VAPYRIN. Schematic representation of the MSP domain as N-terminal barrel-shaped structure, and of the individual ankyrin repeats as pairs of alpha-helices. An additional loop occurring only in monocots (grass-loop) is inserted above repeat 4, and the deletion between repeat 7 and 8 is indicated (gap). This latter feature is common to all VAPYRIN proteins. The percentage of amino acid residues that are identical in at least 11 of the 12 VAPYRINS is given below the MSP domain and the eleven ankyrin repeats. The box highlights repeats 7–10 which contribute to the predicted binding site (compare with Figs. 3 and ​and44).Sequence comparison of the eleven repeats of all the twelve plant species revealed that the individual repeats clustered according to their position in the domain, rather than according to their origin (plant species) (Fig. 2). This shows that the repeats each are well conserved across species, but show little similarity among each other within a given VAPYRIN protein. The higher conservation of repeats 9 and 10 was reflected by the compact appearance of the respective branches, in which the monocot and moss sequences were nested closely with the dicot sequences, compared to other repeats, where the branches appeared fragmented between monocots and dicots, and where the P. patens sequence fell out of the branch as in the case of repeats 4–6 (Fig. 2). Taken together, this points to an old evolutionary origin of the entire ankyrin domain in lower land plants, with no subsequent rearrangement of ankyrin repeats.Open in a separate windowFigure 2Phylogenetic analysis of the individual ankyrin repeats of VAPYRIN. Phylogenetic representation of an alignment of all the 11 repeats of the 12 VAPYRINs compared in Figure 1B and C. The repeats cluster according to their position within the domain, rather than to their origin (plant species). Numbers indicate the position of the repeats within the domain (compare with Fig. 1C). P. patens repeats are highlighted (small circles) for clarity. The monocot repeat 4 sequences (boxed) are remote from the remaining repeat 4 sequences because of the grass loop (compare with Fig. 1C).Ankyrin domains function as protein-protein interaction domains,¹⁷ in which the residues on the surface are involved in the binding of their protein partners.¹⁴ The fact that repeats 9 and 10 exhibited particularly high levels of conservation across species from moss to angiosperms indicated that this region may contain functionally important residues. Within repeat 10, sixteen amino acid positions were identical in >90% of the analyzed species (Fig. 3A and grey bars). Nine of those represent residues that are characteristic for ankyrin repeats (red letters) and determine their typical 3D shape.¹⁷ These residues are considered ankyrin-specific, and are unlikely to be involved in a VAPYRIN-specific function. The remaining seven highly conserved residues in repeat 10, however, are VAPYRIN-specific, since they have been under positive selection, without being essential for the basic structure of the ankyrin repeat. Ankyrin-specific and VAPYRIN-specific residues where identified throughout the entire ankyrin domain (Sup. Fig. 1), and subsequently mapped on a 3-dimensional model of petunia VAPYRIN to reveal their position in the protein (Fig. 3B–G). The ankyrin-specific residues were found to be localized primarily to the interior of the ankyrin domain, with the characteristic glycines (brown) marking the turns between helices and loops (Fig. 3B, D and F, compare with A). In contrast, the VAPYRIN-specific residues were localized primarily on the surface of the ankyrin domain (Fig. 3C, E and G). A prominent clustering of VAPYRIN-specific residues was identified on the concave side of the crescent-shaped ankyrin domain comprising repeats 7–10 close to the gap (Figs. 3G and ​and44). This highly conserved VAPYRIN-specific region contains several negatively and positively charged residues (D, E and K, R, respectively) and aromatic residues (W, Y, F), which may together form a conserved binding site for an interacting protein.Open in a separate windowFigure 33D-Mapping of conserved positions within the ankyrin domain of VAPYRIN. (A) Conserved amino acid residues were evaluated for ankyrin repeat 10 of petunia VAPYRIN as an example. The degree of conservation between the 12 VAPYRINs analyzed in Figures 1B and ​and22 is depicted with grey bars. Average conservation between all the 132 ankyrin repeats of the 12 VAPYRIN sequences is shown with black bars. Residues that are conserved in all 132 repeats (red letters) define the ankyrin consensus sequence, which confers to the repeats their characteristic basic structure.¹⁷ Residues that are >90% conserved but are not part of the basic ankyrin sequence (highlighted with asterisks) are VAPYRIN-specific and may therefore have been conserved because of their specific function in VAPYRIN. Arrows indicate the characteristic antiparallel helices, the turns are marked by conserved glycine residues (underlined; compare with B, D and F). (B–G) 3D-models of the petunia VAPYRIN PAM1. Conserved amino acid residues were color-coded according to their physico-chemical properties (http://life.nthu.edu.tw/∼fmhsu/rasframe/SHAPELY.HTM) with minor modification (see below). In (B, D and F) the ankyrin-specific residues are highlighted (corresponding to the bold letters in Fig. 1A). In (C, E and G), the VAPYRIN-specific residues are highlighted. Note the patch of high conservation on the concave side of the crescent-shaped ankyrin domain between repeats 7–10 next to the gap. (B–E) represent respective side views of the ankyrin domain, (F and G) exhibit the concave inner side of the domain. Color code: Bright red: aspartic acid (D), glutamic acid (E); Yellow: cysteine (C); Blue: lysine (K), arginine (R); Orange: serine (S), threonine (T); Dark blue: phenylalanine (F), tyrosin (Y); Brown: glycine (G); Green: leucin (L), valine (V), isoleucin (I), alanine (A); Lilac: tryptophane (W); Purple: histidine (H); Pink: proline (P).Open in a separate windowFigure 4The highly conserved surface area in domain 8–10 of the ankyrin domain of petunia VAPYRIN. Close-up of the highly conserved region of petunia PAM1 as shown in Figure 3G. Amino acids were color-coded as in Figure 3 and their position in the amino acid sequence is indicated (compare with Sup. Fig. 1).In this context, it is interesting to note that human ankyrin R also contains a binding surface on the concave side of the D34 domain for the interaction with the CBD3 protein.¹⁴ Consistent with an essential function of the C-terminal third of the ankyrin domain, mutations that abolish this relatively short portion of VAPYRIN, have a strong phenotype, indicating that they may represent null alleles.⁹ Based on this collective evidence, we hypothesize that repeats 7–10 are involved in the formation of a protein complex that is essential for intracellular accommodation of rhizobia and AM fungi. Biochemical and genetic studies are now required to identify the binding partners of VAPYRINs, and to elucidate their role in plant endosymbioses.