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https://f1000research.com/articles/7-1842/v1	22 Nov 18	{ "type": "Research Article", "title": "Leopards and giants, tigers and woolly bears: casting a broader net in exploring heparin effects on Lepidoptera wing patterns", "authors": [ "Andrei Sourakov" ], "abstract": "Background: Studies of heparin effects on Lepidoptera wing patterns have been restricted to a small number of species. I report observations from experiments on a broader range of taxa, including first results from swallowtails, tiger moths and microlepidoptera. Methods: Heparin injections were made in prepupal and early pupal stages of the following species representing several Lepidoptera lineages: Junonia coenia, Agraulis vanillae, Asterocampa clyton (Nymphalidae); Heraclides cresphontes, Pterourus troilus, Eurytides marcellus (Papilionidae); Hypercompe scribonia, Estigmene acrea, Hyphantria cunea (Erebidae); and Glyphodes sibillalis (Crambidae). Heparin-induced changes in wing pattern are illustrated, and advantages of using prepupal vs. pupal stages for this type of pharmacological manipulation of wing patterns are discussed. Results: In buckeyes, heparin-induced changes consisted of loss of orange parafocal elements as marginal and submarginal bands shifted basally. In gulf fritillaries, changes in black and silver markings were similar to those found in wild aberrant individuals. In tawny emperor, intermediate and extreme levels of transformation were achieved, pointing to homology of this species’ unusual eyespots to those of other nymphalids. In swallowtails, heparin-induced changes were much more restricted and proved harder to achieve, possibly indicating higher levels of stability and compartmentalization of wing patterns in this butterfly family. In tiger moths, elongation of black markings occurred so that normally distinct spots sometimes merged; in leopard moth, these changes were restricted to areas adjacent to discal spot. In pyraloid moth, changes were mostly restricted to expansion of wing marginal bands and hindwing border. Conclusions: Variation in wing pattern response to heparin found between different species and families in this study warrants further taxonomic widening of exploration of wing pattern formation mechanisms in Lepidoptera. While there are many similarities, there also seem to be very significant differences in the ways wing patterns are formed in different families of butterflies and moths.", "keywords": [ "wing pattern", "butterflies", "moths", "sulfated polysaccharides", "evolution of development" ], "content": "Introduction\n\nThe wing patterns of butterflies and moths are not only physical characteristics that interact with their surroundings according to the laws of physics, such as through the absorption or reflection of heat; the spectacular array and the subtle variations in wing patterns found among ca. 160,000 Lepidoptera species are due to the fact that these patterns are also ornaments, and as such, are there to interact with an observer—whoever that observer may be—contributing not only to natural but also to sexual selection. Hence, understanding how these ornaments evolve and how they are regulated during their development would help understand the process of selection. Altering the expression of a gene ligand, like Wnt, through injecting heparin at the crucial stages of wing pattern development, can provide a glimpse into the mechanisms underlying wing pattern evolution and development.\n\nSince pioneering experiments on the common buckeye, it has been known that the wing pattern of a butterfly can be altered by heparin injection during the pupal stage (Serfas & Carroll, 2005). When, in 2017, I injected heparin into two prepupae, along with several pupae, of the io moth, it was a deviation from standard practice, which called for targeting early pupal stages. In that experiment, the survival rate of the injected prepupae was 100%, while for the pupae, it was 8%, while the two groups achieved similar pattern transformations (Sourakov, 2017). This, combined with the curiosity of how other species would be transformed by heparin injections, prompted me to conduct the experiments described below.\n\nThe common buckeye, Junonia coenia—a nymphalid species on pupae of which heparin-induced wing pattern manipulation was already thoroughly explored by Serfas & Carroll (2005) —offered a good model for examining whether injection at the prepupal stage resulted in different transformations. Additionally, I conducted experiments on several other Lepidoptera species from several families: Nymphalidae: the gulf fritillary (Agraulis vanillae) and the tawny emperor (Asterocampa clyton); Papilionidae: the giant swallowtail (Heraclides cresphontes), the spicebush swallowtail (Pterourus troilus), and the zebra swallowtail (Eurytides marcellus); Erebidae: the leopard moth (Hypercompe scribonia), the fall webworm (Hyphantria cunea) and the acrea moth (Estigmene acrea); and Crambidae: the mulberry leaftier (Glyphodes sibillalis). All of these are highly patterned species common to Florida. Collectively, they offer a good basis for comparison as they represent several different Lepidoptera lineages.\n\n\nMethods\n\nSeveral hundred caterpillars of all species used in this study were collected in Gainesville, Florida, and raised to pupation, with 275 of them used as experimental groups (see Table 1 for numbers per species). Additionally, several unmanipulated individuals were reared in the same conditions for each species as controls. Caterpillars were then reared inside plastic bags or containers on the foliage of their respective hostplants as follows: on Plantago lanceolata (buckeye); on Passiflora incarnata (gulf fritillary); on Celtis laevigata (tawny emperor); on Zanthoxylum clava-herculis and Z. fagara (giant swallowtail); on Cinnamomum camphora and Sassafras albidum (spicebush swallowtail); on Asimina triloba (zebra swallowtail); on Tradescantia ohiensis and Melilotus alba (acrea and leopard moths); on Juglans nigra (fall webworm) on Morus sp. (mulberry leaftier moth). The buckeyes were reared at natural light condition at 27°C; all others, at 24 hour light in the lab at 23°C. The immature stages used to generate the graph in Figure 8 were weighted using Metter Toledo AL104 analytical balances.\n\nHeparin injections were made with either a 10-µl Hamilton syringe or a 0.3-ml hypodermic syringe. In the latter case, the amount was measured out with the 10-µl syringe. The heparin solution was made from porcine-derived heparin sodium salt (manufactured by MP Biomedicals, Inc., supplied by Fisher Scientific) dissolved in distilled water. Different concentrations and volumes of heparin solution were injected. Concentrations and volumes used were different for each species (according to their size), and were sometimes varied in order to achieve variation in response (Table S1) (Sourakov, 2018). Experiments also investigated the effect of temperature on an individual’s response to injection (Table S1) (Sourakov, 2018). When the temperature was lowered from 23°C to 16°C, the experimental animals were placed in this temperature 1-3 hours prior to injection and left there for at least 24 hours afterwards. If a prepupa was injected at this temperature, it was allowed to pupate at 16°C and left an additional 24 hours as pupa at 16°C before being returned to 23°C. For many individuals, the exact time from injection to or after pupation was determined using time-lapse photography with precision of 0.5-1 hour, while for others it was only determined approximately (Table S1) (Sourakov, 2018). While the numbers of control individuals varied depending on availability, from 5 (in case of mulberry leaftiers) to 32 (in case of buckeyes) they were also supplemented by consulting the MGCL collection holdings for these species from the Gainesville area.\n\n\nResults and discussion\n\nCommon buckeye, Junonia coenia. In buckeyes, the heparin-induced changes consisted of the orange parafocal element being lost as the distal marginal and submarginal bands shift basally and overrun it. The width and clarity of the normally cream-colored markings on the dorsal forewing surface is reduced, along with the reduction of the forewing eyespots. This resulted in specimens that are overall less colorful dorsally (Figure 1Ai–Li). Ventrally, the same individuals exhibited a reduction in the size of eyespots and the diffusion of some of the wing pattern elements (Figure 1Aii–Lii). While buckeyes are quite variable in nature, the three “control” groups (non-manipulated, H2O-injected, and wild adults collected from the same locality as caterpillars) were similar to each other. The experimental heparin-injected group, on the other hand, ranged from displaying phenotypes that were not found in any of the control groups (as in Figure 1G–L) to presenting normal coloration (Figure S1) (Sourakov, 2018). An almost complete modification of the pattern as shown in Figure 1G occurred only once and this individual, although it made it out of the pupa, did not appear viable, unable to harden its wings, climb, or fly properly.\n\nHeparin-induced wing pattern changes (right) vs. controls (left) on the dorsal (i) and ventral (ii) surfaces of the common buckeye, Junonia coenia. (A–F) Control group: (A–C) males and (D–F) females injected H2O as prepupae. (G–L) Experimental group: (G–I) Males and (J–L) females injected heparin as prepupae. For both groups, specimens shown are the ones in which the dorsal orange hindwing band was expressed the least. See Table S1 for injection details and Figure S1 for more individuals from both groups.\n\nAnalysis of the wing pattern of all surviving individuals injected as prepupae with heparin (N=21) suggests that injection at the early prepupal stage (as defined in Extended Data) (Sourakov, 2018) is more likely to lead to survival and transformation: among 10 experimental individuals sporting the most transformed wing patterns, three were injected as late prepupae and seven as early prepupae.\n\nThe taxonomy of the genus Junonia in the New World has been complicated by the variability in wing patterns found within and among species and populations. For the widespread species J. coenia this has led to the generation of various taxonomic hypotheses, and modern approaches have recently been used to test them (e.g., Peters & Marcus, 2017). Laboratory-generated aberrations, such as the ones figured in the present study, may be useful for understanding the source of variability found among natural populations and perhaps even help in making taxonomic decisions.\n\nThe Tiger moths: the leopard moth, Hypercompe scribonia, the acrea moth, Estigmene acrea, and the fall webworm, Hyphantria cunea. Among 36 individuals of the leopard and acrea moths that survived injection at different stages of development, transformation was restricted to a single individual in each species injected as a prepupa within 12 hours before pupation (HBP), despite the fact that doses and concentrations injected were quite high and timing varied widely.\n\nThe heparin-induced changes were similar in their manner (Figure 2). The alterations consisted of a very noticeable elongation of the black markings, to the point where some of the normally distinct spots sometimes merged, which happened both dorsally and ventrally. In the leopard moth, the expansion of individual spots was not consistent throughout the forewing, but was restricted to areas of the forewing adjacent to the discal spot, with spots along wing margins remaining unchanged (Figure 2C). Hindwing markings also expanded in response to heparin, especially in the acrea moth (Figure 2B). I examined hundreds of individuals of both species in the collection of the McGuire Center (MGCL) but did not find any with similar phenotypes.\n\n(A, B) The acrea moth Estigmene acrea: (A) control; (B) injected heparin as prepupa (i) dorsal (ii) ventral. (C–E) The leopard moth, Hypercompe scribonia, dorsal view: (C) injected heparin as prepupa; (D–E) typical wild-collected specimens. See Table S1 for injection details.\n\nFor the fall webworm, for which the caterpillars of the entirely white-winged form were available, the experimental individuals that emerged (N=12) were identical to controls (N=40), despite the injection of prepupal and early pupal stages.\n\nThe gulf fritillary, Agraulis vanillae. The gulf fritillary has been one of several model organisms in recent investigations into wing pattern formation (Martin & Reed, 2014; Mazo-Vargas et al., 2017). Martin & Reed (2014, p. 376) showed how heparin injections result in the expansion of Wnt-positive patterns and the reduction or loss of Wnt-negative patterns in this species. This species provided a convenient subject for observing a gradual change in pattern, enabling testing of which developmental stage and temperature are most sensitive to treatment, leading to greater modification of wing pattern. A total of 11 successful manipulations, arranged in order of magnitude of effect on wing pattern (Figure 3Bi), can be correlated with injection stage and dose by consulting Table S1 (Sourakov, 2018). This wing transformation spectrum is contrasted with normal wing pattern that was not altered by injection at 1HBP (Figure 3A), or by pupation at colder temperature (Figure 3M).\n\nHeparin-induced wing pattern changes in the gulf fritillary, Agraulis vanillae (A–L) vs. rare natural aberrations from the MGCL collection (N–O), and control (M); (i) dorsal, (ii) ventral surfaces. See Table S1 for specimen details.\n\nSome of the transformed wing patterns (e.g., Figure 3C, I) are very similar to those of two wild-collected aberrant individuals, which were discovered in the MGCL collection after examining approximately 1000 pinned specimens (Figure 3N–O). These aberrations, while rare, may be of significance for understanding selection mechanisms that lead to divergence of wing patterns in Heliconiinae.\n\nThe tawny emperor, Asterocampa clyton. Recently I reported the successful transformation of a specimen of the tawny emperor butterfly by heparin injection (Sourakov, 2018). That single female individual, in which the dorsal surface of the wings turned almost entirely black, seemed to represent the furthest possible divergence from the normal phenotype.\n\nThis result is replicated again here in a male specimen (Figure 4D), with more accurately measured data concerning the injection (Table S1) (Sourakov, 2018). In addition to replicating the previous results, the main purpose of adding this species to the mix was to obtain intermediates between the normal pattern and the most extreme transformations. Variation in resulting phenotypes was achieved by lowering the dose of heparin and using the prepupal stage in addition to the pupal stage. As a result, two intermediate transformations were achieved (Figure 4B, C): the first injected as a prepupa (Figure 4B) and the second as a pupa (Figure 4C). The individual in Figure 4C is not drastically different from the one in Figure 4D, even though the former was injected with three times less heparin than the latter (Table S1) (Sourakov, 2018). The amount of heparin injected into the prepupa (Figure 4B) was between the doses received by the other two transformed individuals.\n\nNormal wing pattern (A) vs. heparin-induced wing patterns (B–D) in the males of the tawny emperor, Asterocampa clyton; (i) dorsal, (ii) ventral surfaces, (iii) close-up of border eyespots dorsally. See Table S1 for specimen details.\n\nIt is interesting to note that the dorsal hindwing border spots, normally black in this species, rather than disappearing into the black background, as happened in maximally transformed specimens, appeared as orange spots in two of the intermediates (Figure 4(iii)). Marginal eyespots in many Nymphalidae are concentrically organized and serve as models for studies of development, as for example, in Bicyclus anynana, where they have been shown to be positively regulated by Wingless (Wnt) (Özsu et al., 2017). Wnt signaling delimits the boundaries of wing spots, as reviewed by Martin & Courtier-Orgogozo (2017), and is affected by heparin. Figure 4(Aiii–Diii) suggests that, even though the serial border spots of the tawny emperor are not as concentrically organized as in many other nymphalids, they are nevertheless homologous. The marginal bands migrating basally under the influence of heparin are inhibited from invading eyespot centers. One can speculate that these eyespots, like the more concentrically organized ones, are also formed via a signal produced by the few eyespot-organizer cells in the eyespot’s center, as was first described by Nijhout (1980) and recently studied histologically by Iwasaki et al. (2017).\n\nThe giant swallowtail, Heraclides cresphontes. While the giant swallowtail is contrastingly patterned species, and one might expect substantial changes to its pattern resulting from heparin injections, this proved to be far from the case. To see the heparin-induced changes one needs to zoom in on the underside of the hindwing, but even then the transformations are barely noticeable. The normally present red spot in the center of the hindwing located on either side of M3 (Figure 5Aiii, Ciii), may become greatly reduced in heparin-injected individuals (Figure 5Biii, Diii). The number of light-colored scales within the ventral hindwing band that give the band iridescence seem to be replaced by black scales, giving these bands a darker appearance (e.g., Figure 5Aii, Bii). These changes are symmetrical, except in the female in Figure 5E, where asymmetry resulted from an asymmetrical defect in vein development, suggesting the influence veins may have on wing pattern formation. Assessing the twenty surviving individuals that were injected with a variety of doses at different stages, it seems that one should target the window around 5 hours after pupation if one wanted to replicate these results.\n\nNormal male (A) and female (C) vs. heparin treatment (B, D, E) in the giant swallowtail, Heraclides cresphontes: (i) dorsal, (ii) ventral surfaces, (iii) close-up of hindwing band dorsally. Heparin causes increase in numbers of melanic scales within elements composing ventral hindwing band (blue arrow). Heparin also causes reduction of ventral hindwing central red spot (white arrow). Heparin-induced changes are asymmetrical in an individual with asymmetrically defective wing venation (red arrow). See Table S1 for specimen details.\n\nThis species is widespread and can be geographically variable in the characters that demonstrated heparin induced changes (Warren et al., 2013). The present study suggests that both the intraspecific variation in ventral hindwing bands and the variation among species found in the closely-related South American taxa, such as Heraclides paeon, H. homothoas, H. melonius and H. thoas (Tyler et al., 1994 plate 89) would map onto Wnt gene ligand. It also illustrates how wing pattern formation can be compartmentalized in some species, so that variation in gene expression of pattern-mapping genes can affect only some of the compartments, but not others, and it may help to explain the existence of so many species sharing almost identical patterns in the New World.\n\nThe spicebush swallowtail, Pterourus troilus. A total of six individuals of the spicebush swallowtail were injected with heparin, and only two survived (both injected as pupae). There was visible transformation of the wing pattern in one of them (injected 5HAP). The central iridescent patch on the dorsal forewing surface that normally extends to, and sometimes beyond, the border of the large turquoise-colored spots (Figure 6Ai, C, D, E) appears to have shifted basally in the transformed individual (Figure 6Bi, Biii). At the same time, the orange spot located on the costal margin of the hindwing was greatly reduced compared to normal (Figure 6Biii, blue arrow). The corresponding changes occurred on the ventral side of the hindwing (Figure 6Bii).\n\nNormal female of the spicebush swallowtail, Pterourus troilus (A) vs. a female that underwent heparin injection as pupa (B): (i) dorsal, (ii) ventral surfaces. Close-up of hindwing dorsally in heparin-injected specimen (B.iii) is contrasted with that of three typical wild-caught specimens (C–E). Heparin causes contraction of dorsal orange spot (blue arrow). Heparin also causes a shift of iridescent hindwing patch basally (white and red arrows). See Table S1 for specimen details.\n\nNone of the numerous P. troilus specimens that I examined in the MGCL collection sported this phenotype, giving me confidence, despite limited sample size, that it was heparin-induced. The rare aberrations flava and addenda figured in Warren et al. (2013) might be explained by the results presented here.\n\nThe mulberry leaftier moth (Glyphodes sibillalis). This tiny species is relatively large by the standards of micromoths, but it still has a much smaller caterpillar, and hence my expectations of success were low. Nevertheless, among the two surviving experimental individuals, one injected heparin as a prepupa, the other as a pupa, the latter proved to be different not only from the former, but also from the five control specimens. The changes are consistent with heparin-induced changes in other species: the olive-brown hindwing border is wider and darker, and the normally thin and compact submarginal and marginal bands are wider and more diffused on both forewings and hindwings (Figure 7).\n\nHeparin causes expansion of the border area (a). Heparin also causes expansion of marginal band and diffusion of submarginal band (b) and expansion of some of the melanic territories (c). See Table S1 for specimen details.\n\nDifferences in survival by species. It is clear, from the nine species examined in the present study, that heparin treatments are tolerated differently by different species (Table 1). Unlike other species in the present study, very few experimental individuals among the leopard and acrea moths died from heparin injections: 84% of heparin-injected individuals emerged, and even the ones that did not emerge were fully formed inside their pupal cases. Despite the fact that, for the acrea moth, mostly young pupae were injected and, for the leopard moth, almost exclusively prepupae were used, the survival rates were similarly high for both species (Table 1). Compared to some butterfly species, the tiger moths, especially the acrea moth, showed a remarkable resilience to heparin injections, which may be due to their biology, which involves sequestering and synthesizing secondary plant compounds for defense (Singer & Bernays, 2008).\n\nBased on the three swallowtail species used in the present studies, Papilionidae may provide a challenge when it comes to achieving both survival and wing transformation. For instance, one of the species not discussed above, the Zebra swallowtail, Eurytides marcellus, had a zero survival rate, despite attempts to use low concentrations and dosage of heparin at different stages of development and at different conditions. In the giant swallowtail, despite injecting 60 individuals at different stages, visible transformation of wing pattern was difficult to achieve and only a third survived the injection (Table 1). Clearly, more work is needed to optimize my technique; however, considering that these are the first examples from this beautiful family of large, colorful and extremely diverse butterflies with which such manipulations have been conducted, I hope that heparin-induced wing pattern changes described here will contribute to our understanding of the wing pattern evolution as a whole.\n\nWithin the Nymphalidae, the survival varied between species and stages. The gulf fritillary was the most successful species, after the tiger moths, in its ability to survive heparin treatment (Table 1), which is perhaps also due to its ability to feed on and sequester toxic compounds from its host plant. However, unlike the tiger moths, the transformation rate was much higher and more predictable. It seems to survive injections and transform equally well as prepupae and pupae. Among the 100 buckeye individuals used in this experiment, the survival of heparin-injected individuals greatly depended on the developmental stage (Table 1). For unknown reasons, success was achieved only with the prepupal stage: 79% (n=19) of the injected as early prepupae and 45% (n=29) of those injected as late prepupae survived. Despite lowering the dose and varying the stage of injection, as well as conducting injections at lower temperatures, achieving the survival of the tawny emperor was more problematic: only 20% of the individuals survived injections, with 4 additional transformed individuals fully formed but unable to emerge from the pupa. It seems that the odds of a successful transformation was higher when injections were made at about 5HAP.\n\nIs wing pattern transformation stage-sensitive? Heparin injections, for the most part do not interfere with pupation, so, almost all the individuals injected as prepupae and subsequently did not survive died in their pupal stage. It is certainly quite likely that, in most species, at least some wing pattern elements are laid down in the prepupal stage, and hence it is important to explore both prepupae and pupae. The logical question becomes: does heparin linger from the time of injection onwards, or does its action correlate strictly with the time of injection? While the similarities in the outcome of late prepupal and pupal injections in the io moth (Sourakov, 2017) may suggest that there is little difference whether a late prepupa or an early pupa is injected, it may be not the case in every Lepidoptera species. There seemed to be a different pattern of transformations that occurred to buckeyes injected as pupae by Serfas & Carroll (2005) from the buckeyes injected as prepupae in the present study: here, the hindwing eyespots remained practically unchanged, throughout the transformation spectrum (except in one individual in which the pattern was completely overhauled, but which was not viable). Serfas & Carroll (2005), on the other hand, were able to gradually decrease the size of the dorsal hindwing eyespot by increasing the dose of heparin.\n\nAmong 36 heparin injections in tiger moths, including many where the timing would have been perfect to achieve the transformation in Nymphalidae (ca. 5-8HBP), the only obvious wing pattern transformations occurred in two individuals injected as prepupae. This likely suggests that, at least in these species, the effect is stage-sensitive. My unpublished observations from silk moths also suggest that injecting too early in the prepupal stage does not produce the visible transformations that can be achieved in late prepupal and early pupal stages. Hence, I think that the future researchers pursuing studies of wing pattern of the acrea, leopard and perhaps other tiger moths may want to target the time around 12HBP.\n\nHeparin dosage and stages of metamorphosis. Using the methods as described in this preliminary foray made it extremely difficult to provide an accurate estimate of how much heparin was actually being delivered to the system, because of the variation in dosage and, almost undoubtedly because some heparin may be lost as a result of ‘bleeding’ from the injection site. The pressure in a prepupa may help animal expel any foreign object or liquid attempting to penetrate it.\n\nAnother consideration is the changes in weight that a caterpillar undergoes during metamorphosis. My experience with the Io moths, a species in which a normal female can be more than double the weight of a normal male, suggests that the transformation achieved by heparin injections is not only dose- and stage-dependent, but also depends on an animal’s weight. I weighted immature stages of Lepidoptera species that I reared during this study, plus of two additional species of silk moths, the cecropia moth and the imperial moth, that I reared recently. Animals’ weights varied not only with species, but also the same individual’s weight varied greatly depending on the stage of its development (Figure 8). The manner in which weight changed as an animal underwent metamorphosis depended on the family and species it represented. For instance, in butterflies, such as the zebra longwing and gulf fritillary, weight change is minimal, but is more dramatic in the giant swallowtail.\n\nThe slope corresponds to species’ biology: moths spinning more silk lose relatively more weight than their counterparts from the same family that spin less silk. Six leopard moth individuals are included to illustrate intraspecific variation. (BPP - before pupation; LPP - late prepupa).\n\nAmong moths, even within a single family or subfamily, weight change during metamorphosis may vary depending on a species’ biology, specifically silk production by the caterpillar. For instance, among the wooly bears used in the present study, the caterpillar of the acrea moth (also known as saltmarsh caterpillar) used its own hairs to form its cocoon, thus having much less need for silk, which is mostly used to bind these hairs together. Similar-looking leopard moth caterpillar, on the other hand, spun the cocoon entirely from its own silk, adding droplets of bad-smelling repellent for chemical protection. I suspect this is the reason why a leopard moth caterpillar loses relatively more weight during metamorphosis than a saltmarsh caterpillar (Figure 8). As I already pointed out above, intraspecifically, the weight can also vary substantially, as demonstrated by six leopard moth caterpillars in Figure 8.\n\nAmong silk moths, the cecropia moth caterpillar spins a formidable double cocoon, losing ca. 75% of its weight in the process. The imperial moth, on the other hand, though it too belongs to the silk moth family, does not spin a cocoon at all, instead relying for protection on pupating in an underground chamber and on the thick, hard chitin of its pupal case. While its weight loss during the metamorphosis is still significant (ca. 60%), it is noticeably less than in cecropia moth.\n\n\nData availability\n\nRaw data, including raw images and weights of caterpillars and pupa, are available on OSF, DOI: https://doi.org/10.17605/OSF.IO/D2P9H (Sourakov, 2018).\n\nFigure S1. Full spectrum of heparin-induced wing pattern changes in dorsal hindwing of the common buckeye, Junonia coenia (A, B) Control group: (A) males and (B) females injected with H2O as prepupae. (C, D) Experimental group: (C) males and (D) females injected with heparin as prepupae. Wings are arranged left to right demonstrating a gradient in the reduction of the orange band (both groups) and expansion of the marginal bands (experimental group). DOI: https://doi.org/10.17605/OSF.IO/D2P9H (Sourakov, 2018).\n\nPostscriptum-On the term Prepupa, including Figure S2: Morphological recognition of prepupa as a separate stage in Lepidoptera, with further subdivision into early prepupa (epp), prepupa (pp), and pharate pupa (php). (A-B) The common buckeye, Junonia coenia: (A) caterpillar; (B.i) early prepupa; (B.ii) prepupa; (C) The gulf fritillary, Agraulis vanillae: (C.i) early prepupa; (C.ii) prepupa; (C.iii) pharate pupa; (D) the giant swallowtail, Heraclides cresphontes: (D.i) caterpillar; (D.ii) early prepupa; (D.iii) prepupa. DOI: https://doi.org/10.17605/OSF.IO/D2P9H (Sourakov, 2018).\n\nTable S1. Details concerning heparin injections for the specimens illustrated in Figures 1-7. DOI: https://doi.org/10.17605/OSF.IO/D2P9H (Sourakov, 2018).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nI thank Matt Standridge for supplying some of the eggs and larvae of the giant swallowtails raised in the course of this study and Julieta Brambila for providing larvae of the mulberry leaftier moth. Alexandra Sourakov kindly proofread the earlier versions of this manuscript.\n\n\nReferences\n\nIwasaki M, Ohno Y, Otaki JM: Butterfly eyespot organiser: in vivo imaging of the prospective focal cells in pupal wing tissues. Sci Rep. 2017; 7: 40705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin A, Courtier-Orgogozo V: Morphological evolution repeatedly caused by mutations in signaling ligand genes. In Diversity and Evolution of Butterfly Wing Patterns. Springer, Singapore. 2017; 59–87. Publisher Full Text\n\nMartin A, Reed RD: Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems. Dev Biol. 2014; 395(2): 367–378. PubMed Abstract \| Publisher Full Text\n\nMazo-Vargas A, Concha C, Livraghi L, et al.: Macroevolutionary shifts of WntA function potentiate butterfly wing-pattern diversity. Proc Natl Acad Sci U S A. 2017; 114(40): 10701–10706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNijhout HF: Pattern formation on lepidopteran wings: determination of an eyespot. Dev Biol. 1980; 80(2): 267–274. PubMed Abstract \| Publisher Full Text\n\nÖzsu N, Chan QY, Chen B, et al.: Wingless is a positive regulator of eyespot color patterns in Bicyclus anynana butterflies. Dev Biol. 2017; 429(1): 177–185. PubMed Abstract \| Publisher Full Text\n\nPeters MJ, Marcus JM: Taxonomy as a hypothesis: testing the status of the Bermuda buckeye butterfly Junonia coenia bergi (Lepidoptera: Nymphalidae). Syst Entomol. 2017; 42(1): 288–300. Publisher Full Text\n\nSerfas MS, Carroll SB: Pharmacologic approaches to butterfly wing patterning: sulfated polysaccharides mimic or antagonize cold shock and alter the interpretation of gradients of positional information. Dev Biol. 2005; 287(2): 416–424. PubMed Abstract \| Publisher Full Text\n\nSinger MS, Bernays EA: Specialized generalists: behavioral and evolutionary ecology of polyphagous woolly bear caterpillars. Tiger moths and woolly bears: behavior, ecology and evolution of the Arctiidae. Oxford University Press, Oxford. 2008; 103–114. Reference Source\n\nSourakov A: Scientific Note: The Emperor’s new clothes: radical transformation of the wing pattern in Asterocampa clyton caused by heparin. Trop Lepid Res. 2018; 28(1): 29–31. Publisher Full Text\n\nSourakov A: F1000-2018-Sourakov. 2018. http://www.doi.org/10.17605/OSF.IO/D2P9H\n\nSourakov A: Giving eyespots a shiner: Pharmacologic manipulation of the Io moth wing pattern [version 2; referees: 2 approved]. F1000Res. 2017; 6: 1319. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTyler HA, Brown KS, Wilson KH: Swallowtail butterflies of the Americas. Scientific Publishers. 1994; 377. Reference Source\n\nWarren AD, Davis KJ, Stangeland EM, et al.: Illustrated Lists of American Butterflies. [1-XI-2018]. 2013. Reference Source" }	[ { "id": "41054", "date": "13 Dec 2018", "name": "Arnaud Martin", "expertise": [ "Reviewer Expertise Evolutionary Developmental Biology", "Lepidoptera" ], "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\nIntroduction:\n\"Altering the expression of a gene ligand, like Wnt, through injecting heparin at the crucial stages of wing pattern development, can provide a glimpse into the mechanisms underlying wing pattern evolution and development\". Replace \"expression of a gene ligand\" by \"activity of a signaling ligand\". Binari et al1 could be cited here. \"Eurytides marcellus\", replace with \"Protographium\" (as well as elsewhere in the manuscript)\nMethods:\nProvide the Molecular Weight of the commercial source of Heparin sodium sulfate in kDa (shall be indicated on the container)\nTable S1:\nPlease convert all the dosage estimates into ng. For instance, 1uL of 5% = 5ug/100nL corresponds to a 50ug dose. This allows a more direct comparison of all the experiments. What were the conditions used on the species that did not provide meaningful results?\nResults:\nAcrea / Leopard moths. Since heparin tends to mimic the effect of cold-shocks, it would be expected to find natural aberrants of those species that look similar to the heparin treatment. By any chance, are there MGCL specimens that go along similar axes of variation?\nAsterocampa:\nRegarding: \"Marginal eyespots in many Nymphalidae are concentrically organized and serve as models for studies of development, as for example, in Bicyclus anynana, where they have been shown to be positively regulated by Wingless (Wnt) (Özsu et al., 20172). Wnt signaling delimits the boundaries of wing spots, as reviewed by Martin & Courtier-Orgogozo.,20173, and is affected by heparin. Figure 4(Aiii–Diii) suggests that, even though the serial border spots of the tawny emperor are not as concentrically organized as in many other nymphalids, they are nevertheless homologous. The marginal bands migrating basally under the influence of heparin are inhibited from invading eyespot centers.\"\nIt shall be noted 1) that WntA Knock-Outs results in marginal effects in various nymphalids (Mazo-Vargas et al.,20174), shifting the distal Parafocal Elements towards the margin, leaving the hindwing eyespots unaffected, but reducing the size of the WntA-positive forewing eyespots and 2) that the current results in Asterocampa hindwings (and perhaps forewings) seem analagous to the effects of Heparin in Vanessa cardui, as reported in Martin and Reed Dev. Biol. 20145.\nTo me, the Asterocampa heparin results are best explained by analogy with the Vanessa system (also analyzed by Otaki in several articles such as Ataki et al.,20086. Asterocampa shows a similar color inversion effect on hindwing eyespots (both ventral and dorsal) to Vanessa heparin injected specimens. This must be independent of WntA, because WntA KOs do not affect the Vanessa hindwing, and the effect of heparin on those Asterocampa/Vanessa hindwing eyespots could be explained by other morphogens, possibly an eyespot focal Wg signal as proposed in Bicyclus (Özsu et al., 20172), non-Wnt heparin-sensitive morphogens, or a complex interaction dynamics between wing margin signals and eyespot color patterning (the Nijhout book, or some Otaki references goes into classic surgery experiments). Notice however that one could expect an expansion of eyespots upon heparin injection if a focal eyespot Wg signal was in place. Oliver et al.,20127 provide comparative and developmental evidence of complex interactions between the margin and the eyespot positioning process, so I think that overall we are still scratching the surface of the iceberg and the effects of heparin in that area are difficult to interpret.\n\nDiscussion:\nJunonia: provide more comparisons of the results with those reported by Serfas and Carroll. It seems to me that the pre-pupal injection results in less striking effects than the early pupal ones, for instance on the Wg-positive Discalis elements (orange stripes in Junonia forewings)? Weight loss data: please provide more data on the phenomenon of fluid excretion (time, amount?) by the silk-spinning species.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "41144", "date": "21 Dec 2018", "name": "Jeffrey M. Marcus", "expertise": [ "Reviewer Expertise Evolution and development of butterfly color patterns", "insect developmental genetics", "and molecular phylogenetics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript expands on earlier work (including some by Sourakov, himself (Sourakov, 20171) that uses heparin sodium sulfate salt to manipulate color pattern development in Lepidoptera. Most of the prior work had been done in a small number of species in the butterfly family Nymphalidae. This manuscript repeats experiments in the butterfly model system Junonia coenia (Nymphalidae) (Serfas and Carroll, 20052) as controls and compares the results of these experiments to experiments using this technique in additional species of Nymphalid butterflies, as well as species of swallowtail butterflies (family Papilionidae) and of moth species in families Erebidae and Crambidae.\n\nWhile I enjoyed reading the manuscript, there are a number of changes that I would like to suggest to the author:\n\nClarification of abstract:\nIn the methods section of the abstract, the species studied are only listed by their scientific names, while in the results section the species studied are only listed by common name. Consequently, it would be challenging for anyone without an extensive knowledge of lepidopteran nomenclature to easily connect the experimental results with the appropriate species. Suggest replacing the phrase “unusual eyespots” with the phrase “simple eyespots” in the results section of the abstract.\n\nClarification of the introduction:\nAdding heparin sodium sulfate does not alter the expression of Wnt-family ligands (= production of the Wnt proteins by cells), but instead alters their solubility, diffusion in the intercellural space, and activity (by altering their ability to bind to cell-surface receptors) (Fuerer et al. 20103). There is more than one Wnt ligand. As reported in (Martin and Reed, 20144) and as discussed in (Martin and Courtier-Orgogozo, 20175), there are at least 4 different Wnt ligands that are expressed in the developing butterfly wing during color pattern determination, with wingless (wg) and WntA perhaps being the most important. On the whole, it appears that heparin has a much greater effect on phenoytpes associated with WntA signaling than on phenotypes associated with wg signaling.\n\nClarification of methods:\n“Different concentrations and volumes of heparin sodium sulfate solution were injected. Concentrations and volumes used were different for each species (according to their size), and were sometimes varied in order to achieve variation in response (Table S1)”. Please provide some sort of rationale for how dosage was adjusted. Also,Table S1 should include scientific names for each species and most importantly, the sample sizes of each and every trial listed in Table S1.\n\nClarification of results and discussion:\n“Ventrally the same individuals exhibited a reduction in the size of eyespots and the diffusion of some of the wing pattern elements.” Replace “diffusion” (which has clear and specific meaning in chemistry and physics that is not relevant here) with “loss of definition” or similar. Referring to same sentence as R&D#1 immediately above as well as the paragraph that begins “The taxonomy of the genus Junonia in the New World…”. The effects of heparin treatment of color pattern strongly resemble some of the diagnostic phenotypes that distinguish the recently identified cryptic taxon Junonia grisea from J. coenia (including the reduction or elimination of orange parafocal elements on dorsal wing surfaces, reduction in size of dorsal forewing eyespots, and loss of definition of wing pattern elements on ventral wing surfaces (Lalonde and Marcus, 20186). It should also be noted that different New World Junonia taxa, which cannot be distinguished by mitochondrial genotypes are distinguishable from one another by variation at the wingless locus (Borchers and Marcus, 20147; Gemmell et al. 20148; Peters and Marcus 20179; Lalonde and Marcus, 201810; Lalonde et al. 20186). It would be interesting indeed if these species-diagnostic color pattern phenotypes were actually caused by the evolution of Wnt ligand sequences and function. “The normally present red spot in the center of the hindwing located on either side of M3…may become greatly reduced in heparin-injected individuals…” These red spots in Heraclides cresphontes are homologous to the WntA driven patterns in Nymphalid butterflies (see (Martin and Reed 20144) and (Koch and Nijhout, 200211). I would like to see a greater synthesis of the findings of the present work with previous findings from heparin injections in Automeris io (Saturniidae) (Sourakov, 20171), as well as some consideration of what these injections may actually be doing to the mechanism of Wnt signaling. Wnt ligands not only activate signal transduction though the canonical pathway (involving b-catenin), but also through alternative pathways: JNK, and Ca2+. The activation of different downstream pathways is one possible mechanism by which similar ligands could produce different color pattern outcomes, but which signalling pathways are triggered by each of the Wnt signals on lepidopteran wings is currently unknown (Özsu and Monteiro 201712).\n\nI would like to encourage the author to consider these points in his revisions and also to continue his experimental explorations in future work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-1842
https://f1000research.com/articles/9-1237/v1	14 Oct 20	{ "type": "Research Article", "title": "Cross-sectional study of Facebook addiction in a sample of Nepalese population", "authors": [ "Alok Atreya", "Samata Nepal", "Prakash Thapa", "Samata Nepal", "Prakash Thapa" ], "abstract": "Background: Facebook addiction is said to occur when an individual spends an excessive amount of time on Facebook, disrupting one’s daily activities and social life. The present study aimed to find out the level of Facebook addiction in the Nepalese context and briefly discuss the issues associated with its unintended use. Methods: A descriptive cross-sectional study was conducted in the Department of Forensic Medicine of Lumbini Medical College. The study instrument was the Bergen Facebook Addiction Scale typed into a Google Form and sent randomly to Facebook contacts of the authors. The responses were downloaded in a Microsoft Excel spreadsheet and analyzed using Statistical Package for Social Sciences version 16. Results: The study consisted of 103 Nepalese participants, of which 54 (52.42%) were males and 49 females (47.58%). There were 11 participants (10.68%) who had more than one Facebook account. When different approaches were applied it was observed that 8.73% (n=9) to 39.80% (n=41) were addicted to Facebook. Conclusion: When used properly Facebook has its own advantages. Excessive use is linked with health hazards including addiction and dependency. Students who engage more on Facebook may have less time studying, leading to poor academic performance. People need to be made aware of the issues associated with the misuse of Facebook.", "keywords": [ "Addiction", "Crime", "Internet", "Nepal", "Social Networking" ], "content": "Introduction\n\nFounded on 4th February 2004 for communication limited to Harvard students, Facebook today is the most used social networking service (SNS) worldwide. There are 1.59 billion active Facebook users daily and 2.41 billion active Facebook users monthly as of June 20191. The primary motives of Facebook use are to communicate, collaborate and share content2. With such growing popularity and billions of users, there has been a concern of behavioral addiction to this SNS. Facebook addiction is said to occur when an individual spends an excessive amount of time on Facebook over the internet, disrupting one’s daily activities and social life3,4. If five hours or more is spent daily on Facebook then the person is said to be addicted to Facebook5.\n\nThe present study aimed to find out the level of Facebook addiction in a sample of Nepalese population and briefly discuss the issues associated with its unintended use.\n\n\nMethods\n\nA descriptive cross-sectional study was conducted in Department of Forensic Medicine of Lumbini Medical College after obtaining ethical approval from the Institutional Review Committee vide the letter IRC-LMC 01-G/019.\n\nThe Bergen Facebook Addiction Scale (BFAS) is a questionnaire that comprises of six core features of addiction: salience, mood modification, tolerance, withdrawal, conflict, and relapse6. Each of the six-core features consists of three questions, making a total of 18 questions. The final BFAS retained one question for each core element of addiction. Only the scores for questions 1, 5, 7, 11, 13 and 16 determine the level of Facebook addiction. Each question is scored on a 5-point Likert scale using anchors of 1: Very rarely and 5: Very often. Higher scores indicate greater Facebook addiction.\n\nParticipants scoring 4 (often) or 5 (very often) in four out of six questions were considered to be addicted to Facebook6,7. BASF has put forth two scoring schemes to determine Facebook addiction6. As per a polythetic scoring scheme, Facebook addiction was determined by a liberal approach, where a score of 3 or more was observed in at least four of six items; whereas using a conservative approach, a score of 3 or above in all six items determined Facebook addiction by a monothetic scoring scheme6.\n\nConsidering that there are around 1800 people in Lumbini Medical College including students, faculties and staffs, the sample size was calculated using the formula for finite population: n = N * X / (X+(N-1)); taking a confidence level of 95% (Z-score=1.96) and margin of error of 10%, the sample size was calculated to be 92.\n\nThe BAFS was typed into a Google Form and reviewed by all the authors for any mistakes which were then corrected. The link was then shared among the medical and nursing students, doctors, nurses and other health care staff working at Lumbini Medical College Teaching Hospital (LMCTH) through Facebook messenger, WhatsApp and Viber with a request to share the link among their friends or colleagues who were enrolled with LMCTH and were Nepalese citizens (convenient random sampling). The first part of the questionnaire consisted of a statement where it was explained that no financial or material gifts will be provided for completing the questionnaire. The survey did not collect any identifying information of any of the participants and the responses were anonymous. The second part of the questionnaire was a section on consent where the participants had an option to choose whether they voluntarily consented to participate or didn’t consent. The third part of the questionnaire was accessible only to those participants who consented in the second part. The survey didn’t continue for the participants who didn’t consent and the incomplete form submitted. The link was made active on 27 July, 2019. On August 30, 2019 a total of 108 responses were received and the link was disabled from receiving further responses. There were five responses which were incomplete so those were excluded from the study. The obtained responses were downloaded as a Microsoft Excel spreadsheet, which was then exported into SPSS v16 for analysis. Descriptive statistics such as frequency, percentage, mean and standard deviation were used to determine demographic characteristics of the respondents and Facebook addiction (see Underlying data)8.\n\n\nResults\n\nThe study sample consisted of 103 participants, of which 54 (52.42%) were males and 49 females (47.58%). There were 11 participants (10.68%) who had more than one Facebook account (Table 1).\n\nThe majority of participants (n=41, 39.8%) responded that during the last year they often spent a lot of time thinking about Facebook or planned use of Facebook. When asked if they felt an urge to use Facebook more and more, 35 participants (34.0%) felt they never felt such an urge. Only 16 (15.5%) responders used Facebook to forget about their personal problems during the last year. The present study showed that 23.3% of the responders (n=24%) could not cut down their use of Facebook during the last year. When asked whether they became restless or troubled when prohibited from using Facebook, six participants (5.8%) responded that they often felt restless. In 21 participants (20.4%), their use of Facebook had a negative impact on their job/studies. The detailed responses of the participants are presented in Table 2 and Table 3.\n\nThere were ten participants (9.70%) who scored 4 or more in four questions. A 43-year-old male was among one of those seven participants who had multiple Facebook accounts and scored 5 in all the six questions. This was the maximum score one could get as per BFAS, which denotes this individual was severely addicted to Facebook use. As per the conservative approach there were only nine participants (8.73%) who were addicted to Facebook. When the liberal approach was used, there were 41 participants (39.80%) who were addicted to Facebook (Table 4).\n\n\nDiscussion\n\nAlthough the popularity of Facebook is increasing daily, the fact that its use is prone to addiction cannot be denied9. Many recent studies from around the world advocate the potential risk of addiction. There are various assessment tools and diagnostic criteria to investigate SNS addiction; however, BFAS is considered to have good psychometric properties10. Facebook can be used for various purposes like instant messaging, video conferencing, gaming and shopping. Facebook is popular for its content sharing feature. The shared content may be news, personal blogs, pictures and videos, which can be educational, entertaining or explicit.\n\nIn a study conducted among postgraduate medical students in Southern India, it was observed that 26% of the participants were addicted to Facebook and 33% had the possibility of addiction11. The study also concluded that loneliness influenced Facebook addiction.\n\nA Nepalese study on Facebook use conducted among health science students of a private medical college showed that 98.2% were active Facebook users12. The authors of the study concluded that 28.5% of the study participants were unable to reduce their time spent on Facebook and therefore were addicted. In contrast, the present study observed as little as 8.73% of participants addicted to Facebook use. Although the number may seem less, it still raises a concern over the use of SNS in Nepalese context. Excessive use of Facebook by students may lead to poor time management and less time studying, which would result in poor academic performance. Another reason for fewer participants with Facebook addiction in the present study might be the fact that the majority of the participants were health care professionals. There is restricted access to Facebook and other SNS sites in many hospitals. Medical schools including Lumbini Medical College, Palpa; Manipal Teaching Hospital, Pokhara and Nepal Medical College, Kathmandu have blocked access to Facebook from their internet servers.\n\nA study conducted among 355 university students in the Philippines observed that only 15 were Facebook addicts7. A study from the United States showed that nearly 96% of medical undergraduates regularly used Facebook for social life and academic pursuits13. One of the studies conducted in Nepal stated Facebook to be a novel way of communication with members of health professional associations14.\n\nOne of the underlying reasons for excessive use of Facebook may be narcissistic behavior in which the individual is frequently sharing content to gain interactions and positive feedback in an attempt to become popular and admired15. The feeling of happiness tempts the individual to maintain and increase the level of happiness by engaging more on Facebook. Frequent use of Facebook can also lead to a person revealing his/her personal information like place of residence, job, family members etc. People should be made aware of the fact that this information can be used by individuals who take undue advantages or have ulterior motives. The increasing popularity of Facebook and other SNS sites is an indicator of people being more engaged in the online world. It has been observed that loneliness is one of the dominant predictors of social media addiction including Facebook16. We are not far from receiving patients with a complaint of excessive use of SNS sites or the internet in Nepalese hospitals. The clinicians should be aware that treatment in case of Facebook addiction can never be complete abstinence as the internet has become an integral part of social culture. Treatment in such cases should be aimed at controlled use and identifying the underlying cause of compulsive overuse.\n\nThe present study is not without limitations. The study participants were chosen through searching the researcher’s Facebook friend list, and there is a possible sampling bias in our study. The responses were collected anonymously and no identifying information of the responder was recorded; it therefore cannot be known if multiple responses form a same person occurred. Three different approaches were used in the present study that showed a varied addiction rate in the same population. BFAS is based upon use of Facebook, but it doesn’t specify the type of addiction is to technology or content. The convenient sampling, age, gender and occupational imbalance of the present study therefore cannot be generalized to the population of Nepal as a whole.\n\n\nConclusions\n\nFacebook addiction in the Nepalese context as per the present study was at least 8.73%, with a possibility a prevalence of addiction up to 39.80%. When used properly, SNS and the internet have their own advantages. Excessive use is linked with health hazards including addiction and dependency. People need to be made aware of misuse of SNS and the associated issues. Complete abstinence from Facebook or any SNS is difficult to achieve as internet has become integral part of our lives; however, in case of excessive use patients can be advised to control their use. Considering the high popularity of Facebook and other SNS sites in Nepal, it is evident that there is a huge need for future research in this regard.\n\n\nData availability\n\nDRYAD: Cross-sectional study of Facebook addiction in a sample of Nepalese population. https://doi.org/10.5061/dryad.83bk3j9pv8.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nFacebook Newsroom [Internet]. [cited 2019 Nov 18]. Reference Source\n\nThurairaj S, Hoon EP, Roy SS, et al.: Reflections of students’ language usage in social networking sites: Making or marring academic English. The Electronic Journal of e-Learning. 2015; 13(4): 302–16. Reference Source\n\nRana MS, Ahmed O, Hossain MA: Facebook addiction, self-esteem, and life satisfaction of Bangladeshi young people. Dhaka Univ J Psych. 2016; 40: 29–41. Reference Source\n\nMekinc J, Smailbegovic T, Kokic A: Should we be concerned about children using the internet: pilot study. Innov Issues Approaches Soc Sci. 2013; 6(2): 6–20. Publisher Full Text\n\nKaraiskos D, Tzavellas E, Balta G, et al.: P02-232 - Social network addiction: a new clinical disorder? Eur Psychiatry. 2010; 25(Supplement 1): 855. Publisher Full Text\n\nAndreassen CS, Torsheim T, Brunborg GS, et al.: Development of a Facebook Addiction Scale. Psychol Rep. 2012; 110(2): 501–17. PubMed Abstract \| Publisher Full Text\n\nMarcial DE: Are you a Facebook addict? Measuring Facebook addiction in the Philippine University. Int Proc Econ Dev Res. 2013; 66: 12–15. Reference Source\n\nAtreya A, Nepal S, Thapa P: Cross-sectional study of Facebook addiction in a sample of Nepalese population, v3, Dryad. Dataset 2020. http://www.doi.org/10.5061/dryad.83bk3j9pv\n\nGriffiths MD: Facebook addiction: concerns, criticism, and recommendations--a response to Andreassen and colleagues. Psychol Rep. 2012; 110(2): 518–20. PubMed Abstract \| Publisher Full Text\n\nKuss DJ, Griffiths MD: Social Networking Sites and Addiction: Ten Lessons Learned. Int J Environ Res Public Health. 2017; 14(3): 311. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShettar M, Karkal R, Kakunje A, et al.: Facebook addiction and loneliness in the post-graduate students of a university in southern India. Int J Soc Psychiatry. 2017; 63(4): 325–329. PubMed Abstract \| Publisher Full Text\n\nJha RK, Shah DK, Basnet S, et al.: Facebook use and its effects on the life of health science students in a private medical college of Nepal. BMC Res Notes. 2016; 9: 378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGafni R, Deri M: Costs and benefits of facebook for undergraduate students. Interdisciplinary Journal of Information, Knowledge, and Management. 2012; 7: 45–61. Reference Source\n\nChaudhary P, Tuladhar H: Novel ways of improving communication with members of health professional associations. Int J Gynaecol Obstet. 2014; 127 Suppl 1: S15–6. PubMed Abstract \| Publisher Full Text\n\nBrailovskaia J, Schillack H, Margraf J: Facebook Addiction Disorder in Germany. Cyberpsychol Behav Soc Netw. 2018; 21(7): 450–456. PubMed Abstract \| Publisher Full Text\n\nBiolcati R, Mancini G, Pupi V, et al.: Facebook Addiction: Onset Predictors. J Clin Med. 2018; 7(6): 118. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "73468", "date": "03 Nov 2020", "name": "Kishor Adhikari", "expertise": [ "Reviewer Expertise Non communicable disease risk factors", "health system" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: The authors have taken samples from only one medical college of Nepal, so, it may not look correct to state as \"Nepalese population\".\n\nMethod: There is no sampling technique as \"Convenient random sampling\". It is better to keep as convenient sampling.\n\nConclusion: Don't keep many things which are not the findings of your research.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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? Partly", "responses": [ { "c_id": "6141", "date": "04 Dec 2020", "name": "Alok Atreya", "role": "Author Response", "response": "We would like to thank the learned reviewer for the detailed review and positive comments on our manuscript. Based upon the comments the following changes are made: Comment 1 Title: The authors have taken samples from only one medical college of Nepal, so, it may not look correct to state as \"Nepalese population\". Response: The study population for the present study are students (medical and nursing) and employees of Lumbini Medical College, Palpa. The Nepalese students studying in the college are from different parts of the country and so are the employees. However, the whole Nepalese population cannot be represented by this study we have used the term ‘sample of Nepalese population’ in the title. Comment 2 Method: There is no sampling technique as \"Convenient random sampling\". It is better to keep as convenient sampling. Response: Changed as instructed. Comment 3 Conclusion: Don't keep many things which are not the findings of your research. Response: Conclusion is shortened and only the relevant findings are included." } ] }, { "id": "74539", "date": "20 Nov 2020", "name": "Abhishek Das", "expertise": [ "Reviewer Expertise Forensic medicine", "forensic psychiatry", "medical education are my field of research." ], "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 cross sectional study done is presented well with originality.\nThe Abstract reflects the major aspects of the main paper. However, the different approaches mentioned here needs a little elaboration. Inclusion of the keyword \"Facebook\" would be beneficial.\nThe Introduction properly reflects the background of study, but needs to highlight more on existing gap in literature. The level of addiction should be clarified clearly.\nThe Methods section is well written and can be replicated later by others. However the convenience sampling could have been improved by choosing the portion of general Nepali population attending hospital apart from only including those attached to LMCTH. Exactly how many participants approached (google form sent) was not mentioned clearly, so the response rate to BFAS in this study could not be calculated. The response to the SIX core features of the addiction was not mentioned in detail in Results section.\nTwo wrong representations of data noted:\n'BAFS' is to be replaced by BFAS in the first line of last paragraph of methods section. In the description of Tables 2 & 3 on \"who could not cut down their use of Facebook during the last year\" the result should be n=24 without the % sign.\nOverall, this is a well formulated and executed study with authentic results.\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? Partly\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?\nPartly\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": [ { "c_id": "6142", "date": "04 Dec 2020", "name": "Alok Atreya", "role": "Author Response", "response": "We would like to thank the learned reviewer for the detailed review and positive comments on our manuscript. Based upon the comments the following changes are made: Comment 1: The cross-sectional study done is presented well with originality. Response: Thank you for the comment. Comment 2: The Abstract reflects the major aspects of the main paper. However, the different approaches mentioned here needs a little elaboration. Response: The different approaches that were used for e.g. polythetic and monothetic scoring scheme are detailed in the methods and results section so it was removed from here to avoid confusion. The sentence “When different approaches were applied it was observed that 8.73% (n=9) to 39.80% (n=41) were addicted to Facebook.” is reframed to “It was observed that 8.73% (n=9) to 39.80% (n=41) were addicted to Facebook.” Comment 3: Inclusion of the keyword \"Facebook\" would be beneficial. Response: Facebook is added as a keyword. Comment 4: The Introduction properly reflects the background of study, but needs to highlight more on existing gap in literature. The level of addiction should be clarified clearly. Response: The last paragraph is elaborated as: “Excessive use of Facebook and other SNS is linked with health hazards including addiction and dependency. However, limited number of studies in this regard has made it a rarity in Nepalese context. The present study is therefore aimed to find out the level of Facebook addiction in a sample of Nepalese population and briefly discuss the issues associated with its unintended use.” Comment 5: The Methods section is well written and can be replicated later by others. However the convenience sampling could have been improved by choosing the portion of general Nepali population attending hospital apart from only including those attached to LMCTH. Response: We are grateful for the suggestion and will keep it in mind for future works. The study population for the present study are students (medical and nursing) and employees of Lumbini Medical College, Palpa. Although, the Nepalese students studying in the college are from different parts of the country and so are the employees; we have used the term ‘sample of Nepalese population’. Comment 6: Exactly how many participants approached (google form sent) was not mentioned clearly, so the response rate to BFAS in this study could not be calculated. Response: This was one of the limitations of the study. We have added the following sentence as limitation: “As the study participants were requested to share the link among the students and employees of LMCTH, we could not calculate the number people approached and the response rate.” Comment 7: The response to the SIX core features of the addiction was not mentioned in detail in Results section. Response: Table 2 and Table 3 detail the responses on the six core features of addiction. Comment 8: Two wrong representations of data noted: 'BAFS' is to be replaced by BFAS in the first line of last paragraph of methods section. In the description of Tables 2 & 3 on \"who could not cut down their use of Facebook during the last year\" the result should be n=24 without the % sign. Response: Corrected. Comment 9: Overall, this is a well formulated and executed study with authentic results. Response: Thank you." } ] } ]	1	https://f1000research.com/articles/9-1237
https://f1000research.com/articles/9-1393/v1	03 Dec 20	{ "type": "Research Article", "title": "The role of exogenous epidermal growth factor on Ki-67 proliferation marker expression in the submandibular salivary gland of albino rats receiving doxorubicin", "authors": [ "Mohamed Mansy", "Malak Soliman", "Rabab Mubarak", "Mohamed Shamel", "Mohamed Mansy", "Malak Soliman", "Rabab Mubarak" ], "abstract": "Background: This study was conducted to evaluate the role of exogenous epidermal growth factor (EGF) injection on the Ki-67 immuno-expression in submandibular salivary gland tissue of rats receiving doxorubicin (DXR). Methods: A total of 21 two-month-old male albino rats, of 200 g body weight, were divided into three groups: control group; DXR group, the rats received 20 mg/kg body weight DXR as a single intra peritoneal injection; DXR+EGF group, the rats received the same dose of DXR and on the next day they were injected intraperitoneally with 10 µg/kg body weight of EGF daily for one week. Histological sections and immunohistochemical expression of Ki67 sections were examined using a ZEISS Primo Star light microscopy and images taken using Tucsen IS 1000 10.0MP Camera. Results: Ki-67 expression was significantly increased in submandibular salivary glands of rats after DXR injection. However, Ki-67 expression in the glandular tissue was restored to normal levels after EGF injection. Conclusions: EGF preserved glandular architecture after DXR injection and maintained Ki-67 immune-expression within the glandular tissue near to the normal level.", "keywords": [ "Epidermal growth factor", "Ki-67", "Doxorubicin", "submandibular salivary gland" ], "content": "Introduction\n\nKi-67 is a heavy protein of 395 kDa weight. It is a proliferation marker which is highly expressed in various tumors, and has been used for investigations of many cancer types. Ki-67 is controlled by proteolytic pathways and has similar essential properties with other proteins known to regulate the cell cycle (Hofmann & Bucher, 1995). Furthermore, Ki-67 is an important protein for cell division as antisense nucleotides of Ki-67 will stop the division, and it is a vital factor in the formation of ribosomes (Schluter et al., 1993). This is reinforced by the conclusion that Ki-67 immuno-expression associates with the rate of protein production and function of ribosomes (Plaat et al., 1999).\n\nProliferation marker evaluation is of high value in pathological diagnosis and prognosis. It has been reported that Ki-67 has a prognostic character for many forms of malignant tumors, such as lymphomas, breast, prostate and colorectal cancers (Tretiakova et al., 2016). Ki-67 is a protein formed during active phases of the cell mitotic cycle, but is not present in resting cells. Therefore, its expression is used as an assessment tool for tissue proliferation (Faur et al., 2015).\n\nThe division activity measured by Ki-67 has been reported in previous studies, and has a great prognostic significance in different forms of malignancies (Yerushalmi et al., 2010). Ki-67 is a protein linked with cell production and is noticeable in all active phases of the cell cycle cresting at G2 and persisting at low levels after cell cycle exit. It then becomes undetectable in senescent cells (Sobecki et al., 2017).\n\nEpidermal growth factor (EGF) could motivate the production and differentiation of epidermal cells and assist skin renewal and wound healing. The therapeutic effect of EGF in the treatment of thermal injuries is not only confined to rapid activation of the healing process and a decrease in tissue damage, but also decreases the size of the affected area and reduces hyperergic inflammation. EGF has demonstrated its efficacy in thermic injury by stimulating wound healing and decreasing the possibility of purulent septic complication and tissue damage. This process might also involve modulation of the immune system status (Osikov et al., 2014; Parment et al., 2007).\n\nDoxorubicin (DXR) is an important drug for leukemia, Hodgkin's lymphoma and bladder, breast, stomach, lung and ovaries cancer, treatment during chemotherapy (Bielack et al., 1996). Jensen et al. (2002) studied the consequence of chemotherapy on the salivary gland with different solid and hematological tumors. Apart from xerostomia, 50% of the salivary glands of the patients showed ductal dilatation, cyst formation, degenerated acini and inflammatory cell presence. These degenerated salivary glands were markedly detected less than 2 weeks after chemotherapy. DXR motivates reactive oxygen species (ROS) synthesis and depolarizes the membrane potential of the mitochondria. Both excessive ROS synthesis and mitochondrial membrane damage are very important causes of cellular injury (Ghosh et al., 2011).\n\nThis study was conducted to evaluate the role of exogenous EGF injection on the Ki-67 immuno-expression in submandibular salivary gland tissue of rats receiving DXR. Rats were used in this study because they have biology similar to humans and therefore can be a model for human carcinogenicity and recovery.\n\n\nMethods\n\nThis study protocol and the animal care and experimental procedures were approved by the Ethics Committee, Faculty of Dentistry, Cairo University, Egypt (#415). All efforts were made to ameliorate any suffering of the animals by adopting the OECD 423 test guidelines, and all applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\n\nIn total, 21 male adult albino rats, two months old, pathogen free, with an average weight of 200 gm were used. The animals were obtained from and housed in the Animal house, Faculty of Medicine, Cairo University. Sample size calculation was performed using GPower version 3.1.9.2 (University Kiel, Germany) (Faul et al., 2009). The effect size was 0.95 using α level of 0.05 and β level of 0.05, i.e., power = 95%; the estimated sample size (n) was a total of 21 samples for three groups.\n\nThe animals were housed in a controlled environment (temperature 25±2°C, humidity 70–80% and 12hr dark/light cycle) and had free access to food and water. The animals were fed a natural diet and water ad libitum throughout the whole experiment. The rats were acclimatized to their cages for 1 week.\n\nAll 21 rats were given a number (1–21) using a marker pen, then randomized by putting the numbers in an envelope and dividing them into three groups according to the numbers which were taken from the envelope.\n\nThe three groups were as follows:\n\ncontrol group, the rats were kept on a normal diet and did not receive DXR or EGF;\n\nDXR group, the rats received 20 mg/kg body weight DXR as a single intra-peritoneal injection (Ayla et al., 2011);\n\nDXR+EGF group, the rats received the same intraperitoneal dose of DXR (20 mg/kg body weight) and on the next day they were injected intraperitoneally with 10µg/kg body weight of EGF daily for one week (Ohlsson et al., 1997).\n\nThe rats were injected every morning at 9 am in the animal house laboratory of Faculty of Medicine, Cairo university.\n\nThis study was performed to detect changes in the salivary glands after doxorubicin injection and the role of EGF, if any, in reversing any negative changes appearing in the glands. Therefore, histological sections as well as Ki67 immuno expression were used to detect these changes.\n\nThe rats were sacrificed by euthanization by CO2 asphyxiation followed by cervical dislocation when the experiment finished after 1 week.\n\nSubmandibular salivary glands of both sides were dissected out and preparation of specimens for staining procedure was done as follows.\n\nAfter the glands were excised, those of the right side were fixed immediately in 10% neutral buffered formalin. Then, the specimens were washed properly under running water, dehydrated through ascending concentrations of alcohol and transferred to xylene to clear the specimens from alcohol. Then, the glands were embedded in paraffin wax and mounted in the center of the paraffin wax blocks. Sections from paraffin embedded tissues blocks were cut into sections 5-µm thick and mounted on glass slides for histological examination using Samples were processed for regular histopathological examination using H&E stain. Other sections were stained with immuno-peroxidase for immunohistochemical detection of Ki-67 in the glandular tissue using staining reaction containing anti-Ki-67 antibodies (Santa Cruz Biotechnology catalogue # sc-23900).\n\nThe selected sections were studied by ZEISS Primo Star light microscopy and images taken using Tucsen IS 1000 10.0MP Camera in the Oral Biology Lab, Faculty of Dentistry, Cairo University.\n\nThe scoring of the staining reaction of the immunohistochemical parameter (presence of Ki-67) of the different groups was as follows: (-) negative reactivity and (+) positive reactivity.\n\nImage analysis was performed using a computer system software Leica Quin 500 (LEICA Imaging Systems Ltd, Cambridge, England) to assess the area percentage of the immunostaining within the tissues.\n\nThe image analyzer was first calibrated automatically to convert the measurement units (pixels) produced by the image analyzer program into actual micrometer units. Measurement of the area of percentage positive cells was done as previously described (Shi et al., 2007). Briefly, the percentage of positive cells was recorded as an area and area percentage within a standard measuring frame of area 114,342.9 mm2 per 10 fields from different slides. This was done at a magnification of 400X by the light microscope transmitted to the monitor. Areas containing the most uniformly stained tissues were chosen for evaluation. These areas were disguised by blue binary color which could be measured by the computer system. Images were manually corrected for brightness and contrast. Colour thresholding was then performed automatically after which pictures were converted to RGB stack type. Masking of the brown cytokeratin, immuno-stain was performed by blue colour where any brown stain of any intensity was considered positive whereas the background grey stain was considered negative. Area fraction was then calculated automatically representing the area percentage of immune positive cells to the total area of the microscopic field.\n\nImage analysis data representing experimental values of Ki-67 immunostain were given as mean and standard deviation. ANOVA (ONEWAY ANOVA test, n=10, P <0.05) was used to compare the mean area percent of Ki-67 immuno-expression among the specimens of different groups. Tukey’s Multiple Comparison Test (Post Hoc Tukey HSD) was performed to calculate a pair-wise comparison between each group. SPSS 25.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for analysis.\n\n\nResults\n\nHistological examination of the submandibular salivary gland of rats of control group (group I) revealed its main structural components was composed of parenchymal tissue supported by connective tissue stroma (Figure 1A). Histopathological sections of DXR group (group II) showed several pathological changes. The secretory acini appeared with massive cytoplasmic vacuolization, and deformation in the acini and loss of normal cellular orientation were frequently encountered. A clear space separated the parenchymal elements, which might be an index of interstitial edema and extravasated red blood cells (RBCs) in between acini and ducts (Figure 1B).\n\n(A) Control group, regular gland architecture; (B) DXR group, degenerated acini with multiple vacuoles extravasated red blood cells (RBCs) in between acini and ducts; (C) DXR+EGF group, well aligned acini together with blood vessels engorged with RBCs in association with the striated ducts exhibiting good cell alignment and minute areas of small vesicles. H&E staining, orig. mag. X400.\n\nComparing with the histopathological results of DXR group, the DXR+EGF group (group III) sections showed great enhancement in the structural features of the glands. Little evidence of inflammatory condition was present. On the other hand, many blood vessels engorged with RBCs were found in close relation with the striated ducts. Moreover, a rich vascular network was found in association to the excretory ducts (Figure 1C).\n\nThe control group sections showed positive cytoplasmic immunoreactivity for Ki-67 protein in the parenchymal tissue of the glands, which appeared more distinctive in the duct system. Scattered nuclear reactivities were identified for the protein antigen. A few localized focal areas in the secretory acini as well as the endothelial cells of blood vessels expressed the proliferation antigen at higher intensity (Figure 2A). On the other hand, the immunohistochemical findings of the DXR group corroborated with the histological results; Ki-67 protein was localized in the DXR group sections differently to the control group. DXR was found to increase the immuno-expression of Ki-67 protein in the submandibular salivary glands of rats particularly the secretory terminal portions. Wider areas of the acini expressed the antigen and with greater intensity. The expression of the proliferation antigen appeared foamy, probably due to the vacuolar degeneration affecting the glandular tissue. Scattered nuclear and perinuclear reactivities were identified for the protein antigen in this group (Figure 2B).\n\n(A) Control group, positive reaction to Ki-67 protein in the secretory acini and the wall of the blood vessels; (B) DXR group, increased reactivity of Ki-67 in the secretory acini together with the nuclear and perinuclear reactivities; (C) DXR+EGF group, decreased Ki-67 immunoreactivity in the acini. DAB, orig. mag. X400.\n\nThe differences between the control and DXRs groups were statistically significant, as there was a significant increase in the mean area percent of Ki-67 immuno-expression found in DXR group in contrast with the control group (p<0.01; Figure 3).\n\nThe tissue sections of the DXR+EGF group revealed a remarkable decrease in Ki-67 antigen expression in the acinar cells in comparison to the DXR group. Conversely a slight increase in immunoreactivity to the proliferation antigen appeared in the ductal profiles. Nevertheless, the immunoreactivity seemed to be very similar to that of the control group (Figure 2C).\n\nPost Hoc Tukey HSD test comparing between the groups showed that the difference between the control group and DXR+EGF group was not significant (p>0.05). However, a significant increase was recorded between the DXR and control groups, and a significant decrease was recorded between DXR+EGF and DXR groups (p<0.01; Table 1 and Table 2).\n\n\nDiscussion\n\nIn this study, the systemic injection of DXR in rats resulted in pathological structural alterations within the submandibular salivary gland tissue. Atrophied and degenerated acini with multiple cytoplasmic vacuolization were detected. The chemical composition of DXR leads to creation of free radicals and the stimulation of oxidative stress, which is considered the main factor of cellular damage (Saad et al., 2001). DXR also initiates an inequity between free oxygen radicals and antioxidants. This disturbs the oxidant–antioxidant system, leading to tissue damage that is manifested by lipid peroxidation and protein oxidation within the tissue (Karaman et al., 2006).\n\nMitochondrial degeneration possibly is the primary cause of the intracytoplasmic vacuolizations shown repeatedly in parenchymal cells of both acini and ducts. Disturbance in cellular metabolism and sodium ions entering the cell have also been reported to cause this damage. This osmotic effect initiates degradation of large macromolecules within the injured cell and leads to the presence of cytoplasmic vacuoles. Furthermore, other cytoplasmic vacuolations might be due to degeneration of other cell organelles such as Golgi apparatus which appear as empty spaces (Ankily et al., 2020).\n\nIn our study, the submandibular salivary glands of rats injected with EGF after DXR presented great improvement in the gland architecture. Resolution of vacuolar degeneration was noted apart from minute areas of microvesicles detected in the parenchymal tissue. The acini and ducts cells were more alike to those of the control group. The anti-inflammatory response of EGF was evident in this current work and has been confirmed in previously conducted studies. Berlanga et al. (2002) recorded a protective effect of EGF on the intestine from ischemia/reperfusion injury. They noticed the marked reduction of inflammatory infiltrates (mainly neutrophils) in the intestinal tissue after EGF injection. They also registered decreased level of TNF-α (a major pro-inflammatory cytokine), which may contribute to the cytoprotective effect.\n\nThe healing potential of EGF was documented in several studies. It was prevalent in epithelial cell re-epithelialization, proliferation, migration and renewal of gastric glands during renal epithelium regeneration (Flaquer et al., 2010), gastric ulcer healing (Tarnawski & Ahluwalia, 2012), corneal epithelium (Jeon et al., 2018) and salivary glands (Al-Ankily et al., 2018; Shamel et al., 2017).\n\nEGF binding to EGF receptor results in auto phosphorylation of receptor tyrosine kinase and activation of signal transduction pathways that are included in the modulation of cellular division, differentiation and persistence. EGF assists epithelial cell regeneration and plays a vital role in dermal wound healing through motivation of proliferation and migration of keratinocytes (Zeng & Harris, 2014).\n\nKi-67 is localized during active phases of the cell cycle and its expression is used as a sign of proliferation rate (Faur et al., 2015). It is found in all multiplying cells (normal and up-normal cells) and has been shown to be an admirable proliferation marker to detect the growth rate of certain cell populations. It is also used as the proliferation indicator to evaluate several categories of tumors (Brown & Gatter, 2002; Do Prado et al., 2011; Tadbir et al., 2012).\n\nDayan et al. (2002) examined time related alterations in the proliferative capacity of parenchymal cells of human labial salivary gland using Ki-67 as a proliferative marker. They reported positive immuno-expression of Ki-67 within the acini and ducts of the glands in all groups. These findings were in agreement with our results as mild cytoplasmic immunoreactivity for Ki-67 protein was registered in the submandibular salivary gland tissue of the control group. Furthermore, it has been considered that Ki-67 protein localization are not binary as it is continuously decreased during G0 and G1 and is continuously increased from the start of S phase till mitotic exit (Miller et al., 2018).\n\nNonetheless, Birajdar et al. (2014) found Ki‑67 expression in all cases of normal oral epithelium to be mainly presented in the parabasal layer where the numbers of proliferating cells were limited in comparison with the basal cell layer. Furthermore, Hagiwara et al. (2013) detected Ki-67 mainly in the parabasal cells and infrequently in the basal cells in the normal squamous epithelium.\n\nKi-67 proliferation index was found prominently reduced following chemotherapy treatment, showing the anti-proliferative effect on tumors (Miller et al., 2003 and Miller et al., 2006; Lee et al., 2008). An unexpected finding in the current study was the significant increase in the expression of Ki-67 in the submandibular salivary glands of rats, particularly the secretory terminal portions, secondary to DXR in comparison with control group (p<0.01).\n\nSasaki et al. (1987) studied Ki-67 in HeLa S3 cells (human cell line derived from cervical cancer cells), and showed an increase in Ki-67 antigen after treatment with DXR, as well as its continuous expression throughout the cell cycle. They hypothesized that this is due to the maintaining response of Ki-67 antigen in the cell cycle; interference in DNA replication might cause a reactive enhancement of the Ki-67 protein.\n\nKausch et al. (2003) found that the expression of Ki-67 is increased at G2/M, which is exactly the period during which DXR induces apoptosis. They suggested that DXR could have an inhibitory effect on Ki-67 protein production, which may induce apoptosis. However, cancer cells, in an attempt to survive this effect, increase their mRNA to produce more protein. Ultimately, the production of protein by the cell and induction of apoptosis by DXR reaches an equilibrium, the result of which has been a lack of change in the protein after DXR treatment.\n\nThese results were also constant with findings in human hepatocellular carcinoma in which DXR treatment caused the acceleration of cell cycle transition; at an early time point allowing cell cycle continuance, but finally leading to cell cycle arrest (Choi et al., 2012).\n\nAccording to Etemad-Moghadam et al. (2013) a significant increase occurred in Ki-67 mRNA following incubation of cancer cells with DXR, but there was no change in the expression of its protein. However, they failed to explain the exact function and role of Ki-67 in proliferation and cell cycle.\n\nChemotherapy targets rapidly proliferating cells that are closest to blood vessels but poorly penetrated tumor cells located distal to functional blood vessels (hypoxic regions). Hypoxic cells do not respond to treatment because of the cytotoxic effects generated by oxygen-dependent free radicals. Surviving hypoxic cells in intervals between treatments might re-oxygenate and proliferate from enhanced supply of nutrients released from digestion of dead cells close to the blood vessels. Saggar & Tannok (2015) noted that DXR resulted in the highest increase in Ki-67 cells in reoxygenated surviving hypoxic cells.\n\nHultman et al. (2018) found that post DXR treatment the BE (2)-C (neuroblastoma cell line derived from human bone marrow) tumor growth presented a remarkable increase in Ki-67-index (from 43% to 64%; p<0.01), thus indicating a move towards cycling cells by application of DXR. Tredan et al. (2007) previously determined the same hypothesis and found in vitro that quiescent (G0) tumor cells enter cell cycle after DXR treatment.\n\nUnexpectedly, we found that Ki-67 proliferation marker expression decreased significantly in the DXR+EGF group in comparison to the DXR group (p<0.001). Weak to mild cytoplasmic immunoreactivity for Ki-67 protein was shown in the parenchymal tissue of the glands of the EGF supplemented group in a manner matching its expression in the control group. Although the expression was a bit stronger than the control, the statistical correlation was not significant (P>0.05). The immunohistochemical results might be correlated with the ultrastructure findings, as binucleation was frequently encountered in DXR group displaying high expression of the proliferation marker, while being unidentified with EGF treatment of low antigen expression (Mansy et al., 2020).\n\nComparable findings were reported by Fatimah et al. (2012), as they found EGF significantly decreased the pluripotent genes expression of cultured human amnion epithelial cells. It is likely that the mitogenic EGF did not favor abnormal proliferation, as had been unexpectedly detected in the current investigation secondary to DXR. In a previous study, a disruption in normal expression of EGF was found correlated with improved proliferation and differentiation of medial cells in developing palate and resulted in cleft palate in rat embryo (Abbott & Bimbaum, 1990).\n\n\nConclusions\n\nWe conclude that EGF has a cytoprotective and reparative effect against DXR induced changes on salivary gland tissue in rats. DXR injection significantly increased Ki-67 immunoexpression in the glandular tissue. However, exogenous EGF preserved the immunohistochemical expression of Ki-67 in the glands or restored it to approximately to the normal level.\n\n\nData availability\n\nFigshare: The Role of Exogenous EGF on Ki-67 proliferation marker expression on Submandibular Salivary Gland of Albino Rats Receiving Doxorubicin, https://doi.org/10.6084/m9.figshare.13042625.v4 (Shamel, 2020).\n\nThis project contains the following underlying data:\n\n- Original, unedited light microscopy images in TIFF format\n\n- Original, unedited Ki67 immuno-expression images in TIFF format\n\n- Area percentage of Ki-67 immuno-expression for all 21 rats in excel sheet\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbbott BD, Bimbaum LS: TCDD-induced altered expression of growth factors may have a role in producing cleft palate and enhancing the incidence of clefts after coadministration of retinoic acid and TCDD. 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Reference Source\n\nMiller I, Min M, Yang C, et al.: Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence. Cell Press. 2018; 24(5): 1105–1112.e5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiller WR, Dixon JM, Macfarlane L, et al.: Pathological features of breast cancer response following neoadjuvant treatment with either letrozole or tamoxifen. Eur J Cancer. 2003; 39(4): 462–468. PubMed Abstract \| Publisher Full Text\n\nMiller WR, White S, Dixon JM, et al.: Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole. Br J Cancer. 2006; 94(7): 1051–1056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOhlsson B, Jansen C, Ihse I, et al.: Epidermal Growth factor Induces Cell Proliferation in Mouse Pancreas and Salivary glands. Pancreas. Department of Surgery, University Hospital. Lund. Sweden. 1997; 14(1): 94–98. PubMed Abstract \| Publisher Full Text\n\nOsikov MV, Telesheva LF, Likhacheva AG: Effect of Local Application of Epidermal Growth Factor on Innate Immunity and Cell Composition of Destruction Focus in Experimental Thermal Injury. Bull Exp Biol Med. 2014; 157(3): 307–10. PubMed Abstract \| Publisher Full Text\n\nParment K, Zetterberg A, Ernerudh J, et al.: Long-term immunosuppression in burned patients assessed by in vitro neutrophil oxidative burst (PhagoburstW). Burns. 2007; 33(7): 865–871. PubMed Abstract \| Publisher Full Text\n\nPlaat B, Kole A, Mastik M, et al.: Protein synthesis rate measured with L-[1– 11C] tyrosine positron emission tomography correlates with mitotic activity and MIB-1 antibody-detected proliferation in human soft tissue sarcomas. Eur J Nucl Med. 1999; 26(4): 328–332. PubMed Abstract \| Publisher Full Text\n\nSaad SY, Najjar TA, Al-Rikabi AC: The preventive role of deferoxamine against acute doxorubicin-induced cardiac, renal and hepatic toxicity in rats. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamel M: The Role of Exogenous EGF on Ki-67 proliferation marker expression on Submandibular Salivary Gland of Albino Rats Receiving Doxorubicin. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.13042625.v4\n\nShamel M, Ankily M, Bakr M: Epidermal growth factor restores normal levels of myosin expression in submandibular salivary glands of rats treated with botulinum toxin. Journal of Advanced Medical and Dental Sciences Research. 2017; 5(1): 5–9. Reference Source\n\nShi SR, Liu C, Young L: Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods. Biotech Histochem. 2007; 82(6): 301–309. PubMed Abstract \| Publisher Full Text\n\nSobecki M, Mrouj K, Colinge J, et al.: Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels. Cancer Res. 2017; 77(10): 2722–34. PubMed Abstract \| Publisher Full Text\n\nTadbir AA, Pardis S, Ashkavandi ZJ, et al.: Expression of Ki67 and CD105 as proliferation and angiogenesis markers in salivary gland tumors. Asian Pac J Cancer Prev. 2012; 13(10): 5155– 5159. PubMed Abstract \| Publisher Full Text\n\nTarnawski AS, Ahluwalia A: Molecular mechanisms of epithelial regeneration and neovascularization during healing of gastric and esophageal ulcers. Curr Med Chem. 2012; 19(1): 16–27. PubMed Abstract \| Publisher Full Text\n\nTredan O, Galmarini CM, Patel K, et al.: Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst. 2007; 99(19): 1441–54. PubMed Abstract \| Publisher Full Text\n\nTretiakova MS, Wei W, Boyer HD, et al.: Prognostic value of Ki67 in localized prostate carcinoma: a multi-institutional study of >1000 prostatectomies. Prostate Cancer Prostatic Dis. 2016; 19(3): 264–270. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYerushalmi R, Woods R, Ravdin PM, et al.: Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 2010; 11(2): 174–183. PubMed Abstract \| Publisher Full Text\n\nZeng F, Harris RC: Epidermal growth factor, from gene organization to bedside. Semin Cell Dev Biol. 2014; 28: 2–11. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "75760", "date": "05 Jan 2021", "name": "Mushfiq Hassan Shaikh", "expertise": [ "Reviewer Expertise Head and Neck Cancer", "Molecular Biology", "Immunotherapy", "Genome editing", "Cancer genomics", "Translational research." ], "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\nIs the study design appropriate and is the work technically sound? Answer: The study design is okay and the work is technically sound. However, reviewer has a concern about the experiment length. The authors have performed the drug experiment on rats for 7 days. Can the authors provide reference of such study length is used in any other previous studies? If so, please add those references in the manuscript.\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Answer: The statistical analysis and its interpretation was appropriate, however, the reviewer feels that authors should re-plot figure 3 using statistical software.\n\nAre the conclusions drawn adequately supported by the results? Answer: The conclusion drawn adequately, however need some addition and modification. the reviewer has discussed this in the report.\n\nReviewer’s Report: This is an interesting study. Authors in this manuscript presented their study about the role of exogenous epidermal growth factor (EGF) on Ki-67 proliferation marker expression in the submandibular salivary gland on albino rats receiving doxorubicin. To understand the role of EGF in submandibular salivary gland, authors have selected albino rats for this study. The expression pattern of Ki-67 proliferation marker was tested in albino rats treated with doxorubicin, using immunohistochemistry (IHC) technique. The manuscript is structurally organized, however, the writing appears to lack clarity in several places. In the manuscript, especially in the ‘Discussion’ section, the authors have discussed, and referred to different interesting topics, however, in several places failed to correlate those information with the current study. This makes those information irrelevant to mention unless authors make some effort to show the relevance. Therefore, the reviewer feels that rewriting and reorganising the ‘discussion’ section in succinct manner would be needed to enhance the quality of the manuscript. The reviewer has provided some suggestions below for the modification of the ‘discussion’ section. Similar suggestion would apply for the ‘Methods’ section, as the section is unnecessarily too long and in some cases lack proper scientific language use. The authors should consider rewriting the ‘methods’ section in succinct form as well. As the authors have demonstrated changes in the rat tissue after the injection of EGF, which is encouraging, but what does it means, that is, what is the significance of doing this study? The authors should consider adding few sentences about the significance of this study in the manuscript. The authors have performed this study on rat’s epithelium, but drawn comparisons with human epithelial cells, which are considered two different species. Have the authors performed investigation and observed similar findings in human tissue? If not, then the authors should carefully conclude the study with their findings and make relevance with the human. Furthermore, the authors have performed the drug experiment on rats for 7 days, the reviewer is concerned about the length of the experiment. Can the authors provide reference of such study length is used in any other previous studies? If so, please add those references in the manuscript.\n\nAlthough this manuscript might have met the criteria for publication on this journal should the following several concerns be addressed properly.\nThe title of the manuscript looks too long, authors should consider meaningful shortening of title.\n\nThe background of the abstract needs more clarity. Rather than just copying the title here, authors should add two sentences on the background and significance of the study.\n\nUnder the ‘methods’ section of the abstract, what did the authors mean by ‘immunohistochemical expression of Ki67 sections’? What is this ‘Ki67 section’? Please clarify.\n\nUnder the ‘results’ section of the abstract, ‘However, Ki-67 expression in the glandular tissue was restored to normal levels’. What did the authors mean by ‘normal levels’? Similar term ‘normal level’ was used in Conclusion. What is the normal level of Ki67 expression? Please, clarify.\n\nIn the first paragraph of the ‘Introduction’ section, authors mentioned, Ki67 is ‘used for investigations of many cancer types’, can the authors clarify for what sort of investigations? Furthermore, ‘Ki-67 has similar essential properties with the other proteins known to regulate cell cycle’, can the authors explain, what are those essential properties?\n\nIn the third paragraph of the ‘Introduction’ section, ‘The division activity measured by Ki-67…..’, the authors should explain, what this ‘division activity’ meant here?\n\nIn the fifth paragraph of the ‘Introduction’ section, the sentence, ‘Apart from xerostomia, 50% of the salivary glands of the patients showed ductal dilation, cyst formation, degenerated acini and inflammatory cell presence.’ Did authors mean those conditions were seen in normal healthy patient or in cancerous patients ? It is confusing here, please clarify.\n\nIn the sixth paragraph of the ‘Introduction’ section, ‘….. the role of exogenous EGF injection on the Ki-67 immuno-expression in submandibular salivary gland….’ , authors should consider rewriting this sentence, as this sounds confusing, especially, it sounds like role of EGF injection on Ki-67 expression.\n\nIn the first paragraph of the ‘Methods’ section, authors mentioned that they have followed institutional guidelines for animal study. Can the authors mention in detail what guideline their institution follows, is it Declaration of Helsinki guidelines ? If so, please mention that.\n\nIn the fourth paragraph of the ‘methods’ section, did the authors kept all 21 rats in one case of separate cages ? Please, clarify.\n\nThe authors should consider rewriting the ‘methods’ section in succinct but mention all necessary details, as this section is unnecessarily too long and lacks proper scientific language use.\n\nMany places across the manuscript, authors used the term ‘Ki67-immuno expression’. Please use, just Ki-67 expression, which will be sufficient and sounds better. Furthermore, authors should use a consistent term throughout the manuscript not interchange between ‘Ki-67 immuno expressions’ and ‘Ki-67 immuno reactivity’, as it becomes confusing. For example, in the fourth paragraph of the ‘results section’, authors mentioned Ki-67 immuno-expression, but in Figure 3, it was Ki-67 immuno-reactivity.\n\nIn the 2nd paragraph of the ‘Results’ section, ‘DXR+EGF’ group (group III) sections showed great enhancement in the structural features of the gland’, what did the authors mean by ‘enhancement in the structural features of the gland’ here ? Were there increment in the number of structures or enlargement of structures ? Please, clarify.\n\nIn the 4th paragraph of the ‘Results’ section, ‘DXR was found to increase the immune-expression of Ki-67 protein in the submandibular salivary gland……..’ What did the author mean in this sentence, is not clear? Did the authors mean DXR increased the expression of Ki-67 ? If so, how or by which mechanism?\n\nThe authors should consider to remake figure 3, as this, at current state, does not provide much of information and rather confusing. Is the control here indicate 100% ? If so, what is Area % less than 8 ? Y axis numbers or percentages are confusing. The reviewer suggest the authors could use statistical programs such as, Graph Pad Prism, SPSS or R programming to plot this figure properly. Typically, Microsoft Excel is not considered as an ideal stat platform for publication. Furthermore, please add figure legend after title of the figure for Figure 3.\n\nIn table 1, authors mentioned ‘Area percentage’ but inside the table no percentage sign. Please, add percentage sign where appropriate.\n\nIn the 5th paragraph of the ‘Results’ section, it reads, ‘increase in immune-reactivity to the proliferation antigen….’. Can authors clarify which ‘Proliferation antigen’ they are referring here ?\n\nMany places across the manuscript, authors used ‘not significant (p>0.05)’. Use of this form is a bit confusing while reading, therefore, reviewer suggests the author should only use p-value across the text if anything is significant, such as, (p<0.05). Alternatively, mention the exact p-value, in case of non-significant cases.\n\nUnder the ‘Discussion’ section, 4th, 5th and 6th paragraphs do not fit well in discussion section, authors may consider moving them to introduction section but in more briefly manner.\n\nThe paragraph 8 of the discussion section, authors referred about the Ki-67 expression in normal oral epithelium. How the authors justify the relevance with the current study providing the above information in this paragraph?\n\nThe paragraph 9 and 10 in the discussion section should be merged as one paragraph.\n\nIn paragraph 12 of the discussion section, authors starts the paragraph with ‘These results were also consistent….’. What did the authors mean by ‘These results’ here? Which results authors referring to? Please, clarify as the whole sentence is unclear.\n\nIn paragraph 13 of the discussion section, authors talked about Ki-67 miRNA expression in another study. How the authors justify the relevance with the current study providing the above information in this paragraph?\n\nIn paragraph 14 of the discussion section, ‘Chemotherapy targets rapidly……functional blood vessel’ , this sentence needs reference. Please, add reference.\n\nIn paragraph 17 of the discussion section, authors highlights the EGF effects in pluripotent gene expression. Please, justify the relevance of the above mentioned topic with the current study.\n\nIn Conclusion, please write in few sentences about the justification and significance of this current study.\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? Partly\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?\nPartly\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? Partly", "responses": [] }, { "id": "76739", "date": "07 Jan 2021", "name": "Marwa Abdul-Salam Hamied", "expertise": [ "Reviewer Expertise Molecular basis of Head and Neck cancers" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Mansy et al. is clearly and accurately presented. They analyzed whether epidermal growth factor effect on ki-67 proliferation marker or not in the submandibular gland parenchyma of albino rats receiving DXR. The submandibular salivary glands of rats injected with EGF after DXR showed great improvement in the glandular tissue. It could be considered a novel treatment for human carcinogenicity and recovery. The title and abstract are appropriate for the content of the text. The figures clearly showed histopathological features and ki-67 expression. Furthermore, the article is well constructed, the experiments were well conducted, and the analysis was well performed.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "76741", "date": "08 Jan 2021", "name": "Mahmoud Serag", "expertise": [ "Reviewer Expertise Dentistry", "Oral Health", "Prosthodontics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research by Mansy et al. is an interesting study which examined the effect of Epidermal growth factor on salivary gland of albino rats receiving doxorubicin detecting ki67 expression as a proliferation marker. The title of the study is suffiecient and reflects the purpose of the study. The methods sections was more than sufficient and I think there is more than enough details that it can be shortened and only referencing is enough. Histological and immunohistochemical results figures were clear. The manuscript is structurally organized and well written but needed minor modifications in some areas as in the discussion which needed some correlation between other studies and the current study. Conclusions were drawn adequately and the overall manuscript is well constructed and meets the criteria for indexing.\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/9-1393
https://f1000research.com/articles/9-1390/v1	03 Dec 20	{ "type": "Research Article", "title": "Benchmark assessment of molecular geometries and energies from small molecule force fields", "authors": [ "Victoria T. Lim", "David F. Hahn", "Gary Tresadern", "Christopher I. Bayly", "David L. Mobley", "Victoria T. Lim", "David F. Hahn", "Gary Tresadern", "Christopher I. Bayly" ], "abstract": "Background: Force fields are used in a wide variety of contexts for classical molecular simulation, including studies on protein-ligand binding, membrane permeation, and thermophysical property prediction. The quality of these studies relies on the quality of the force fields used to represent the systems. Methods: Focusing on small molecules of fewer than 50 heavy atoms, our aim in this work is to compare nine force fields: GAFF, GAFF2, MMFF94, MMFF94S, OPLS3e, SMIRNOFF99Frosst, and the Open Force Field Parsley, versions 1.0, 1.1, and 1.2. On a dataset comprising 22,675 molecular structures of 3,271 molecules, we analyzed force field-optimized geometries and conformer energies compared to reference quantum mechanical (QM) data. Results: We show that while OPLS3e performs best, the latest Open Force Field Parsley release is approaching a comparable level of accuracy in reproducing QM geometries and energetics for this set of molecules. Meanwhile, the performance of established force fields such as MMFF94S and GAFF2 is generally somewhat worse. We also find that the series of recent Open Force Field versions provide significant increases in accuracy. Conclusions: This study provides an extensive test of the performance of different molecular mechanics force fields on a diverse molecule set, and highlights two (OPLS3e and OpenFF 1.2) that perform better than the others tested on the present comparison. Our molecule set and results are available for other researchers to use in testing.", "keywords": [ "force field", "molecular modeling", "OpenFF", "OPLS", "molecular mechanics", "molecular dynamics", "quantum mechanics" ], "content": "Introduction\n\nThe study of chemical and biological systems relies on an accurate assessment of the energetics and geometries of the systems. Many computational methods serve to help investigate these systems, ranging from more accurate, higher cost quantum mechanical techniques to more approximate methods which compromise accuracy in favor of increased eﬃciency. Classical mechanics-based calculations fall into the latter group, and have an advantage over more theoretically rigorous calculations of being able to model larger systems over longer time timescales1–4.\n\nThe modeling and simulation of molecular systems in classical mechanical calculations typically requires a force field, a set of energy functions and associated parameters comprising the potential energy function. This potential energy function defines interactions between components in the system based on the coordinates of its particles5,6.\n\nForce fields have a long history of development. Strategies for force field development vary in terms of the chemical space covered, the types of data used for training, and the approach to optimize parameters given a set of input data7–10. The training data used to develop a force field usually includes input data from both experimental and reference quantum mechanical (QM) calculations. This finite amount of input data is carefully chosen to be representative of the systems for which the force field is designed. The limit of accuracy for some force field is measured by its ability to reproduce experimental observables, such as hydration free energies. When experimental evidence is unavailable, the force field can be assessed with respect to quantum mechanical data, for instance its ability to reproduce QM geometries and relative energies. Given the complexity of force field development, including multidimensional input data, various functional forms, and approaches to chemical perception11, force fields vary in how accurately they can compute properties of interest. Indeed, many examples serve to highlight the limitations of force fields12–16.\n\nOur focus in this work is on force fields for small molecules, which are instrumental in drug discovery; for instance, evaluating binding free energies and modeling ligand binding poses. Relatively few literature studies evaluate force field accuracy on general small drug-like molecules, in contrast to force fields for proteins17–25, nucleic acids26–28, carbohydrates29–32, and other specific chemical systems33–45. On small molecules, these studies comprise predictions of solvation free energies46,47, strain energies48 experimental osmotic coeﬃcients49, partition coeﬃcients50,51, conformer energies52–56, conformer geometries55,57, and robustness of parameterization57. Most of these studies assess four or fewer force fields on molecule sets up to several hundreds of molecules. We present a broader assessment of general small molecule force fields on a large, diverse library of drug-like compounds and evaluate how accurately these force fields perform. We use QM data as a valuable source of information for force field assessment and to explore chemical space relatively quickly and easily.\n\nIn this work, we benchmarked small molecule force fields with respect to quantum mechanical results. We assessed nine force fields belonging to four families: the General Amber Force Field, first and second generations (GAFF58 and GAFF259); the Merck Molecular Force Field, initial and “static” versions (MMFF9460–64 and MMFF4S56,65); the third extended version of the Optimized Potentials for Liquid Simulations Force Field (OPLS3e66); and the SMIRKS-based force fields from the Open Force Field Initiative (SMIRNOFF99Frosst67 and its successor OpenFF “Parsley”68, versions 1.0, 1.1, and 1.2). For a dataset of 22,675 molecular structures of 3,271 small molecules, we conducted molecular mechanics (MM) energy minimizations using force fields and evaluated optimized geometries and energies, compared with reference to quantum mechanical data. We also identified particular chemical groups that represent systematic outliers in the force field-optimized geometries and energies. This work provides a general understanding of the strengths of different small molecule force fields and identifies areas of improvement for future force field development.\n\n\nMethods\n\nWe obtained the molecule set in this work from QCArchive69 from the dataset labeled OpenFF Full Optimization Benchmark 1 (accessed November 11, 2019), which was created for the purpose of benchmarking OpenFF-1.070,71. An initial preprint of this work was posted after benchmarking OpenFF-1.0, but subsequently we were able to include OPLS3e results and added benchmarking of OpenFF-1.1 and 1.2. It is important to note, then, that this dataset was not curated to present any force field in a particular light; it was selected for benchmarking OpenFF-1.0 and has been retained as-is for the present comparison. However, OpenFF-1.2 marked a substantial refit and used an expanded training set of molecules, selection of which was at least partially informed by benchmarking of OpenFF-1.072. These training set changes meant we had to remove some structures from our benchmark set to ensure there was no overlap between training and test sets. Particularly, we removed 2398 structures from 419 molecules which were used for training the more recent OpenFF-1.2.\n\nOverall, the benchmark set was chosen to include a broad range of drug-like compounds71,73. This QCArchive dataset contains QM geometry-optimized structures and energies at the B3LYP-D3BJ/DZVP level of theory74–78. This method and basis set were chosen by the Open Force Field initiative to provide reasonably accurate conformational energies and geometries at moderate computational cost52,53.\n\nIn our dataset, we organized molecular structures such that conformers of the same molecule were grouped together if they have the same absolute (non-isomeric) graph. Importantly, we do not use the SMILES string listed in the QCArchive DataFrame to represent the molecule itself, because the identity of the molecule may change during QM geometry optimization due to changes in bonding/tautomerization, such as shown in Figure 1. Molecules with different tautomerization states, which have different chemical connectivity, are treated as distinct molecules in our study. While two molecular structures may start QM optimizations from the same connectivity, we only use their final geometries to identify and distinguish molecules based on their connectivity. We grouped together all structures in the dataset whose final geometries yielded the same canonical isomeric SMILES string, as evaluated by OEMolToSmiles from the OpenEye OEChem Python toolkit79. The structures were then organized into conformer sets as perceived by OEChem’s OEAbsCanonicalConfTest. This dataset organization procedure takes into account any molecular identity changes during QM optimization, such as if two molecules no longer had the same tautomerization state after QM optimization or if two different molecules ended up in the same tautomerization state. We ensured that what we identified as a molecule, and all of its given conformers, contained the same chemical connectivity.\n\nOn the left hand side, we show the Lewis structure and three associated conformers of an example molecule from the QCArchive OpenFF Full Optimization Benchmark 1 dataset. Yellow circles highlight the regions of potential tautomerization changes. The QCArchive SMILES labels are colored in red. The right hand side shows the structures after QM optimization. The canonical isomeric SMILES labels representing the optimized molecules are colored in blue. Only the middle structure retains the original tautomeric identity. In our dataset, the geometries on the right hand side would be analyzed as distinct molecules.\n\nThe resulting QM geometries were used as input structures for gas phase energy minimizations using the following small molecule force fields: GAFF58, GAFF259, MMFF9460–63, MMFF4S56,65, OPLS3e66, SMIRNOFF99Frosst67, and Parsley68. The SMIRNOFF99Frosst version used here is SMIRNOFF99Frosst-1.1.0.offxml. SMIRNOFF99Frosst is a SMIRKS Native Open Force Field (SMIRNOFF) and descends from the AMBER parm99 force field as well as Merck-Frosst’s parm@frosst. Its successor is the OpenFF Parsley force field, for which three versions (1.0, 1.1, and 1.2) were evaluated, specifically openff_unconstrained-1.0.0-RC2.offxml (OpenFF-1.0), openff_unconstrained-1.1.1.offxml (OpenFF-1.1) and openff_unconstrained-1.2.0.offxml (OpenFF-1.2).\n\nEach structure was assigned AM1 Mulliken-type partial charges with bond-charge corrections (AM1-BCC charges)80,81 from the electrostatically least-interacting functional group technique. The partial charges were generated using the openmoltools wrapper82 to OpenEye’s oequacpac charging engine79 calling OEAM1BCCELF10Charges.\n\nTo assign force field parameters to each molecule, we used antechamber and tleap59 via openmoltools82 for the GAFF2 force fields. Parameter assignment as well as energy minimization for the MMFF94S force fields were handled using OpenEye oeszybki79. The custom OPLS3e charge and parameter assignment was performed in two steps using Schrodinger Maestro (v. 2020-183). First, ligprep84 was used to convert ligands to Maestro format with settings to avoid modifying protonation or tautomeric states. Then ffbuilder was used to check for missing parameters and launch torsional drives with constrained minimization at the B3LYP/6-31G* level followed by single-point M06-2X/cc-pVTZ(-f) calculations. New OPLS3e parameters were derived for 1096 dihedrals, at a computational cost of about 100 CPU cores for 2 weeks to run high level DFT torsion fitting. This made the OPLS3e calculations substantially more costly; this may be in part because some of the benchmark set originates from eMolecules and consists of rather diverse and in some cases unusual chemistry which is not well captured by OPLS3e without additional parameterization.\n\nWe used the Open Force Field toolkit for SMIRNOFF99Frosst and Parsley, in all cases applying pre-assigned charges as described above. For the minimizations with OPLS3e, Schrodinger’s macromodel was used with the PRCG algorithm with a gradient tolerance of 0.05 kcal/mol. All other energy minimizations were completed in OpenMM85 using the LBFGS algorithm with an energy tolerance of 5.0e-9 kJ/mol and 1500 maximum number of iterations.\n\nWe removed any molecular structure that was not successfully parameterized with all force fields. This set consisted of 721 structures that were unable to be parameterized by GAFF or GAFF2, 522 structures that raised an error during OpenMM setup through the Open Force Field toolkit, and 50 which had various OpenEye charging or stereochemical perception errors. Our pruned set going into energy minimization contained 22,675 structures from 3,271 molecules with unique chemical connectivity. Corresponding files containing QM geometries and energies, SMILES strings and depictions are deposited on GitHub, benchmarkff/molecules/set_v03_non_redundant/86. The repository also contains the structures removed due to parameterization or setup errors (in the benchmarkff/molecules/issues directory) and the structures removed due to overlap with the OpenFF-1.2 training set (in the benchmarkff/molecules/set_overlapping directory).\n\nWe compared the energy-minimized geometries and energies for each force field with respect to the QM reference data by computing the following metrics: relative energy difference (ddE), root-mean-square deviation of atomic positions (RMSD), and torsion fingerprint deviation (TFD)87–89. The relative energy difference (ddE) between the FF and QM energy for the ith conformer of a specific molecule was computed using the following expression:\n\nwhere the 0th conformer is defined as the conformer with the lowest QM energy for the given molecule.\n\nMolecules may change conformation after energy minimization, which may lead to lower agreement between FF and QM energies for minimizations beginning from a particular conformer. To address this potential issue, we performed a conformer matching process for each FF structure which considered the final optimized geometries and energy differences. We ensured that every MM conformer was within 1.0 Å RMSD of a QM reference structure. The QM reference conformer was removed from analysis if there were no FF conformers that matched it within 1.0 Å RMSD. Furthermore, if a molecule ends up with two of the “same” FF-minimized conformers compared to a QM reference structure, we only keep the FF conformer with the lowest RMSD score while any redundant conformers are removed from analysis. For this reason, the number of total molecular structures for each force field will likely differ after conformer matching as the intricate conformational energy landscapes are represented differently by various QM methods and force fields. Then, the mean signed deviation (MSD) was computed over all N conformers of each molecule with Equation 3, iterating over the relative energy dE of each conformer i. The reference conformer with dE = 0 was removed from the MSD calculation. The molecule MSDs were then represented in violin plots to compare among all force fields.\n\nTo compare FF geometries with QM geometries, we used RMSD and TFD scores. The RMSD values, calculated with OpenEye OERMSD, took into account hydrogen atoms, symmetry-related transformations, and overlaid structures to yield the lowest possible RMSD. TFDs were computed using the RDKit Python library. We evaluated each of these three metrics individually and looked for potential correlations between energies and structures in terms of agreement with reference QM data.\n\nWe identified specific OpenFF-1.2 parameters which were overrepresented in high TFD regions. First, we collected all molecules having TFD scores above a visually determined cutoff of 0.12. We considered only molecules with distinct chemical connectivity. For each molecule in the high TFD group, we took the unique set of all parameters applied to the molecule. Thus, while a parameter may be applied multiple times to a single molecule, it would count only one time for that molecule. A parameter may be included multiple times when considering the entire TFD subset if it is applied to more than one molecule in the subset. For each parameter i in the force field, we computed its representation ratio as the fraction of molecules which apply that parameter:\n\nThis ratio was calculated for the high TFD subset as well as for the full set of molecules. To identify whether a parameter would be found more likely in the high TFD subset than the full molecule set, we compared the two representation ratios between the subset and full set using the one-sample Z-test for proportions. The population proportion for some parameter was designated as its representation ratio in the full molecule set, and the sample proportion was assigned to be the parameter’s representation ratio in the high TFD subset. We took the 95% confidence intervals from this Z-test to be the error bars for the representation ratios of the high TFD subset. Parameters having 20 or fewer molecules in the high TFD subset were excluded from further analysis and plotting due to inconclusive results from small sample sizes.\n\nThe complete Python code used for the setup, FF minimizations, and analysis of this work is open sourced and available on Github at https://github.com/MobleyLab/benchmarkff90. An earlier version of this article can be found on chemRxiv (doi: https://doi.org/10. 26434/chemrxiv.12551867.v2).\n\n\nResults and Discussion\n\nHere, we present and discuss our results comparing several general small molecule force fields against reference QM data. We are interested in two major categories of comparison – energetic agreement and geometric agreement. Particularly, an ideal force field will yield the same energy minima or optimized geometries as the QM energy landscape, with no additional minima, and the relative energies of those minima will agree between QM and MM. Thus, to assess performance in these two categories, we computed relative conformer energies and compared these between MM and QM, as well as assessed geometric agreement of MM optimized geometries with those from QM. We also identified specific parameters for the improvement of future versions of the OpenFF small molecule force field.\n\nOur study relies on the assumption that force field accuracy can be evaluated using gas phase energies and geometries. One of the greater goals of force field science, such as that of the Open Force Field Initiative, is building force fields that will work well in the condensed phase (e.g., small molecules in solution or binding to biomolecules). That being said, we make our assumption based on two key observations. First, force fields—especially those in the AMBER family—are usually fitted to reproduce gas phase conformational energies and geometries58. This means that we are testing these force fields on properties they are fitted to reproduce. Second, bonded parameters are not expected to change significantly on transfer to the condensed phase. Rather, non-bonded interactions are particularly important in condensed phase simulations. Of the non-bonded interactions, electrostatics models are often polarized beyond what would be expected in the gas phase in order to reproduce condensed-phase properties, and Lennard-Jones parameters can be tuned to reproduce condensed phase properties (as has been a particular focus of the OPLS force fields91,92). Even when these are done, force fields retain bonded terms parameterized to reproduce QM geometries and energetics, further emphasizing the importance of testing in such a context. We therefore believe our assumption is reasonable and that this work warrants investigation.\n\nWe start our force field benchmark analysis by comparing FF energies to QM energies. Here, since our choice of reference energy for MM is arbitrary, we choose to compare relative conformer energies. For any given molecule, an ideal force field would have relative energies for different conformers in MM that agree with those for the same conformers in QM. For the differences in relative conformer energies that we computed—that is, the difference between the MM relative conformer energies and the QM relative conformer energies—a FF with greater agreement to QM should have more values around or at 0 kcal/mol, and a FF with lower agreement with QM would exhibit a broader distribution of values that are further away from 0 kcal/mol.\n\nThe relative conformer energies of all molecular structures in our dataset with the nine force fields were generally within ±50 kcal/mol of the energies of the most favorable QM conformers (Table 1), and 95% of the relative conformer energies were within 11 kcal/mol. However, GAFF had outlying energies that were several orders of magnitude beyond this range (row 1 of Table 1). These energies were traced back to six molecules (62 conformers thereof) shown in Figure 2. These molecules all contain a polar hydrogen atom which, after geometry optimization, overlaps with its parent atom. The spurious overlap of these hydrogen atoms, and associated energy extremes, is due to a missing van der Waals parameter in GAFF. In GAFF2 (and SMIRNOFF99Frosst and subsequent OpenFF force fields11,67,68), hydroxyl hydrogens no longer have zero Lennard-Jones parameters, which seems to eliminate the problem for these molecules. Similar collapse of hydroxyl groups in close proximity has been observed previously in force fields with zero LJ parameters for hydroxyl hydrogens11.\n\nEnergy units are in kcal/mol.\n\na With outliers removed\n\nThe right hand side depicts the QM and FF geometries for phosphoenolpyruvic acid. The GAFF structure shows a representative overlap of a polar hydrogen atom with its connected parent atom due to a missing van der Waals parameter. On the left hand side, the overlapping hydrogen for the six molecules are denoted by cyan asterisks.\n\nAfter excluding the 62 GAFF outliers, the ddE energies are histogrammed in Figure 3 and Extended data, Figure S.193. The difference between MM relative conformer energies and QM relative conformer energies exhibit very similar distributions for all force fields. All distributions appear asymmetric, having a skew towards more negative ddE values than positive ones, indicating that the conformer energy differences may be underpredicted by MM compared to QM. Force fields of the same family tend to be more consistent with each other (GAFF and GAFF2, MMFF94 and MMFF94S), see Extended data, Figure S.193. From these results, the qualitative ordering of force fields from lowest to highest agreement with QM energies goes as SMIRNOFF99Frosst < MMFF94 ∼ MMFF94S < GAFF ∼ GAFF2 ∼ OpenFF-1.0 ∼ OpenFF-1.1 < OpenFF-1.2 < OPLS3e. In other words, the peak size around ddE = 0 kcal/mol (the fraction of molecules described particularly well) is greatest for OPLS3e, closely followed by OpenFF-1.2. OPLS3e predicts 55.3 ± 0.3% of conformers within 1 kcal/mol of QM, with OpenFF-1.2, GAFF2, and MMFF94S identifying 54.8 ± 0.3%, 51.3 ± 0.3%, and 47.0 ± 0.3% respectively. By this metric, OPLS3e and OpenFF-1.2 seem to exhibit roughly similar performance, with the other force fields performing somewhat worse. Figure 3b illustrates the progress made within the OpenFF family of force fields. The predecessor SMIRNOFF99Frosst performs worst of all investigated force fields and is improved upon by the first releases OpenFF-1.0 and OpenFF-1.1, which show intermediate performance. Finally, the most recent release OpenFF-1.2 indicates further improvement.\n\nEach molecular structure, including different conformers of the same molecule, is counted separately. Since the global minimum molecular structures were set to zero deliberately and add a constant offset to the central bin, they are removed from the counts. A force field having higher agreement with QM would have a higher bin centered at ddE = 0 kcal/mol. (a) compares the latest release of all four force field families. (b) shows the four histograms belonging to the OpenFF family of force fields. OpenFF-1.0 (purple) and OpenFF-1.1 (light blue) overlap in the central bin. The corresponding graph comparing histograms of all calculated force fields can be found in the Extended data, Figure S.193.\n\nGiven that two conformers starting from the same geometry may optimize to two distinct conformers after FF minimization, we took another approach to analyzing energy distributions, only considering the FF conformers that correspond to a QM counterpart. A FF conformer is deemed to have a “match” with a QM conformer if its RMSD is less than or equal to 1 Å (see more details in Methods). The number of matched conformers for each force field are: 20,815 (GAFF), 20,836 (GAFF2), 20,674 (MMFF94), 20,684 (MMFF94S), 21,961 (OPLS3e), 16,177 (SMIRNOFF99Frosst), 19,103 (OpenFF-1.0), 17,965 (OpenFF-1.1), 21,428 (OpenFF-1.2). The mean signed deviation of the matched conformer energies are shown as violin plots in Figure 4. The violin plots are scaled such that each violin has the same area. This figure shows that the mean signed deviation of relative conformer energies is also fairly consistent between different force fields as seen in Figure 3. Upon closer inspection, the violins for OPLS3e and OpenFF-1.2 are slightly wider around 0 kcal/mol (and narrower elsewhere), signifying marginally higher agreement with QM energies. Equivalent results for an RMSD threshold of 0.3 Å to the QM structure is shown in the Extended data, Figure S.293. With this lower RMSD criteria, the number of structures within the cutoff is roughly halved compared to a threshold of 1 Å while the ranking of force fields remains unaltered. Note that this conformer filtering step was only used for analyzing the energies in the violin plots, and other results throughout this work do not rely on matched conformers.\n\nThe energy MSDs only take into account structures matched within 1 Å of the QM reference structure, so there are minor differences in the amount of data used to plot each violin (see text). To correct for this, each plot was scaled to the same area. The vertical axis is shown on a logarithmic scale. An overlay of the violin plots on the right panel better shows the subtle distinctions between the force fields in the most populated region, near zero error. An equivalent graph with an RMSD threshold of 0.3 Å is shown in the Extended data, Figure S.293.\n\nWe next examine agreement between FF-optimized geometries and those from QM, as calculated by each molecule’s RMSD and TFD scores with reference to the parent QM-optimized geometries. While RMSD is the more common metric, it may depend on the molecule size, complicating interpretation of geometric agreement94,95. In contrast, TFD was designed to be more independent of molecule size in order to compare molecular conformations more meaningfully87. This can help offset issues with RMSD where larger, more ﬂexible molecules can contribute the most to RMSD. The TFD score between two molecular structures is evaluated by computing, normalizing, and Gaussian weighting the (pseudo)torsion deviation for each bond and ring system. While TFD is normalized from 0 to 1, RMSD is unbounded. Both RMSD and TFD are similar in that a higher value signifies lower agreement between the geometries of two molecules. A FF which yields optimized geometries closer to those of QM would have generally smaller RMSD/TFD values. We calculated RMSD and TFD scores for all MM optimized geometries with respect to QM geometries. We plotted this data in histograms in Figure 5.\n\nHistograms of the RMSD (a, c) and TFD (b, d) values between force field structures as compared to QM structures. Values closer to zero indicate higher geometric similarity for both RMSD and TFD. Panels (a) and (b) compare the families of force fields (GAFF2, MMFF94s, OPLS3e, and OpenFF-1.2). Panels (c) and (d) compare the force fields of the OpenFF family (Smirnoff99Frosst, OpenFF-1.0, OpenFF-1.1, and OpenFF-1.2). The corresponding graphs with histograms of all force fields are shown in the Extended data, Figure S.393.\n\nIn terms of geometry agreement, we observed similar results between the RMSD and TFD plots. The ranking of the force fields is mostly the same as with the ddE rankings above, with OPLS3e performing best followed by the latest open force field release, OpenFF-1.2. One major difference is the ranking of MMFF94S over GAFF2, while the latter had better agreement with QM in terms of ddE. The OpenFF force fields show clear improvement with newer versions by having higher densities close to zero and also by having tails successively reduced. Although SMIRNOFF99Frosst had a non-negligible density at RMSD > 2 Å, virtually all structures optimized with OpenFF-1.2 agree with the QM structures with RMSD < 2 Å (Figure 5c). Both TFD and RMSD distributions show qualitatively the same ranking of force fields, whereas the quantitative differences appear to be of different magnitudes. For example, MMFF94S is very close to GAFF2 in terms of RMSD (Figure 5a). According to TFD, MMFF94S appears to be closer to OpenFF-1.2, with GAFF2 having less agreement with QM (Figure 5b).\n\nFrom the histograms, we can identify areas for force field refinement of molecular geometries by analyzing molecules with significant conformational differences from the QM reference (molecules with TFD values > 0.12), and in particular by focusing on parameters which occur more frequently than expected in such molecules. Parameters which are overrepresented in molecules with significant deviations are more likely to be responsible for such deviations. To assess this, we computed the representation ratio (Equation 4) for each OpenFF-1.2 force field parameter in both the high TFD molecule subset as well as in the full set of molecules. We estimated whether each parameter was applied more frequently in the high TFD subset compared to the full set by computing the one-sample Z-test for proportions. Figure 6 shows the results for a subset of the OpenFF-1.2 force field parameters, wherein the parameters of interest have a statistically significantly higher representation ratio in the high TFD subset within a 95% confidence interval. These parameters are listed in Table 2 for the complete OpenFF-1.2 force field, and likely warrant further investigation as a possible cause of deviations from the QM reference. The complete set of OpenFF-1.2 representation ratio plots are placed in the Extended data, Figures S.4-S.693.\n\nThe blue bars represent the parameter ratios from the full molecule set, and the red bars represent the parameter ratios from only the set of molecules with TFD values greater than 0.12. Error bars denote the 95% confidence interval determined from the one-sample Z-test for proportions. Parameters which are estimated to be overrepresented in molecules with high TFDs have statistically significant differences between the full set and high TFD set of parameter ratios (also see Table 2). Parameters with statistically significant differences in this plot are a1, a2, a7, a8, a9, a17, a18, and a19.\n\nThese parameters show statistically significant differences (p < 0.05) in representation ratios of the high TFD molecules compared to ratios of the full molecule set. Refinement of these parameters may address conformational differences in MM-optimized molecular geometries compared to QM-optimized geometries.\n\nWe then sought to determine if there was a dependence between the relative energies and geometries. Scatter plots of ddE versus RMSD/TFD are shown for all force fields in Figure 7. Each structure in our dataset is plotted as a single point. The ddE values are plotted on a logarithmic scale. We include in the Extended data, Figure S.7 analogous plots with ddE represented on a linear scale93. Given tens of thousands of points on each plot leading to many overlapping points, we applied a color gradient from red to blue to represent regions from low to high density, respectively. Similar to the data represented as one-dimensional histograms (Figure 3 and Figure 5), a higher density of points at the origin indicates results in better agreement with the reference QM data. There seems to be no general correlation between the energies and geometries. However, using this visualization we identified particular chemical moieties that represent outlying energies or geometries (vide infra).\n\nThe points are colored by the interpolated density of points in a certain area. Blue indicates region of high density, that is, high compactness of points in that area. A force field having better agreement in both relative energies and geometries with the QM reference would have more points around the origin (ddE = 0, TFD = 0), though it is presumably possible for a force field to improve along one axis without improving along the other. The vertical axis is represented on logarithmic scale; the same plots with linear scaling can be found in the Extended data, Figure S.793.\n\nIn this diverse set of molecules, we point out three particular moieties, those containing an N-N single bond (3824 structures), those containing an azetidine ring (543 structures), and a highly substituted octahydrotetracene (50 structures). These subsets are highlighted for OpenFF-1.2 in Figure 8 (see Extended data, S.9 for other force field results93). Molecules containing an N-N single bond have a wide spread of energies with several ddE outliers between -10 to -20 kcal/mol. Structures with azetidine revealed both energy and geometry outliers in the Amber and OpenFF force field families. Lastly, the substituted octahydrotetracene scaffold was found to be challenging to all force fields in reproducing QM energies (an example is presented in the Extended data, Figure S.893). These moieties represent systematic outliers that can be used in future studies investigating particular shortcomings of force fields or improving future versions of force fields. Indeed, some of these issues have been a focus of fitting of the OpenFF 1.1 and 1.2 force fields96. We have calculated the average and standard deviation statistics of ddE and TFD for the whole set of structures and the subsets containing these moieties. The results are listed as Extended data, Table 1 and Figure S.1093. Both the spread and average of the distributions of the subset are generally larger than the ones of the whole set, emphasizing that these moieties are challenging to be parameterized. For the OpenFF family of force fields, a clear improvement in these statistics can be seen for the newer versions, especially for the N-N moiety (both TFD and ddE) and the octahydrotetracene (in terms of ddE).\n\nColors highlight particular chemical groups that appear to be systematic outliers in energies or geometries. On the right hand side, we show a figure with high TFD and low ddE as circled in the scatter plot. The QM structure is in purple, and the force field structure is colored in silver. Analogous plots for all other force fields are shown in the Extended data, Figure S.993.\n\n\nConclusions\n\nIn this work, we presented a large-scale analysis of nine small molecule force fields in terms of their relative conformer energies and geometries compared to reputable QM data. Amongst the force fields (GAFF, GAFF2, MMFF94, MMFF94S, OPLS3e, SMIRNOFF99Frosst, OpenFF-1.0, OpenFF-1.1, and OpenFF-1.2), OPLS3e performed best in terms of reproducing QM conformer energies and geometries. However, it is worth noting the higher computational cost of the high level DFT torsion fitting for generating the optimal OPLS3e parameters (likely in part due to the diversity of the present molecule set), whereas with the other force fields this step was rapid.\n\nThe OpenFF versions showed improvements in both metrics with each new version, and the latest OpenFF-1.2 appears to be approaching the degree of accuracy of OPLS3e, at least on this dataset. This is despite the extra dihedral parameter fitting with OPLS3e. Thus OpenFF-1.2 seems to be positioned as the best open source/free small molecule force field in this study, as OPLS3e is proprietary.\n\nOther aspects of interest included the ability of MMFF94 and MMFF94S to capture QM geometries better than several other force fields, but still not as well as OPLS3e or OpenFF-1.2, especially when using a more size-independent geometry measure. Finally, we identified particular chemical moieties that were systematic outliers in terms of relative energies or geometries. These N-N, azetidine, and octahydrotetracene-like compounds represent potential areas for improvement in future force field development.\n\nOur work also highlights the progress the Open Force Field Initiative has made towards its goal of producing high quality public, open force fields built with infrastructure which enables rapid parameterization. Particularly, the series of OpenFF force fields presented here demonstrate marked improvements in accuracy over a relatively short time, and these improved force fields are available to everyone. One key challenge going forward will be to continue improving treatment of problematic areas of chemical space and expanding coverage. In parallel, future OpenFF updates will include improved treatment of torsions (via Wiberg bond order-based parameter interpolation97 which was recently implemented in our toolkit) and better handling of trivalent nitrogen geometries98 which we hope will boost performance further.\n\nBeyond these specific conclusions, we believe the general strategies employed here for benchmarking force field performance will be useful far more broadly than this specific study. Particularly, comparing performance by both geometric and energetic measures is particularly important, as the analysis we have done demonstrates. Additionally, the availability of a large amount of public data in QCArchive facilitates straightforward large scale benchmarking in a way it has not been done previously.\n\nWe share our Python code comprising the setup, minimization, and analysis of this research on Github, available at: https://github.com/MobleyLab/benchmarkff90.\n\n\nData availability\n\nZenodo: Molecular geometries and energies from quantum mechanical calculations and small molecule force field evaluations. https://dx.doi.org/10.5281/zenodo.424785999.\n\nZenodo: Supporting Information: Molecular geometries and energies from quantum mechanical calculations and small molecule force field evaluations. http://dx.doi.org/10.5281/zenodo.429920093\n\nThis project contains the following extended data:\n\nHistograms for all force fields regarding energies of conformers, RMSD and TFD relative to QM reference data for all force fields investigated in this work\n\nPlots similar to those in Figure 7 with linear scaling of the vertical axis\n\nPlots in the same manner of Figure 8 for all force fields in this work\n\nAverage and standard deviation statistics of relative energies and TFDs for different (sub)sets of structures\n\nAn example of one of the octahydrotetracene-based structures having high deviation in ddE\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nCode availability\n\nSource code used in conducting the modeling, analysis and plots is available on GitHub, with the specific version used here archived on Zenodo.\n\nSource code available from: https://github.com/mobleylab/benchmarkff\n\nArchived source code at time of publication: https://dx.doi.org/10.5281/zenodo.425269490\n\nLicense: MIT", "appendix": "Acknowledgements\n\nThe authors thank the Open Force Field Initiative researchers for their contributions in project planning and feedback, especially Caitlin Bannan, Jordan Ehrman, Jessica Maat, Lee-Ping Wang, and Hyesu Jang. 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PubMed Abstract \| Publisher Full Text\n\nBeauchamp K, Rustenburg A, Rizzi A, et al.: OpenMolTools.\n\nSchrödinger: Schrödinger Release 2020-1: Maestro. 2020. Reference Source\n\nMadhavi Sastry G, Adzhigirey M, Day T, et al.: Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des. 2013; 27(3): 221–34. PubMed Abstract \| Publisher Full Text\n\nEastman P, Swails J, Chodera JD, et al.: OpenMM 7: Rapid Development of High Performance Algorithms for Molecular Dynamics. PLoS Comput Biol. 2017; 13(7): e1005659. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim VT: BenchmarkFF. 2020. Reference Source\n\nSchulz-Gasch T, Sch¨arfer C, Guba W, et al.: TFD: Torsion Fingerprints as a new measure to compare small molecule conformations. J Chem Inf Model. 2012; 52(6): 1499–1512. PubMed Abstract \| Publisher Full Text\n\nEhrman JN, Bannan CC, Lim VT, et al.: Improving Force Fields by Identifying and Characterizing Small Molecules with Parameter Inconsistencies. 2019. Publisher Full Text\n\nEhrman J, Lim VT, Bannan CC, et al.: Improving Small Molecule Force Fields by Identifying and Characterizing Small Molecules with Inconsistent Parameters. ChemRxiv. 2020. Reference Source\n\nLim VT, Mobley DL, Hahn DF: MobleyLab/benchmarkff: Version 1.0.0: Used in paper. 2020. http://www.doi.org/10.5281/zenodo.4252694\n\nJorgensen WL, Maxwell DS, Tirado-Rives J: Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J Am Chem Soc. 1996; 118: 11225–11236. Publisher Full Text\n\nDodda LS, Cabeza de Vaca I, Tirado-Rives J, et al.: LigParGen Web Server: An Automatic OPLS-AA Parameter Generator for Organic Ligands. Nucleic Acids Res. 2017; 45(W1): W331–W336. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim VT, Hahn D, Tresadern G, et al.: Supporting Information: Molecular geometries and energies from quantum mechanical calculations and small molecule force field evaluations (Version 1.0.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4299200\n\nReva BA, Finkelstein AV, Skolnick J: What is the probability of a chance prediction of a protein structure with an rmsd of 6 A? Fold Des. 1998; 3(2): 141–147. PubMed Abstract \| Publisher Full Text\n\nSargsyan K, Grauffel C, Lim C: How Molecular Size Impacts RMSD Applications in Molecular Dynamics Simulations. J Chem Theory Comput. 2017; 13(4): 1518–1524. PubMed Abstract \| Publisher Full Text\n\nJang H: Update on Parsley Minor Releases (Openff-1.1.0, 1.2.0). 2020. Publisher Full Text\n\nStern C: Capturing Non-Local through-Bond Effects When Fragmenting Molecules for QC Torsion Scans. 2020. Publisher Full Text\n\nMobley DL: Current Status of OpenFF and Our Near-Term Roadmap. 2020. Publisher Full Text\n\nLim VT, Hahn DF, Tresadern G, et al.: Molecular geometries and energies from quantum mechanical calculations and small molecule force field evaluations. 2020. http://www.doi.org/10.5281/zenodo.4247859" }	[ { "id": "75766", "date": "22 Dec 2020", "name": "Sereina Riniker", "expertise": [ "Reviewer Expertise Computational chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: The authors describe the benchmarking of different general force fields for small organic molecules against QM reference data in terms of gas-phase geometries and energies. A large dataset of 3271 molecules with 22675 conformers was assembled and is freely available. Although the force fields are typically aimed for condensed-phase systems and the dataset provides gas-phase data, such a benchmarking set and the accompanying analysis is highly valuable for the force-field community. Python code is also available on Github. From the results, it is encouraging to see that systematic improvement of force fields is possible. Minor revisions are suggested below to further improve the clarity and quality of the article.\nSuggested Revisions:\nDataset: The authors had to remove the 419 molecules used for training of OpenFF-1.2 from the QCArchive dataset to ensure an unbiased test set. Did this lead to a certain enrichment or depletion of particular functional groups in the remaining test set?\n\nQM reference data: How much variation in the QM ddE would you expect for different functionals/basis sets? In other words, what “error” (or uncertainty) is associated with the QM calculations? An indication of the variation between different QM methods is given in Faraday Discuss., 195, 497-520 (2016).\n\nThe authors compare the ddE between the QM and MD approaches. However, can we expect the deviations to be systematic?\n\nTable 1: It would be interesting to see the min and max ddE for GAFF after the removal of the six molecules with collapsing hydroxyl groups (maybe in parentheses after the original values?).\n\nFig. 3 (and Fig. S1): I think there should be a sufficiently large number of data points to choose a smaller bin size for the histograms.\n\nFig. 7: It appears that there is a straight line around ddE = 0, i.e. conformers with a large variety in TFD all have a ddE of zero. Is there an explanation for this?\n\nThe TFD does not give equal weights to all torsions in a molecule: (1) all bonds in rings are combined to a single value in the torsion fingerprint, and (2) torsions in the centre of the molecule are weighted more than terminal torsion angles. In the calculation of ddE, on the other hand, all deviations contribute similarly to the energy difference. Could this be one of the explanations that there is basically no correlation between higher ddE values and higher TFD values? I would suggest to consider a torsional-angle RMSD as an alternative measure for the structural differences.\n\nHow important is the targeted torsion fitting in OPLS3e for its performance? In other words, would you expect the performance of e.g. OpenFF-1.2 to improve significantly with custom torsions?\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": [] }, { "id": "77769", "date": "04 Feb 2021", "name": "Sreyoshi Sur", "expertise": [ "Reviewer Expertise Computational Chemistry and Computational Biophysics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, the authors have looked at how closely do the molecular mechanics and quantum mechanics optimized data match for small molecules in gas phase. In order to achieve this they used two parameters:\n\nConformer relative energies, calculated using different forcefields and the corresponding quantum mechanically calculated energies.\n\nEnergy minimized geometric structures evaluated using the forcefields and the QM calculations.\n\nThey provided a list of 3271 molecules with 22675 different structures. Molecules were selected based on unique chemical connectivity and whether all the molecules can be parametrized by GAFF, GAFF2, MMFF94, MMFF4S, OPLS3e, SMIRNOFF99Frosst, and OpenFF Parsley (1.1,1.2,1.3). Molecules that were part of training set for the forcefield OpenFF Parsley was removed for evaluation too. For every evaluated structure, first the authors compared each molecules conformers QM calculated and the MM energies. Their results show that for a greater number of molecules OPLS 3e evaluated energies were closest to the QM energies, followed by OpenFF 1.2. They also evaluated MM energy for the conformers which most closely matched the energy minimized QM structures. In this case also OPLS3e and OpenFF 1.2 performed better than the rest of the molecules. To compare the 3D structures, two metrics were used, RMSD and Torsion fingerprint deviation. Using these two metrics very slight changes were observed in terms of the relative energies and the two forcefields which match closest to QM remain the same (OPLS 3e and OpenFF 1.2).\n\nComments: The paper addresses most of the concerns in terms of comparing small molecule's structure and energies w.r.t different MM forcefields. The authors have provided their GitHub repository consisting of all the test data and their code which was used to obtain the results. This enables the reader to reproduce the results of the paper and also have a workflow to run some of these QM vs MM comparisons with completely new sets of molecules.\n\nQuestions/Minor revisions for the authors:\nFor Figure 7, I would suggest rearranging the plots so that they are next to each other and thus easy to compare.\n\nFor Figure 7, the authors can also show a difference plot between OPLS3e and OpenFF 1.2 and between OPLS3e and MMFF945, then the subtle changes between OpenFF1.2 and MMFF945 around ddE=0 compared to OPLS3e will be more prominent.\n\nFor the 3271 molecules chosen, what is the overall chemical diversity of these molecules?\n\nThese small molecules are usually used as ligands to proteins, can the authors comment on how these improvements in the OpenFF 1.2 will impact binding affinity calculations?\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/9-1390
https://f1000research.com/articles/9-1389/v1	03 Dec 20	{ "type": "Brief Report", "title": "Phylogenetic analysis of the betacoronavirus S1 subunit", "authors": [ "Irina Zyrianova" ], "abstract": "The ongoing pandemic outbreak of coronavirus disease 2019 (COVID-19) has been caused by the new betacoronavirus (BetaCoV) severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Together with other epidemic outbreaks of BetaCoV infectious diseases (Severe Acute Respiratory Syndrome (SARS) in 2002-2003 in China and Middle East Respiratory Syndrome (MERS) in 2012 in the Middle East, which have been caused by SARS-CoV and MERS-CoV, respectively), these events have generated interest in the coronaviruses (CoVs). Although many phylogenetic analyzes have been reported at a gene or protein level, there is no study as yet encompassing the many sequences publicly available for BetaCoVs, including those that have been manipulated in the lab. In this study, the phylogenetic analysis of 679 different S1 protein sequences of BetaCoVs from a total of 1595, which are publicly available in GenBank from the beginning of the pandemic event to April 2020, has been carried out. The S1 subunit is one part of the S (spike) protein, one of three CoV envelope proteins. The S1 subunit contains a host cell receptor binding domain. This domain is essential in the initiation of the infectious process. Therefore, its phylogenetic analysis is very important for studying CoV evolution. The phylogenetic analysis of BetaCoV S1 protein presented herein shows the evolutionary history of BetaCoVs from bovine CoV to SARS-CoV-2.", "keywords": [ "S protein", "S1 subunit", "betacoronaviruses", "bovine coronavirus", "MERS-CoV", "SARS-CoV", "SARS-CoV-2", "phylogenetic analysis" ], "content": "Introduction\n\nAccording to the International Committee on Taxonomy of Viruses (ICTV; 2019, Release #35), betacoronaviruses (BetaCoVs) have been classified as belonging to Riboviria realm, Orthornavirae kingdom, Pisuviricota phylum, Pisoniviricetes class, Nidovirales order, Cornidovirineae suborder, Coronaviridae family, Orthocoronavirinae subfamily, and Betacoronavirus genus; species/subspecies of BetaCoVs are listed in Figure 1.\n\nThe following abbreviations are applied: lhc – the lab host cells, lhm – the lab host mouse; and SARS-CoV – Severe acute respiratory syndrome coronavirus, PCoV – Pangolin coronavirus, BatSARSL-CoV – Bat SARS-like coronavirus, BatCoV – Bat coronavirus, BetaCoV – Betacoronavirus, RoCoV – Rodent coronavirus, RtCoV – Rat coronavirus, RouBatCoV – Rousettus bat coronavirus, HCoV – Human coronavirus, HBetaCoV – Human betacoronavirus, HECoV – Human enteritis coronavirus, EriHedCoV – Erinaceus Hedgehog coronavirus, PipBatCoV – Pipistrellus bat coronavirus, HypBatCoV – Hypsugo bat coronavirus, MERS-CoV – Middle East respiratory syndrome coronavirus, TylBatCoV – Tylonycteris bat coronavirus, PhiaffCoV – Rhinolophus affinis coronavirus, CoV-Neo – Coronavirus Neoromocia, EqCoV – Equine coronavirus, MHV – Murine hepatitis virus, MuCoV – Murine coronavirus, PHEV – Porcine hemagglutinating encephalomyelitis virus, RbCoV – Rabbit coronavirus, DcCoV – Dromedary camel coronavirus, CamCoV – Camel coronavirus, CRCoV – canine respiratory coronavirus, BCoV – Bovine coronavirus, WatbuCoV – Waterbuck coronavirus, GirCoV – Giraffe coronavirus. The stars designate protein sequences deduced from nucleotide sequences using the GeneRunner program. The numbers in front of sequence annotation are the unique sequence numbers for each S/S1 sequence in the batch for each BetaCoV species for more comfortable use. Using data from 12–17.\n\nThe ongoing pandemic outbreak of coronavirus disease 2019 (COVID-19) with pneumonia symptoms has been caused by a new BetaCoV, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2; originally named as 2019-nCoV)1,2. Two other BetaCoVs, SARS-CoV and MERS-CoV, have caused epidemic outbreaks of infectious diseases - Severe Acute Respiratory Syndrome (SARS) 2002–2003 in China and Middle East Respiratory Syndrome (MERS) in 2012 in the Middle East, respectively. All these outbreaks are severe or even fatal human diseases2,3. The other three human BetaCoVs (human coronavirus OC43, NL63, and HKU1; HCoV-OC43, HCoV-NL63, and HCoV-HKU1), usually cause cold symptoms2,3.\n\nThe rest of BetaCoVs primarily infect nonhuman mammals, among which bovine coronavirus (BCoV) is the most significant for the farming industry all over the world. Although, BCoV infected cattle have low mortality, they suffer from calf diarrhea (winter dysentery), respiratory symptoms, and substantial losses in milk and meat production4. Another BetaCoV, porcine hemagglutinating encephalomyelitis virus (PHEV), which is the causative agent of neurological and digestive disease in pigs, is also significant for farmers. Although it remains poorly studied because of its low clinical prevalence reported so far, it could lead to an animals' fatal end, causing significant harm to the swine industry5. Another BetaCoV is equine coronavirus (EqCoV), which causes diarrhea in foals and impacts the horse breeding industry6. There is also canine respiratory coronavirus (CRCoV), which is found in pet animals, associated with mild to severe canine infectious respiratory disease7.\n\nSeveral small mammal BetaCoVs have been discovered to date. Among them are rodent coronavirus (RoCoV), rabbit coronavirus (RbCoV), and hedgehog coronavirus (HedCoV)8. There is also the more studied murine hepatitis virus (MHV), which causes hepatitis, enteritis, respiratory diseases, and encephalomyelitis in the central nervous system in mice and rats9. Furthermore, there are different bat coronaviruses (BatCoV), which have well-adapted hosts (different species of bats) in the natural environment. These BetaCoVs should be specially noted as they are suggested to be the origin of MERS-CoV, SARS-CoV, and SARS-CoV-210,11.\n\nThe S (spike) protein is one of three viral envelope proteins. It is considered a member of the class I viral membrane fusion proteins, including those from the influenza virus, human immunodeficiency virus (HIV), and Ebola virus. The S protein is involved in the initiation of the infectious process. It acts as an intermediary of viral and host cell membrane fusion and is a significant inducer of host immune responses12–15. The S protein assembles into trimmers and folds so that it sticks out from the membrane surface to form spikes; hence its name: spike protein. The virion surface looks like a corona (Latin for crown) because of these spikes, and this feature became the reason for the name coronaviruses12–15.\n\nIn most species of CoVs, the S protein is cleaved into two approximately equal size subunits, S1 and S2. The S1 subunit contains a host cell receptor binding domain (RBD). There are detected N-terminal domain (NTD) and C-terminal domain (CTD) in the S1 subunit, and one or both of which function as RBD. NTDs are responsible for binding the sugar (O-ac-Sia – 9-O-acetyl sialic acid) or the protein receptor CEACAM1 (the carcinoembryonic antigen-related adhesion molecule 1), and CTDs are responsible for recognizing protein receptors ACE2 (angiotensin-converting enzyme 2 – the zinc peptidase) and DPP4 (the dipeptidyl peptidase 4 – the serine peptidase), CD209L, and CD209 – the immunoglobulin-like cell adhesion molecule12–17. The schematic structure of the Betacoronavirus S protein is shown in Extended data18.\n\nThe S1 subunit is the most divergent region of the S protein, and the S1 RBD is the principal determinant of species and tissue susceptible to infection12. Therefore, its phylogenetic analysis is very important for studying coronavirus evolution. Although many phylogenetic analyzes have been reported for S or S1 on a genetic or protein level, no study had been made for all publicly available S1 protein sequences of all known BetaCoVs. In this study, the data of BetaCoV S1 protein sequence phylogenetic analysis has been presented. The S protein sequences used have been collected from GenBank before April 2020.\n\n\nMethods\n\nA total of 1595 S protein sequences, which are publicly available in GenBank from the beginning of the pandemic event to April 2020, have been used in this study. Some S protein sequences have been deduced from the corresponded nucleotide sequences using the GeneRunner program. Only 679 different of them have been implemented to the phylogenetic analysis since identical sequences do not contribute to phylogenetic relationships.\n\nIdentical S and S1 protein sequences have been found using the ClustalW option of the MEGA X (Version 10.0.5) program19 and excluded from the phylogenetic analysis. All identical S and S1 protein sequences are available as Underlying data20. S1 ends for SARS-CoV-2 (-RRAR685), SARS-CoV (-SLLR667), MERS-CoV (-RSVR751), HCoV-OC43 (-RRSR757), HCoV-HKU1 (-RRKRR760), MHV-A59 (-RRAHR721), BCoV-Quebec (-RRSRR768), BatCoV-HKU4 (-STFR749), and BatCoV-HKU-5 (-RVRR745) have been determined according to Millet and Whittaker (2014) and James et al. (2020)21,22. The rest of the S1 subunit ends have been deduced using the S1 sequence alignment in ClustalW (see Underlying data23).\n\nPhylogenetic analysis has been performed with the MEGA X (Version 10.0.5) program19, using the Maximum Likelihood method and the JTT matrix-based model24 with 1000 bootstrap replications and uniform rates among sites. The analysis of 679 different sequences of the S1 subunit has been implemented in two steps. In the first step, different phylogenetic trees have been constructed for each BetaCoV species/subspecies, or several BetaCoV species/subspecies have been combined into the one primary tree25–39; except for human enteritis coronavirus (HECoV), for which all different protein sequences have been used in the summarised tree. RbCoV, RoCoV, rat (Rt) CoV, and HedCoV have been included together in the primary tree34. Also, SARS-CoV-2 and pangolin coronavirus (PCoV) have been combined into the one primary tree39. Bat CoVs have been divided into four groups by alignment; the primary tree has been constructed for each of them35–38. In the second step, the sequences have been selected from primary trees, and a summarized tree has been constructed. As can be seen from each phylogenetic tree (see Underlying data25–39) one or more sequences have been selected from each phylogenetic clade. The sequences have been chosen as follows: if the separated clade consists of several sequences, then the sequences that have been found closer to the branching point of the entire clade are selected; if the clade consists only of one sequence, this sequence is taken into the summary tree. The summary bootstrap consensus tree have been inferred from 1000 replicates. The percentage of replicate trees in which the associated taxa clustered together is shown next to the branches. Percentages ≥50% are shown (Figure 1).\n\n\nResults\n\nFigure 1 shows that all S1 subunits of BetaCoVs species are originated from BCoV S1. The BCoV group also includes other ruminant BetaCoVs, which do not have an individual detached clade. There is an intermediate branch of human enteritis coronavirus among the BCoV group. It confirms the transmission of HECoV from bovine.\n\nFurthermore, the phylogenetic clade of CRCoV and HCoV-OC43 has been separated from BCoVs, and then four clades have been detached one after another. These are Dromedary camel (Dc) CoV, PHEV, EqCoV, and RoCoV. They are followed by the clade consisting of MHV and HCoV-HKU1, which has several intermediates of RtCoVs.\n\nAfter that, the group of MERS-CoV, BatCoV-HKU-4, and BatCoV-HRU-5 separate, and has an intermediate group of HedCoV. The MERS-CoV is already one of the particularly dangerous BetaCoVs for humans40. This group is followed by the group of RouBatCoVs (Rousettus bat coronaviruses).\n\nFinally, the SARS-CoV clade is formed. It is divided into two phylogenetic branches. One consists of SARS-CoVs and BatSARSL-CoVs (Bat SARS-like coronaviruses). Another consists of SARS-CoV-2s, PCoVs, and BatSARSL-CoVs. This clade could be named as that of the pandemic BetaCoVs, because of SARS-CoV-2.\n\nThus, we see in Figure 1 that the evolution of BetaCoV S1 proceeds from BCoV, which is not dangerous for humans, to SARS-CoV and SARS-CoV-2, which are especially hazardous for humans.\n\n\nSummary\n\nThe phylogenetic analysis carried out in this study has shown that the evolution of S1 of BetaCoVs begins from BCoV, which is not dangerous for humans, and then, passing through BetaCoVs of dogs (CRCoV), camels (DcCoV), pigs (PHEV), horses (EqCoV), rodents (RoCoV, MHV, RtCoV) and hedgehogs (HedCoV) leads to SARS-CoV and SARS-CoV-2, which are already particularly dangerous for humans. Therefore, we shouldn't underestimate the potential danger of BCoV.\n\n\nData availability\n\nFigshare: 100% homology sequences of Betacoronaviruses S protein and S1 subunit, https://doi.org/10.6084/m9.figshare.12962378.v420.\n\nFigshare: The S1 sequence alignment of Betacoronaviruses, https://doi.org/10.6084/m9.figshare.1310689423.\n\nFigshare: The phylogenetic analysis of the Bovine coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12956963.v525.\n\nFigshare: The phylogenetic analysis of the Human coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12957932.v426.\n\nFigshare: The phylogenetic analysis of the Severe Acute Respiratory Syndrome-related coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12957977.v527.\n\nFigshare: The phylogenetic analysis of the Middle East Respiratory Syndrome coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12957989.v428.\n\nFigshare: The phylogenetic analysis of the Murine hepatitis virus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958004.v429.\n\nFigshare: The phylogenetic analysis of the Canine respiratory coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958028.v430.\n\nFigshare: The phylogenetic analysis of the Porcine hemagglutinating encephalomyelitis virus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958091.v431.\n\nFigshare: The phylogenetic analysis of the Equine coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958112.v432.\n\nFigshare: The phylogenetic analysis of the Dromedary camel coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958136.v433.\n\nFigshare: The phylogenetic analysis of the small mammal BetaCoV coronavirus S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958172.v434.\n\nFigshare: The phylogenetic analysis of the Bat coronavirus (HKU3 group) S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958184.v435.\n\nFigshare: The phylogenetic analysis of the Bat coronavirus (HKU4 group) S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958193.v536.\n\nFigshare: The phylogenetic analysis of the Bat coronavirus (HKU5 group) S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958196.v537.\n\nFigshare: The phylogenetic analysis of the Bat coronavirus (HKU9,10 group) S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.12958208.v438.\n\nFigshare: The phylogenetic analysis of the Severe Acute Respiratory Syndrome-related coronavirus 2 S1 subunit protein sequence, https://doi.org/10.6084/m9.figshare.13102889.v239.\n\nFigshare: The schematic structure of the betacoronavirus S protein, https://doi.org/10.6084/m9.figshare.12951413.v218.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe author would like to thank Ilya Artemin – interpreter.\n\n\nReferences\n\nZhu N, Zhang D, Wang W, et al.: A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–733. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNadeem MS, Zamzami MA, Choudhry H, et al.: Origin, Potential Therapeutic Target and Treatment for Coronavirus Disease (COVID-19). Pathogens. 2020; 9(4): 307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYe ZW, Yuan S, Yuen KS, et al.: Zoonotic origins of human coronaviruses. Int J Biol Sci. 2020; 16(10): 1686–1697. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanno T, Ishihara R, Hatama S, et al.: A long-term animal experiment indicating persistent infection of bovine coronavirus in cattle. J Vet Med Sci. 2018; 80(7): 1134–1137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMora-Díaz JC, Piñeyro PE, Houston E, et al.: Porcine Hemagglutinating Encephalomyelitis Virus: A Review. Front Vet Sci. 2019; 6: 53. 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Nat Rev Microbiol. 2019; 17(3): 181–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhou P, Yang XL, Wang XG, et al.: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798): 270–273. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasters PS: The Molecular Biology of Coronaviruses. Adv Virus Res. 2006; 66: 193–292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu G, Wang Q, Gao GF: Bat-to-human: spike features determining ‘host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol. 2015; 23(8): 468–478. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi F: Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol. 2016; 3(1): 237–261. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSatarker S, Nampoothiri M: Structural Proteins in Severe Acute Respiratory Syndrome Coronavirus-2. Arch Med Res. 2020; 51(6): 482–491. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmraie R, Napoleon MA, Yin W, et al.: CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells. bioRxiv. 2020; 2020.06.22.165803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaskalska A, Dabrowska A, Szczepanski A, et al.: Membrane Protein of Human Coronavirus NL63 Is Responsible for Interaction with the Adhesion Receptor. J Virol. 2019; 93(19): e00355–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZyrianova I: The schematic structure of the betacoronavirus S protein. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12951413.v2\n\nKumar S, Stecher G, Li M, et al.: MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6): 1547–1549. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZyrianova I: 100% homology sequences of Betacoronaviruses S protein and S1 subunit. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12962378.v4\n\nMillet JK, Whittaker GR: Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 2015; 202: 120–134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJaimes JA, André NM, Chappie JS, et al.: Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. J Mol Biol. 2020; 432(10): 3309–3325. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZyrianova I: The S1 sequence alignment of Betacoronaviruses. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.13106894\n\nJones DT, Taylor WR, Thornton JM: The rapid generation of mutation data matrices from protein sequences. Bioinformatics. 1992; 8: 275–282. PubMed Abstract \| Publisher Full Text\n\nZyrianova I: The phylogenetic analysis of the Bovine coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12956963.v5\n\nZyrianova I: The phylogenetic analysis of the Human coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12957932.v4\n\nZyrianova I: The phylogenetic analysis of the Severe Acute Respiratory Syndrome-related coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12957977.v5\n\nZyrianova I: The phylogenetic analysis of the Middle East Respiratory Syndrome coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12957989.v4\n\nZyrianova I: The phylogenetic analysis of the Murine hepatitis virus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958004.v4\n\nZyrianova I: The phylogenetic analysis of the Canine respiratory coronavirus S1 subunit protein sequence. figshare. Figure. 2020. https://doi.org/10.6084/m9.figshare.12958028.v4\n\nZyrianova I: The phylogenetic analysis of the Porcine hemagglutinating encephalomyelitis virus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958091.v4\n\nZyrianova I: The phylogenetic analysis of the Equine coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958112.v4\n\nZyrianova I: The phylogenetic analysis of the Dromedary camel coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958136.v4\n\nZyrianova I: The phylogenetic analysis of the small mammal BetaCoV coronavirus S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958172.v4\n\nZyrianova I: The phylogenetic analysis of the Bat coronavirus (HKU3 group) S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958184.v4\n\nZyrianova I: The phylogenetic analysis of the Bat coronavirus (HKU4 group) S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958193.v5\n\nZyrianova I: The phylogenetic analysis of the Bat coronavirus (HKU5 group) S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958196.v5\n\nZyrianova I: The phylogenetic analysis of the Bat coronavirus (HKU9,10 group) S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12958208.v4\n\nZyrianova I: The phylogenetic analysis of the Severe Acute Respiratory Syndrome-related coronavirus 2 S1 subunit protein sequence. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.13102889.v2\n\nYuan L, Tang Q, Cheng T, et al.: Animal models for emerging coronavirus: progress and new insights. Emerg Microbes Infect. 2020; 9(1): 949–961. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77185", "date": "02 Feb 2021", "name": "Houcemeddine Othman", "expertise": [ "Reviewer Expertise Bioinformatics" ], "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 by Irina Zyrianova provides phylogenetic evidence about the evolution of SARS-CoV-2 from bovine coronavirus. Although the topic is interesting and could lead to establishing better monitoring strategies for beta-coronaviruses, the author could have discussed the results in a more elaborated way. The experimental protocol is not clear and many ambiguities need to be clarified to the reader.\nAlso, the phylogenic analysis is only reported for the spike protein. It is known that the evolution rate of the protein S is higher than the entire genome. In this regard, getting general conclusions about the evolutionary history would not be appropriate.\nOther minor comments are described below:\nAbstract:\n\"including these that have been manipulated in the lab\" - It is not clear what the author means exactly by this.\n\n\"there is no study as yet encompassing the many sequences publicly available for BetaCoVs\" - I don't think this is accurate. Maybe the author could soften the claim a bit or be more specific given that other works tried to analyze the evolutionary history of SARS-CoV-21,2.\n\n\"The S1 subunit is one part of the S (spike) protein, one of three CoV envelope proteins.\" - perhaps reformulate?\nFigure 1: It should be referenced in the Results section and not the Introduction. A table can be useful to show the species/subspecies of BetaCovs.\nParagraph 4, Introduction: I am not sure why the author mentioned the hepatitis virus.\nParagraph 5, Introduction: A reference is needed for the first sentence.\nParagraph 6, Introduction: \"one or both of which function as RBD\", NTD and CTD are independent of RBD. Check the original paper describing the structure of the spike protein3.\nParagraph 1, Methods:\n\"since identical sequences do not contribute to phylogenetic relationships\", maybe \"Do not contribute to adding information about the evolutionary relationship of the taxa\".\n\nCan the author please specify if the set of sequences are nucleotide or protein sequences? It is better to study the phylogeny using nucleotide sequences, as it is more capable to discriminate the evolutionary events at short periods. Although it should use one of the alignment algorithms that allows for taking into consideration the coding nature of these sequences to reduce the alignment errors.\nParagraph 2, Methods:\nIt is not clear what the author did here. Is it a redundancy removal stage? If so wasn't that done in the previous paragraph?\n\nHow could ClustalW be used to do that?\n\nWhat is the meaning of \"deducing the ends\" and what is the reason behind that?\nI do not recommend using two stages to build the tree. I can understand that this has been done to ease the figure, but there are many software and packages available to collapse the clades without losing too much information. Also, I noticed that some branches lack the bootstrap values including the divergence between the BCoVs and CrCovs and HCoV OC43.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "6341", "date": "11 Nov 2021", "name": "Irina Zyrianova", "role": "Author Response", "response": "The paper by Irina Zyrianova provides phylogenetic evidence about the evolution of SARS-CoV-2 from bovine coronavirus. Although the topic is interesting and could lead to establishing better monitoring strategies for beta-coronaviruses, the author could have discussed the results in a more elaborated way. The experimental protocol is not clear and many ambiguities need to be clarified to the reader. Also, the phylogenic analysis is only reported for the spike protein. It is known that the evolution rate of the protein S is higher than the entire genome. In this regard, getting general conclusions about the evolutionary history would not be appropriate. The answer is: I am not getting general conclusions about the evolutionary history of BetaCoVs: It is said in my manuscript: \"...the evolution of BetaCoV S1 proceeds from BCoV, …, to SARS-CoV and SARS-CoV-2,...” But the reviewer suggests that I am not very far from such conclusion, because the S (especially S1 part) very important protein and gene for the evolution history of CoVs, as it is mentioned by me in introduction. In this regard we should more pay attention to the phylogenetic problem: more genes or more taxa? A. Rokas and S.B. Carroll (2005)4 said that “increasing gene number may not lead to increasing phylogenetic accuracy”. Other minor comments are described below: Abstract: \"including these that have been manipulated in the lab\" - It is not clear what the author means exactly by this. The answer is: One of the meanings of “manipulation” in the Oxford dictionary is “the control of someone or something in order to get an advantage, often unfairly or dishonestly”. In my manuscript, the meaning is precisely according to this interpretation: the controlling of nucleotide or protein sequences in the labs to get some advantages for experimental needs. \"there is no study as yet encompassing the many sequences publicly available for BetaCoVs\" - I don't think this is accurate. Maybe the author could soften the claim a bit or be more specific given that other works tried to analyze the evolutionary history of SARS-CoV-21,2. The answer is: If the reviewer thinks that I am not accurate, they should dive into a reference. I have no task to pay attention only to the evolutionary history of SARS-CoV-2 but on all BetaCoVs. \"The S1 subunit is one part of the S (spike) protein, one of three CoV envelope proteins.\" - perhaps reformulate? The answer is: I think the meaning is precise: S protein is one of three CoV envelope proteins. Figure 1: It should be referenced in the Results section and not the Introduction. A table can be useful to show the species/subspecies of BetaCoVs. The answer is: Yes, I thought about such table, but I have decided that it would be redundant information because all species represented on Figure 1. Paragraph 4, Introduction: I am not sure why the author mentioned the hepatitis virus. The answer is: Murine hepatitis virus is the BetaCoV. Paragraph 5, Introduction: A reference is needed for the first sentence. The answer is: The reference is in the first sentence and is as follows: (ICTV; 2019, Release #35). Paragraph 6, Introduction: \"one or both of which function as RBD\", NTD and CTD are independent of RBD. Check the original paper describing the structure of the spike protein3. The answer is: It is mentioned in the reference14 to my manuscript: \"One or both of these S1 domains potentially bind receptors and function as the receptor-binding domain (RBD)\" Paragraph 1, Methods: \"since identical sequences do not contribute to phylogenetic relationships\", maybe \"Do not contribute to adding information about the evolutionary relationship of the taxa\". The answer is: I think both these sentences mean the same. Can the author please specify if the set of sequences are nucleotide or protein sequences? It is better to study the phylogeny using nucleotide sequences, as it is more capable to discriminate the evolutionary events at short periods. Although it should use one of the alignment algorithms that allows for taking into consideration the coding nature of these sequences to reduce the alignment errors. The answer is: It is said in Paragraph 1, Methods: “A total of 1595 S protein sequences…..”. The reviewer may be right that it is better to study the phylogeny using nucleotide sequences, but it is a fundamental question, which has the origin in Fisher’s theorem of natural selection.5 According to the central dogma of molecular biology DNA-RNA-Protein, it could be said that proteins carry “fitness effects” of nucleotide mutations. “In terms of Fisher’s primary thesis, we cannot overstate the essential role of new mutations and their fitness effects.” 5 Proteins are functions of nucleotide sequences so that it could be estimated phylogenetic relationships using protein sequences. Paragraph 2, Methods: It is not clear what the author did here. Is it a redundancy removal stage? If so wasn't that done in the previous paragraph? The answer is: I do not understand what the reviewer means. How could ClustalW be used to do that? The answer is: What is exactly the reviewer's meaning? What is the meaning of \"deducing the ends\" and what is the reason behind that? The answer is: Maybe it is better to say \"defining the ends\". The reason behind this is that some S1 subunit ends had not been known, so it is used known S1 ends to resolve not known S1 ends by alignment in ClustalW. I do not recommend using two stages to build the tree. I can understand that this has been done to ease the figure, but there are many software and packages available to collapse the clades without losing too much information. Also, I noticed that some branches lack the bootstrap values including the divergence between the BCoVs and CrCovs and HCoV OC43. The answer is: Yes, I would prefer to do this without two stages to build the tree. The problem rests on insufficient computer memory, and this rests on the necessary funding. Using this method of making trees, I am trying to draw the researchers' attention to the need to look for the cause of the existing problems of viral diseases in early phylogenetic history for those viruses without waiting for the financiers to think of allocating more funds for such research. Furthermore, the lack of bootstrap values for some branches is also one problem of the meager funding of research on virus sequencing. Low values here are associated with incomplete statistics on sequences. Simply put, there is not a sufficient number of sequences, there is not an adequate resolution of branches. Is the work clearly and accurately presented and does it cite the current literature? - No. The answer is: I have answered the above comments. Is the study design appropriate and is the work technically sound? - Partly. The answer is: It is a brief report and not the full article. Are sufficient details of methods and analysis provided to allow replication by others? - Partly. The answer is: It is a brief report and not the full article. If applicable, is the statistical analysis and its interpretation appropriate? - Partly. The answer is: It is a brief report and not the full article. Are all the source data underlying the results available to ensure full reproducibility? - Yes. Are the conclusions drawn adequately supported by the results? - No. The answer is: I have answered the above comments. It is a brief report and not the full article. References 1. Li T, Liu D, Yang Y, Guo J, et al.: Phylogenetic supertree reveals detailed evolution of SARS-CoV-2. Scientific Reports. 2020; 10 (1). Publisher Full Text 2. Jaimes J, André N, Chappie J, Millet J, et al.: Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. Journal of Molecular Biology. 2020; 432 (10): 3309-3325 Publisher Full Text 3. Wrapp D, Wang N, Corbett K, Goldsmith J, et al.: Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020; 367 (6483): 1260-1263 Publisher Full Text References for answer 4. A. Rokas, S.B. Carroll More Genes or More Taxa? The Relative Contribution of Gene Number and Taxon Number to Phylogenetic Accuracy. Mol. Biol. Evol. 2005; 22(5):1337–1344. doi:10.1093/molbev/msi121 5. W. F. Basener, J.C. Sanford J. The fundamental theorem of natural selection with mutations. Math. Biol. 2018; 76:1589–1622. https://doi.org/10.1007/s00285-017-1190-x" } ] }, { "id": "93539", "date": "20 Sep 2021", "name": "Peng Zhou", "expertise": [ "Reviewer Expertise Coronavirus", "Bat biology", "pathogen discovery" ], "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\nOverall, the study contributed little to the relevant research area. The phylogeny of betaCoV is clearly demonstrated by several researchers. In addition, the conclusions of this paper are not accurate:\nYou can't simple state if one virus is dangerous or not just based on their position in a tree, or if they are more famous. In fact, even within sarbecovirus that caused two large-scale pandemics, many of them don't infect people. They are not more dangerous to human society than BCoV\n\nI don't understand how the author concludes, \"It confirms the transmission of HECoV from bovine\". If you include more alpha CoV, you are sure to have different opinion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "7178", "date": "22 Sep 2021", "name": "Irina Zyrianova", "role": "Author Response", "response": "Step by step answers. Overall, the study contributed little to the relevant research area. The answer is: If my research has attracted the attention of scholars like you (CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China), I am already glad that it touched your established opinion in the research. The phylogeny of betaCoV is clearly demonstrated by several researchers. The answer is: I wrote in my work: ”Although many phylogenetic analyzes have been reported for S or S1 on a genetic or protein level, no study had been made for all publicly available S1 protein sequences of all known BetaCoVs.” So, it means that I said as you exactly said, but you ignored the fact that new sequences are included that had become available in the first months 2020 after the pandemic caused by BetaCoV SARS-CoV-2. In addition, the conclusions of this paper are not accurate: 1. You can't simply state if one virus is dangerous or not just based on their position in a tree, or if they are more famous. In fact, even within sarbecovirus that caused two large-scale pandemics, many of them don't infect people. They are not more dangerous to human society than BCoV. The answer is: I wrote in my work: \"Thus, we see (Figure 1) that the evolution of BetaCoV S1 proceeds from BCoV, which is not dangerous for humans, to SARS-CoV and SARS-CoV-2, which are especially hazardous for humans.\" Please do not speculate on what is not in my conclusions. I do not simply state about dangerousness of viruses or not just based on their position in a tree, or even because they are more famous. I only said that BCoV is not dangerous for humans, and SARS-CoV and SARS-CoV-2 are especially hazardous for humans. It is based on the fact that SARS-CoV and SARS-CoV-2 cause the epidemic and the pandemic, respectively, but BCoV is not. Or you consider that species of viruses that cause epidemics or even pandemics are not dangerous. You said that \"In fact, even within sarbecovirus that caused two large-scale pandemics, many of them don't infect people. They are not more dangerous to human society than BCoV.\" Yes, it could be, but if some of them become reasons for epidemics or even pandemics, all species of these individual representatives, which could influence human health as SARS-CoV or SARS-CoV-2, become hazardous for humans at all as species. 2. I don't understand how the author concludes, \"It confirms the transmission of HECoV from bovine\". If you include more alpha CoV, you are sure to have a different opinion. The answer is: In my work, conclusions are made solely on the data provided in this work, namely on phylogenetic analysis of BetaCoVs, and not on other genera AlphaCoVs, GammaCoVs, and DeltaCoVs, which have separated evolutionary branches from BetaCoVs and evolution and transmission of the representatives from each of these genera (AlphaCoVs, BetaCoVs, GammaCoVs, and DeltaCoVs) should be investigated first inside each genus separate, and only then to compare them with each other. We can see from the tree in my work that HECoV evolves from BCoV. So, we should conclude that HECoV does not have its own evolution history but from BCoV. Based on the close relationship of cows on farms with humans, there is close natural contact between humans and cows. Consequently, the adaptation of BCoV to humans, with the origin of HECoV, and transmission should be in this process. So, this conclusion about transmission is a consequence of the evolutionary position of this virus and the well-known uncontroversial situation of contact between humans and cows. Other comments are described below: Is the work clearly and accurately presented and does it cite the current literature? Partly. The answer is: What is exactly not clear and accurate? Your answer “Partly” isn't justified. Is the study design appropriate and is the work technically sound? No. The answer is: What is exactly not appropriate in my design? What is not technical? Your answer “No” isn't justified. Are all the source data underlying the results available to ensure full reproducibility? Partly. The answer is: Which data are not available? Your answer “Partly” isn't justified. Are the conclusions drawn adequately supported by the results? No. The answer is: I answered on this above, and consider that answer “No” isn't justified. In conclusion, I consider that the reviewer has done the unsubstantiated and superficial review-report of my work." } ] } ]	1	https://f1000research.com/articles/9-1389
https://f1000research.com/articles/9-1388/v1	03 Dec 20	{ "type": "Software Tool Article", "title": "isa4j: a scalable Java library for creating ISA-Tab metadata", "authors": [ "Dennis Psaroudakis", "Feng Liu", "Patrick König", "Uwe Scholz", "Astrid Junker", "Matthias Lange", "Daniel Arend", "Dennis Psaroudakis", "Feng Liu", "Patrick König", "Uwe Scholz", "Astrid Junker", "Matthias Lange" ], "abstract": "Experimental data is only useful to other researchers if it is findable, accessible, interoperable, and reusable (FAIR). The ISA-Tab framework enables scientists to publish metadata about their experiments in a plain text, machine-readable format that aims to confer that interoperability and reusability. A Python software package (isatools) is currently being developed to programmatically produce these metadata files. For Java-based environments, there is no equivalent solution yet. While the isatools package provides a lot of flexibility and a wealth of different features for the Python ecosystem, a package for JVM-based applications might offer the speed and scalability needed for writing very large ISA-Tab files, making the ISA framework available in an even wider range of situations and environments. Here we present a light-weight and scalable Java library (isa4j) for generating metadata files in the ISA-Tab format, which elegantly integrates into existing JVM applications and especially shines at generating very large files. It is modeled after the ISA core specifications and designed in keeping with isatools conventions, making it consistent and intuitive to use for the community. isa4j is implemented in Java (JDK11+) and freely available under the terms of the MIT license from the Central Maven Repository (https://mvnrepository.com/artifact/de.ipk-gatersleben/isa4j). The source code, detailed documentation, usage examples and performance evaluations can be found at https://github.com/IPK-BIT/isa4j.", "keywords": [ "ISA-Tab", "FAIR data", "reproducible research", "metadata", "Java", "object-oriented programming", "framework" ], "content": "Introduction\n\nIn recent years, the question of how to publish research data has increasingly come into the limelight of discussions among scholars, funders, and publishers1. Wilkinson et al.2 establish a set of principles to ensure that data are shared in a way that is useful to the community and worthwhile for data producers: Data should be findable, accessible, interoperable, and reusable (FAIR) – not only by humans but also by computers. In some scientific fields, there are well-curated, consistent, and strongly integrated databases that provide easy access for both humans and machines, such as Genbank and UniProt for nucleotide and protein sequences3,4. Other areas, like plant phenotyping, do not yet have central databases or established file formats and things become especially difficult when data from different domains need to be published in conjunction. The Investigation-Study-Assay (ISA) framework and the corresponding ISA-Tab file format5 provide a clearly defined, machine-readable, and extensible structure for explanatory metadata that bundles common elements while keeping data in separate files using appropriate formats. Several communities have already created specific standards (such as MIAPPE6 or MIAME7) and infrastructure8 based on the ISA framework. Furthermore, tools have been developed for validating, converting, and manually crafting ISA-Tab metadata7,9,10. However, given the ever-increasing volume of research data generated in high-throughput experiments, the manual creation of metadata is simply not feasible in many situations. A Python package called isatools for programmatically generating ISA-Tab metadata is currently under development (https://isatools.readthedocs.io) featuring methods to parse, validate, build, and convert ISA files. It also offers a feature to create sample collection and assay run templates according to a specified experimental design which can be useful when planning an experiment. Building ISA-Tab files, isatools provides great flexibility and ease of use: users can create and connect ISA objects in arbitrary order and degree of detail and isatools automatically determines the appropriate formatting when the ISA-Tab text is rendered.\n\nNaturally, this flexibility requires isatools to keep the whole object structure in memory and resolve the optimal path through the object chain when the content is serialized. This can notably impact performance when describing large and complex studies including a high number of replicates and attributes, as for instance required by the MIAPPE standard for plant phenotyping experiments. This could make it challenging to use isatools in interactive and time-sensitive applications. Additionally, in the majority of cases, the desired file structure is already clear beforehand based on such community standards or your own decision of what needs to be documented, so this flexibility is often not needed. We therefore set out to develop a solution that focuses on high performance and scalability, and which would integrate well into JVM-based data publishing ecosystems. The library, called isa4j, addresses these goals by providing interfaces for exporting ISA-formatted metadata not only to files, but also to any data stream provided by the application (e.g. a HTTP response stream in a web application) and using an iterative approach for creating ISA-Tab files: Instead of loading all records into memory and writing them in one go, an output stream is opened, a single record is created, flushed out into the stream, and then immediately dropped again from memory. This guarantees memory usage to remain constant so that isa4j imposes no limit on the size of the generated metadata and is able to process datasets too big to fit into memory. The output stream can also be picked up by the application and piped into further processing steps, such as calculating checksums or compressing the ISA-Tab content. In exchange, the user needs to structure rows consistently as headers cannot be modified once they are written. The schema in Figure 1 shows the exemplary integration of isa4j into different application scenarios for supporting the FAIR data sharing paradigm. In this article, we explain how isa4j can be used to generate ISA-Tab metadata and compare it to isatools in performance and scalability regarding both quantity and complexity of ISA-Tab entries.\n\nHeterogeneous data sources like SQL and NoSQL databases, laboratory information management systems (LIMS) and application programming interfaces (API) that store data and metadata of scientific experiments can be fed into isa4j to integrate and transform this data to output complying with the ISA specifications: The isa4j library can, for example, be embedded in command line interface (CLI) applications to create ISA-Tab files in a batch processing manner. It may also be embedded in web services to create ISA-Tab files on the fly via an API based on specific user requirements. ISA-Tab files created with CLI applications could be uploaded to public research data repositories for long-term storage and web applications as graphical user interfaces would allow low-barrier interactive access to experimental data. Both examples demonstrate how isa4j can be used for FAIR data sharing.\n\n\nMethods\n\nisa4j is implemented in Java (JDK11+) and can therefore also be used with other JVM-based languages like Groovy or Kotlin. It uses the Gradle Build Tool (https://gradle.org) to resolve dependencies and create arti-facts. Logging is realized via the framework-agnostic SLF4J library (http://www.slf4j.org/) so that isa4j works with a variety of logging libraries. The object-oriented Java class structure is modelled according to the published ISA specifications (https://isa-specs.readthedocs.io) to make isa4j intuitive to use and keep consistency with other ISA applications. The Ontology and OntologyAnnotation classes allow linking characteristics, units, and other metadata to established vocabularies such as those collected by the OBO Foundry11.\n\nisa4j is not an application itself but a software library providing methods for generating ISA-Tab metadata in JVM-based applications or scripts. As a result, operation requires at least a basic level of coding skills in Java or another JVM-based language. When using a build tool like Maven or Gradle, isa4j can simply be added as a dependency to be downloaded from the Central Maven Repository (https://mvnrepository.com/artifact/de.ipk-gatersleben/isa4j). Otherwise, the JAR file can be downloaded from there and manually included in the class path. To use isa4j’s logging feature, one of the SLF4J bindings needs to be included the same way (http://www.slf4j.org/manual.html).\n\nYou can then import isa4j classes and start building Investigation, Study, and Assay files. For examples and details on the code interface itself, please consult the current project page (https://github.com/IPK-BIT/isa4j) as things may change in future versions and we do not want to confuse you with potentially outdated information.\n\nScalability of isa4j was assessed and compared to the Python isatools API in two dimensions: number of entries and complexity of entries.\n\nAt the simplest complexity level (Minimal), Study file rows consisted only of a Source connected to a Sample through a Process, and that Sample connected to a DataFile through another Process in the Assay File, with no Characteristics, Comments, or other additional information (6 columns in total). At the second degree of complexity (Reduced), a Characteristic was added to the Sample in the Study File, and the Assay File was expanded to include an intermediary Material Object (11 columns). The third and final level of complexity (Real World) was modelled after the MIAPPE v1.1 compliant real-world metadata published for a plant phenotyping experiment (https://doi.org/10.5447/IPK/2020/3, 119 columns). Exemplary ISA-Tab output for each of the three complexity levels can be found at https://ipk-bit.github.io/isa4j/scalability-evaluation.html#complexity-levels.\n\nFor each complexity level, CPU execution time was measured for writing a number of n rows in Study and Assay File each, starting at 1 and increasing in multiplicative steps up to a million rows. Every combination of complexity level and number of rows was measured for 5 consecutive runs in isatools and 15 runs for isa4j (here results varied more) after a warm-up of writing 100 Real World complexity rows. Additionally, memory usage was measured for realistic complexity in 5 separate runs after CPU execution time measurements.\n\nAll evaluations were carried out on a Linux server with two Intel Xeon E5-2697 v2 CPUs running at 2.70 GHz, 256 GB DDR3 RAM running at 1600 MHz and CentOS 7.8.2003. isatools was evaluated under Python 3.7.3 [Clang 11.0.0 (clang-1100.0.33.16)] using isatools version 0.11 and memory-profiler version 0.57 for measuring RAM usage. isa4j was evaluated under AdoptOpenJDK 11.0.5. For both libraries, a memory consumption baseline was calculated after the warm-up runs and an additional Garbage Collector invocation. This baseline consumption was subtracted from all subsequent memory consumption values as we wanted to measure purely the memory consumed by the ISA-Tab content, not libraries and other periphery1. The actual code generating the files and measuring time and memory usage for Python isatools2 and isa4j3 can be found on the isa4j GitHub repository.\n\n\nResults\n\nFigure 2 shows the performance of both libraries at increasing file size for three different levels of complexity. isa4j consistently takes up less CPU execution time than isatools for all tested scenarios, reducing the time required for writing 1 million rows of Real World complexity from 8.6 hours to 43 seconds.\n\nTop: CPU execution time at 3 different row complexity levels; Real World complexity was modelled after MIAPPE v1.1 compliant ISA-Tab metadata generated for a plant phenotyping experiment. Small colored lines on the left mark the highest point of each curve to help estimate the maximum value. Bottom: Memory consumption for isa4j and isatools along the same x-axis.\n\nThe emphasis on being useful especially in large-scale datasets is further amplified by isa4j’s memory usage stability: While there is no notable increase for either library up to a volume of 25 rows, starting at about 250 rows, isatools memory consumption increases linearly with the number of rows being formatted, resulting in a maximum consumption of 15.8 GB for one million rows. isa4j memory consumption remains stable at about 0.5 MB independently of the number of rows written, demonstrating that the iterative technique of formatting and writing the rows had the desired effect.\n\nWe have integrated isa4j into the BRIDGE portal, which is a visual analytics and data warehouse web application hosting data of 22621 genotyped and 9527 phenotyped germplasm samples of barley (Hordeum vulgare L.)12. The underlying data was derived from the study of Milner et al.13. isa4j was integrated to allow the MIAPPE-compliant16 export of customized subsets of phenotypic data of germplasm samples together with the corresponding passport data14 in the ISA-Tab format. These subsets can be derived from germplasm selections identified by the user during exploratory data analysis. In the ISA-Tab export dialog, the user can choose whether the associated plant images should be physically contained as files in the resulting ZIP file or whether they should only be linked as URLs to a version of the images available online. Due to the support of streaming in isa4j, the phenotypic data export module of BRIDGE is able to export large ZIP archives of several gigabytes with low main memory consumption of the web server. Another advantage over non-streaming approaches is that the download can start without delay and that no temporary files have to be created on the server. The process flow concept is shown in Figure 3.\n\nThe BRIDGE web application uses isa4j to create ISA-Tab files from experimental data stored in a relational database management system (RDBMS). The data consists of passport data14 describing the basic characteristics of a germplasm sample (such as accession number and location of origin) on the one hand, and of phenotypic data, which systematically describe phenotypic characteristics of the individual germplasm samples, on the other. Phenotypic images that are stored as binary files on a separate file server are linked from the ISA-Tab files and are included in the final ISA-Tab ZIP archive.\n\n\nDiscussion\n\nWe have created a library for programmatically generating ISA-Tab metadata files in JVM-based environments and shown that it is considerably more performant and scalable than the existing Python based solution. It has been integrated into a large-scale data warehouse web software to validate practical feasibility and provide an example of how the library could help make ISA-Tab metadata available in time-sensitive applications.\n\nCPU execution time appears to have a roughly linear relationship with the number of rows being written at n > 250 but this is only valid as long as isatools memory consumption does not surpass what the system can provide. Exceeding that, additional time for swapping from and to the hard disk will be required. There may also be further non-linear effects due to optimization steps, such as the compilation to native machine code some JVMs perform for frequently used code parts. Lastly, exact CPU time requirements will naturally depend on the specific system in use but the overall relationships and proportions shown here should hold true for all situations.\n\n\nConclusions\n\nThe presented isa4j library provides a simple interface to create and export ISA-Tab metadata and can be seamlessly integrated into existing JVM-based pipelines, desktop tools or web applications. isa4j is less flexible than the Python-based isatools as it does not allow one to change the file structure after streaming has started, but the desired ISA-Tab configuration is often known beforehand, making this a peripheral limitation. In exchange, isa4j provides significantly better performance, especially for large datasets. We hope that this library will make the ISA framework available to an even wider audience and range of situations and help make published research data more interoperable and reusable for others. As a next step, we are going to begin developing a specialized isa4j extension for plant phenotyping experiments, isa4j-miappe, intended to make it even easier for researchers in the field to ensure their metadata comply with the community standard. If you would like to contribute or develop an isa4j extension for your own community, please feel free to get in touch with us.\n\n\nData availability\n\nRaw performance measurement data can be found at https://raw.githubusercontent.com/IPK-BIT/isa4j/master/docs/performance_data.csv (archived: Zenodo, IPK-BIT/isa4j: isa4j-1.0.4, http://doi.org/10.5281/zenodo.427516815).\n\n\nSoftware availability\n\nSoftware available from: https://mvnrepository.com/artifact/de.ipk-gatersleben/isa4j\n\nSource code available from: https://github.com/IPK-BIT/isa4j Archived source code as at time of publication: http://doi.org/10.5281/zenodo.427516815\n\nLicense: MIT", "appendix": "Footnotes\n\n1 Baseline memory consumption was approximately 100 MB for isatools and 11 MB for isa4j.\n\n2 https://github.com/IPK-BIT/isa4j/blob/master/src/test/resources/de/ipk_gatersleben/bit/bi/isa4j/performanceTests/isatools_performance_test.py\n\n3 https://github.com/IPK-BIT/isa4j/blob/master/src/test/java/de/ipk_gatersleben/bit/bi/isa4j/performanceTests/PerformanceTester.java\n\n\nReferences\n\nBarend M: Invest 5% of research funds in ensuring data are reusable. Nature. 2020; 578(7796): 491–491. PubMed Abstract \| Publisher Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJJ, et al.: The FAIR guiding principles for scientific data management and stewardship. Sci Data. 2016; 3(1): 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenson DA, Cavanaugh M, Clark K, et al.: GenBank. Nucleic Acids Res. 2018; 46(D1): D41–D47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe UniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2014; 43(Database issue): D204–D212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSansone S, Rocca-Serra P, Field D, et al.: Toward interoperable bioscience data. Nat Genet. 2012; 44(2): 121–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPapoutsoglou EA, Faria D, Arend D, et al.: Enabling reusability of plant phenomic datasets with MIAPPE 1.1. New Phytol. 2020; 227(1): 260–273. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzález-Beltrán A, Neumann S, Maguire E, et al.: The risa r/bioconductor package: integrative data analysis from experimental metadata and back again. BMC Bioinformatics. 2014; 15 Suppl 1(Suppl 1): S11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaug K, Salek RM, Conesa P, et al.: MetaboLights—an open-access general-purpose repository for metabolomics studies and associated meta-data. Nucleic Acids Res. 2012; 41(Database issue): D781–D786. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocca-Serra P, Brandizi M, Maguire E, et al.: ISA software suite: supporting standards-compliant experimental annotation and enabling curation at the community level. Bioinformatics. 2010; 26(18): 2354–2356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaguire E, Gonzalez-Beltran A, Whetzel PL, et al.: OntoMaton: a bioportal powered ontology widget for google spreadsheets. Bioinformatics. 2010; 29(4): 525–527. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith B, Ashburner M, Rosse C, et al.: The OBO foundry: coordinated evolution of ontologies to support biomedical data integration. Nat Biotechnol. 2007; 25(11): 1251–1255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKönig P, Beier S, Basterrechea M, et al.: BRIDGE - a visual analytics web tool for barley genebank genomics. Front Plant Sci. 2020; 11: 701. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilner SG, Jost M, Taketa S, et al.: Genebank genomics highlights the diversity of a global barley collection. Nat Genet. 2018; 51(2): 319–326. PubMed Abstract \| Publisher Full Text\n\nAlercia A, Diulgheroff S, Mackay M: FAO/Bioversity Multi-Crop Passport Descriptors V.2.1 [MCPD V.2.1]. 2015. Reference Source\n\nPsaroudakis D, Arend D: IPK-BIT/isa4j: isa4j-1.0.4 (Version isa4j-1.0.4). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4275168" }	[ { "id": "77273", "date": "16 Feb 2021", "name": "Massimiliano Izzo", "expertise": [ "Reviewer Expertise Applied Computer Science", "Software Engineering" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 isa4j, an optimised Java-based library to generate and serialise ISA-TAB metadata. I find particularly interesting that isa4j supports writing the ISA-metadata output on streams as well as files, as this can be very useful when building modern client-server applications. isa4j has an interesting approach of loading into memory only one row at the time, hence limiting memory consumption. The memory consumption comparison with isatools makes a good argument for using isa4j for certain large scale experiment.\nI am curious to know whether isa4j-generated ISA-TABs comply with the ISA-TAB validation rules, also with respect to the configuration files for specific assays (latest version can be found here. There are a few discrepancies with respect to the official ISA-TAB specifications: for instance, Processes cannot have names in \"isa4j\" and as a consequence \"Assay Name\" or synonymous columns are missing. Characteristic categories are treated as strings in isa4j, while are OntologyAnnotations in the ISA-API. I might have missed other, minor, discrepancies. I would suggest adding more equivalence tests with existing datasets to align more this library with isatools.\nThe developers don’t seem to have put the tests into continuous integration; I think it would be worth doing so.\nIn any case, I think isa4j is a useful tool with strong performance, that will be very helpful to produce ISA-TAB metadata from a variety of large-scale experiments with noteworthy performances and low resources consumption.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6473", "date": "18 Mar 2021", "name": "Dennis Psaroudakis", "role": "Author Response", "response": "Dear Massimiliano, Thank you for the kind and qualified review! It’s always gratifying when a reviewer actually evaluates the product and doesn’t just limit themselves to the manuscript. isa4j does deviate from the specifications as you’ve described. The reason is that we modeled its output after isatools and apparently Process names and Characteristic Category ontology attributes only show up in ISA JSON output but not in ISA-Tab. Since isa4j can currently only generate ISA-Tab we decided to simplify the model in order not to confuse the user. If you ever do include these attributes in your ISA-Tab output please let us know and we’d be happy to follow suit with isa4j so it stays consistent. Whether isa4j’s output complies with any configuration file will be entirely up to the user, isa4j does not do any validation in that regard so the user needs to make sure his output makes sense. The planned isa4j-miappe extension will hopefully make that easier for plant phenotyping experiments. We do have continuous integration tests with GitHub actions but apparently they were quite well hidden, neither you nor Nils Hoffmann found them. We have added a badge on the README to make them more visible." } ] }, { "id": "79165", "date": "08 Mar 2021", "name": "Nils Hoffmann", "expertise": [ "Reviewer Expertise Bioinformatics", "mass spectrometry of small molecules", "data standardization." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 JAVA-based implementation of the Investigation-Study-Assay (ISA) framework for the structured description of biological experiments, their protocols and their results. They position their implementation as a complement to the existing Python-based implementation that uses a complete in-memory model of the ISA data structures before writing them to the actual output files. For large studies, e.g. for whole populations or large cohorts, this can mean that memory and CPU requirements are very demanding.\nThus, the authors implemented their JAVA library to write out lines as they arrive, requiring that the user fix their data format description before starting to write out to the final files. Therefore, their implementation's memory complexity scales constant with the number of rows to be written, as each row can be created ad-hoc and then written out to the target file. In order to underline this advantage over the Python-based library, the authors created different benchmarks, illustrating the memory and CPU time usage of each library for a collection of different study designs with increasing levels of complexity, highlighting the significant speed and memory advantage of their implementation.\nFinally, the authors demonstrate the practical feasibility of their library through integration into the BRIDGE web portal where they employ isa4j to generate ISA-tab files on the fly for the studies stored in BRIDGE.\nThe support for ISA-tab in programming languages other than Python, especially with a focus on performance, is a timely and needed addition. For JAVA, the graphical client ISACreator was previously developed, but has not seen any significant updates throughout the last few years. Specifically, where neither the flexibility of the Python-based ISA tools nor a graphical user interface for a predefined ISA format are required, the isa4j library can be a valuable, performant, yet still validating tool to generate ISA-tab files in many different life-sciences domain, such as metabolomics, proteomics, genomics, etc. Thus, it addresses a current need and does this in a well designed and performant way.\nMinor comments:\nThe manuscript states, that the library is available from mvnrepository.com, while the GitHub page states that it is available from Maven Central, please update the manuscript accordingly.\n\nThe online documentation to set up a custom isa4j project should also mentions that an slf4j logging implementation needs to be on the class path, e.g. for Gradle: implementation 'org.slf4j:slf4j-simple:1.7.30'\n\nThe documentation at https://ipk-bit.github.io/isa4j/getting-started/investigation-file.html in section \"Add them as investigation contacts\" is syntactically not correct (missing semicolons ';' ), also the method findByName does not exist in version 1.0.4 of the library (getByName does).\n\nI would recommend to set up a continuous integration system linked to the GitHub repository (GitHub actions, Travis, CircleCI, etc.) to make sure that the source code is buildable and automatically tested.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6474", "date": "18 Mar 2021", "name": "Dennis Psaroudakis", "role": "Author Response", "response": "Dear Nils, Thank you for your diligent and very well-structured review, and especially for checking the documentation in such detail; we would probably not have noticed these mistakes ourselves for a long time. We have corrected them and are also linking to mvnrepository.com on the GitHub page now, that seemed easier than submitting a whole new manuscript version just for that one change. We do have continuous integration tests with GitHub actions but Massimiliano Izzo did not see them either, apparently they were quite well hidden. We have added a badge on the README to make them more visible." } ] } ]	1	https://f1000research.com/articles/9-1388
https://f1000research.com/articles/9-1387/v1	02 Dec 20	{ "type": "Review", "title": "Review of the nutritional composition, medicinal, phytochemical and pharmacological properties of Citrus reticulata Blanco (Rutaceae)", "authors": [ "Collen Musara", "Elizabeth Bosede Aladejana", "Silas Mufambi Mudyiwa", "Collen Musara", "Silas Mufambi Mudyiwa" ], "abstract": "Citrus reticulata Blanco is a moderately-sized fruit tree widely used as herbal medicine worldwide. The nutritional composition, medicinal uses, phytochemistry and pharmacological properties of C. reticulata were critically reviewed in the current study. The literature linked to C. reticulata properties was obtained from multiple internet sources including Elsevier, Google Scholar, SciFinder, Web of Science, Pubmed, BMC, Science Direct, and Scopus. Ethnopharmacological research identified antioxidants such as vitamin C, carotenoids and phenolic compounds, also a source of sugars, organic acids, amino acids, pectins, minerals and volatile organic compounds as components of C. reticulata. As a medicinal plant, C. reticulata is used for the treatment of dyspepsia, gastro-intestinal distension, cough with profuse phlegm, hiccup and vomiting. The crude extracts of C. reticulata fruits have depicted anti-inflammatory, anticholesterolemic, analgesic, antiasthmatic, antiscorbutic, antiseptic, antitussive, carminative, expectorant, stomachic. With more people becoming nutrition-conscious, there has been an increase in the demand for the use of citrus fruits and their by-products as traditional medicines for conventional healthcare in developing countries.", "keywords": [ "Citrus reticulata", "medicinal", "nutrition", "pharmacological", "photochemistry" ], "content": "Introduction\n\nCitrus reticulata Blanco is a large species belonging to the family Rutaceae, with various varieties and hybrids1. It includes popular citrus types such as Satsumas, Clementines, Tangerines2 and the Mediterranean mandarin3,4. Tangerine is a group of orange-coloured citrus fruits consisting of mandarin hybrids5, although the term tangerine is used interchangeably with mandarin.\n\nMandarins, like other citrus species, are indigenous to the subtropical and tropical zones of Asia, particularly China and Cochin-China6,7. Some researchers have reported that mandarins, alongside other citrus species, evolved in a region including Vietnam, South China, India and Japan8,9. They are now widely cultivated around the world in the warm temperate and tropical areas8,10–12. Mandarins account for 22–25 per cent of world citrus production among the commercially cultivated citrus species9,13,14. The major citrus growing regions of the world and their estimated output is shown in Figure 1.\n\nThis figure has been reproduced from Mahato et al. under the terms of the Creative Commons Attribution 4.0 International license (CC-BY-4.0)9.\n\nMandarins, including both monoembryonic and polyembryonic cultivars and many interspecific hybrids, are the most diversified category of citrus fruits15,16. Nevertheless, a remarkable similarity has been documented between mandarin cultivars at molecular and isoenzymatic levels17–19. There are 36 mandarin species, according to Tanaka20, while Swingle recognized only three species, one of them being C. reticulata consisted of 34 species of Tanaka’s system21. Chromosome studies have shown that the genus has a stable chromosome number of 2n=18, except for a few polyploids, with a small number of chromosome markers in the conventional karyotype22. The medicinal uses, phytochemistry and pharmacological properties of C. reticulata were examined in the current report.\n\nThe mandarin plant is a spiny, evergreen, bushy shrub growing 2-8 m tall23 with most varieties averaging 7.5 m13. The tree has a dense top with slender branches bearing dark green, lance-shaped leaves with a prominent midrib. Petioles are narrowly winged or slightly margined. This tree bears white scented flowers followed by oval to flattened, sweet-fleshed, golden fruits13. The mandarin fruit resembles other oranges, is smaller and more oblate than oranges, round in shape, orange in colour, sweet in taste, with a thin, loose, easy-to-peel skin and can be easily damaged by cold. The fruit is up to 8 cm in diameter with easily separable segments2.\n\nFlowering is induced through low-temperature stress or soil water deficit stress24,25. Under subtropical climates, flowering is an annual event occurring during spring26–28. In tropical areas, flowering is a continuous event, mostly determined by moisture availability from sufficient rain or water supply27–29 while in temperate regions it occurs with the onset of winter30.\n\n\nMethods and justification of the study\n\nThe literature search was performed from March 2020 to June 2020. A mixed-method review approach which involved combining quantitative and qualitative research was used to compile the review. Information on nutritional composition, medicinal uses, phytochemical and pharmacological properties of C. reticulata was gathered from textbooks, theses and online research articles from databases such as Elsevier, Google Scholar, Scopus, Science Direct, SciFinder, PubMed, BMC, and Web of Science. These data sources were chosen based on the topic covered and the main search key terms included “taxonomy, botany, distribution, nutritional composition, ethnobotanical uses, biological and chemical properties” in relation to C. reticulata. Search terms were set to be in the title, keywords and abstract. To avoid too much filtering of literature, the terms were searched individually. Focusing, on its multipurpose roles, C. reticulata production and utilization can be a catalyst for the development of rural households and community livelihoods. It is therefore imperative to document its nutritional composition, medicinal use and pharmacological properties. With more people becoming nutrition-conscious, demand for citrus fruits and their by-products has grown even in developing countries.\n\nMandarin is a rich source of vitamins C and A, proteins, dietary fibres and essential minerals such as calcium, potassium, phosphorus and magnesium. Also, they contain minute quantities of vitamins B1, B2, B3, B5, B6, B9 and E8,13. On average, 100 g of mandarin orange consists of 85% water (85.2 g), 13% carbohydrates (13.34 g), 0.81 g protein, 0.38 g dietary fibre and 0.31 g fat8,13. Table 1 shows the nutritional composition of raw mandarins.\n\nThe table has been reproduced with permission from Liu et al.8.\n\nThe sugars, acids, carotenoids, polyphenols, limonoids and vitamins in C. reticulata determine the flavour of the fruit. The vitamins, fibre and health-boosting plant compounds like flavonoids provide many health benefits to humans who eat the fruits and related by-products. For example, the vitamin B complex helps prevent infections, helps promote cell health, energy levels, proper nerve functions, hormone and cholesterol production, and cardiovascular health. In particular, mandarin fruits are rich in β-cryptoxanthine, xanthophyll with pro-vitamin A activity31.\n\nMandarins are well-accepted by consumers owing to their pleasant flavours and abundant phytochemicals. With more people becoming nutrition-conscious, there has been a growing demand for citrus fruits like mandarins, and their by-products13.\n\nThe chemical constituents of C. reticulata peel, juice and fruit are shown in Table 2. The peel has high magnesium and carotenoid content32. Methyl-N-methyl anthranilate, a natural antinociceptive compound, has been isolated from mandarin leaves33. Secondary metabolites such as terpenoids, flavonoids and phenolics compounds act as deterrents to insects and microbial attack34.\n\n\nEssence oil and aroma\n\nMandarin is a source of essential oils which are characterized by a fresh-juice fragrance that is widely used in citrus juice products as a natural flavoring agent8,35. The essential oils contain volatile compounds, mainly aldehydes, limonene, ketones, esters, alcohols, terpenes, β-myrcene, 3-carene and α-pinene which provides the distinctive aromas and tastes of citrus fruits1,36,37. Limonene, preceded by γ-terpinene, p-cymene, alpha-pinene and myrcene, is the most abundant compound in mandarin essential oil1,38–43.\n\nThe essential oils are greatly utilized as fragrance materials in beverages, foods, medical formulations, perfumery, toiletries and other cosmetic products44. To some extent, they can also be used as traditional medicine2,9,45. The Chinese use the dried peel of the fruit in the regulation of ch’I (energy/vitality) and to enhance digestion. The leaves and juvenile twigs are a source of essential oil called 'petitgrain oil'23.\n\n\nMedicinal uses\n\nThe edible part of the raw mandarin fruit possesses antioxidants such as vitamin C, carotenoids and phenolic compounds. The fruit is also a rich source of amino acids, sugars, organic acids, amino acids, pectins, minerals and volatile organic compounds46–51. These constituents are essential for the proper functioning of the body by protecting it against chronic diseases and providing basic nutrition52. The dietary fibre and phenolic compounds in mandarins are useful in the formulation of functional foods32. Mandarin fruit also contains coumarins, for instance, bergapten which sensitizes the skin to sunlight53.\n\nThe fruit has been reported to possess laxative, aphrodisiac, antiemetic, astringent and tonic properties54,55 while the fruit peel regulates skin moisture, softens hard and rough skin and cleanses oily skin56. Traditionally, it is also used as a stomachic and carminative56–58. Both the pericarp and endocarp are anticholesterolemic, analgesic, antiseptic, antiasthmatic, anti-inflammatory, antiscorbutic, antitussive, carminative, expectorant and stomachic57,67. Therefore, they are used in the treatment and management of dyspepsia, gastro-intestinal distension, cough with profuse phlegm, hiccup and vomiting58,67. The unripened green exocarp is used in the treatment of chest pains and hypochondrium, gastro-intestinal distension, swelling of the liver and spleen and cirrhosis of the liver. The seed is analgesic and carminative, thus used in the treatment of hernia, lumbago, mastitis and pain or swellings of the testes67.\n\n\nPharmacological properties of C. reticulata\n\nThe ethanolic extract of mandarin fruit shell, a traditional herbal medicine used for gastric ulcer treatments in China, showed activity against five clinical strains of Helicobacter pylori at the minimum inhibitory concentration (MIC) close to 60 µg/mL68. The essential oil of this plant has shown antimicrobial activity with the zone of inhibition varying from 9.16 to 27.63 mm against Escherichia coli, Listeria innocua, Methicillin-Resistant Staphylococcus aureus, S. aureus and Candida albicans43. In a comparative study, the peel ethanol extract of C. reticulata inhibited the growth of all the Gram-positive bacteria tested, with the highest zone of inhibition of 20.33 ± 1.527 mm against Bacillus spp. However, the juice extract showed more activity against the Gram-negative bacteria with a maximum zone of inhibition of 11.33 ± 1.154 mm against Klebsiella pneumonia69. Zainab et al.70 also reported that the peel extract of C. reticulata exhibited a high zone of inhibition against S. aureus (28 mm) while E. coli, S. typhi and P. aeruginosa showed resistance to the peel extracts. The presence of flavanones in the peel of C. reticulata could be responsible for the efficacy of the peel extract than that of the juice71. Yashaswini and Arvind72 carried out a study to determine the antibacterial potential of C. reticulata var. Kinnow peel extracts against pathogenic strains of S. aureus, E. coli, P. aeruginosa and K. pneumonia. The acetone extract inhibited the growth of K. pneumonia and E. coli with a MIC value of 68.75 µg/mL and maximum zone of inhibition of 7.93 mm and 7.75 mm against K. pneumonia and E. coli respectively.\n\nSultana et al.57 have reported that the volatile oil of C. reticulata peel possesses antimicrobial activities against Escherichia coli, Staphylococcus aureus, Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, and Candida albicans. Thus, suggested that the volatile oil could be useful for the treatment of skin disorders and the therapy can be incorporated into the cosmetic formulation. Based on the findings of this review, the essential oil, juice and peel extracts of C. reticulata may possess beneficial antibacterial agents that can be exploited in controlling unwanted bacterial infections. The peel oils of mandarins exhibit toxic insecticidal and antibacterial properties8.\n\nKang et al.73 have reported that the methanol extracts (100 g/mL) of C. reticulata peel showed increased apoptosis on SNU-C4, human colon cancer cells through Bax-related caspase-3 activation, thus, suggested the use of C. reticulata on colon cancer patients. In an in vitro study, two flavone glucosides isolated from the mandarin fruit peel showed differentiation-inducing activity in mouse myeloid leukaemia cells (M1), and the cells exhibited phagocytic activity74. In addition, hexane and dichloromethane bark extracts of C. reticulata assayed against human lung adenocarcinoma cell line A549, human breast adenocarcinoma cell line MCF7, human Caucasian prostate adenocarcinoma cell line PC3, and one normal human prostate cell line PNT2 revealed that the extracts possess good apoptosis-inducing activity against the human cancer cell lines. Thus, the authors concluded that the hexane or dichloromethane extract of the bark of C. reticulata is a good crude drug treatment against lung, breast and prostate cancer. However, further in vitro and in vivo testing would be required before any recommendation of this drug can be given75.\n\nThe anticancer potential of Citrus medica (2 morphotypes), C. sinensis, C. maxima, C. limon and C. reticulata peels were investigated using in vitro assays and in vivo cancer models76. The finding depicted that both the extracts and EOs of C. reticulata peels had significant activity against Dalton’s Lymphoma Ascites (DLA) cell line in an MTT assay. The peel oil showed 91.9% and 100% cell death at 25 and 50 µg/mL, respectively, while the water extract showed 49.8% cell death at 5 µg/mL and 100% cell death at 25 and 50 µg/mL respectively. The in vivo study revealed that mice pre-treated with C. reticulata peel extract were significantly (50%) protected from DLA compared to post-treated mice (33%) without any obvious toxic symptoms. The volatiles (essential oils, limonoids) and non-volatiles (mainly polymethoxy flavones) in Citrus peels have been recognized as their bioactive/anticancer constituents76,77.\n\nThe antiproliferative activity of limonoids extracted from C. reticulata was evaluated against a series of human cancer cell lines62. Limonoids exhibited significant growth inhibitory effects at high concentration of 100 µg/mL against human breast cancer cell lines (MCF-7). However, it could not inhibit leukaemia (HL-60), ovary (SKOV-3), cervix (HeLa), stomach (NCI-SNU-1) and liver (Hep G2) cancer cells lines62.\n\nGbaj et al.78 evaluated the anxiolytic potentials of methanol and aqueous peels extracts of C. reticulata in Libya using an elevated plus-maze. The result revealed that the peel extracts exhibited significant anxiolytic activity. In addition, the anxiolytic effect of naringin has been confirmed in 6-8 weeks old mice weighing 30 to 35 g79.\n\nHassan et al.64 investigate the protective effect of the ethanol extract of the aerial part of C. reticulata cultivated in Saudi Arabia against genotoxicity induced by benzo(a)pyrene (BaP) in mice using the comet assay. In the mice treated with BaP, there was a significant increase in the DNA fragmentation in the liver tissues of male mice and an increased rate of DNA damage in mice blood cells. However, the liver and blood cells of the mice treated with ethanol extract demonstrated significant protection by inhibiting the rate of DNA damage. It was concluded that the aerial part of C. reticulata could be useful to reduce the genotoxicity induced by hazardous chemical agents64. The presence of flavonoid compounds and various secondary metabolites could be responsible for the protective effect, pharmacological and therapeutic properties of C. reticulata80–82.\n\nBoudries et al.43 investigated the antioxidant activities of C. reticulata, C. reticulata cultivar Wilking and C. clementine from Algeria using 1,1-diphenyl-2-picrylhydrazil (DPPH) and reducing power. The essential oil (EO) of C. reticulata exhibited the strongest DPPH free radical-scavenging activity in a dose-dependent manner, followed by clementine and wilking EOs. Also, in a concentration-dependent manner, the EO of C. reticulata showed the greatest reducing power followed by wilking and clementine EOs. According to Junior et al.83, the antioxidant nature of the citrus essential oils in terms of free radical scavenging may be due to the antioxidant activity of limonene, which was the main constituent of the oil. The peel of C. reticulata was evaluated for antioxidant activity, the results displayed prominent, concentration-dependent free-radical scavenging activity on stable DPPH free radicals and reactive hydroxyl radicals84.\n\nAlso, the fruit peel of C. reticulata, Zingiber officinale and Sesamum indicum were investigated for their antioxidant activities using the DPPH radical scavenging technique. The findings revealed that Z. officinale had the highest antioxidant activity followed by C. reticulata, and S. indicum. The antioxidant activity of these plants could be attributed to a wide variety of constituents, such as the flavonoid content which are considered as major biological antioxidants85.\n\nRincon et al.32 suggested the use of tangerine peel in reducing the risk of cardiovascular diseases and some associated with lipid oxidation.\n\nThe protective effect of the essential oils of C. reticulata on isoniazid induced hepatotoxicity in Wistar rats was investigated86. About 50 gm/kg, p.o. of isoniazid was administered for 30 days in order to induced liver damage in the rats. A total of 200 mg/kg, p.o.of the essential oil was administered daily for 30 days, while the standard group received Liv5286. The result revealed a significantly elevated level of ALT, AST, bilirubin and a decreased total protein content in the rats treated with only isoniazid as compared to the group that do not received isoniazid. However, a significant reduction in all the biochemical parameters was observed in the rats treated with the essential oil and Liv5286.\n\nIn an in vitro study, the effect of tangeretin (a flavonoid isolated from tangerine juice) on hydroxylation of midazolam, a CYP3A4 probe was evaluated using human liver microsomes and recombinant CYP3A4. The finding revealed that tangeretin is a potent and regioselective stimulator of midazolam 1'-hydroxylation and complementary DNA-expressed CYP3A487. However, further studies are required as the authors have indicated that tangerine juice might not have a clinical effect on CYP3A-mediated drug metabolism in humans.\n\nOmer et al.65 investigated the antihypercholesterolemic potential of the crude ethanolic extracts of C. reticulata fruit peel in an in vivo study. The findings revealed that daily administration of 250 mg/kg and 500 mg/kg doses of the extracts to the albino rats for four weeks produced a reduction in serum low-density lipoprotein-cholesterol, total cholesterol and triglycerides levels. Also, a significant elevation in serum high-density lipoprotein-cholesterol was observed, thus, indicating their cardioprotective effects and potential as therapeutic antihypercholesterolemic agents65. Hence, the efficacy of C. reticulata peels extracts on the observed lipid profile parameters might be attributed to the presence of polymethoxylated flavones which occur in the fruit peels.\n\nAccording to a study by Apraj and Pandita66, both hot and cold alcoholic extracts of C. reticulata exhibited strong anti-collagenase and anti-elastase activity, indicating its anti-ageing ability. However, further study is required to determine whether the extracts can be incorporated into skincare products as anti-wrinkle agents.\n\nThe oral administration of C. reticulata extracts up to 200 mg/kg has been reported to be safe78. Also, Li et al.68 reported that a single oral dose of 16 g/kg of naringin does not produce acute oral toxicity in rats.\n\nWorldwide, the mandarin fruit is eaten as fresh produce88. It is peeled and eaten plain, used in salads, desserts and main dishes or cooked in puddings, cakes and confectionery. The peel, pulp and seeds are often discarded as waste or they can be processed into animal feed88.\n\nMany by-products, including pectin, dried pulp, molasses, marmalades, candied peel, peel seasoning, purees, beverage bases, alcohol, bland syrup, citric acid, seed oil and flavonoids can be obtained from mandarin fruits4,36,89. The dried peel or rind has a sweet-spicy flavour often used as a flavouring in cakes4 or as a spice for cooking, baking, drinks or candy90. Rind powder extract, a rich source of phenolic compounds having free radical scavenging activity can be used as an anti-oxidant in meat products88.\n\n\nConclusion\n\nC. reticulata is an important plant which contains some compounds and nutritional values that are of great health importance. The fruit is rich in antioxidants and phenolic compounds, sugars, organic acids, amino acids, pectins, minerals and volatile organic compounds. These substances are essential for the proper functioning of the body by protecting it against chronic diseases, provides basic nutrition and useful in the formulation of functional foods. Based on research carried out, the fruits and peels of C. reticulata have been reported to possess neuropharmacological, hepatoprotective, anticancer, antimicrobial, antigenotoxicity, antioxidant activities, antihypercholesterolemic and cardiovascular effects. The usage of C. reticulata was shown to be broad, ranging from dietary applications to the treatment of terminal medical conditions, thus, it is imperative to carry out more research on the toxicity of this plant. Since the peels contain bioactive constituents of pharmacological importance, further investigations should be conducted to investigate whether it could be boiled and consumed orally, establish dosage ranges for safe consumption and evaluate target-organ toxicity.\n\n\nData availability\n\nNo data are associated with this article", "appendix": "References\n\nSawamura M, Thi Minh Tu N, Onishi Y, et al.: Characteristic odor components of Citrus reticulata Blanco (ponkan) cold-pressed oil. Biosci Biotechnol Biochem. 2004; 68(8): 1690–1697. PubMed Abstract \| Publisher Full Text\n\nBoughendjioua H, Boughendjioua Z: Chemical composition and biological activity of essential oil mandarin (Citrus reticulata) cultivated in Algeria. Int J Pharm Sci Rev Res. 2017; 44(1): 179–184. Reference Source\n\nOrtiz JM: Botany: Taxonomy, morphology and physiology of fruits, leaves and flowers. In: Dugo G and Di Giacomo A. Citrus: The genus Citrus. New York:Taylor and Francis; 2002; 16–35. 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Flavours and fragrances: Chemistry, bioprocessing and sustainability. Berlin: Springer-Verlag; 2007; 43–86.\n\nImbesi A, De Pasquale A: Citrus species and their essential oils in traditional medicine. In: Dugo G andDiandDi Giacomo A. Citrus: The genus Citrus. New York: Taylor and Francis; 2002; 577–601.\n\nYe XQ, Chen JC, Liu DH, Jiang P, et al.: Identification of bioactive composition and antioxidant activity in young mandarin fruits. Food Chem. 2011; 124(4): 1561–6. Publisher Full Text\n\nMatsumoto H, Ikoma Y: Effect of different postharvest temperatures on the accumulation of sugars, organic acids, and amino acids in the juice sacs of Satsuma mandarin (Citrus unshiu. Marc) fruit. J Agric Food Chem. 2012; 60(39): 9900–9. PubMed Abstract \| Publisher Full Text\n\nZhang YM, Sun YJ, Xi WP, et al.: Phenolic compositions and antioxidant capacities of Chinese wild mandarin (Citrus reticulata. Blanco) fruits. Food Chem. 2014; 145: 674–80. PubMed Abstract \| Publisher Full Text\n\nRodrigo MJ, Cilla A, Barbera R, et al.: Carotenoid bioaccessibility in pulp and fresh juice from carotenoid-rich sweet oranges and mandarins. Food Funct. 2015; 6(6): 1950–9. PubMed Abstract \| Publisher Full Text\n\nAntoine S, Pailly O, Gibon Y, et al.: Short- and long-term effects of carbohydrate limitation on sugar and organic acid accumulation during mandarin fruit growth. J Sci Food Agric. 2016; 96(11): 3906–14. PubMed Abstract \| Publisher Full Text\n\nChen DL, Huang YR, Liang HL, et al.: Column chromatographic extraction for quickly separating the volatiles, flavonoids, and pectin from tangerine peel. Sep Sci Technol. 2016; 51(3): 485–93. Publisher Full Text\n\nAl-Snafi AE: Nutritional value and pharmacological importance of citrus species grown in Iraq. IOSR J Pharm. 2016; 6(8): 76–108. Publisher Full Text\n\nBown D: Encyclopaedia of herbs and their uses. London: Dorling Kindersley; 1995. Reference Source\n\nChopra RN, Nayar SL, Chopra IC: Glossary of Indian medicinal plants (Including the Supplement). New Delhi: Council of Scientific and Industrial Research; 1986.\n\nAnonymous: The wealth of India: A dictionary of Indian raw materials and industrial products. New Delhi: National Institute of Science Communication and Information Resources; 2000; 65–69. Reference Source\n\nKhan MA, Ali M, Alam P: Phytochemical investigation of the fruit peels of Citrus reticulata Blanco. Nat Prod Res. Formerly Natural Product Letters. 2010; 24(7): 610–620. PubMed Abstract \| Publisher Full Text\n\nSultana HS, Ali M, Panda BP: Influence of volatile constituents of fruit peels of Citrus reticulata Blanco on clinically isolated pathogenic microorganisms under in–vitro. Asian Pac J Trop Biomed. 2012; 2(3): S1299–S1302. Publisher Full Text\n\nApraj VD, Pandita NS: Evaluation of skin anti-aging potential of Citrus reticulata Blanco peel. Pharmacognosy Res. 2016; 8(3): 160–168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWingerath T, Stahl W, Sies H: Beta-Cryptoxanthin selectively increases in human chylomicrons upon ingestion of tangerine concentrate rich in beta-cryptoxanthin esters. Arch Biochem Biophys. 1995; 324(2): 385–390. PubMed Abstract \| Publisher Full Text\n\nGranado F, Olmedilla B, Blanco I, et al.: Major fruit and vegetable contributors to the main serum carotenoids in the Spanish diet. Eur J Clin Nutr. 1996; 50(4): 246–250. PubMed Abstract\n\nIrwig MS, El Sohemy A, Baylin A, et al.: Frequent intake of tropical fruits that are rich in beta-cryptoxanthin is associated with higher plasma beta-cryptoxanthin concentrations in Costa Rican adolescents. J Nutr. 2002; 132(10): 3161–3167. PubMed Abstract \| Publisher Full Text\n\nTian Q, Miller EG, Ahmad H, et al.: Differential inhibition of human cancer cell proliferation by citrus limonoids. Nutr Cancer. 2001; 40(2): 180–184. PubMed Abstract \| Publisher Full Text\n\nBalamurugan P, Rajkumar AR, Prasad MP: Comparative phytochemical analysis of Rutaceae family (Citrus Species) extracts. Int J Sci. 2014; 148–150. Reference Source\n\nHassan AZ, Ahmed KM, Abu-Gabal NS, et al.: Phytochemical and genotoxicity studies of Citrus reticulata aerial part in mice. Egyptian Pharmaceutical Journal. 2017; 16(2): 87. Publisher Full Text\n\nOmer SS, Elsiddig IM, Mohammed AE, et al.: Phytochemical screening, antioxidant activity and lipid profile effects of Citrus reticulata fruit peel, Zingiber officinale. Rhizome and Sesamum indicum seed extracts. Intern J Medical Health Biomed Bioeng Pharm Eng. 2015; 9: 797–804. Reference Source\n\nApraj VD, Pandita NS: Pharmacognostic and phytochemical evaluation of Citrus reticulata Blanco peel. Int J Pharmacogn Phytochem Res. 2014; 6(26): 328–331. Publisher Full Text\n\nYeung HC: Handbook of Chinese herbs and formulas. Los Angeles: Institute of Chinese Medicine; 1985; 1. Reference Source\n\nLi Y, Xu C, Zhang Q, et al.: In vitro anti-Helicobacter pylori. action of 30 Chinese herbal medicines used to treat ulcer diseases. J Ethnopharmacol. 2005; 98(3): 329–333. PubMed Abstract \| Publisher Full Text\n\nShakya A, Luitel P, Kumari R, et al.: Comparative study of antibacterial activity of juice and peel extract of citrus fruits. Tribhuvan University J Microbiol. 2019; 7(6) : 82–8. Publisher Full Text\n\nZainab AGC, Kadhim NK: Antimicrobial activity of different aqueous lemon extracts. J Appl Pharm Sci. 2013; 3(6): 74–78. Reference Source\n\nLevaj B, Dragovic V, Bursac D, et al.: Determination of flavonoids in pulp and peel of mandarin fruits. Agric Conspec Sci. 2009; 74(3): 221–225. Reference Source\n\nYashaswini Y, Arvind: Antimicrobial Properties of Orange (Citrus reticulata var. Kinnow). Peel Extracts against Pathogenic Bacteria. Int J Curr Microbiol App Sci. 2018; 7(3): 737–746. Publisher Full Text\n\nKang SA, Park HJ, Kim MJ, et al.: Citri reticulataeViridereticulataeViride pericarpium extract induced apoptosis in SNU-C4, human colon cancer cells. J Ethnopharmacol. 2005; 97(2): 231–235. PubMed Abstract \| Publisher Full Text\n\nSugiyama K, Umehara M, Kuroyanagi A, et al.: Studies on the differentiation inducers of myeloid leukemic cells from citrus species. Chem Pharm Bull (Tokyo). 1993; 41(4): 714–719. PubMed Abstract \| Publisher Full Text\n\nPLANTMEDS: Potential antimalarial and anticancer lead compound discovery from Cameroonian medicinal plants. 2018. Reference Source\n\nNair A, Kurup SRR, Nair AS, et al.: Citrus peels prevent cancer. Phytomed. 2018; 50: 231–237. Publisher Full Text\n\nKoolaji N, Shammugasamy B, Schindeler A, et al.: Citrus peel flavonoids as potential cancer prevention agents. Curr Dev Nutr. 2020; 4(5): nzaa025. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGbaj MA, Sadawe IA, Meiqal NM, et al.: Evaluation of neuropharmacological activities of methanolic and aqueous extracts of Citrus reticulata (Rutaceae) fruit peels. Am J Biomed Sci Res. 2019; 2(4): 131– 135. Publisher Full Text\n\nFernandez SP, Nguyen M, Yow TT, et al.: The flavonoid glycosides, myricitrin, gossypin and naringin exert anxiolytic action in mice. Neurochem Res. 2009; 34(10): 1867–1875. PubMed Abstract \| Publisher Full Text\n\nBorokini TI, Omotayo FO: Phytochemical and ethnobotanical study of some selected medicinal plants from Nigeria. J Med Plant Res. 2012; 6(7): 1106–1118. Publisher Full Text\n\nBoligon AA, Sagrillo MR, Machado LF, et al.: Protective effects of extracts and flavonoids isolated from ScutiabuxifoliaReissek against chromosome damage in human lymphocytes exposed to hydrogen peroxide. Molecules. 2012; 17(5): 5757–5769. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl-Rafie HM, Mohammed RS, Hamed MA, et al.: Phytochemical and biological studies of total ethanol and petroleum ether extracts of Terminalia bentzoe. (L.) leaves. Int J Pharmacogn Phytochem Res. 2016; 8: 592–603. Reference Source\n\nJunior EF, Souza P, Nascimento J, et al.: Antinociceptive and antiinflammatory properties of the ethanolic extract of Pouteria ramiflora. roots. Latin Amer J Pharm. 2009; 28(6): 812–818. Reference Source\n\nTumbas VT, Ćetkovic GS, Đilas SM, et al.: Antioxidant activity of mandarin (Citrus reticulata) peel. Acta Periodica Technologica. 2010; 41: 195–203. Publisher Full Text\n\nBravo L: Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutr Rev. 1998; 56(11): 317–333. PubMed Abstract \| Publisher Full Text\n\nKangralkar VA, Gavimath CV, Venkatesh V, et al.: Protective effect of essential oils of Citrus reticulata on isoniazid induced hepatotoxicity in wistar rats. Intern J Pharm Appl. 2010; 1(2): 59–61.\n\nBackman JT, Maenpaa J, Belle DJ, et al.: Lack of correlation between in vitro and in vivo studies on the effects of tangeretin and tangerine juice on midazolam hydroxylation. Clin Pharmacol Ther. 2000; 67(4): 382–390. PubMed Abstract \| Publisher Full Text\n\nRafiq R, Kaul SA, Sofi N, et al.: Nayik Citrus peel as a source of functional ingredient: A review. J Saudi Soc Agric Sci. 2018; 17: 351–358.\n\nBraddock RJ: By-products of citrus fruit. Food Technol. 1995; 49(9): 74–77. Reference Source\n\nMorton JF: Mandarin orange. In: Fruits of warm climates. Miami; 1987. Reference Source" }	[ { "id": "75652", "date": "26 Jan 2021", "name": "Uma Rani Sinniah", "expertise": [ "Reviewer Expertise Plant science on agronomy", "biochemistry and phytochemisty" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well written for the purpose intended. All the subject matter namely nutritional composition, medicinal, phytochemical and pharmacological properties as highlighted in the title of this article has been adequately reviewed and cited.\nSecondary metabolites in plants are of major interest and are often taught in plant biochemistry courses. The content of secondary metabolites differ in different plant species and the use of such plant phytochemical for various purposes such as antibacterial, antimicrobial, anti cancer, anti-inflammatory etc, also differ among the plant species. This paper provides a comprehensive outlook and will be a good specimen to demonstrate the types of secondary metabolites present and its bioactive compounds in relation to pharmacological activities. The article can be a one stop reference on the properties of Citrus reticulata up to 2020.\nJust a few minor typos are present, which are highlighted here.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "6305", "date": "26 Jan 2021", "name": "Elizabeth Bosede Aladejana", "role": "Author Response", "response": "Thank you for taking the time to evaluate our article. All the grammatical errors would be corrected. Thank you." } ] }, { "id": "100915", "date": "13 Jan 2022", "name": "Maan B. Rokaya", "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\nManuscript is relevant for indexing, but it needs major changes. Please see the comments below:\nAbstract\nAuthors have not mentioned why this review is necessary. Is it because this species is important as medicine, food or any other things? Please write aims.\n\nIn the abstract, mention how many compounds were found until now. How many diseases are treated? What pharmacological studies are carried out?\n\nWhat is the conclusion of your review, which is quite not clear in abstract?\nIntroduction\nI suggest authors to start with importance of plant species as medicine and food. Then mention about Citrus reticulata which is used as food and medicine. Mention some figures about uses, production and trade. Then in separate section mention about varieties in brief (too much detail is not required). Introduction should be followed by aims of your study. I guess it will be wise to see some good review papers if you want to write up to the indexable standard.\n\nBotanical description is not very necessary but if you want to keep, make it brief.\n\nJustification of the study should be at the end of Introduction not as separate section.\nMethod\nLiterature review was from March 2020-June 2020. Rather than this, mention how many literatures were considered and mention the oldest and newest literature. Again, please see some review papers to write your text in indexable way.\nNutritional composition\nWere these components from the same species or from same species but with different varieties? Need to mention this very clearly.\n\nTable 1 is redundant.\nChemical constituents, oil and aroma\nNot in detail and needs much more elaboration. From which plant parts were they extracted? Mention in detail. I know it is mentioned in Table 2 but it should be written in detail. Please categorize compounds into different groups so that it will be easy for readers to follow.\n\nIn the beginning mention how many compounds have been isolated so far.\n\nGive chemical structures of all the compounds.\n\nAntioxidant property is considered as property with no pharmacological relevance (Gafner, 2018). So, discuss according to this.\nPharmacological properties\nIn this section, only information about extracts and results are mentioned, which is not enough. It is also necessary to write about what methods were used. The doses, type of experimental animal/cell/bacteria, controls (positive/negative if any) are required. Please write results in detail but remember that it should be concise. Also make a table to show all these. Please follow some good review papers on specific plant species published elsewhere and to help improve your manuscript.\n\nConclusions should directly link to your findings. Write about its importance and future prospective. What are the study gaps and what more is needed?\nOverall, this manuscript should undergo Major revision.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1387
https://f1000research.com/articles/9-1094/v1	04 Sep 20	{ "type": "Research Article", "title": "Biocuration - mapping resources and needs", "authors": [ "Alexandra Holinski", "Melissa L. Burke", "Sarah L. Morgan", "Peter McQuilton", "Patricia M. Palagi", "Alexandra Holinski", "Melissa L. Burke", "Sarah L. Morgan" ], "abstract": "Background: Biocuration involves a variety of teams and individuals across the globe. However, they may not self-identify as biocurators, as they may be unaware of biocuration as a career path or because biocuration is only part of their role. The lack of a clear, up-to-date profile of biocuration creates challenges for organisations like ELIXIR, the ISB and GOBLET to systematically support biocurators and for biocurators themselves to develop their own careers. Therefore, the ELIXIR Training Platform launched an Implementation Study in order to i) identify communities of biocurators, ii) map the type of curation work being done, iii) assess biocuration training, and iv) draw a picture of biocuration career development. Methods: To achieve the goals of the study, we carried out a global survey on the nature of biocuration work, the tools and resources that are used, training that has been received and additional training needs. To examine these topics in more detail we ran workshop-based discussions at ISB Biocuration Conference 2019 and the ELIXIR All Hands Meeting 2019. We also had guided conversations with selected people from the EMBL-European Bioinformatics Institute. Results: The study illustrates that biocurators have diverse job titles, are highly skilled, perform a variety of activities and use a wide range of tools and resources. The study emphasises the need for training in programming and coding skills, but also highlights the difficulties curators face in terms of career development and community building. Conclusion: Biocurators themselves, as well as organisations like ELIXIR, GOBLET and ISB must work together towards structural change to overcome these difficulties. In this article we discuss recommendations to ensure that biocuration as a role is visible and valued, thereby helping biocurators to proceed with their career.", "keywords": [ "Biocuration", "Career development", "Training needs", "Biocuration tools", "Bioinformatics", "Life Sciences" ], "content": "Introduction\n\nTogether with bioinformatics, curated databases have become an essential part of modern molecular biology. Databases such as Ensembl1, COSMIC2 and PomBase3 are accessed daily by thousands of people worldwide, illustrating that well-structured life science data, available to all in public repositories, are fundamental to research. Often, these biological databases are annotated by biocurators whose work, in a simplified explanation, is to i) collect scientific data, ii) verify and validate the information collected, iii) add value by structuring it in a logical, consistent and relevant manner and iv) integrate it into databases. Bourne and McEntyre4, in their homage to the profession, considered biocurators as the “museum cataloguers of the Internet age”. We believe biocurators are more than this, they are integral to the modern life sciences and pivotal to good data management. They are the guardians of the integrity and FAIRness of life sciences data.\n\nThe art and science of biocuration helps make data Findable, Accessible, Interoperable and Reusable (FAIR)5; it places data into context, makes it more interoperable, adding unique identifiers, licences, and structured descriptions alongside other appropriate metadata. In this respect, biocuration shares some aspects of data stewardship, but there are also some differences. Data stewardship involves improving the FAIRness of a dataset, but doesn’t include adding value to the data, via curation and integration. In many ways, biocuration begins where data stewardship finishes, with data stewards helping researchers to prepare their data for biocurators to add value through integration into appropriate databases.\n\nThe European life sciences infrastructure for biological information (ELIXIR; https://elixir-europe.org) is an inter-governmental organisation that brings together bioinformatics resources across Europe and helps researchers to find, analyse, and exchange life science data. In 2016, the ELIXIR Data Platform put in place a process to identify European data resources that are of fundamental importance to research in the life sciences6. Many of these resources are manually curated and ELIXIR recognises the substantial added value that this brings to these resources. Manual biocuration is a highly valued task, and the biocuration efforts of a resource are an important indicator of its quality.\n\nThe International Society for Biocuration (ISB; https://www.biocuration.org) counts over 250 members working in over 82 institutions around the globe. The 27th annual Nucleic Acids Research database issue and molecular biology database collection lists 1613 databases, including 59 new databases7. Biocurators not only work in well-known databases but also in ad-hoc, often behind the scenes projects where biocuration is needed, be they in academia, non-governmental organisations or private companies. A large, diverse body of individuals and teams are involved in biocuration across ELIXIR. It is anticipated that a number of these may not identify themselves as biocurators per se – this could be due to them being unaware of this as a position or career path, or that biocuration is not the main aspect of their job. In this article, we use a broad definition of ‘biocurator’ to encompass anyone who carries out biocuration tasks as part of their work regardless of whether it is the primary focus of their role.\n\nThe ELIXIR Training Platform (TrP), one of the five infrastructure pillars of ELIXIR, aims to strengthen national bioinformatics programmes, grow bioinformatics capacity and competences across ELIXIR member states and empower researchers to use ELIXIR’s services and tools (https://elixir-europe.org/platforms/training). A large number of biocurators work within the ELIXIR Nodes, and the ELIXIR TrP requires a clear picture of who they are, their profiles, what resources they work on, the tools that they use and, in particular, their training needs. The last survey of biocurators was conducted several years ago in 2011, and in an international context where ELIXIR and the Global Organization for Bioinformatics Learning, Education and Training (GOBLET, https://www.mygoblet.org) did not yet exist. An up-to-date profile of the landscape of biocuration would allow these organisations and employers to take action to recognise and value biocuration, and to plan future training.\n\nIn this research article we describe the outcomes of an ELIXIR Biocuration Implementation Study that aims to i) identify communities of biocurators within ELIXIR and worldwide, ii) review and map the kind of biocuration work being done, for which databases and life science/health domains, iii) assess the capacity requirements for new biocurators and training provision available, and iv) draw a picture of the biocuration career development. We outline a set of recommendations to ensure that the biocuration career path can be even more valued. We urge the community to ensure that the knowledge and skills offered by existing biocurators are shared across ELIXIR Nodes and beyond, and to develop training so it is available to those areas and disciplines where biocurators needs are in highest demand.\n\n\nMethods\n\nIn order to map the landscape of biocuration, we first ran a pilot survey with staff at the Wellcome Genome Campus, near Cambridge, UK. The pilot was used to test the survey questions with a smaller audience so that they could be fine-tuned to enable us to undertake the required analysis. This pilot included questions (see Extended data: Pilot survey questions8) about the nature of the respondents’ work (job title, location and type of organisation), the tools and resources used in their day-to-day work, training that the respondents have received, and additional training needs. It was sent to all campus staff as we had the specific aim of capturing individuals who may not identify as biocurators but who carry out biocuration-related tasks as a part of their role. The pilot survey was open between 12 December 2018 and 18 January 2019 with reminders sent ten days and one day before the final deadline.\n\nAdditionally, we met with four EMBL-European Bioinformatics Institute (EMBL-EBI) biocurators who had replied to the pilot survey to discuss their responses in more detail. The conversations were carried out in person by Alexandra Holinski and Melissa Burke at EMBL-EBI and took the form of a guided conversation (see Extended data: Questions to guide conversations with biocurators8) with answers captured and transcribed directly, rather than recording and transcribing later.\n\nThe final survey (see Extended data: Global survey questions8) was a revised version of the pilot. Revisions were made where questions led to overly general and broad answers and to introduce relevant topics raised in the conversations. This survey was disseminated globally by making use of Wellcome Genome Campus, ELIXIR, International Society for Computational Biology (ISCB), ISB, the SIB Swiss Institute of Bioinformatics and GOBLET mailing lists. It was also publicised via Twitter and LinkedIn with reminders and retweets sent at regular intervals. To reach biocurators in industry, the survey was promoted at the EMBL-EBI Industry Programme quarterly meeting and was disseminated through their mailing list. This global survey was open between 4 March 2019 and 1 May 2019. Both the pilot and global survey were conducted online using SurveyMonkey (https://www.surveymonkey.co.uk).\n\nTwo workshops were organised to present and discuss the results of the global survey. The workshops were designed to actively capture the participants’ feedback on the survey results and their thoughts on several key questions. In both workshops attendees were asked to note down their answers to the questions on post-it notes that were collected on whiteboards. The slides presented in the workshops are publicly available9–12.\n\nThe first workshop was held on 7 April at the 11th ISB Biocuration Conference 2019 in Cambridge, UK. Twenty-eight people attended the workshop where we presented the interim survey results collected between 4 and 22 March 2019. In this workshop we directed the following questions to the participants: i) How did you get into the field? Have you left? What do you do now? ii) How long did it take you to feel “fully” trained as a biocurator? iii) What skill/piece of knowledge was the most difficult to learn when becoming a biocurator? iv) How would you encourage others into the field of biocuration? v) How can we engage with others whose role is partially biocuration; or those sitting outside of traditional academic/research centres? vi) What career support would you like to see for those in biocuration; where would you like your skills to take you?\n\nThe second workshop was conducted on 19 June 2019 at the ELIXIR All Hands Meeting in Lisbon, Portugal. Twenty-one people attended the workshop in which we presented the full results of the survey. The participants were asked to comment on the following questions: i) How can ELIXIR support biocurators? ii) What are your experiences with/thoughts on community biocuration? iii) Anything else you would like to tell us?\n\nIn our analysis, we focus on the outcomes of the global survey, the conversations with EMBL-EBI biocurators and the two workshops. Analysis of all data was performed by A.H. and M.B. Where free text answers were provided in the global survey, themes across the answer sets were initially independently identified and categorised by A.H. and M.B. These individual sets were then combined to form the final answer sets provided below. For example, for the question ‘Please provide a short description of your current work.’ the answers were sorted into categories based on the tasks mentioned. A.H and M.B took a similar approach for analysing the guided conversations. Themes across the answers were independently identified and categorised and the individual sets were combined. For the word clouds, responses that make no sense were removed from the word lists, e.g. fff or wsx. The responses were edited for case, e.g. Bionformatician vs. bioinformatician, to ensure that each term is only displayed once in the word cloud. Word clouds were created with WordClouds.com (https://www.wordclouds.com/). It is worth noting that the outcomes of the pilot survey do not differ greatly from the results of the global survey.\n\nOur study was designed to collect data on biocuration practices and training needs, with only a limited set of sensitive personal data (email and name) which were not to be used as part of the reporting set. Given the nature of our study, we therefore deemed it unnecessary to undergo formal ethical review via an ethics committee. For participants, all details relating to the involvement in the study, including how the information they provided would be used and where results would be reported, was provided in both an invitation email and at the start of the survey. Participant consent was presumed based on their completion of the survey.\n\nThis study was conducted in accordance with GDPR. Directly identifiable data (e.g. name, email address) was not collected from all respondents as this was an optional element of the survey. The nature of a number of the responses provided does however mean that it may be possible to identify individuals in specific positions due to their unique job titles and affiliated institutions. Given this potential for identification we are not providing raw data outputs from the survey and transcripts from the guided conversations.\n\n\nResults\n\nThe global survey received 212 responses with most from Europe and the USA (Figure 1).\n\nThe map represents the countries in which those biocurators who responded to the survey work. The colour shading in the map indicates the number of respondents from each country.\n\nRespondents work in 91 different organisations, with 84% of respondents belonging to academic institutions. Eight percent of the respondents work in an industrial organisation and 1% span both academia and industry (see Underlying data: Global_survey_deidentified8). Among the named academic institutes, 25% of respondents belong to international biocuration hubs, including EMBL-EBI, the Wellcome Sanger Institute and SIB. In free text answers, respondents indicated that they work across a wide range of life science domains, with the most commonly mentioned being bioinformatics, genomics, genetics, biology and biochemistry (Figure 2).\n\nThe size of the text in the word cloud is related to how often particular life science domains were mentioned by respondents. The colours were chosen to create a better contrast between the words.\n\nThe survey also shows that the job titles of respondents are diverse, including uncommon titles, such as knowledge engineer, data editor, repository manager, and data wrangler (the diversity of job titles is reflected in the word cloud Figure 3A). Even for biocuration hubs, such as the EMBL-EBI, a large variety of job titles was reported (Figure 3B).\n\nThe figure shows all responses (A) and responses filtered by respondents from EMBL-EBI (B). The size of the text in the word cloud is related to how often particular job titles were mentioned by respondents. The colours were chosen to create a better contrast between the words.\n\nThis diversity of job titles makes biocurator positions difficult to identify, and this may impede the building of a sustainable community. In both the guided conversations and workshops, biocurators emphasised that the inconsistency of job titles hampers their career planning. Biocurators may not easily find relevant job adverts or in fact identify with them based on the title alone, and employers may have difficulty in determining what skills and knowledge to associate with which job title.\n\nThe survey shows that biocurators perform a diverse range of activities and that biocuration tasks are undertaken in a variety of roles. From a list of biocuration activities, the tasks most often selected in the survey were data annotation (69 of 166 respondents - 42%), ontology and controlled vocabulary application (62 of 166 respondents - 37%), data analysis (48 of 166 respondents - 29%), and literature curation (47 of 166 respondents - 28%) (see Underlying data: Global_survey_deidentified8). In addition, many respondents indicated in free text that they also take part in database, pipeline and method development, web-interface design, help-desk and user requests, teaching, outreach, team management, grant writing, and ontology development (see Underlying data: Global_survey_deidentified8). This variety of tasks makes it difficult to define a clear biocuration profile.\n\nParticipant responses to the survey, our conversations with biocurators and the workshops also make it clear that the precise nature of biocuration methods employed depends on the area in which they work. This is reflected in the 400 different tools listed in the survey (see Underlying data: Tools and resources8) as being used in biocuration activities. These resources can be broadly clustered into public databases and tools, literature search tools, ontology services, scripting languages and in-house biocuration tools; many of them are specific to the biocurators’ roles and particular life science domains in which they work. Biocurators that we spoke to and workshop participants emphasised that this makes common biocuration pipelines difficult to identify and implement, and complicates efficient knowledge exchange and community building amongst biocurators.\n\nIn our conversations with biocurators and at the workshops, biocurators emphasised that it can take a long time to be trained as a biocurator and most agreed that biocuration is a continuous learning process. According to the survey, 46 of 164 (28%) respondents said that they have received training in biocuration, while the rest said that they have not received any training (see Underlying data: Global_survey_deidentified8). Training was mainly received in the form of one-to-one teaching and self-directed training, or via face-to-face courses (Figure 4). Respondents view one-to-one and self-directed training as the most impactful forms. The in-house nature of this training combined with the specific nature of biocuration tasks suggests that biocuration knowledge gained in one role is not necessarily easily transferable to another.\n\nRespondents could choose several answers. This question was answered by 44 respondents. The percentage of respondents who chose the respective training type is also indicated in the figure.\n\nFrom the responses provided, we collated a list of face-to-face and online training courses, materials and providers (see Underlying data: Biocuration training course list8), which may be relevant to biocurators. This course collection is not exhaustive and may again reflect the nature of the informal 1:1 and in-house training many respondents said they had received.\n\nIn the survey, from free-text answers, the most commonly indicated training needs are programming/scripting/coding (mentioned in 18 of 134 responses - 13%), development and use of ontologies (mentioned in 16 of 134 responses - 12%), and database management (mentioned in 10 of 134 responses - 7%) (see Underlying data: Global_survey_deidentified8). Survey respondents and biocurators that we spoke to highlighted that computational skills, such as programming/scripting/coding and extracting relevant data from the literature, are decisive for a successful career development yet are the most difficult to learn. Other skills that were highlighted as being essential for a successful biocuration career include biological knowledge (mentioned in 41 of 124 responses - 33%), attention to detail (mentioned in 23 of 124 responses - 19%), patience (mentioned in 11 of 124 - 9%), communication (mentioned in 8 of 124 responses - 6%) and curiosity (mentioned in 8 of 124 responses - 6%) (see Underlying data: Global_survey_deidentified8). These views were reiterated and emphasised by biocurators that we spoke to individually and at the workshops.\n\nOur conversations with biocurators and the workshop discussions make it clear that biocurators typically enjoy their jobs. It is an attractive and rewarding career for skilled individuals who like data analysis, scientific writing and communication. Biocurators who participated in our study stated that the job offers the opportunity to move away from the lab but still work in a research environment without experiencing the pressure of publishing papers and offers a good work-life balance. The outcomes from the conversations and workshops, however, also suggest that biocuration is an invisible career; many people within the scientific community are unaware of biocuration as a career and undervalue the impact of biocuration. All four EMBL-EBI biocurators that we spoke to as well as some workshop participants commented that they had not previously heard of the role before becoming a biocurator. Some also felt that biocuration is underappreciated, which is reflected in difficulties obtaining funding for biocuration work.\n\nIn summary, our findings show that biocurators are a highly skilled group of individuals, however a lack of sustainable community building contributes to the fact that new biocurators are difficult to recruit, and that existing biocurators are unaware of biocuration training and future career opportunities and may feel isolated in their job.\n\n\nDiscussion and recommendations\n\nModern science, whatever the discipline, is turning into something of a data science. This seems particularly true of the life sciences, where the explosion in omics data, followed by an increase in publications and linked datasets, has led to a massive overall increase in freely available structured and unstructured biological data. Biocuration, the action of combining data from multiple sources, and adding value by converting them into a coherent, consistent and structured interoperable dataset, has never been more essential to enable the understanding, digestion and interpretation of these data.\n\nIn this ELIXIR Implementation Study, we asked biocurators from across the world for their thoughts on the state of biocuration, both in terms of the day-to-day technicalities - what tools they use, what skills they need - and the more long range subjects of training and career opportunities. We acknowledge that the survey may not have reached all biocuration communities due to, for example, the preference of certain social media channels in different countries or the lack of awareness of ISB and ELIXIR in certain (geographical) communities. Nevertheless, we believe that the results are a fair representation of the varied views and experiences of biocurators worldwide. Our study highlights the diversity of roles and experience within the biocuration community but also reinforces the need for improved recognition of this work and career.\n\nThe day-to-day activities of biocurators are amazingly diverse and include both biocuration and non-biocuration tasks such as training, outreach, website design and project management. As noted by Samili and Vita13, this highlights that biocurators possess many transferable skills in addition to their scientific and biocuration expertise, characteristics that are highly valued by employers. Our survey shows that biocurators in different resources work quite differently, using different tools and following different workflows and pipelines. There are some efforts to encourage biocurators in different resources to streamline their work by following the same biocuration workflows14. Steps have been taken to encourage this streamlining through the addition of biocuration-related databases, curation tools and standard metadata records to FAIRsharing9,11 (an ELIXIR Recommended Interoperability Resource and the ELIXIR Registry of Standards). However, it is clear from our survey that the diversity of biocuration practices and the data curated mean that a universal biocuration tool or workflow are still some time away.\n\nHaving said that, some commonalities in the experience of biocurators can be found. For instance, the clear need for more training on programmatic or coding skills, such as Python, and data modelling skills such as the creation, maintenance and use of ontologies. This is in line with a 2011 survey of biocurators carried out by the ISB in which 55% of respondents thought that better training in computer languages would be beneficial for their jobs, and 43% indicated that they would benefit from better training in bioinformatics15. Similarly, in response to a 2017 survey of biocuration training needs conducted during the development of the Postgraduate certificate in Biocuration at the University of Cambridge, 32% of respondents rated ‘Application of programming to curation tasks - e.g. scripting in Python as their top training need (S.L. Morgan, EMBL European Bioinformatics Institute; personal communication). From our discussions, it became clear to us that it is essential for biocurators to learn coding skills, not only to be able to wrangle data and perform tasks more efficiently but to facilitate discussion with database developers and gain a greater understanding of how the data they curate is represented in a database.\n\nIn our survey, 28% of respondents indicated that they had received biocuration training and some training courses were specifically named by respondents. It is possible that this relatively low figure is due to the way in which respondents interpreted the question. For example, respondents might not necessarily equate “training in a programming language”, “1:1 training” or “self-directed training” as “biocuration training”. However, a low level of formal training was also reported in response to the survey of biocuration training needs conducted during the development of the Postgraduate certificate in Biocuration at the University of Cambridge. In that survey, 12% of respondents had received training in the form of formal short courses while 88% had received informal 1:1 training in work (S.L Morgan, personal communication). The specific nature of biocuration tasks observed in our survey calls for a shift in the way that we teach biocuration skills. There is a need to develop bioinformatics training that is targeted to biocurators (e.g. programming skills for biocuration related tasks vs programming for research related tasks). Ensuring that all biocurators are up to speed with the latest biocuration and data stewardship best practices should increase the standard and consistency of biocuration while providing transferable skills that improve career mobility.\n\nThe lower reported level of formal training suggests that a potential challenge for biocurators is in being able to find available and/or appropriate training courses. An absence of training courses will also affect community building, with fewer opportunities for biocurators to meet, network and share expertise. To facilitate expanded training for biocurators, training providers should make their courses and training materials easier to find, for example by sharing them in portals such as ELIXIR’s TeSS (the ELIXIR Training and Events Portal; https://tess.elixir-europe.org/)12, and by adopting FAIR principles to ensure that they are appropriately tagged and described16. This would allow democratisation of training efforts such that they expand out from centres of excellence (such as the EMBL-EBI or SIB) to independent biocurators. Some training events and materials listed in the survey have already been indexed in TeSS12, which will allow the identification of gaps in training provision.\n\nOur study also highlights the need for improved career support. This sentiment seems to have changed little from the 2011 ISB survey, in which 82% of respondents felt concerned about future work opportunities15. It can take many years of training and experience to become a biocurator, yet currently, salaries and more importantly job security, often do not reflect this investment. Key barriers to career progression highlighted in our survey are the diversity of job titles, which can make relevant opportunities difficult to find (and diverse in their nature), alongside a lack of recognition of the skills and the role of biocurators both by those performing biocuration activities and in the wider scientific community.\n\nBiocurators that participated in the workshop discussions indicated that one way to address these challenges is to establish stronger links between academia and industry to create a wider and more diverse biocuration community, facilitate knowledge exchange and enable career planning. Another way biocurators can be helped is through appropriate credit, both in terms of citation of the resources they maintain and personal credit through nanopublications, annotation credits and other attributions displayed and linked via their OrCID17.\n\nUltimately, we perhaps need a new model of careers in biocuration (especially for those individuals for whom biocuration is their primary role), such as those being developed for Research Software Engineers18,19 and Data Stewards20. In these settings, centralised teams of research software engineers or data stewards have been created, and this has led to improved knowledge exchange, peer support and career sustainability19,20. For example, within a University environment, a pool of dedicated biocurators could be created, ready to be deployed where need and experience allows, and supported with local funding, so the biocurators themselves have job security.\n\nWe urge funding agencies to recognise the importance of biocuration and to fund both databases and biocuration training activities appropriately. In addition, we recommend that both academia and industry provide clearer career structures for scientists whom biocuration is their primary activity, given their role and impact will only grow. We call on everyone, from the ISB, ELIXIR, academia, industry, and biocurators themselves, to work together to create a more integrated global community of biocurators, to share expertise, training initiatives and best practices.\n\n\nConclusion\n\nBiocuration activities have a vital role in the life science data ecosystem, but are often undervalued. These skills, both innate and taught, are highly prized and fundamental not only to the preservation and FAIRness of life science data but are also transferable to other data science and knowledge management roles. The issue we face is one of education. Sometimes scientists performing biocuration activities do not realise the value and rarity of their skills and in many cases researchers and managers do not realise or appreciate the work that biocurators perform. To future-proof this career and the valuable work that biocurators do, organisations, employers and biocurators themselves must act as champions for biocuration and advocate for structural changes. We believe organisations like ELIXIR, ISB and GOBLET have the mandate to actively support the biocurators in their scientific endeavours, work towards raising awareness about the impact that biocuration has within different scientific communities and to convince team leaders of the importance and value of skilled biocuration work. Organisations can also act to facilitate, encourage and support knowledge exchange between biocuration communities. Biocurators themselves have a role to play in championing the work that they do, engaging with the researchers who rely on their expertise and in training the future generation of biocurators. By doing this, organisations and individuals can contribute to the creation of a sustainable and visible biocuration community. We hope the work we have presented here continues this educative process as we continue to define the profession of biocuration.\n\n\nData availability\n\nThe nature of a number of the responses provided means that it may be possible to identify individuals in specific positions due to their unique job titles and affiliated institutions. Given this potential for identification we are not providing raw data outputs from the survey. Instead we provide the de-identified data on Zenodo from the global survey.\n\nThe nature of the responses provided in the guided conversations and the fact that all four participants are staff of EMBL-EBI mean that it may be possible to identify the individuals. Given this potential for identification, we do not provide the transcripts but a themed summary of the responses. Information that may lead to the identification of individuals has been redacted.\n\nData relating to the workshops can be found in the slide sets from these workshops: https://doi.org/10.7490/f1000research.1116798.19 and https://doi.org/10.7490/f1000research.1117413.110.\n\nZenodo: Biocuration - mapping resources and needs - Underlying data, http://doi.org/10.5281/zenodo.39917378.\n\nThis project contains the following underlying data:\n\nGlobal_survey_deidentified.xlsx (de-identified responses to the global survey)\n\n○ This file includes de-identified responses to the survey questions. Responses that may lead to the identification of respondents have been redacted. Free text responses to questions 6, 14 and 15 have been categorised into tasks, topics and skills, respectively.\n\nBar graphs of global survey.xlsx (quantitative responses to multiple choice questions in the global survey. For some questions, respondents could choose more than one option)\n\nTools and resources.xlsx (Tools and resources used for biocuration work and listed by the respondents of the global survey)\n\nBiocuration training course list.xlsx (formal training courses listed by respondents of the global survey)\n\nThemed summary of the responses given in the guided conversations - deidentified.docx\n\nZenodo: Biocuration - mapping resources and needs - Underlying data, http://doi.org/10.5281/zenodo.39917378.\n\nThis project contains the following underlying data:\n\nPilot survey questions.docx (questionnaire sent to staff of Wellcome Genome Campus)\n\nQuestions to guide conversations with biocurators.docx (conversation guide outlines the type of questions to be asked)\n\nGlobal survey questions.docx (globally disseminated questionnaire revised on the basis of the pilot survey)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe would like to acknowledge all the scientists who took part in the study, as well as our colleagues from the ELIXIR Training, Data and Interoperability Platforms, ISB and GOBLET.\n\n\nReferences\n\nYates AD, Achuthan P, Akanni W, et al.: Ensembl 2020. Nucleic Acids Res. 2020; 48(D1): D682–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTate JG, Bamford S, Jubb HC, et al.: COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res. 2019; 47(D1): D941–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLock A, Rutherford K, Harris MA, et al.: PomBase 2018: user-driven reimplementation of the fission yeast database provides rapid and intuitive access to diverse, interconnected information. Nucleic Acids Res. 2019; 47(D1): D821–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBourne PE, McEntyre J: Biocurators: Contributors to the World of Science. PLoS Comput Biol. 2006; 2(10): e142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurinx C, McEntyre J, Appel R, et al.: Identifying ELIXIR Core Data Resources. F1000Res. 2016; 5: ELIXIR-2422. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRigden DJ, Fernández XM: The 27th annual Nucleic Acids Research database issue and molecular biology database collection. Nucleic Acids Res. 2020; 48(D1): D1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolinski A, Burke ML, Morgan SL, et al.: Biocuration - mapping resources and needs - Underlying Data (Version 2) [Data set].Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3991737\n\nHolinski A, Burke M, Morgan S, et al.: Mapping the landscape of biocuration - where are the biocurators and what do they need? F1000Res. 2019. http://www.doi.org/10.7490/f1000research.1116798.1\n\nHolinski A, Burke M, Morgan S, et al.: Mapping the landscape of biocuration - where are the biocurators and what do they need? F1000Research. 2019. http://www.doi.org/10.7490/f1000research.1117413.1\n\nMcQuilton P: FAIRsharing.org - mapping the landscape of databases, standards and policies (describing, linking and assessing their FAIRness). F1000Res. 2019. Publisher Full Text\n\nPalagi PM, McQuilton P, Beard N: TeSS: the ELIXIR training portal. F1000Res. 2019. Publisher Full Text\n\nSalimi N, Vita R: The Biocurator: Connecting and Enhancing Scientific Data. PLoS Comput Biol. 2006; 2(10): e125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVenkatesan A, Karamanis N, Ide-Smith M, et al.: Understanding life sciences data curation practices via user research. F1000Res. 2019; 8: 1622. http://www.doi.org/10.12688/f1000research.19427.1\n\nBurge S, Attwood TK, Bateman A, et al.: Biocurators and Biocuration: surveying the 21st century challenges. Database. 2012; 2012: bar059. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcia L, Batut B, Burke ML, et al.: Ten simple rules for making training materials FAIR. PLoS Comput Biol. 2020; 16(5): e1007854. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThessen AE, Woodburn M, Koureas D, et al.: Proper Attribution for Curation and Maintenance of Research Collections: Metadata Recommendations of the RDA/TDWG Working Group. Data Sci J. 2019; 18(54): 1–11. Publisher Full Text\n\nHettrick S: A not-so-brief history of Research Software Engineers \| Software Sustainability Institute. 2016. Reference Source\n\nBaxter R, Hong NC, Gorissen D, et al.: The Research Software Engineer. 2012. Reference Source\n\nTeperek M, Cruz MJ, Verbakel E, et al.: Data Stewardship Addressing Disciplinary Data Management Needs. Int J Digit Curation. 2018; 13(1): 141–9. Publisher Full Text" }	[ { "id": "72815", "date": "20 Oct 2020", "name": "Tanya Berardini", "expertise": [ "Reviewer Expertise biocuration", "community engagement", "data resource sustainability" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 details the analysis of a global survey of biocuration work using the results of several in person interviews, online SurveyMonkey polls, and a couple of workshops to identify biocurators and the types of work that they perform. It is apparent that the community is diverse not just in training, background,and job titles, but also in the skill sets and tools that are employed in day to day tasks. I appreciate the details provided by the study and the emphasis on the need for more broader scientific engagement to understand and appreciate this type of work.\nI recommend acceptance without revisions and hope that the indexing and broad dissemination of this paper will result in a greater appreciation for and support of the biocuration community.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "72814", "date": "26 Oct 2020", "name": "Luana Licata", "expertise": [ "Reviewer Expertise Biocuration", "Molecular and Causal interactions", "Community Standard Formats" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presents the result of an implementation study on biocuration needs. The article describes the results coming from the analysis of an online SurveyMonkey polls, filled out by over 200 curators from 33 countries around the globe and integrated with the results of few interviews with a subset of curators and with the outcomes of two questionnaires performed during two workshops.\nThe implementation study aimed to define four points;\nIdentify communities of biocurators\n\nMap the type of curation work being done\n\nAssess biocuration training\n\nDraw a picture of biocuration career development.\nThe global survey was useful to identify biocuration communities, the types of curation work and to understand the training needs while the interviews and workshops questionnaires were useful to outline career development and communities need.\nIn the paper, the study is well explained and the conclusion well described.\n\nHowever, I think the study design miss some aspects that would have make the result more comprehensive and consistent.\nWhile the global survey analysis has been performed on a wide and geographically well distributed biocuration community, the interviews analysis not. In my opinion, the guided conversations and the selection of biocurators would have benefited from the involvement of a wider number of curators, such as for example, curators coming from different academia and research institutes where the figure of biocurator is less recognised and supported compared to the EMBL-EBI. The interview of a wider number of curators, it could have highlighted and arisen further needs and pain points.\n\nMoreover, the paper described two questionnaires performed during two workshops held during the 11th ISB Biocuration Conference 2019 and the ELIXIR All Hands Meeting 2019 but the results of these surveys are not explained in full detail and are not available in the supplementary data. There is only a link to the slides presented in the workshops. It would be very useful a summary table or a figure on the results of the two workshops.\n\nNevertheless, I think that the conclusions arising from this study are well drawn and pointed out the main needs of the biocuration community. I particularly liked the final recommendation and suggestions. I believe that this article will help to increase the appreciation and understanding of biocuration work by scientific communities and the support by Academia and Research Institutes.\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? Partly\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6139", "date": "02 Dec 2020", "name": "Alexandra Holinski", "role": "Author Response", "response": "Dear Luana Licata, Thank you for taking the time to review our article and for your valuable feedback on our project. We would like to address two of your comments: 1) “While the global survey analysis has been performed on a wide and geographically well distributed biocuration community, the interviews analysis not. In my opinion, the guided conversations and the selection of biocurators would have benefited from the involvement of a wider number of curators, such as for example, curators coming from different academia and research institutes where the figure of biocurator is less recognised and supported compared to the EMBL-EBI. The interview of a wider number of curators, it could have highlighted and arisen further needs and pain points.” The guided conversations served to explore the questions asked in the pilot survey in more detail and to identify additional relevant topics for inclusion in the global survey. We agree that one-to-one interviews with a wider number of curators would have been useful, however due to time constraints we were unable to do so. To efficiently gather more diverse and detailed insight into the biocuration landscape we made use of group discussions during the workshops held at ISB 2019 and the ELIXIR all hands 2019 meetings as described in the manuscript. These workshops included a global audience of biocurators. By using this approach we were able to gather a broader set of ideas through the workshops than would have been possible through one-to-one interviews. 2) “Moreover, the paper described two questionnaires performed during two workshops held during the 11th ISB Biocuration Conference 2019 and the ELIXIR All Hands Meeting 2019 but the results of these surveys are not explained in full detail and are not available in the supplementary data.” In both workshops we did not perform questionnaires but held group discussions in which we asked attendees to note down their answers to specific questions on post-it notes which we collected on whiteboards. This is outlined in the “Methods” (paragraph: Workshops at ISB Biocuration Conference 2019 and ELIXIR All Hands Meeting 2019). Photographs and transcripts of the post-it notes are included in the slides from the workshops (referenced in the article). We understand that this is not clearly specified in the “Methods”, and have revised this section accordingly. We hope that our responses help to clarify the rationale behind the study and welcome any further comments. Kind regards, The authors" } ] } ]	1	https://f1000research.com/articles/9-1094
https://f1000research.com/articles/9-604/v1	15 Jun 20	{ "type": "Case Report", "title": "Case Report: Simultaneously diagnosed gastric adenocarcinoma and pernicious anemia – a classic association", "authors": [ "Syed Kamran", "Mattias K. Dilling", "Nathaniel A. Parker", "Joel Alderson", "Nathan D. Tofteland", "Quoc V. Truong", "Syed Kamran", "Mattias K. Dilling", "Joel Alderson", "Nathan D. Tofteland", "Quoc V. Truong" ], "abstract": "Primary gastric cancer remains one of the most prevalent malignancies worldwide. Often patients remain asymptomatic until it is detected at an advanced stage with a poor prognosis. Thus, it’s characteristically difficult to initially diagnose until it becomes late stage, at which point prognosis becomes poor. Pernicious anemia is a classic risk factor for the development of primary gastric cancer, but is uncommonly seen in clinical practice. Over time, patients who produce the autoantibodies to intrinsic factor that cause pernicious anemia typically will present initially with clinically significant megaloblastic anemia and peripheral neuropathy. However, patients can also present with more nonspecific signs and symptoms. Thus, clinicians should remain vigilant as circulating anti-intrinsic factor antibodies only worsen the disease over time and increase the risk of developing primary gastric cancer. This report not only presents the rare concurrent diagnosis of pernicious anemia and gastric cancer, but also aims to increase clinical awareness of these two conditions’ classic association because early diagnosis and treatment significantly impacts morbidity and mortality.", "keywords": [ "Autoimmune gastritis", "Parietal cells", "Stomach cancer", "Gastric adenocarcinoma", "Pernicious anemia" ], "content": "Introduction\n\nDespite the decline in gastric cancer incidence rates over the past several decades, it remains one of the most common and fatal malignancies worldwide. Yearly, over one million cases are diagnosed with an estimated 780,000 annual mortality incidence1. The high mortality of gastric cancer is in part attributed to late initial diagnosis2. The diagnosis of gastric carcinoma often is delayed because up to 80% of patients are asymptomatic during the early stages of stomach cancer3. Weight loss, abdominal pain, nausea and vomiting, early satiety, and esophageal reflux-like symptoms are often late signs of tumor progression4. By the time many symptoms develop, the disease is almost invariably too far advanced for curative procedures2. Classic physical exam findings, such as organomegaly (e.g. stomach, liver) or regional lymphadenopathy (e.g. Virchow’s node, Sister Mary Joseph’s nodule) should raise suspicions for a gastric malignancy3. More common risk factors for development of gastric cancer include Helicobacter pylori infection, tobacco smoking, heavy alcohol use, age, diet, and non-Caucasian ethnicity3,4.\n\nAlthough less common, pernicious anemia (PA) remains a classic risk factor for primary gastric cancer. The condition is defined as autoimmune destruction of the intrinsic factor (IF) glycoprotein, or destruction of the gastric body and fundal parietal cells that produce IF5. Since IF plays a crucial role in the transportation and absorption of vitamin B12, the product of this deleterious autoimmune process is the characteristic megaloblastic anemia6. Importantly, PA represents 20% – 50% of the adult cases of vitamin B12 deficiency worldwide7.\n\nPA often has an insidious onset, which can make early diagnosis difficult. Clinically, patients commonly present initially with nonspecific symptoms such as fatigue, weakness, and mild paresthesias7. However, unintentional weight loss can occur in 50% of patients5. Chronically severe vitamin B12 deficiency can lead not only lead to worse neurologic dysfunctions, but also glossitis and gastrointestinal issues6. Clinically significant vitamin B12 deficiency manifests as neurologic dysfunction in 74% of patients8. The classic late-stage neurologic complication of vitamin B12 deficiency is subacute combined degeneration of the posterior and lateral columns of the spinal cord due to demyelination. However, peripheral neuropathy and non-neurologic manifestations are more common5.\n\nIndividuals with PA have long been suggested to be at increased risk for gastric cancer9. The pathogenesis of gastric cancer arising from PA is thought to be due chronic inflammation with extensive atrophy of the gastric mucosa, leading to increased risk of progression to gastric neoplastic lesions10. This report presents the uncommon, but classic association, of PA with primary gastric cancer. When patients present with abdominal symptoms, unintentional weight loss, megaloblastic anemia, and low vitamin B12 levels, we should consider the presence of PA and its associated complication of primary gastric cancer.\n\n\nCase report\n\nA 61-year-old Hispanic female retail worker, for whom the only pertinent past medical history was intermittent social alcohol consumption, presented to the emergency department with epigastric pain. Symptom onset began three weeks prior to her initial presentation and had been progressively worsening. Her chief complaints were associated with heartburn, decreased appetite, weakness, and 60-pound unintentional weight loss over the past six months. The patient attributed all of the symptoms to her physically and mentally demanding occupation. She denied a personal history of nausea, vomiting, dysphagia, hematemesis, hematochezia, melena, or screening colonoscopy. Her family history was negative for any gastrointestinal diseases or malignancies.\n\nVital signs and measurements were unremarkable. A detailed physical examination was nonrevealing. Serum laboratory analysis was notable for a significant anemia, as well as a low vitamin B12 and iron deficiency (Table 1). She was urgently managed with a restrictive transfusion strategy by one unit of leukoreduced packed red blood cells, which improved her symptoms and hemoglobin. Abdominopelvic imaging was obtained by computerized tomography (CT) scans with contrast, which were primarily equivocal (Figure 1). Subsequently, she underwent esophagogastroduodenoscopy (EGD) for further evaluation. Esophageal findings included moderate esophagitis and salmon-colored mucosa at the gastroesophageal junction suggestive of Barrett's esophagus. Beyond the esophagus, a large 7-cm hiatal hernia was evident. An extensive, deep ulcer was noted to involve the entirety of the incisura and pre-pyloric area, as well as extended along the lesser curvature (Figure 2). Multiple biopsies of the ulcer were obtained to evaluate for malignancy, as well as random gastric biopsies to evaluate for Helicobacter pylori colonization. Helicobacter pylori immunohistochemical (IHC) stain was ultimately negative. Due to the tumor's size, gross appearance, ulcerations, and bleeding erosions noted on EGD a malignant process was suspected. Based on the suspicions for an underlying malignant gastric neoplasm and the presence of a vitamin B12 deficiency, serum serologic testing for PA was obtained. Laboratory testing was positive for anti-IF confirming the diagnosis of PA (Table 1).\n\nNonspecific gastric fold thickening in the fundus is observed (arrow). An incidental finding in the liver was noted by a small focal hypoattenuation in the middle segment of the left lobe of the liver adjacent to the fissure for ligamentum teres (arrowhead). This nodule was confirmed as PET-negative on later PET/CT studies.\n\n(From proximal to distal.) (A) Esophagitis. (B) Gastro-esophageal junction. (C) More esophagitis, and a tongue of columnar mucosa. (D) Normal gastric cardia. (E) Normal gastric fundus. (F) Cavernous ulcer along the incisura (lesser curvature) with debris, food particles, and some central exudates (G) Continued ulcer description. At the 12 o'clock position the scope is originating from the gastric cardia and fundus region. At 3 o'clock is the expected location of the pylorus. However, due to size and extent of the ulcer typical anatomy and landmarks were considerably distorted making visualization of the fundus from that particular EGD position not possible. At 6 o'clock the ulcer is shown to extend along the incisura. At 9 o'clock food debris is seen along the greater curvature.\n\nCorrected reticulocyte count and reticulocyte index for her age and gender suggests an inappropriate marrow response (reticulocyte index < 2) likely relating to the patient’s nutritional deficiencies, instead of bone marrow abnormalities. The low vitamin B12 level supports the lack of sufficient vitamin B12 in the blood. Serum IF-blocking antibody testing was not obtained on admission, but after EGD, which was positive and confirmed the diagnosis PA.\n\nMicroscopically, antral mucosa demonstrated mild chronic gastritis. Histopathology revealed a diffuse-type, invasive poorly differentiated adenocarcinoma. IHC staining was only positive for pancytokeratin. The malignant-appearing cells lacked immunoreactivity for synaptophysin, CD45, and HER2 (Figure 3). Together with histopathology and this IHC profile gastric adenocarcinoma was confirmed. She underwent a positron emission tomography (PET) scan for staging. The PET scan showed a localized but advanced gastric neoplasm not associated with any regional PET-avid lymphadenopathy. No obvious metastatic disease was present (Figure 4). Thus, she was diagnosed with locally advanced Stage IB gastric adenocarcinoma (Figure 5).\n\nPET from skull to mid-thigh reveals extensive, diffuse hypermetabolism throughout the gastric wall compatible with a PET-avid infiltrating gastric neoplasm. No metastatic disease is apparent. Scattered areas of contrast uptake within the bowel are likely physiologic and limit evaluation for lesions. Contrast uptake within the brain and genitourinary system are physiologic.\n\nPresented according to CARE guidelines.\n\nShe was started on intravenous vitamin B12 and iron replacement (dose given in Figure 5). Her remaining symptoms improved slowly while she observed in post-operative period with twice-daily oral 40 mg pantoprazole and daily polyethylene glycol. She continued to improve following intravenous vitamin B12 and iron replacement and transitioned to a scheduled oral cyanocobalamin 1000 µg daily regimen. Oral iron supplementation was discouraged due to the propensity of oral iron medication to be significantly irritative to the gastric mucosal lining. She was dismissed from the hospital and established care with a local oncologist. Due to the nature of her disease, chemotherapy was recommended. The patient initiated treatment with the FLOT regimen (oxaliplatin, leucovorin, docetaxel, fluorouracil) for her advanced, invasive poorly differentiated gastric adenocarcinoma (Figure 5). However, the patient did not tolerate chemotherapy well and only was able to endure one cycle. She wished to stop all therapies and was transitioned to hospice care. Two weeks after stopping chemotherapy, the patient expired.\n\n\nDiscussion\n\nThe prevalence of PA is 0.1% in the general population, and approximately 2% in patients older than 60 years of age7. The highest prevalence is seen in Northern Europeans, specifically those in Scandinavia and the United Kingdom11. Several autoimmune diseases, such as type 1 diabetes mellitus, vitiligo, and autoimmune thyroid disease, have all been reported to have an increased association with the development of PA7. A diagnosis of PA is made when patients have a low vitamin B12 level and are positive for anti-IF antibody or anti-parietal cell antibody, or low vitamin B12 level in the presence of atrophic gastric mucosa seen on histopathology11. PA is often clinically diagnosed prior to any subsequent development of cancer. Concurrent diagnosis of PA and gastric cancer, as with our patient, is exceedingly rare and often observed in patients who present late in the course of their disease.\n\nHaving a clinical awareness to the presence of a vitamin B12 deficiency is often sufficient to begin the workup. However, reliance on biochemical evidence alone to suggest low vitamin B12 is not recommended. Various occult malignancies can falsely elevate vitamin B12. Thus, a vitamin B12 deficiency can be masked. Given the underlying pathophysiology of PA, patients with a known history of autoimmune conditions are at an increased risk to develop anti-IF autoantibodies. Anti-parietal cell antibodies are present in around 90% of patients with PA, but have low specificity. Conversely, anti-IF antibodies are found in around 60% of patients with PA, but are considered much more specific for the disease11. Similar to most serologic tests, autoantibody testing for IF has a low sensitivity. In contrast, specificity remains high and the presence of anti-IF autoantibodies is diagnostic for PA12.\n\nBased on the American Society of Gastrointestinal Endoscopy, following the diagnosis of PA endoscopy, evaluation by EGD is recommended, especially if gastrointestinal symptoms are present13. However, only about 25% of patients diagnosed with PA patients undergo subsequent upper endoscopic screening13. If endoscopic findings are suspicious for a neoplastic process, tissue sampling can aid in the diagnosis of a gastric malignancy. Commonly, gastric specimens are immunoreactive to markers for carcinoma, such as pancytokeratin. However, the diagnosis can be challenging since primary gastric adenocarcinoma cells commonly exhibit partial synaptophysin immunoreactivity14.\n\nThe mainstay treatment for PA involves parenteral replenishment. Due to its autoimmune nature, treatment is typically needed indefinitely. High-dose oral or sublingual vitamin B12 therapy can also be used, provided a good adherence to treatment and a positive response is seen15. Intranasal formulations are not recommended due to the higher cost, side effects, and inconsistent absorption. Following definitive surgical mangement by gastrectomy, indefinite treatment with parenteral vitamin B12 is still appropriate15.\n\nResearch into anti-IF autoantibodies has been ongoing as a possible immunotherapy target. While no immunotherapeutic agent currently exists, experts have proposed a specific clone of CD4+ T-cells to be involved in the destruction of the gastric cells in PA and autoimmune gastritis16. The finding of these T-cells could lay the groundwork for developing new immunotherapies against the T-cells in future studies, especially in PA patients in whom the anti-IF autoantibodies are not present.\n\nThe relative risk for gastric adenocarcinoma in PA patients is as high as 6.8 (95% CI: 2.6–18.1)8. After the initial diagnosis of PA, the risk of developing gastric cancer increases. The association between PA and gastric cancer becomes strongest 6 years after the first record of PA (OR: 2.53). In contrast, if PA is discovered and treated early, gastric cancer is less likely to develop (OR: 1.77)10. Thus, clinicians should remain vigilant as unnoted circulating anti-IF autoantibodies worsen PA and increase the risk of patients developing gastric cancer overtime10,12.\n\nOnce gastric cancer develops and is ultimately diagnosed following histopathologic investigation, the prognosis often varies based on several factors. Overall, proximal tumors near the gastroesophageal junction and cardia have a poorer prognosis17. Gastric cancer has a male predominance and a higher mortality rate in men1. Compared to surgical intervention alone or concomitantly with chemotherapy, chemotherapy and radiation increases the chances of achieving complete remission. If the patient is willing and able to undergo surgical management, upper GI endoscopic resection or gastrectomy is recommended. Following surgical intervention there are currently no randomized trials to help guide posttreatment surveillance strategies18. The National Comprehensive Cancer Network suggests a risk-stratified surveillance strategy that is tailored to individual patients. No specific guidance is provided, but factoring tumor staging and if a patient underwent endoscopy or gastrectomy is recommended19. Vitamin B12 and iron must be closely monitored along with bone health, especially in female patients who have undergone a total gastrectomy. Follow-up PET/CT scans can be considered as they are indicated clinically20.\n\n\nConclusion\n\nPrimary gastric cancer remains one of the most prevalent malignancies worldwide and is associated with poor outcomes. This is likely due to patient’s remaining asymptomatic until late-stage progression. Thus, early detection is paramount. PA is uncommonly seen in clinical practice, but remains a classic risk factor for the development of primary gastric cancer.\n\nIdentifying pertinent physical exam features and pairing them with lab findings of a vitamin B12 deficiency can be crucial steps in uncovering the gastric cancer early. In severe cases such as this patient, it is important to obtain proper radiographic imaging and an EGD. Unfortunately, only a minority of patients diagnosed with PA undergo subsequent gastric cancer screening with upper endoscopy. Thus, there is a need for increased clinical awareness of these two conditions’ classic association.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nRawla P, Barsouk A: Epidemiology of gastric cancer: global trends, risk factors and prevention. Prz Gastroenterol. 2019; 14(1): 26–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaconi G, Manes G, Porro GB: Role of symptoms in diagnosis and outcome of gastric cancer. World J Gastroenterol. 2008; 14(8): 1149–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLayke JC, Lopez PP: Gastric cancer: diagnosis and treatment options. Am Fam Physician. 2004; 69(5): 1133–40. PubMed Abstract\n\nZali H, Rezaei-Tavirani M, Azodi M: Gastric cancer: prevention, risk factors and treatment. Gastroenterol Hepatol Bed Bench. 2011; 4(4): 175–85. PubMed Abstract \| Free Full Text\n\nPernicious Anemia. 2019; Accessed: May 6, 2020. Reference Source\n\nAslinia F, Mazza JJ, Yale SH: Megaloblastic anemia and other causes of macrocytosis. Clin Med Res. 2006; 4(3): 236–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndres E, Serraj K: Optimal management of pernicious anemia. J Blood Med. 2012; 3: 97–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHealton EB, Savage DG, Brust JC, et al.: Neurologic aspects of cobalamin deficiency. Medicine. 1991; 70(4): 229–45. PubMed Abstract \| Publisher Full Text\n\nVannella L, Lahner E, Osborn J, et al.: Systematic review: gastric cancer incidence in pernicious anaemia. Aliment Pharmacol Ther. 2013; 37(4): 375–382. PubMed Abstract \| Publisher Full Text\n\nMurphy G, Dawsey SM, Engels EA, et al.: Cancer risk after pernicious anemia in the US elderly population. Clin Gastroenterol Hepatol. 2015; 13(13): 2282–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBizzaro N, Antico A: Diagnosis and classification of pernicious anemia. Autoimmun Rev. 2014; 13(4–5): 565–8. PubMed Abstract \| Publisher Full Text\n\nGreen R: Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood. 2017; 129(19): 2603–11. PubMed Abstract \| Publisher Full Text\n\nPritchard DM, Hooper M: Letter: gastric cancer and pernicious anaemia - only a minority of UK pernicious anaemia patients have had a gastroscopy. Aliment Pharmacol Ther. 2016; 43(10): 1106–7. PubMed Abstract \| Publisher Full Text\n\nBasturk O, Tang L, Hruban RH, et al.: Poorly differentiated neuroendocrine carcinomas of the pancreas: a clinicopathologic analysis of 44 cases. Am J Surg Pathol. 2014; 38(4): 437–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDevalia V, Hamilton MS, Molloy AM, et al.: Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol. 2014; 166(4): 496–513. PubMed Abstract \| Publisher Full Text\n\nToh BH, Chan J, Kyaw T, et al.: Cutting edge issues in autoimmune gastritis. Clin Rev Allergy Immunol. 2012; 42(3): 269–78. PubMed Abstract \| Publisher Full Text\n\nPetrelli F, Ghidini M, Barni S, et al.: Prognostic role of primary tumor location in non-metastatic gastric cancer: a systematic review and meta-Analysis of 50 studies. Ann Surg Oncol. 2017; 24(9): 2655–68. PubMed Abstract \| Publisher Full Text\n\nMorgan D: Early gastric cancer: Treatment, natural history, and prognosis. UpToDate. In TW Post, Feldman M, Tanabe K, Soybel D, Robson K, & Savarese D (ed): UpToDate, Waltham, MA; 2020. Reference Source\n\nNCCN clinical practice guidelines in oncology. 2020. Accessed: January 17, 2020. Reference Source\n\nSmyth EC, Verheij M, Allum W, et al.: Gastric cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2016; 27(suppl 5): v38–v49. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66908", "date": "24 Jul 2020", "name": "Faustina N A. Sackey, Ph.D., FMWI", "expertise": [ "Reviewer Expertise Oncology", "Nephrology", "Developmental Heart Defects." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “Case Report: Simultaneously diagnosed gastric adenocarcinoma and pernicious anemia – a classic association” presented a case about a 61-year-old woman who suffered both pernicious anemia and gastric cancer, and even after various treatments later succumb due to advancement of the disease. The manuscript was well-written with all the essential details regarding the appropriate tests leading to the patient’s diagnosis and treatment. However, I have the following concerns:\n1. Title:\nPlease keep it simple. My suggestion: “Simultaneous diagnosis of gastric adenocarcinoma and pernicious anemia – a classic association.”\n\n2. Abstract:\nIt would be great to include the disease prevalence or mortality ranking since an Abstract is what readers would glance first and must represent the text. Once prevalence is mentioned, statistics must be indicated. There is GLOBOCAN 2018 statistics about the global ranking of prevalence and mortality to check and include.\n\n3. Introduction:\nWhat is the median overall survival (OS) of gastric cancer since the disease is largely asymptomatic? Please check and add.\n\n4. Discussion:\nI know the authors made mention of autoimmune destruction of the intrinsic factor (IF) glycoprotein leading to the perturbation of Vitamin D absorption. Is this the only pathogenesis of gastric cancer, or are there other possibilities? Please check and include so that readers would have the full picture of the etiology of the disease.\nSince the authors said part of their aims of the study is to increase clinical awareness of the classic association of PA and gastric cancer, due to the asymptomatic nature of gastric cancer, early diagnosis is quite challenging. Create one more paragraph in the Discussion to suggest remedies that would curtail identification of the disease in advance stages. Examples:\nCut-off age to perform an annual screening of individuals, as it is commonly done for colorectal cancer. Family history of PA and gastric cancer should aid in identifying high-risk individuals and be screened for epidermal growth factor receptor (EGFR) overexpression, a potent biomarker for poor prognosis, and expressions of both vascular endothelial growth factor A (VEGFA), and mutations of the P53 tumor suppressor gene (TP53). Screening high-risk individuals for the pathogen, H. pylori infections.\nLastly, it will be excellent for the authors to use their experience to come up with feasible and preferred treatment options like surgical resection performed as total or subtotal gastrectomy; if chemotherapy is to be used, what drugs? 5-FU/leucovorin? Targeted therapy: Ramucirumab, a monoclonal antibody against vascular endothelial growth factor receptors 2, anti-EGFR antibody, nimotuzumab, together with vitamin B12 therapy to resolve the anemia.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "67562", "date": "27 Jul 2020", "name": "Talha Badar", "expertise": [ "Reviewer Expertise Malignant Hematology---> Leukemia/myeloid disorder and stem cell transplant/cellular therapy." ], "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\nKamran et al. presented a case report and literature review of gastric cancer with concurrent diagnosis of PA. My comments are below:\n\nBased on my assessment of this report, one could easily argue that this could be someone with history of GERD due to hiatal hernia leading to Barrett's esophagus leading to GE junction adenocarcinoma. The authors should focus on differential diagnosis of conditions leading to GE junction/gastric adenocarcinoma.\n\nPrior history of GERD? (Especially with barret esophagus findings, hiatal hernia), eating habits? Occupational and family history? All to be known in patients with gastric cancer to look for possible etiology for cancer diagnosis.\n\nCould it be a GE junction tumor, extending to stomach?\n\nWhat is sensitivity and specificity of the IF antibody assay?\n\nWhy was FLOT chemotherapy used, why not upfront Sx for Stage 1b disease and adjuvant therapy based on surgical pathology? This should be explained in more detail.\n\nFigure 5 is good, I suggest to mention the timeline in weeks/days/months from presentation --> diagnosis --> treatment --> transition to hospice.\n\nIntroduction, paragraph 3, the third sentence needs better structuring.\n\nThe references need to be better formatted, especially 5, 13 and 19.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "5785", "date": "30 Jul 2020", "name": "Nathaniel Parker", "role": "Author Response", "response": "Based on my assessment of this report, one could easily argue that this could be someone with history of GERD due to hiatal hernia leading to Barrett's esophagus leading to GE junction adenocarcinoma. The authors should focus on differential diagnosis of conditions leading to GE junction/gastric adenocarcinoma. - Comment acknowledged. This request is out of the scope for this article, and does not align with the article's purpose. Prior history of GERD? (Especially with barret esophagus findings, hiatal hernia), eating habits? Occupational and family history? All to be known in patients with gastric cancer to look for possible etiology for cancer diagnosis. - All pertinent and/or available information has been presented and detailed in the case description section. Could it be a GE junction tumor, extending to stomach?- All pertinent and/or available information has been presented and detailed in the case description section. Also, this is unlikely based on the EGD images and summary of the report detailed in the article. What is sensitivity and specificity of the IF antibody assay?- Please see Description section, paragraph 3, sentence 6.Why was FLOT chemotherapy used, why not upfront Sx for Stage 1b disease and adjuvant therapy based on surgical pathology? This should be explained in more detail. - All pertinent and/or available information has been presented and detailed in the case. Figure 5 is good, I suggest to mention the timeline in weeks/days/months from presentation --> diagnosis --> treatment --> transition to hospice. - Comment acknowledged. The authors decline to make the suggested change based on the differences in figure style presentation not providing additional value and clarity to the overall figure and article. Introduction, paragraph 3, the third sentence needs better structuring. - Comment acknowledged. The authors decline to make the suggested change. This sentence is adequate and its current structure is justified. The references need to be better formatted, especially 5, 13 and 19. - Comment acknowledged. The authors decline to make the suggested change because the current format of this reference was made by the Editors." } ] }, { "id": "74105", "date": "10 Nov 2020", "name": "Martyn Hooper MBE", "expertise": [ "Reviewer Expertise Founder and current Executive Chairman of the Pernicious Anaemia Society - reg. charity no. 1147839" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Pernicious Anaemia Society's helpline often receives calls from newly diagnosed patients who want to know how likely they are to develop stomach cancer. It is the third most common subject for calls.\nThis paper is to be commended for addressing the issues surrounding Pernicious Anaemia and Stomach Cancer, however, the numbers and percentages given in relation to PA are disputable simply because the assays used to assess the B12 status of patients is so unreliable. Consequently any attempt to estimate how prevalent B12 Deficiency is amongst the general population is difficult to say the least. Then there's the problem of what levels constitutes a deficiency or, even more problematic, a sub-clinical deficiency. This problem often leads to patients spending many years making return visits to primary care doctors and presenting with a wide range of symptoms - 30% of the PA Society waited between two and five years before being given an explanation of their worsening symptoms, with 14% waiting over ten years. Perhaps the 'Active B12 ' trust (holotranscobalamin) along with Homocysteine and Methylmalonic Acid might be considered if the patient's clinical picture is discordant with the serum B12 results.\nThere is another problem worth mentioning - only around 40-50% of patients presenting with the symptoms of Pernicious Anaemia will have any enlarged red blood cells and many won't have any anaemia. If the patient is assessed as being deficient in B12 the assay to determine whether the deficiency is due to autoimmune pernicious anaemia is also seriously flawed - something addressed by the Guideline from the British Committee for Standards in Haematology referenced by the authors. With there being little chance of the return of the Schilling Test anytime soon clinicians should be aware of the failings of the competitive binding luminescence assay to diagnose PA.\nIt's interesting to note that members of the Pernicious Anaemia Society report that 82% of members have some gastrointestinal problems. The most common is sudden unaccountable bouts of diarrhoea which patients treat using strong probiotics although it can take up to a month for the patient to find any relief from their symptoms. The Pernicious Anaemia Society is campaigning to have all newly diagnosed patients to automatically be referred for endoscopy and colonoscopy to detect any neuro-endocrine tumours which patients are particularly susceptible to.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-604
https://f1000research.com/articles/9-1262/v1	21 Oct 20	{ "type": "Research Article", "title": "Activation of the Keap1-Nrf2 pathway by specioside and the n-butanol extract from the inner bark of Tabebuia rosea (Bertol) DC", "authors": [ "Sandra Catalina Garzón-Castaño", "Francisco Javier Jiménez-González", "Luz Angela Veloza", "Juan Carlos Sepúlveda-Arias", "Francisco Javier Jiménez-González", "Luz Angela Veloza" ], "abstract": "Background: A large number of chemical compounds exert their antioxidant effects by activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2. The aim of this study was to evaluate the activation of the Keap1-Nrf2 pathway by both the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC and specioside isolated from this extract. Methods: The antioxidant activity of the extract and specioside isolated from the inner bark of T. rosea were evaluated using the oxygen radical absorbance capacity (ORAC) and the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques, whereas their effects on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The effects of the compound and the extract on activating the Keap1-Nrf2 pathway were evaluated using a Nrf2 Transcription Factor Assay kit. Induction of the Nrf2-mediated antioxidant response genes HMOX-1 and NQO1 was evaluated by real-time PCR. The protective effects against H2O2-induced oxidative stress in HepG2 cells was determined as the percent protection using the MTT method. Results: Both the n-butanol extract and specioside exhibited activity at low concentrations without affecting cellular viability, since the cell viability was greater than 80% after 24 hours of exposure at each tested concentration. In addition, Nrf2 dissociated from Keap1 after treatment with the n-butanol extract at a concentration of 0.25 µg/mL after 4 hours of exposure. An increase in the Nrf2 level in the cytoplasm after 4 hours of exposure to 2 μM specioside was observed. Nrf2 levels stabilized in the nucleus 12 hours after stimulation with both specioside and the extract. After 6 hours of stimulation, both the extract and specioside induced the expression of HMOX-1 and NQO1. Conclusion: The n-butanol extract from the inner bark of T. rosea and specioside produced protective effects against H2O2-induced oxidative stress in HepG2 cells.", "keywords": [ "Tabebuia rosea", "specioside", "Bignoniaceae", "extracts", "Nrf2", "antioxidant agents" ], "content": "Abbreviations\n\nAAPH, 2,2’-Azobis(2-amidinopropane)dihydrochloride; ALA, alpha-lipoic acid; CUR, curcumin; ORAC, oxygen radical absorbance capacity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone\n\n\nIntroduction\n\nOver the years, plants have been used to treat several diseases, are considered an important source of biologically active natural products, and many have been used for the synthesis of various drugs. The natural products present in nature have great diversity in terms of their chemical structures and physicochemical properties, in addition to their biological activities. The Tabebuia genus belongs to the Bignoniaceae family, which includes more than 800 species and is considered as one of the most abundant family of plants in the neotropics1. The genus Tabebuia is distributed from Mexico to South America and has been used in traditional medicine. Several bioactive molecules such as saponins, quinones, tannins and alkaloids2,3 found in Tabebuia species have anti-inflammatory and antioxidant activities4–6. There is a wide distribution of the species Tabebuia rosea throughout Colombia, which has motivated the study of its therapeutic properties. The antioxidant capacity of extracts obtained from T. rosea has not been widely studied, although this activity has been demonstrated in other species of the Tabebuia genus.\n\nOxidative stress is the result of an imbalance between the production of reactive oxygen species (ROS) and the cellular antioxidant capacity, inducing oxidative damage, which plays a role in the development of premature aging, chronic diseases and cancer7–11. In addition, oxidative stress contributes to pathogenesis in many neurodegenerative diseases, such as Parkinson's, Alzheimer's, Huntington's and amyotrophic lateral sclerosis, where increases in oxidative markers have been found12,13. Cells respond to oxidative stress through various defense mechanisms, such as the elimination of free radicals and the generation of antioxidant molecules mediated by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Nrf2 is a redox-sensitive transcription factor that plays a major role in cell defense against oxidative stress. Nrf2 belongs to a family of basic proteins with a leucine zipper domain (bZip)14. Under normal conditions, Nrf2 is localized in the cytoplasm and is inhibited by the Keap1 protein (Kelch ECH associating protein 1) and therefore degraded in the proteasome. In the presence of oxidative stress, Nrf2 translocates to the nucleus. Once there, it binds to the ARE site and induces the expression of antioxidant enzymes such as NAD(P)H quinone oxidoreductase (NQO1) and heme oxygenase 1 (HMOX-1). This response is important to modulate the homeostatic balance of the cells15–20. A large number of molecules activate transcriptional regulatory mechanisms to induce their antioxidant activity.\n\nNatural product research, guided by ethnopharmacological knowledge, has made significant contributions to drug innovation by providing new chemical structures and understanding of their mechanisms of action. Considering the potential health benefits and the possible pharmacological effects of extracts obtained from T. rosea, its abundance in Colombia and the few investigations regarding its antioxidant properties and the molecular mechanisms involved in its activity, the aim of this study was to evaluate the mechanism responsible for the in vitro antioxidant activity of the n-butanol extract obtained from the inner bark of T. rosea (Bertol) DC.\n\n\nMethods\n\nThe inner bark of T. rosea (Bertol) DC was collected at the Universidad Tecnológica de Pereira campus in April 2011. The plant was identified at the Colombian National Herbarium (voucher no. COL 582577). The collection and processing of the material were covered by collection permission number 1133/2014 issued by the National Environmental Licensing Authority (ANLA) of Colombia.\n\nExtracts were obtained as previously described21,22. Plant material was dried and macerated in analytical grade methanol for 48 hours. This was followed by homogenization, filtration, and concentration under vacuum using a vacuum rotary evaporator (Heidolph, Laborota model) at 40°C to obtain the crude extract. The crude extract was dissolved in 400 mL of distilled water and underwent liquid–liquid extraction with increasing polarity solvents: n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and n-butanol (all solvents were of analytical grade). Each extract was vacuum dried by a vacuum rotary evaporator. Endotoxin levels in the extracts were assayed using the Limulus Amebocyte Lysate Test (E-Toxate kit, Sigma Chemical Co., Saint Louis, MO, USA, Cat No. ET0200-1KT). The n-butanol extract was kept refrigerated at 4°C in an amber tube protected from light, heat, air and moisture. For each of the biological assays, the extract was dissolved in 0.1% DMSO (dimethyl sulfoxide, Merck, Darmstadt, Germany, Cat No. 1029521000).\n\nThe butanolic extract was concentrated with rotary evaporation under reduced pressure, obtaining a dark brown extract (12.5 g). The butanolic extract (8.0 g) was subjected to separation by column chromatography (CC) on Diaion® HP-20 (Mitsubishi Chemical Corp.), with a water-isopropanol elution gradient (90:10 to 10:90), obtaining subfractions A-J. Tr-1 (25.9 mg) was isolated from subfraction D with a semipreparative HPLC-DAD system (Hitachi-Merck) in reversed-phase (LiChrocart 250-10, LiChrospher 100; 10 µm, Merck) by isocratic elution with H2O-ACN (70:30% v/v) containing 1% v/v CH3COOH. Specioside (Tr-1, Figure 1) was obtained as a dark brown amorphous solid (m.p. 142-162°C). The FTIR spectrum displayed absorption bands attributable to carbonyl groups (νC=O 1698 cm-1), hydroxyl groups (νO-H 3383 cm-1), and aromatic rings (νC=C 1605 cm-1).\n\nFull assignments from the 1H and 13C NMR spectra were made through the use of 1H-1H COSY, HSQC and HMBC experiments. All the experiments were performed on a 400 MHz Agilent spectrometer (125.6 MHz for 13C); using deuterated methanol as solvent. The 1H NMR spectrum showed two olefinic protons at δH 6.37 (H-3, dd) and δH 4.98 (H-4, dd), characteristic of the iridoid nucleus. This structure was confirmed by correlations shown in the HMBC spectrum with carbons at δC 140.95 (C-3) and δC 101.50 (C-4). In addition, two olefinic protons at δH 7.67 (1H, d, J = 16.0 Hz, H-7”) and δH 6.38 (1H, d, J = 15.9 Hz, H-8”) suggested the presence of a trans conformation, which is characteristic of a p-coumaroyl skeleton. The p-coumaroyl structure was confirmed by the observation of two signals at δH 7.48 (2H, d, J = 8.7 Hz, H-2”, H-6”) and δH 6.81 (2H, d, J = 8.7 Hz, H-3”, H-5”), characteristic of an AA’XX’ system; these data were confirmed by the 13C NMR spectrum, which exhibited eight carbon signals, including carbonyl carbon δC 164.49 (C-9’’), which was attributed to the p-coumaroyl ester. The presence of anomeric protons at δH 4.79 (1H, d, J = 7.9 Hz, H-1’), and methine signals at δH 3.42–3.23 (4H, m) are characteristic of a sugar moiety. Analysis of the 1D and 2D NMR spectra in addition to comparisons with literature data for glucoside analogs suggested that the saccharide portion was a glucose moiety. Characteristic 1H NMR, 13C NMR, COSY, HSQC and HMBC spectra are supplied as Extended data23.\n\nThe oxygen radical absorbance capacity was determined using the method described by Ou et al. with some modifications24. 2,2’-Azobis(2-amidinopropane)dihydrochloride (AAPH) and sodium fluorescein stock solutions were prepared in a 75 mM, pH 7.0 phosphate buffer solution. Thirty-one microliters of each sample was diluted in 187 µL of fluorescein (120 nM) and incubated at 37°C for 10 min. The reaction was started by the addition of 31 µL of AAPH (143 mM) to reach a final volume of 249 µL per well. The extract was evaluated at concentrations of 0.25, 0.5, 1 and 2 µg/mL. Specioside and catalposide25 (Sigma Chemical Co., Saint Louis, MO, USA, Cat No. SMB00094-1MG) as well as the controls (α-lipoic acid26,27, curcumin28,29 and quercetin30,31) were evaluated at concentrations of 0.5, 1, 2 and 4 µM. A Trolox® standard curve was prepared. Changes in fluorescence were measured with a Varian Cary Eclipse 1.1 fluorescence spectrophotometer at 2 min intervals for 120 min with emission and excitation wavelengths of 515 and 493 nm, respectively. The excitation slit width was 5 nm, as was the emission slit width32,33. The antioxidant capacity was calculated as the area under the curve (AUC)34 and is expressed as µmol Trolox® equivalents per liter (µmol TE/L).\n\nThe antioxidant activity of the extract and compounds was also evaluated by the DPPH method using the methodology described by Brand-Williams et al. with some modifications35. Thirty microliters of samples and controls were prepared at concentrations ranging from 0.25 to 2 µg/mL and 0.5 – 4 µM, respectively, and mixed with 2 mL of a methanol solution of DPPH (20 μg/mL DPPH, 5 × 10−5 mol/L); each mixture was agitated and kept in the dark for 30 min at RT. The absorbance was measured at 517 nm in a Shimadzu UV–1700 spectrophotometer. Ascorbic acid (5 – 200 μg/mL) was used for the standard curve. Each experiment was repeated three times, and the antioxidant capacity was calculated as the percent inhibition36.\n\nThe human hepatocarcinoma cell line (HepG2; ATCC; CRL-11997) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Glutamax (GIBCO/BRL, USA, Cat No. 10564-011) and 10% heat-inactivated FBS (GIBCO, Cat No. 16140071), 200 µg/mL penicillin, 200 µg/mL streptomycin, 400 µg/mL neomycin (GIBCO, Cat No. 15640-055), 5 µg/mL amphotericin and 1 mM sodium pyruvate (all from Sigma Chemical Co., Saint Louis, MO, USA, Cat Nos. A9528-50MG and S8636-100ML, respectively). Cells were maintained at 37°C in a 5% CO2 atmosphere.\n\nThe viability of HepG2 cells in the presence of the extracts and compounds was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method37. Cells (5×104 cell/well) were treated with the extracts (0.25, 0.5 and 1 µg/mL) and compounds - 0.5, 1 and 2 µM specioside, catalposide and controls: α-lipoic acid (ALA), curcumin (CUR) and 2-trifluoromethyl-2ʹ-methoxychalcone38 (CHAL), diluted in DMSO and incubated for 24 hours. After treatment, the medium was discarded, and 200 μL of DMEM containing 0.5 mg/mL MTT (Sigma Chemical Co., Saint Louis, MO, USA, Cat No. M2128-500MG) was then added to each well. The plates were incubated for 4 hours at 37°C in a 5% CO2 atmosphere. The medium was discarded, and 100 μL of DMSO was then added to solubilize the formazan crystals. The absorbance was measured with an ELISA microplate reader at 492 nm (ELx800; BioTek Instruments Inc., USA). The percent viability was calculated based on the nontreated control. Three independent assays were performed, each in triplicate.\n\nA total of 1×106 HepG2 cells/well were cultured in DMEM. The medium was discarded, and the cells were exposed to the extract (0.25 or 1 µg/mL), compounds or controls (0.5 or 2 µM) for 0, 4, 12 or 24 hours. After exposure, cells were harvested and used for the simultaneous extraction of nuclear and cytosolic proteins following the specifications included in the Nuclear Extraction Kit (Abcam, Cambridge, UK, ab113474). Total protein was quantified using the BCA Protein Quantification Kit (Abcam, Cambridge, UK, ab102536). Nrf2 was detected by using the Nrf2 Transcription Factor Assay Kit (Colorimetric, Abcam, Cambridge, UK, ab207223) following the manufacturer’s instructions. The absorbance of each well was measured at 450 nm in an ELx800 BioTek microplate reader.\n\nHepG2 cells (3×105 cells/well) were treated with extract (0.25 or 1 µg/mL), compounds or controls (0.5 or 2 µM) for 0, 4, 6 or 8 hours. After treatment, mRNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Maryland, USA, Cat No. 74134). mRNA was quantified with a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA). The expression of the HMOX-1 and NQO1 genes21,39–41 was evaluated by RT-qPCR using predesigned TaqMan Gene Expression Assays (Hs01110250_m1 and Hs01045993_g1) and the TaqMan® RNA-to-CTTM 1-Step Kit (Applied Biosystems, Foster City, CA, Cat No. 4331182). The run method was holding at 48°C for 15 min, 95°C for 10 min and cycling (40 cycles) at 95°C for 15 sec and 60°C for 1 min. β-actin (Applied Biosystems, Foster City, CA, ref. 4325788) was used as an endogenous control.\n\nHydrogen peroxide (H2O2) was employed as a stressor agent in order to evaluate the protective capacity of the extract (0.25 and 1 µg/mL) and compounds (0.5 and 2 µM). The controls used were ALA and CUR, since these compounds have been reported to have protective effect against oxidative stress mediated by Nrf242,43. Cells were grown to a density of 5×104 cells/well in DMEM and incubated for 24 hours under a 5% CO2 atmosphere at 37°C. The medium was discarded, and the cells were exposed for 12 hours to different concentrations of the extract (0.25 or 1 µg/mL), compounds and controls (0.5 or 2 µM). Subsequently, the medium was discarded, and one of the plates was exposed to 0.98 µM H2O2 (previously determined concentration) and the second plate was used as a control. After 24 hours, 200 μL of MTT (0.5 mg/mL, Sigma) was added to both plates followed by incubation at 37°C for 4 hours. The medium was discarded, and 100 µL of 99.8% DMSO (Sigma) was added to solubilize the formazan crystals. The absorbance was measured in an ELISA microplate reader at 570 nm with correction at 630 nm (ELx800; BioTek Instruments Inc., USA). The percent inhibition was calculated.\n\nEach experiment was performed at least in duplicate. The results are expressed as the mean ± SD of at least three independent experiments. Statistical analysis was performed using the Kruskal-Wallis test, and a p-value less than 0.05 was considered statistically significant. The statistical tests were applied using GraphPad Prism, version 5.02 (GraphPad Software, San Diego, CA, USA).\n\n\nResults\n\nThe antioxidant activity of the n-butanol extract from the inner bark of T. rosea, the isolated compound specioside and the catalposide iridoid compound reported from the Bignoniaceae family were evaluated using the ORAC and DPPH techniques44. The results showed that there was a tendency for the activity in the ORAC assay to be higher when the extract or compound concentration increased, displaying a concentration-dependent relationship, except for the control compound ALA. In addition, specioside, catalposide and the n-butanol extract displayed the best antioxidant activity, and this activity was significantly higher than that induced by ALA (Table 1, p <0.05), whose percent DPPH inhibition was low (<25%). Specioside displayed the best antioxidant activity, followed by catalposide, quercetin, curcumin, α-lipoic acid and finally the n-butanol extract (Table 1). The results from the MTT assay indicated that neither the n-butanol extract from the inner bark of T. rosea nor the pure compounds affected the viability of HepG2 cells, since the viabilities were all greater than 80% after 24 hours of exposure.\n\nTE, Trolox equivalents.; ORAC, oxygen radical absorbance capacity; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. All experiments were carried out in triplicate. Data represent the mean ± SD. Kruskal Wallis, Dunn's multiple comparisons test. ap<0.01, bp<0.0001 compared with control (α-Lipoic acid). cp<0.05 compared with extract and control (α-Lipoic acid).\n\nNrf2 detection enabled comparisons of the basal Nrf2 status in both the cytosol and the nucleus. It also allowed for comparison of their associated changes after exposure to the n-butanol extract (0.25 and 1 µg/mL) and the pure compounds specioside and catalposide (0.5 and 2 µM) in HepG2 cells. The results showed an increase in Nrf2 levels in the cytosol and nucleus after 4 hours of exposure (Figure 2) to the extract (0.25 µg/mL) and the controls ALA, CUR and CHAL at the lowest concentration (0.5 µM), showing a significant difference (p <0.05). By increasing the concentration of the n-butanol extract to 1 µg/mL and the pure compounds and controls to 2 µM, increases in the Nrf2 levels were observed in all samples after 4 hours of exposure (Figure 3), and this increase was maintained at 12 hours for specioside, catalposide and the n-butanol extract (p <0.05). As shown in Figure 3b, the Nrf2 levels in the nucleus increased 4 and 12 hours after exposure to the pure compounds and the n-butanol extract. In addition, the level of the protein after 12 hours of exposure to specioside were significantly higher (p<0.05) than that after exposure to the control CUR after 4 hours. This increase was measured in relation to the basal level (nonexposed cells).\n\nNrf2 levels in cytosol (a) and nucleus (b) after 0, 4, 12 and 24 hours of exposure to 0.5 mM specioside, catalposide, controls and 0.25 µg/mL n-butanol extract. Kruskal-Wallis, Dunn's post hoc. * p<0.05, p<0.01, * p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.\n\nNrf2 levels in cytosol (a) and nucleus (b) after 0, 4, 12 and 24 hours of exposure to 2 mM specioside, catalposide, controls and 1 µg/mL n-butanol extract. Kruskal Wallis, Dunn's post hoc. * p<0.05, p<0.01, * p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.\n\nThe levels of expression of the HMOX-1 and NQO1 genes were evaluated by qRT-PCR and quantified using the 2-ΔΔCt method. The results indicate that the molecules specioside and catalposide (0.5 µM) and the n-butanol extract (0.25 µg/mL) increased the expression levels of HMOX-1 (>1.5-fold change) and NQO1 (>1.4-fold change) after 6 hours of exposure (Figure 4). As shown in Figure 4a, the pure compounds significantly increased the expression levels of HMOX-1 (p<0.05) compared to the controls CUR and H2O2. The relative expression level of the NQO1 gene increased significantly after treatment with specioside compared with the control ALA (Figure 4b). At higher concentrations (1 µg/mL for the extract and 2 µM for the pure compounds and controls), the expression levels of HMOX-1 and NQO1 increased after 6 to 8 hours of exposure to specioside, catalposide and the n-butanol extract (Figure 5). A significantly higher expression level of HMOX-1 was observed after 8 hours of exposure to specioside, catalposide and the n-butanol extract compared with the controls (CUR, CHAL and H2O2, p <0.05, Figure 5a). The relative expression level of NQO1 significantly increased after exposure to specioside, catalposide and the n-butanol extract compared to the control CHAL (p <0.01, Figure 5b).\n\nRelative HMOX-1 (a) and NQO1 (b) mRNA levels after 0, 4, 6 and 8 hours post-exposure to 0.5 µM (pure compounds), 0.25 µg/mL (n-butanol extract) and induction of oxidative stress with 0.98 mM (H2O2). Kruskal Wallis Dunn's post hoc. * p<0.05, p<0.01, * p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.\n\nRelative HMOX-1 (a) and NQO1 (b) mRNA levels after 0, 4, 6 and 8 hours post-exposure to 2 µM (pure compounds), 1 µg/mL (n-butanol extract) and 0.98 mM (H2O2). Kruskal Wallis, Dunn's post hoc * p<0.05, p<0.01, * p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.\n\nThe protective effects of the n-butanol extract and the pure compounds against H2O2-induced oxidative stress was evaluated after 24 hours of exposure to H2O2. The results showed that exposure to the n-butanol extract and pure compounds reduced cell viability to 60 - 70% after H2O2 exposure (Figure 6a). A significant protective effect (p<0.05) was observed with the n-butanol extract (0.25 µg/mL) and specioside (0.5 and 2 µM), greater than 10%, compared to the control CHAL (2 µM). Additionally, exposure of cells to catalposide at the same concentration (2 µM) displayed a higher protective effect than that of the control CUR and specioside (Figure 6b). These results indicate that the n-butanol extract, specioside, catalposide, ALA and CUR induced protective effects in HepG2 cells against H2O2-induced oxidative stress at 0.98 mM.\n\na. Percentage of cell viability. b. Protective effect. Kruskal-Wallis, Dunn's post hoc. * p<0.05, p<0.01, * p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.\n\n\nDiscussion\n\nOxidative stress is the result of an imbalance between the production of reactive oxygen species (ROS) and the cellular antioxidant capacity and plays a critical role in the development of different neurodegenerative diseases and cancer12,45. Plants are an important source of biologically active natural products, many of which are also models for the synthesis of drugs. Compounds in nature reveal great diversity in terms of chemical structure and biological properties46. Studies carried out by Ghosh et al. and Fitmawati et al. concluded that medicinal plants are the best sources of phytochemicals and bioactive compounds that are useful for the development of drugs, antioxidants and those showing antidegenerative effects47,48. Several natural compounds exert their antioxidant effects through the activation of the key transcriptional regulation mechanism of Nrf2, allowing the coordinated expression of antioxidant enzymes such as NQO1 and HMOX-1. The modulation of the Keap1-Nrf2 pathway is important to maintain the homeostatic balance of the cell49.\n\nThe antioxidant activity results from the n-butanol extract by the ORAC method, which measures the oxygen radical scavenging capacity, showed a concentration-dependent relationship, obtaining the best activity (p <0.05) at 2 µg/mL. There are no reports in the literature regarding the evaluation of the antioxidant capacity of T. rosea extracts using the ORAC technique. Studies carried out to determine the hydroxyl scavenging radical capacity from species in the Tabebuia genus include those from the crude extracts of the leaves from Tabebuia chrysantha G. Nicholson. These studies indicated that the methanolic and aqueous extracts have a significant effect on the uptake of hydroxyl radicals (between 57–86%). Moreover, it was detected that the extracts of T. chrysantha can also act to decrease the production of these radicals50. The antioxidant activity reported by Kwak et al. for iridoid glycosides showed that specioside and catalposide had potent antioxidant activity by the ORAC method51. When evaluating the antioxidant capacity by the DPPH method, it was observed that at 0.25-2 µg/mL (0.5-4 µM pure compound), an inhibition percentage of 50% was not obtained, and specioside and CUR showed concentration-dependent behavior. The results indicated that the extract of the inner bark of T. rosea and the pure compounds show the absence of the DPPH radical scavenging activity. Franco Ospina et al. reported the low antioxidant activity of the extracts from the inner bark of T. rosea using the DPPH assay. A study of the methanolic extract from the flowers, leaves, roots and inner bark of T. pallida showed antioxidant potential using the FRAC and DPPH methods52. Comparative analysis of the ethanolic extracts of T. rosea from flowers shows strong antioxidant activity against DPPH and hydroxyl radicals. On the other hand, the report by Joubouhi et al. on iridoid compounds found radical scavenging ability against DPPH and ABTSˑ+53.\n\nAlong with the previous results, the in vitro antioxidant activity was carried out with 0.25 and 1 µg/mL extract and 0.5 and 2 µM pure compounds. Activation of the Keap1-Nrf2 pathway revealed the ability of the extract and compounds (specioside and catalposide) to activate this transcription factor, with an increase in the levels of the protein in the nucleus after treatment with the extract and pure compounds. Several natural antioxidant compounds, such as curcumin, sulforaphane and resveratrol, have been reported as electrophilic regulators of the activation of the Keap1-Nrf2 complex. In addition, they are also used for the treatment of different pathologies, such as type 2 diabetes, asthma, and cancer54. The translocation of Nrf2 to the nucleus allows the expression of antioxidant response genes such as HMOX-1 and NQO1. The results show how the compounds and extract increase the expression levels of HMOX-1 and NQO1. A study carried out by our research group evaluated the antioxidant capacity of the ethyl acetate extract from the inner barks of T. rosea and T chrysantha, in which the capacity of the extracts to activate Nrf2 translocation (after 4 hours of exposure) was reported to induce the expression of NQO121. Glycosylated iridoid compounds such as aucubin55, catalposide and verproside (the main compounds found in the ethyl acetate fraction of Veronica ciliata)56 showed a protective effect mediated by Nrf2, increasing the expression levels of the gene and the antioxidant protein NQO1. Ma et al. reported that aucubin positively regulates Nrf2 translocation and induces the response of phase II antioxidant enzymes such as HMOX-1, NQO1 and SOD, considering aucubin a promising candidate to prevent oxidative stress that induces testicular damage57. Moon et al. evaluated the ability of catalposide to induce the expression of HMOX-1 and its protein in a concentration- and time-dependent manner and found that exposure of neuro-2A cells to catalposide generates a protective effect against H2O2-induced oxidative stress, increasing the levels of the enzyme HMOX-158.\n\nIn order to evaluate the protective effects of the extract and compounds, the concentration of H2O2 (0.98 mM) that induced death by oxidative stress in 50% of the cells was determined. The concentration found in this study for the HepG2 cell line is similar to previous reports59. The results show that the extract and compounds exert a protective effect against oxidative stress induced by H2O2. The extract, specioside and catalposide had protective effects of more than 10%. The results obtained for catalposide are similar to those reported by Moon et al. Catalpol, another glycosylated iridoid, showed a protective effect against oxidative stress induced by H2O2 in a primary astrocyte culture60. Wang et al. evaluated the Nrf2-mediated neuroprotective capacity of swertiamarin, a glycosylated secoiridoid, and reported increases in the levels of the antioxidant proteins NQO1 and HMOX-1 in addition to an increase in the Nrf2 protein at the nuclear level61. The only report of the protective effects of specioside was made by Asthana et al., in which the ability of the compound to modulate antioxidant enzymes such as CAT and SOD was evaluated in a Caenorhabditis elegans model62.\n\nThis study is the first report of the in vitro protective effects of the extract of the inner bark of T. rosea against oxidative stress induced by H2O2.\n\n\nConclusion\n\nThe present study indicates that the n-butanol extract from the inner bark of T. rosea and its isolated compound specioside have promising antioxidant activity. Both biocompounds have the ability to activate the Keap1-Nrf2 pathway, inducing the expression of HMOX-1 and NQO1, and generating a protective effect against H2O2-induced oxidative stress in the HepG2 cell line. These results reinforce the importance of these plants in the search for new antioxidant molecules.\n\n\nData availability\n\nOpen Science Framework: Activation of the Keap1-Nrf2 pathway Tabebuia. https://doi.org/10.17605/OSF.IO/HW6X944.\n\nThis project contains the following underlying data:\n\n- Dataset 1 Nrf2 Levels Figure 2.csv\n\n- Dataset 2 Nrf2 Levels Figure 3.csv\n\n- Dataset 3 Relative HMOX-1 and NQO1 mRNA levels Figure 4.csv\n\n- Dataset 4 Relative HMOX-1 and NQO1 mRNA levels Figure 5.csv\n\n- Dataset 5 Relative HMOX-1 and NQO1 mRNA levels Figure 6.csv\n\nOpen Science Framework: Garzon_et_al_Nrf22020_Supplementary .pdf. https://doi.org/10.17605/OSF.IO/TRVB223.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBittencourt NS Jr, Pereira EJ Jr, de Souza São-Thiago P, et al.: The reproductive biology of Cybistax antisyphilitica (Bignoniaceae), a characteristic tree of the South American savannah-like “Cerrado” vegetation. 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Publisher Full Text\n\nSepúlveda-Arias JC, Garzón-Castaño SC, jimenez-González FJ, et al.: Garzon_et_al_Nrf22020_Supplementary .pdf. http://www.doi.org/10.17605/OSF.IO/TRVB2\n\nOu B, Hampsch-Woodill M, Prior RL: Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe. J Agric Food Chem. 2001; 49(10): 4619–26. PubMed Abstract \| Publisher Full Text\n\nMoon MK, Choi BM, Oh GS, et al.: Catalposide protects Neuro 2A cells from hydrogen peroxide-induced cytotoxicity via the expression of heme oxygenase-1. Toxicol Lett. 2003; 145(1): 46–54. PubMed Abstract \| Publisher Full Text\n\nShay KP, Michels AJ, Li W, et al.: Cap-independent Nrf2 translation is part of a lipoic acid-stimulated detoxification stress response. Biochim Biophys Acta. 2012; 1823(6): 1102–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHwang S, Byun JW, Yoon JS, et al.: Inhibitory Effects of α-Lipoic Acid on Oxidative Stress-Induced Adipogenesis in Orbital Fibroblasts From Patients With Graves Ophthalmopathy. Medicine (Baltimore). 2016; 95(2): e2497. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalogun E, Hoque M, Gong P, et al.: Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J. 2003; 371(Pt 3): 887–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzalez-Reyes S, Guzman-Beltran S, Medina-Campos ON, et al.: Curcumin pretreatment induces Nrf2 and an antioxidant response and prevents hemin-induced toxicity in primary cultures of cerebellar granule neurons of rats. Oxid Med Cell Longev. 2013; 2013: 801418. 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Publisher Full Text\n\nCao G, Prior RL: Measurement of oxygen radical absorbance capacity in biological samples. Methods Enzymol. 1999; 299: 50–62. PubMed Abstract \| Publisher Full Text\n\nBrand-Williams W, Cuvelier ME, Berset C: Use of a free radical method to evaluate antioxidant activity. LWT - Food Science and Technology. 1995; 28(1): 25–30. Publisher Full Text\n\nChen Z, Bertin R, Froldi G: EC50 estimation of antioxidant activity in DPPH assay using several statistical programs. Food Chem. 2013; 138(1): 414–20. PubMed Abstract \| Publisher Full Text\n\nMosmann T: Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983; 65(1-2): 55–63. PubMed Abstract \| Publisher Full Text\n\nKumar V, Kumar S, Hassan M, et al.: Novel Chalcone Derivatives as Potent Nrf2 Activators in Mice and Human Lung Epithelial Cells. J Med Chem. 2011; 54(12): 4147–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuerta-Olvera SG, Macías-Barragán J, Ramos-Márquez ME, et al.: Alpha-lipoic acid regulates heme oxygenase gene expression and nuclear Nrf2 activation as a mechanism of protection against arsenic exposure in HepG2 cells. Environ Toxicol Pharmacol. 2010; 29(2): 144–9. PubMed Abstract \| Publisher Full Text\n\nKoriyama Y, Kamiya M, Takadera T, et al.: Protective action of nipradilol mediated through S-nitrosylation of Keap1 and HO-1 induction in retinal ganglion cells. Neurochem Int. 2012; 61(7): 1242–53. PubMed Abstract \| Publisher Full Text\n\nYap WH, Khoo KS, Ho ASH, et al.: Maslinic acid induces HO-1 and NOQ1 expression via activation of Nrf2 transcription factor. Biomedicine & Preventive Nutrition. 2012; 2(1): 51–8. Publisher Full Text\n\nBalogun E, Hoque M, Gong P, et al.: Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element. Biochem J. 2003; 371(Pt 3): 887–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoriyama Y, Nakayama Y, Matsugo S, et al.: Protective effect of lipoic acid against oxidative stress is mediated by Keap1/Nrf2-dependent heme oxygenase-1 induction in the RGC-5 cell line. Brain Res. 2013; 1499: 145–57. PubMed Abstract \| Publisher Full Text\n\nSepúlveda-Arias JC, Garzón-Castaño SC, Jiménez-González FJ, et al.: Activation of the Keap1-Nrf2 pathway Tabebuia. 2020. http://www.doi.org/10.17605/OSF.IO/HW6X9\n\nNa HK, Surh YJ: Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1. Free Radic Biol Med. 2014; 67: 353–65. PubMed Abstract \| Publisher Full Text\n\nOlga RLS: Flora andina y amazónica: un aporte a su conocimiento químico. Reference Source\n\nGhosh P, Das C, Biswas S, et al.: Phytochemical composition analysis and evaluation of in vitro.medicinal properties and cytotoxicity of five wild weeds: A comparative study. F1000Res. 2020; 9: 493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFitmawati F, Resida E, Kholifah SN, et al.: Phytochemical screening and antioxidant profiling of Sumatran wild mangoes ( Mangifera spp.): a potential source for medicine antidegenerative effects. F1000Res. 2020; 9: 220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDi Domenico F, Foppoli C, Coccia R, et al.: Antioxidants in cervical cancer: Chemopreventive and chemotherapeutic effects of polyphenols. Biochim Biophys Acta. 2012; 1822(5): 737–47. PubMed Abstract \| Publisher Full Text\n\nOspina LF, Aragón DM, Vergel NE, et al.: Anti-inflammatory and antioxidant activities of Phenax rugosus. (POIR.) WEDD and Tabebuia chrysanta. G. NICHOLSON. Vitae. 2011; 18(1): 49–55. Reference Source\n\nKwak, Kim HJ, Lee KH, et al.: Antioxidative iridoid glycosides and phenolic compounds from Veronica peregrina. Arch Pharm Res. 2009; 32(2): 207–13. PubMed Abstract \| Publisher Full Text\n\nRahman MM, Islam MB, Biswas M, et al.: In vitro. antioxidant and free radical scavenging activity of different parts of Tabebuia pallida. growing in Bangladesh. BMC Res Notes. 2015; 8: 621. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSobiyana P, Anburaj G, Manikandan R: Comparative Analysis of the in vitro. antioxidant antivity of Tabebuia rosea. and Tabebuia argentea. J Pharmacogn Phytochem. 2019; 8(1): 2673–2677. Reference Source\n\nRobledinos-Antón N, Fernández-Ginés R, Manda G, et al.: Activators and Inhibitors of NRF2: A Review of Their Potential for Clinical Development. Oxid Med Cell Longev. 2019; 2019: 9372182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQiu YL, Cheng XN, Bai F, et al.: Aucubin protects against lipopolysaccharide-induced acute pulmonary injury through regulating Nrf2 and AMPK pathways. Biomed Pharmacother. 2018; 106: 192–9. PubMed Abstract \| Publisher Full Text\n\nLu Q, Tan S, Gu W, et al.: Phytochemical composition, isolation and hepatoprotective activity of active fraction from Veronica ciliata against acetaminophen-induced acute liver injury via p62-Keap1-Nrf2 signaling pathway. J Ethnopharmacol. 2019; 243: 112089. PubMed Abstract \| Publisher Full Text\n\nMa B, Zhang J, Zhu Z, et al.: Aucubin, a natural iridoid glucoside, attenuates oxidative stress-induced testis injury by inhibiting JNK and CHOP activation via Nrf2 up-regulation. Phytomedicine. 2019; 64: 153057. PubMed Abstract \| Publisher Full Text\n\nMoon MK, Choi BM, Oh GS, et al.: Catalposide protects Neuro 2A cells from hydrogen peroxide-induced cytotoxicity via the expression of heme oxygenase-1. Toxicol Lett. 2003; 145(1): 46–54. PubMed Abstract \| Publisher Full Text\n\nSubramaniam SR, Ellis EM: Esculetin-induced protection of human hepatoma HepG2 cells against hydrogen peroxide is associated with the Nrf2-dependent induction of the NAD(P)H: Quinone oxidoreductase 1 gene. Toxicol Appl Pharmacol. 2011; 250(2): 130–6. PubMed Abstract \| Publisher Full Text\n\nBi J, Jiang B, Liu JH, et al.: Protective effects of catalpol against H2O2-induced oxidative stress in astrocytes primary cultures. Neurosci Lett. 2008; 442(3): 224–7. PubMed Abstract \| Publisher Full Text\n\nWang H, Wei W, Lan X, et al.: Neuroprotective Effect of Swertiamain on Cerebral Ischemia/Reperfusion Injury by Inducing the Nrf2 Protective Pathway. ACS Chem Neurosci. 2019; 10(5): 2276–86. PubMed Abstract \| Publisher Full Text\n\nAsthana J, Yadav AK, Pant A, et al.: Specioside ameliorates oxidative stress and promotes longevity in Caenorhabditis elegans. Comp Biochem Physiol C Toxicol Pharmacol. 2015; 169: 25–34. PubMed Abstract \| Publisher Full Text" }	[ { "id": "73506", "date": "27 Oct 2020", "name": "Jaime Martin‐Franco", "expertise": [ "Reviewer Expertise Organic Synthesis", "natural products and spectral analysis by RMN" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPlants are an important source of biologically active natural products, many of them also constitute models for the synthesis of numerous drugs. The genus Tabebuia includes about 100 species and is the largest genus in the Bignoniaceae family. It is important to indicate that molecules present in the inner bark of some species of this genus (used in traditional medicine) have important pharmacological potential. A large number of chemical compounds exert their antioxidant effects through the activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). In cells exposed to oxidative stress. Nrf2 is released and translocated to the nucleus and activates the antioxidant response. The aim of this study was to evaluate the mechanism responsible for the in vitro antioxidant activity of the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC. Specioside was isolated and elucidated previously in the Research Group Polifenoles at Universidad Tecnológica de Pereira from the n-butanol extract obtained from the inner bark of T. rosea. The antioxidant activity of specioside was evaluated using the ORAC and DPPH techniques. The effect of specioside and the extract on the viability of HepG2 cells was determined using the MTT method. The effect of the compound and the extract on the translocation of Nrf2 to the nucleus was evaluated using the Nrf2 Transcription Factor Assay Kit (abcam), according to the manufacturer's instructions. Molecular docking was carried out using the AutoDock software. The antioxidant activity indicated that both the extract and the isolated compounds have antioxidant activity as measured by the ORAC (oxygen radical absorbance capacity) technique. The cell viability was determined using the MTT assay. The results indicate that specioside exhibits its activity at low concentrations without affecting the viability of the cells since viability was greater than 90% after 24 hours of exposure. They allowed performing in vitro tests to evaluate the effect of the isolated compounds on the activation of the Nrf2 signaling pathway in HepG2 cells. Nrf2 is dissociated from Keap1 by the n-butanol extract at a concentration of 0.5 μM after 4 hours of exposure, showing an increase in both cytosolic and nuclear Nrf2 levels with a significant difference (p <0.05) in comparison to the basal levels. Specioside at a concentration of 2 μM did increase the Nrf2 levels in the cytoplasm after 4 hours of exposure and was stabilized in the nucleus at 12 hours after stimulation with specioside and extract (significant difference when compared to the basal levels, p <0.05). The molecular docking analysis showed the interaction of the iridoid group of the ligand with the residue Tyr572 and Ser602 of Keap1 to be important for the interaction with Nrf2. In the analysis of the 1H NMR spectrum of the specioside shown in Fig. 1. the authors should write E instead of trans and configuration instead of conformation.\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": [ { "c_id": "6110", "date": "24 Nov 2020", "name": "Juan Carlos Sepúlveda-Arias", "role": "Author Response", "response": "Dear Dr. Martin-Franco, Thank you very much for you comments. We did change \"trans conformation\" with \"E configuration\"." } ] }, { "id": "73587", "date": "09 Nov 2020", "name": "Luca Rastrelli", "expertise": [ "Reviewer Expertise Food chemistry", "natural products", "analytical chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 indicates that the n-butanol extract from the inner bark of T. rosea and its isolated compound specioside have promising antioxidant activity.\nThis work is well presented and easy to read. The experiments and the structural characterization have been conveniently described and the analyses were performed by appropriate methods. There is sufficient discussion of the results obtained. It merits being indexed after minor revision. Detailed remarks on the text are as follows:\nExtraction and isolation: The authors obtain 12.5 g of BuOH extract, please report the quantities of bark extracted to calculate the yield. Figure 1: Report catalposide structure.\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": [ { "c_id": "6111", "date": "24 Nov 2020", "name": "Juan Carlos Sepúlveda-Arias", "role": "Author Response", "response": "Dear Dr. Rastrelli. Thank you very much for your comments. In the \"Plant material, extract preparation and specioside isolation\" section, we did include the quantity of bark extracted and the yields. In Figure 1 we did include the catalposide structure." } ] } ]	1	https://f1000research.com/articles/9-1262
https://f1000research.com/articles/9-1164/v1	22 Sep 20	{ "type": "Research Article", "title": "Immunohistochemical analysis of stem cells from human exfoliated deciduous teeth seeded in carbonate apatite scaffold for the alveolar bone defect in Wistar rats (Rattus novergicus)", "authors": [ "Tania Saskianti", "Alexander Patera Nugraha", "Chiquita Prahasanti", "Diah Savitri Ernawati", "Ketut Suardita", "Wibi Riawan", "Tania Saskianti", "Alexander Patera Nugraha", "Diah Savitri Ernawati", "Ketut Suardita", "Wibi Riawan" ], "abstract": "Background: Stem cells from human exfoliated deciduous teeth (SHED) seeded in carbonate apatite scaffold (CAS) may have multiple functions that could be used to regenerate the alveolar bone defects. The purpose of this study is to examine the ability of SHED and CAS in alveolar bone defects using an immunohistochemical analysis. Methods: ten three-month-old healthy male Wistar rats (R. novergicus) that weighed between 150–250 grams (g) were used as animal models. A simple blind random sampling method was used to select the sample that was assigned to the study group for CAS and SHED seeded in CAS (n=5). The animal study model of the alveolar bone was established by extracting the anterior mandible teeth. Rodent anesthesia was applied to relieve the pain during the procedure for all test animals. Immunohistochemistry was performed after seven days to facilitate the examination of the receptor activator of NF-κβ ligand (RANKL), osteoprotegrin (OPG), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin, and osteopontin expression. The data was analyzed using the unpaired t-test (p<0.01) and Pearson’s correlation test (p<0.05). Results: The OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin expressions were higher in SHED seeded in CAS than CAS only with a significant difference between the groups (p<0.01). Furthermore, the RANKL expression was lower in SHED seeded in CAS compared to CAS only. There was a strong reverse significant correlation between OPG and RANKL expression (p<0.05). Conclusions: The number of osteogenic marker expressing cells, such as OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin, increased. However, RANKL expression in the alveolar bone defects that were implanted with SHED seeded in CAS did not increase after seven days.", "keywords": [ "Bone Defect Socket", "Carbonate Apatite Scaffold", "Medicine", "Osteogenic Ability", "Stem Cell from Human Exfoliated Deciduous Teeth" ], "content": "Introduction\n\nPeriodontitis is the second most prevalent oral disease after dental caries1. Approximately 743 million people globally suffer from periodontitis, and this figure has increased by 57.3% over the last ten years2,3. Globally, the losses that are due to reduced productivity caused by severe periodontitis are estimated to be 53.99 million United States (US) dollars annually3,4. Periodontitis is common in Indonesia5. A previous study that was conducted by the Health Ministry of Republic of Indonesia in Basic Health Research (Riset Kesehatan Dasar or RISKESDA) in 2018 showed that there was a 74.1% prevalence of periodontitis6. The rate of periodontitis varies in each country, but together with dental caries, periodontitis is the main reason for tooth loss in adults1. Periodontitis should be treated appropriately because, if it is neglected, it can become severe and lead to the loss of the affected alveolar bone and resorption7. Low socio-economic conditions in certain populations will increase the prevalence and extent of the tooth loss, which can result in an alveolar bone defect due to the limited access to dental treatment8. Tooth extraction due to periodontitis can lead to alveolar bone resorption and the destruction of the alveolar bone components9. The resorption of the alveolar bone or a reduction in the jawbone dimensions might occur as a result of severe periodontitis that is left untreated or neglected9–11. The presence of periodontal disease, irrational or traumatic dental extraction, periapical root fractures or alveolectomies during dental extractions are considered risk factors for or the etiology of an alveolar bone defect12. An alveolar bone defect can be problematic for dental rehabilitation due to the placement of dental prosthethics13. Osseointegrated of dental implants with sufficient initial stability requires adequate bone quality and quantity. Moreover, it is suggested that socket preservation is performed to enhance the success of the osseointegrated dental implants14.\n\nIn the dental medicine field, the management and rehabilitation of alveolar bone defects has long been viewed as a challange15. To overcome alveolar bone defects, dentists must consider bone grafting surgeries for socket preservation to obtain an adequate bone density, volume, quality, and geometry for the implant placement. This will enable osseointegration of the dental implant15.\n\nThere have been many attempts to overcome alveolar bone defects, such as bone grafts, platelet rich fibrin (PRF), mesenchymal stem cells, hematopoetic stem cells, and herbal medicine16–23. Bone grafts are still not effective; therefore, alternative tissue engineering approaches are required24.\n\nThe current most promising treatment for an alveolar bone defect is through regenerative medicine, which uses tissue engineering. This tissue engineering involves three components, and is therefore referred to as triad tissue engineering: growth factors, stem cells, and a scaffold25,26. Mesenchymal stem cells (MSCs) can differentiate into various cells, such as osteogenic, adipogenic and chondrogenic differentiations27. The oral cavity provides a rich source of MSCs. MSCs, such as gingiva mesenchymal stem cells (GMSCs), dental pulp stem cells (DPSCs), and stem cells from human exfoliated deciduous teeth (SHED), can be easily isolated and obtained from the oral cavity tissue using minimally invasive procedures comparted to those needed for bone marrow mesenchymal stem cells (BM-MSCs)28–31.\n\nSHED is one of the MSCs from the oral cavity that can be used to regenerate damaged tissue, such as an alveolar bone defect24. SHED is capable of differentiating and proliferating. Moreover, to optimally facilitate SHED proliferation, cell growth, and differentiation, a biocompatible cell carrier or scaffold is necessary32. Carbonate apatite is a biomaterial that is commonly used as a scaffold. Carbonate apatite has been clinically proven to be a good bone scaffold for the regenerative medicine33.\n\nThe hypothesis of this study is that the number of osteogenic markers expressing cells, such as OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin, would increase in the alveolar bone defects seven days after being implanted with SHED seeded in carbonate apatite scaffold (CAS), with the exception of the receptor activator of NF-κβ ligand (RANKL) expression. Osteocalcin, osteopontin, ALP, RUNX are the osteogenic differentiation markers of SHED. CAS can facilitate the osteogenic differentiation of SHED in vitro. Meanwhile, RANKL / OPG ratio are well-known as markers that can be used to predict the success of bone remodeling. Some growth factors are secreted by SHED, such as TGF-β and VEGF, which have an important role in supporting bone formation and controlling the inflammation process17–24,32–34.\n\nWistar rats (Rattus novergicus) were selected as the animal models because many studies have used this animal to study the effect of medication on the alveolar bone defects21–24. Additionally, these rats are not aggressive, and they are easy to handle and observe. This made them suitable animal models to induce the response of the human tissue system. Furthermore, the purpose of this study is to examine the ability of SHED and CAS in the alveolar bone defects using an immunohistochemical analysis.\n\n\nMethods\n\nAll experimental procedures involving animals were carried out in accordance with the guidelines from the National Health Institute on the care and use of laboratory animals to ameliorate any suffering for the animals.\n\nThis study was an experimental laboratory design. A post-test-only control group study design was conducted. The formulation that used to calculate the sample size in this study was\n\nsample size = 2SD2(Zα/2+ Zβ)2/d2 where the standard deviation (SD) = 1.1; Zα/2 = Z 0.05/2 = Z0.025 =1.96 (from Z table) at type 1 error of 5%; Zβ = Z0.20 = 0.842 (from Z table) at 80% power; d = effect size = 1.94. The number of samples, which was five trial animals in each group. The sample in each group was randomly chosen by giving each trial animal a tag number. Following that, the researcher randomly chose the tag numbers.\n\nThe SHED were obtained from deciduous teeth using the following criteria: #83, #73 deciduous tooth, free of caries, no root resorption, and a vital and intact pulp was obtained through tooth extraction from a healthy, 7–10 years-old pediatric patient who underwent orthodontics treatment. The healthy deciduous tooth was extracted from healthy pediatric patients undergoing orthodontics treatment performed at the Dental Hospital, Universitas Airlangga, Surabaya, Indonesia. Patient anonymity was maintained and written informed consent was obtained from the patient’s parents. Ethical approval was obtained from the Universitas Airlangga, Faculty of Dental Medicine ethics committee (171/HRECC.FODM/VIII/2017) that covered for both human sampling and the animal procedures.\n\nThe dental pulp cavity was opened using drills under aseptic condition. The dental pulp was isolated with a broach then washed three times with phosphate-buffered saline (PBS). Dental pulp tissue was minced into small pieces (≤0.5 mm) in 10-cm culture dishes digested in a solution of 3 mg/mL collagenase type I (no cat. CLS-01, Worthington Biochem, Freehold, NJ) and 4 mg/mL Dispase® II (cat no. 42613-33-2, Sigma Aldrich, USA) for 1 h at 37°C. Dulbecco′s Modified Eagle′s Medium (cat no. D5030, Merck, US), was utilized to culture the dental pulp from the deciduous tooth. Fetal bovine serum (FBS, catcat no. F2442, Merck, US) with 20% concentration, five milimeter L-glutamine (cat no. 5030081, Gibco Invitrogen®, 25, USA), 100 U/ ml penicillin-G, 100 ug/ml streptomycin, and 100 ug/ml kanamycin (cat no. 15140163, Gibco Invitrogen®, 25, USA) was added34.\n\nEvery four days, the medium was changed to eliminate the unattached cell on the culture plate and the cells were maintained up to four passages. Phosphate Buffer Saline was used to wash the cells to eliminate debris. Trypsin-EDTA 0.05% was applied to detach the cells and transfer them onto a bigger culture plate. After the cells reached 70–80% confluence was obtained, the SHED cells in the 4 passaged were then prepared for the next step of the study24,32,34.\n\nTen three-month-old healthy male Wistar rats (R. novergicus) that weighed between 150–250 grams (g) were used as animal models and were obtained from the Research Center of Faculty of Dental Medicine, Universitas Airlangga (UNAIR) Surabaya, Indonesia.\n\nAll experimental procedures involving animals were carried out in keeping with guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals to ameliorate any suffering of animals35. The animal models were acclimatized for a week at a temperature of 21–23 °C with controlled humidity (50 ± 5%) in a 12-hour artificial light cycle (8 am to 8 pm) to help them to adapt to the same conditions, as they had various origins. All the rats were located individually in polycarbonate cages (0.90 × 0.60 × 0.60 m). Furthermore, every animal model was fed with standard pellet, and water was provided ad libitum with the husk replaced every three days. All animal models were routinely inspected and observed regarding their food consumption and fecal characteristics20.\n\nRodent anesthesia of 0.1 mL/10 grams body weight (BW) (160095, Kepro™, Netherlands), and xylazine (160096, Xyla™, Netherlands) (ketamine dose 35 mg/kg body weight and xylazine five mg/kg body weight) was administered intramuscularly on the gluteus muscle to ameliorate the pain during the procedure of inducing the alveolar bone defects on the animal models. Sterile needle holder clamps were used to extract the anterior teeth of the mandibular to induce the alveolar bone defects in the animal models36.\n\nAfter the alveolar bone defect was induced, the transplantation of the SHED seeded in CAS or CAS only was performed in the afflicted area. Before being placed in a 24-well tissue culture plate and prepared for the experimental group, a 20 ml suspension of SHED at passage four to five with a density of 106 cells was seeded into CAS (no cat AKD 20602410125, GAMACHA, Swayasa Prakarsa Company, Indonesia). The dose was determined based on the evidence from a previous in vivo study, which was 106 cells per sample34. To perform the interrupted suture to fix the wound after transplantation, a 5.0 suture monofilament was used24,32,34.\n\nSeven days post transplantation, all the animal models were terminated using an overdosed rodent anesthesia with an intravenous injection of 100 mg/kg BW (Pentobarbital, 1507002, Pubchem, USA). We used this euthanasia method to ameliorate animal suffering that arises from the termination process. After the termination of animal study, we collected the afflicted alveolar bone samples for further histological analysis. The animal model’s head was cut from the back by sterile sharp surgical scissors (metzenbaum scissors fine tips, no cat. 3565, Medesy, Maniago, Italy) and tweezer (Tweezer de bakey mini, no cat. 1007/10-TO, Medesy, Maniago, Italy), exposing the anterior of the mandible allowing the afflicted alveolar bone sample to be obtained. Before sample collection, all the animals were observed for any general toxicity probability, including edema or death, and measured had their body weight (using a digital scale, ZB22-P, Zieis®, USA). All these measurements were done by a single blinded observer. The afflicted tissue was then extracted and immersed in 10% neutral buffer formalin for fixation.\n\nThe sample was decalcified and immersed in 10% EDTA (cat no. 17892, Ajax Finechem, Thermo Fisher Scientific; Taren Point, Australia). Following that, the samples were underwent tissue processing overnight (Leica TP1020, USA), prior to embedding in molten paraffin wax (HistoCore Arcadia H - Heated Paraffin Embedding Station, Leica, USA). Sections were cut at 5 µm rotary microtome (RM2235, Leica, USA). Paraffin ribbons were flattened in a water bath at 40°C and collected onto polysine microscope slides (Thermo Scientific) prior to drying at 60°C for 16 hr (Sakura Heater, Tokyo, Japan)37.\n\nImmunohistochemistry staining was conducted using a 3.3'-diaminobenzidine stain kit (DAB) (cat noD7304-1SET, Sigma Aldrich, US). Antibody monoclonal (AbMo) of RANKL 1:500 dilution (cat. no sc-377079), osteoprotegrin (OPG) 1:500 dilution (cat. no sc-390518), runt-related transcription factor 2 (RUNX2) 1:500 dilution (cat. no sc-390351), transforming growth factor-β (TGF-β) 1:500 dilution (cat. no sc-130348), vascular endothelial growth factor (VEGF) 1:500 dilution (cat. no sc-7269), alkaline phosphatase (ALP) 1:500 dilution (cat. no sc-271431), osteocalcin (cat. no sc-365797)) 1:500 dilution, and ostepontin (cat. no sc-21742) 1:500 dilution were used in this study (Santa Cruz Biotechnology™, US). The observation and examination of the number of the RANKL, OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin expressions in the periodontal tissue were performed manually by two observers in five perspective fields of view by utilizing Nikon H600L light microscope (Japan) at 400x magnification. We also provide 200x and 1000x magnification of each marker for context (Nikon, Japan)37.\n\nThe Statistical Package for Social Science (SPSS) 20.0 version (IBM corporation, Illinois, Chicago, United State) software was used in this study to analyze the data. To compare the significant differences between the groups in the RANKL, OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin expressions, a t-test was employed (p<0.01). The OPG and RANKL expressions’ association was examined using Pearson’s correlation test (p<0.05).\n\n\nResults\n\nThe transplantation of SHED seeded in CAS or CAS only at selected doses did not lead to any general toxicity, edema, death or changes in body weight of the rats (see underlying data38). The expressions of OPG, RUNX2, TGF- β, VEGF, ALP, osteocalcin, and osteopontin in SHED seeded in CAS were greater than in the CAS only group. In comparison, the RANKL expression was lower in SHED seeded in CAS compared to CAS only (see Figure 1, Figure 2, Figure 3, Figure 439–46). There was a significant increase in OPG, RUNX2, TGF-β VEGF, ALP, osteocalcin, and osteopontin expressions and decreased RANKL expression in SHED seeded in CAS compared to CAS only (p<0.01). There was a significant strong reverse correlation between the OPG and RANKL expressions (p<0.01) (Table 1).\n\nImmunohistochemistry with antibody monoclonal (AbMo) and DAB were performed to examine the (A) RANKL and (B) OPG expressions. The positive cells were stained brown (black box) with a 200x, 400x, and 1000x magnification using a light microscope. The number of osteoblasts expressing (C) RANKL and (D) OPG in the alveolar bone of the rats.\n\nImmunohistochemistry with antibody monoclonal (AbMo) and DAB were performed to examine the (A) VEGF and (B) TGF-β expressions. The positive cells were stained brown (black box) with 200x, 400x, and 1000x magnification using the light microscope. The number of osteoblasts expressing (C) VEGF and (D) TGF- β in the alveolar bone of the rats.\n\nImmunohistochemistry with antibody monoclonal (AbMo) and DAB were performed to examine the (A) RUNX2 and (B) ALP expressions. The positive cells were stained brown (black box) with 200x, 400x, and 1000x magnification using the light microscope. The number of osteoblasts expressing (C) RUNX2 and (D) ALP in the alveolar bone of the rats.\n\nImmunohistochemistry with antibody monoclonal (AbMo) and DAB were performed to examine the (A) osteocalcin and (B) osteopontin expressions. Positive cells were stained brown (black box) in 200x, 400x, 1000x magnification by using the light microscope. The number of osteoblasts expressing (C) osteocalcin and (D) osteopontin in the alveolar bone of the rats.\n\nInformation: significant at p value > 0.05; *significant at p value < 0.01. RANKL - receptor activator of NF-κβ ligand, OPG - osteoprotegrin, TGF- β - transforming growth factor-β, VEGF - vascular endothelial growth factor, RUNX2 - runt-related, transcription factor 2, ALP - alkaline phosphatase, OSC - osteocalcin, OSP osteopontin, SHED- stem cells from human exfoliated deciduous teeth, CHA - carbonate hydroxyapatite\n\n\nDiscussion\n\nSevere alveolar defect has become a problem for both the patients and clinicians, especially regarding dental implant placement and ossteointegration15.\n\nThis experimental study confirms the hypothesis that the transplantation of SHED seeded in CAS could increase the number of osteogenic markers expressing cells, such as OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin, but not the RANKL expression in the bone defects after seven days in comparison to the CAS group17–20,22,32. This result supports the theory that SHED possess functions that can enhance OPG to bind to RANKL, which results in the inhibited osteoclastogenesis34. There is a strong reverse significant correlation between OPG and RANKL expressions in this study. The SHED with the scaffold increases the OPG expression meanwhile, decreases the RANKL expression, which is supported by the previous study by Prahasanti et al.34\n\nCAS plays an important role in supporting SHED proliferation and differentiation24,32. RUNX2, ALP, osteocalcin, and osteopontin are osteogenic differentiation markers of MSCs. These markers are essential and important for the analysis of osteoblastogenesis and bone regeneration17,18,20. ALP expression increases due to the signaling bone morphogenic protein (BMP), RUNX2, osterix system, and Wnt cascade interacting with each other. The increased expression of RUNX can enhance ALP expression17,18. Several growth factors also stimulate the activation of the MSCs’ osteogenic differentiation, such as VEGF and TGF- β. TGF-β significantly increases the expression of the early-phase osteogenic differentiation marker genes47. VEGF is associated with all the bone formation steps, including mesenchymal condensation48. VEGF has a direct influence on the MSC osteogenic differentiation through the regulation of osteoprogenitors using the angiocrine function. VEGF recruits immune cells to the osteogenic niche49.\n\nThe osteogenic microenvironment in defective alveolar bone can induce SHED to differentiate into bone cells, especially osteoblast24,32,34. The activation of the osteorix and RUNX2 systems can stimulate the expression of osteocalcin and osteopontin17. OSC is a secreted protein that is dependent on Vitamin K, a macromolecule with a role in bone mineralization18. Osteopontin plays a pivotal role in bone remodeling, regulating osteoclastogenesis, osteoclast activity, and differentiation. Osteopontin maintains the bone mineral matrix inorganic components of bone, such as hydroxyapatite, Ca(PO4)(OH)2. Osteopontin, which is expressed in osteoblasts, is responsible for bone remodeling in bone homeostasis20. Both osteocalcin and osteopontin are important for bone maturation because they are major non-collagenous proteins that are involved in bone matrix organization and deposition.\n\nOsteocalcin and osteopontin are produced during bone formation49. Both of them control––either directly and/or indirectly–the mass, mineral size, and orientation50–52. Both proteins also play structural roles in the bone and determine the bone’s propensity to fracture53. This is in accordance with our findings, as it states that there is a significant enhancement of the OPG, RUNX2, TGF- β, VEGF, ALP, osteocalcin, and osteopontin expressions, and the decreased RANKL expression is more significant in Group II than Group I. Bone regeneration is a complex process that requires highly orchestrated interactions between different cells and signals to form the new mineralized tissue54. MSCs have the ability to differentiate into osteoprogenitors and osteoblasts, as well as to form the calcified bone matrix55.\n\nSHED have the potential to play a significant role in tissue engineering and regenerative medicine. A previous study by Nakajima et al. declared that SHED, in comparison to the hDPSCs or hBMSCs group, produce the largest osteoid and collagen fibers. Furthermore, SHED transplantation possess a potential and sufficient ability for bone regeneration to repair the bone defect56,57.\n\nThe limitations of this study were that the observation and evaluation were performed seven days post transplantation of SHED seeded in CAS on the animal model, and only an immunohistochemical examination was performed. Further studies will be necessary to evaluate the changes in the alveolar bone and periodontal tissue post transplantation of SHED seeded in CAS in the alveolar bone defect animal models. With a longer observation time, further studies using methods, such as qRT-PCR and/or the western blot analysis, could be conducted to estimate the expression of bone molecular markers. Future studies are also required to confirm the effective dose of the used biomaterials when it is ready to be applied in the clinical study of humans.\n\n\nConclusion\n\nIn conclusion, the expression of OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin increases significantly with treatment with SHED seeded in CAS. Moreover, the RANKL expression in the alveolar bone defect did not increase in SHED seeded in CAS as documented immunohistochemically.\n\n\nData availability\n\nFigshare: RANKL. https://doi.org/10.6084/m9.figshare.12609986.v139\n\nThis project contains the following underlying data:\n\n= CAS RANKL 200x.jpg (Expression of RANKL at 200x magnification in the CAS group)\n\n= CAS RANKL 400x.jpg (Expression of RANKL at 400x magnification in the CAS group)\n\n= CAS RANKL 1000x.jpg (Expression of RANKL at 1000x magnification in the CAS group)\n\n= CAS SHED RANKL 200x.jpg (Expression of RANKL at 200x magnification in the CAS SHED group)\n\n= CAS SHED RANKL 400x.jpg (Expression of RANKL at 400x magnification in the CAS SHED group)\n\n= CAS SHED RANKL 1000x.jpg (Expression of RANKL at 1000x magnification in the CAS SHED group)\n\nFigshare: OPG. https://doi.org/10.6084/m9.figshare.12609983.v140\n\nThis project contains the following underlying data:\n\n= CAS OPG 200x.jpg (Expression of OPG at 200x magnification in the CAS group)\n\n= CAS OPG 400x.jpg (Expression of OPG at 400x magnification in the CAS group)\n\n= CAS OPG 1000x.jpg (Expression of OPG at 1000x magnification in the CAS group)\n\n= CAS SHED OPG 200x.jpg (Expression of OPG at 200x magnification in the CAS SHED group)\n\n= CAS SHED OPG 400x.jpg (Expression of OPG at 400x magnification in the CAS SHED group)\n\n= CAS SHED OPG 1000x.jpg (Expression of OPG at 1000x magnification in the CAS SHED group)\n\nFigshare: RUNX2. https://doi.org/10.6084/m9.figshare.12610478.v141\n\nThis project contains the following underlying data:\n\n= CAS RUNX2 200x.jpg (Expression of RUNX2 at 200x magnification in the CAS group)\n\n= CAS RUNX2 400x.jpg (Expression of RUNX2 at 400x magnification in the CAS group)\n\n= CAS RUNX2 1000x.jpg (Expression of RUNX2 at 1000x magnification in the CAS group)\n\n= CAS SHED RUNX2 200x.jpg (Expression of RUNX2 at 200x magnification in the CAS SHED group)\n\n= CAS SHED RUNX2 400x.jpg (Expression of RUNX2 at 400x magnification in the CAS SHED group)\n\n= CAS SHED RUNX2 1000x.jpg (Expression of RUNX2 at 1000x magnification in the CAS SHED group)\n\nFigshare: TGF-Beta. https://doi.org/10.6084/m9.figshare.12610487.v142\n\nThis project contains the following underlying data:\n\n= CAS TGF-β 200x.jpg (Expression of TGF-β at 200x magnification in the CAS group)\n\n= CAS TGF-β 400x.jpg (Expression of TGF-β at 400x magnification in the CAS group)\n\n= CAS TGF-β 1000x.jpg (Expression of TGF-β at 1000x magnification in the CAS group)\n\n= CAS SHED TGF-β 200x.jpg (Expression of TGF-β at 200x magnification in the CAS SHED group)\n\n= CAS SHED TGF-β 400x.jpg (Expression of TGF-β at 400x magnification in the CAS SHED group)\n\n= CAS SHED TGF-β 1000x.jpg (Expression of TGF-β at 1000x magnification in the CAS SHED group)\n\nFigshare: VEGF. https://doi.org/10.6084/m9.figshare.12610484.v143\n\nThis project contains the following underlying data:\n\n= CAS VEGF 200x.jpg (Expression of VEGF at 200x magnification in the CAS group)\n\n= CAS VEGF 400x.jpg (Expression of VEGF at 400x magnification in the CAS group)\n\n= CAS VEGF 1000x.jpg (Expression of VEGF at 1000x magnification in the CAS group)\n\n= CAS SHED VEGF 200x.jpg (Expression of VEGF at 200x magnification in the CAS SHED group)\n\n= CAS SHED VEGF 400x.jpg (Expression of VEGF at 400x magnification in the CAS SHED group)\n\n= CAS SHED VEGF 1000x.jpg (Expression of VEGF at 1000x magnification in the CAS SHED group)\n\nFigshare: ALP. https://doi.org/10.6084/m9.figshare.12610493.v144\n\nThis project contains the following underlying data:\n\n= CAS ALP 200x.jpg (Expression of ALP at 200x magnification in the CAS group)\n\n= CAS ALP 400x.jpg (Expression of ALP at 400x magnification in the CAS group)\n\n= CAS ALP 1000x.jpg (Expression of ALP at 1000x magnification in the CAS group)\n\n= CAS SHED ALP 200x.jpg (Expression of ALP at 200x magnification in the CAS SHED group)\n\n= CAS SHED ALP 400x.jpg (Expression of ALP at 400x magnification in the CAS SHED group)\n\n= CAS SHED ALP1000x.jpg (Expression of ALP at 1000x magnification in the CAS SHED group)\n\nFigshare: Osteocalcin. https://doi.org/10.6084/m9.figshare.12610481.v145\n\nThis project contains the following underlying data:\n\n= CAS osteocalcin 200x.jpg (Expression of osteocalcin at 200x magnification in the CAS group)\n\n= CAS osteocalcin 400x.jpg (Expression of osteocalcin at 400x magnification in the CAS group)\n\n= CAS osteocalcin 1000x.jpg (Expression of osteocalcin at 1000x magnification in the CAS group)\n\n= CAS SHED osteocalcin 200x.jpg (Expression of osteocalcin at 200x magnification in the CAS SHED group)\n\n= CAS SHED osteocalcin 400x.jpg (Expression of osteocalcin at 400x magnification in the CAS SHED group)\n\n= CAS SHED osteocalcin 1000x.jpg (Expression of osteocalcin at 1000x magnification in the CAS SHED group)\n\nFigshare: Ostepontin. https://doi.org/10.6084/m9.figshare.12610490.v146\n\nThis project contains the following underlying data:\n\n= CAS osteopontin 200x.jpg (Expression of osteopontin at 200x magnification in the CAS group)\n\n= CAS osteopontin 400x.jpg (Expression of osteopontin at 400x magnification in the CAS group)\n\n= CAS osteopontin 1000x.jpg (Expression of osteopontin at 1000x magnification in the CAS group)\n\n= CAS SHED osteopontin 200x.jpg (Expression of osteopontin at 200x magnification in the CAS SHED group)\n\n= CAS SHED osteopontin 400x.jpg (Expression of osteopontin at 400x magnification in the CAS SHED group)\n\n= CAS SHED osteopontin 1000x.jpg (Expression of osteopontin at 1000x magnification in the CAS SHED group)\n\nFigshare: Raw Data Bone Molecular Markers. https://doi.org/10.6084/m9.figshare.12610499.v158\n\nThis project contains the following underlying data:\n\n= Raw Data Molecular Marker.xlsx (The raw data of molecular markers examined by means of IHC analysis)\n\nFigshare: Animal Body Weight. https://doi.org/10.6084/m9.figshare.12610502.v138\n\nThis project contains the following underlying data:\n\n= Animal Body Weight.xlsx (Animal Body Weight, pre and post test)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nEdman K, Öhrn K, NordstrÖm B, et al.: Trends over 30 years in the prevalence and severity of alveolar bone loss and the influence of smoking and socio-economic factors–based on epidemiological surveys in Sweden 1983 – 2013. 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Surabaya: Airlangga University Press. 2014; 2: 38.\n\nNugraha AP, Narmada IB, Ernawatı DS, et al.: The aggrecan expression post platelet rich fibrin administration in gingival medicinal signaling cells in Wistar rats (Rattus novergicus) during the early osteogenic differentiation (in vitro). Kafkas Univ Vet Fak Derg. 2019; 25(3): 421–425. Reference Source\n\nNugraha AP, Narmada IB, Ernawati DS, et al.: Gingival mesenchymal stem cells from Wistar rat’s gingiva (Rattus novergicus) - Isolation and characterization (in vitro study). J Int Med Res. 2018; 11(2): 694–699. Reference Source\n\nSuciadi SP, Nugraha AP, Ernawati DS, et al.: The efficacy of human dental pulp stem cells in regenerating submandibular gland defects in diabetic Wistar rats (Rattus novergicus). Research J Pharm and Tech. 2019; 12(4): 1573–1579.\n\nMiran S, Mitsiadis TA, Pagella P: Innovative dental stem cell-based research approaches: The future of dentistry. Stem Cells Int. 2016; 2016: 7231038. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaskianti T, Ramadhani R, Budipramana ES, et al.: Potential proliferation of stem cell from human exfoliated deciduous teeth (SHED) in carbonate apatite and hydroxyapatite scaffold. J Int Dent Med Res. 2017; 10(2): 350–353. Reference Source\n\nAlhasyimi AA, Pudyani PP, Asmara W, et al.: Enhancement of post-orthodontic tooth stability by carbonated hydroxyapatite-incorporated advanced platelet-rich fibrin in rabbits. Orthod Craniofac Res. 2018; 21(2): 112–118. PubMed Abstract \| Publisher Full Text\n\nPrahasanti C, Subrata LH, Saskianti T, et al.: Combined hydroxyapatite scaffold and stem cell from human exfoliated deciduous teeth modulating alveolar bone regeneration via regulating receptor activator of nuclear factor-Κb and osteoprotegerin system. Iran J Med Sci. 2019; 44(5): 415–421. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan B: Guidelines on the care and use of animals for scientific purposes. National Advisory Committee for Laboratory Animal Research. 2004. Reference Source\n\nKhoswanto C: A new technique for research on wound healing through extraction of mandibular lower incisors in Wistar rats. Euro Dent J. 2019; 13(2): 235–237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSavi FM, Brierly GI, Baldwin J, et al.: Comparison of Different Decalcification Methods Using Rat Mandibles as a Model. J Histochem Cytochem. 2017; 65(12): 705–722. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNugraha AP: Animal Body Weight. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12610502.v1\n\nNugraha AP: RANKL. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12609986.v1\n\nNugraha AP: OPG. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12609983.v1\n\nNugraha AP: RUNX2. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12610478.v1\n\nNugraha AP: TGF-Beta. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12610487.v1\n\nNugraha AP: VEGF. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12610484.v1\n\nNugraha AP: ALP. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12610493.v1\n\nNugraha AP: Osteocalcin. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12610481.v1\n\nNugraha AP: Osteopontin. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12610490.v1\n\nIgarashi Y, Chosa N, Sawada S, et al.: VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes. Int J Mol Med. 2016; 37(4): 1005–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGe Q, Zhang H, Hou J: VEGF secreted by mesenchymal stem cells mediates the differentiation of endothelial progenitor cells into endothelial cells via paracrine mechanisms. Mol Med Rep. 2018; 17(1): 1667–1675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBailey S, Karsenty G, Gundberg C, et al.: Osteocalcin and osteopontin influence bone morphology and mechanical properties. Ann N Y Acad Sci. 2017; 1409(1): 79–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStein GS, Lian JB, Owen TA: Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J. 1990; 4(13): 3111–23. PubMed Abstract \| Publisher Full Text\n\nBoskey A, Gadaleta S, Gundberg C, et al.: Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone. 1998; 23(3): 187–96. PubMed Abstract \| Publisher Full Text\n\nThurner PJ, Chen CG, Ionova-Martin S, et al.: Osteopontin deficiency increases bone fragility but preserves bone mass. Bone. 2010; 46(6): 1564–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoundarik A, Diab T, Sroga G, et al.: Dilatational band formation in bone. Proc Natl Acad Sci U S A. 2012; 109(47): 19178–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrosso A, Burger MG, Lunger A, et al.: It takes two to tango: Coupling of angiogenesis and osteogenesis for bone regeneration. Front Bioeng Biotechnol. 2017; 5: 68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsao YT, Huang YJ, Wu HH, et al.: Osteocalcin mediates biomineralization during osteogenic maturation in human mesenchymal stromal cells. Int J Mol Sci. 2017; 18(1): 159. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakajima K, Kunimatsu R, Ando K, et al.: Comparison of the bone regeneration ability between stem cells from human exfoliated deciduous teeth, human dental pulp stem cells and human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun. 2018; 497(3): 876–882. PubMed Abstract \| Publisher Full Text\n\nLeyendecker JA, Gomes PCC, Lazzaretti FT, et al.: The use of human dental pulp stem cells for in vivo bone tissue engineering: A systematic review. J Tissue Eng. 2018; 9: 2041731417752766. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNugraha AP: Raw Data Bone Molecular Markers. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12610499.v1" }	[ { "id": "71798", "date": "08 Oct 2020", "name": "Niswati Fatmah Rosyida", "expertise": [ "Reviewer Expertise Biomaterial in Orthodontic", "Biological Tooth Movement" ], "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 of this study have investigated the ability of SHED and CAS in alveolar bone using immunohistochemical analysis. Based on the study results, the authors have concluded that all the variables increased on Day 7, while RANK expression decreased. The results are certainly interesting. The paper is very well organized, easy to follow, having a novelty and impact. Minor concerns about the study are listed as follows:\nIntroduction\nPlease reduce the explanation about periodontitis as a causative factor on the bone defect, moreover this study does not use the periodontitis model.\n\nPlease add the information about the problem or the success of the SHED and CAS combination from current or past research.\nMethod\nThere is no information regarding the experimental animal grouping. The authors should mention it.\n\nPlease explain why there is no control group without treatment SHED in CAS, or SHED group only.\nDiscussion\nThere was no discussion why the histological analysis only was done only on 7 day ater treatment, what the reason?\n\nCAS have proved induce the healing from the previous study. The success of this formulation is caused by the combination of SHED and CAS, but in the discussion, there is lack of CAS role. Please add the explanation.\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": [ { "c_id": "6140", "date": "02 Dec 2020", "name": "Alexander Patera Nugraha", "role": "Author Response", "response": "The authors thank the Honorable Reviewer 1 for her excellent review of the paper and in-depth suggestions made to improve the quality of the paper. Followings are the responses to the suggestions of the Honorable Reviewer. The valuable suggestions and corrections are incorporated cautiously, and the paper is made clearer while revising the manuscript. Introduction Please reduce the explanation about periodontitis as a causative factor on the bone defect, moreover this study does not use the periodontitis model. * Please kindly check the new version of our paper. We have been reduced the explanation about periodontitis as a causative factor on the bone defect due to we used the tooth extraction induce alveolar bone defect model in rats. Please add the information about the problem or the success of the SHED and CAS combination from current or past research. This study novely was the transplantation of SHED seeded in CAS could increase the number of osteogenic markers expressing cells, such as OPG, RUNX2, TGF-β, VEGF, ALP, osteocalcin, and ostepontin, but not the RANKL expression in the bone defects after seven days in comparison to the CAS group. The previous researchs about combination of SHED and CAS ameliorate alveolar bone defect post tooth extraction is still limited. Method There is no information regarding the experimental animal grouping. The authors should mention it. Ten wistar rats were assigned into two groups respectively; CAS group and CAS+SHED group. Please explain why there is no control group without treatment SHED in CAS, or SHED group only. The limitations of this study were that the observation and evaluation were performed on transplantation of SHED seeded in CAS and CAS only on the animal model. Discussion There was no discussion why the histological analysis only was done only on 7 day after treatment, what the reason? The limitations of this study were that the observation and evaluation were performed seven days post transplantation of SHED seeded in CAS on the animal model, and only an immunohistochemical examination was performed. Further studies will be necessary to evaluate the changes in the alveolar bone and periodontal tissue post transplantation of SHED seeded in CAS in the alveolar bone defect animal models. With a longer observation time, further studies using methods, such as qRT-PCR and/or the western blot analysis, could be conducted to estimate the expression of bone molecular markers. CAS have proved induce the healing from the previous study. The success of this formulation is caused by the combination of SHED and CAS, but in the discussion, there is lack of CAS role. Please add the explanation. CAS plays an important role in supporting SHED proliferation and differentiation. The osteogenic microenvironment in defective alveolar bone may induce SHED seeded in CAS to differentiate into bone cells, especially osteoblast." } ] }, { "id": "72008", "date": "03 Nov 2020", "name": "Lidia Audrey Rocha Valadas", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn general the main topic of the manuscript is ambitious and interesting. The paper is well designed and supported with updated references. The introduction is written in clear language, covering a large number of relevant issues. Methods show all details of the methodology used to perform the research but I suggest to provide more details about the sample. Please provide the number and place of approval of your project's ethical opinion. Results are precise and Discussion supports them.\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/9-1164
https://f1000research.com/articles/9-1385/v1	01 Dec 20	{ "type": "Software Tool Article", "title": "Semi-automated analysis of dot blots using ImageJ/Fiji", "authors": [ "Anna H. Klemm" ], "abstract": "Commercially available dot blots provide a set of specific antibodies spotted on membranes in a given pattern. If the target analyte is present in the solution that the membrane is incubated with, the detection reaction will result in a chemiluminescence signal which is recorded by film or scanner. In order to know which analytes were detected, the analysis consists of measuring the intensity of the recorded signal on each spot. Manually measuring the entire array (typically ~200 spots) is unreliable and tedious. Fully automatic registration of the blot membrane to the template pattern often fails since there might be only very few positive spots on the membrane. This article presents an ImageJ/Fiji macro that requires minimal user input to perform a robust iterative registration of an adjustable template mask representing the spot pattern to the recorded blot. Once the template mask is matched to the dot blot, the spot intensity of each dot is measured and reported in a results table.", "keywords": [ "Dot Blot", "FIJI", "ImageJ macro", "template registration", "intensity measurements" ], "content": "Introduction\n\nA dot blot is a common technique in molecular biology to query the presence of an analyte in a solution. Commercially available dot blots contain a set of so-called capture antibodies spotted to a membrane in a given pattern, with each spot representing one specific antibody. Binding of the analyte to the capture antibody is detected by a detection antibody and chemiluminescence signals are recorded by film or scanner, similar as in classical western blots. Dot blots typically contain ~200 spots to be analyzed. For automatic quantification of the dot intensities a mask reflecting the spot pattern needs to be registered to the blot. Common registration algorithms are either intensity-based, as in e.g. StackReg1, or rely on the automatic identification of prominent features present in both images (e.g. Linear Stack Alignment with SIFT2)1,2. A scanned dot blot can often not be aligned to the mask with either method, especially when only a few analytes in the dot blot give positive signals. For algorithms that depend simply on feature detection, the pattern of the mask is too dense and regular to be successfully registered to few sparse dots. The intensity-based algorithm fails for similar reasons. As a result, open source tools currently available for analyzing dot blots either strongly rely on user input3 or assume full regular patterns4, which is not always the case. The presented ImageJ Macro DotBlot_Analysis.ijm relies on a few (3–5) landmarks selected by the user to give a first estimate of the transformation between the scanned dot blot and a template pattern. It then automatically searches for more landmarks and performs the final registration and measurements. It runs within the free and open-source software Fiji5.\n\n\nMethods\n\nThe macro expects two images: the scanned dot blot and a mask image. The mask image is a representation of the spot pattern. After starting the macro, the user will be asked to select the names of the mask and dot blot images, as well as enter an expected spot size in pixel and a prominence value (discussed under Implementation). The user then needs to mark 3–5 corresponding dots (=landmarks) in the scanned dot blot and in the mask. DotBlot_Analysis.ijm uses the user-set landmarks to register the mask image to the scanned blot in an iterative process. After registration the macro creates selections for each spot. The user has the possibility to manually adjust the selections by activating a selection within the ROI manager, adjusting it and pressing “update” to save the new position. After this the macro performs a background subtraction and measures the intensities of each spot. The final output of the macro is a result table that lists intensity measurements for each spot. For quality control purposes, the macro also saves the selections within the ROI Manager as a zip-file and a control-image with all spot selections overlaid over the inverted background-subtracted dot blot. The zip-file can be re-opened in Fiji by drag&drop: all selections will be listed in the ROI Manager after re-opening.\n\nThe mask is a schematic representation of the spot pattern and needs to be registered to the scanned blot by translation, rotation and scaling (affine transformation). The macro registers the mask in two steps. In the first step it uses the 3–5 landmarks set by the user. The macro runs the plug-in “Landmark Correspondences”3 by Stephan Saalfeld, which matches the two sets of dots to determine the transformation and applies the transformation to the mask. In a second registration step, the macro automatically searches for more landmarks, in order to achieve an even finer registration. The landmarks are determined with the following computational steps:\n\n1. Gaussian-filtering of the scanned, inverted dot blot. Signals are thereby smoothed and single signals are transformed to a smooth intensity distribution with (ideally) one single intensity maximum in the center of the spot.\n\n2. Activation of the outlines of the single spots, as detected after the first registration step.\n\n3. Search for local maxima on the gaussian-blurred image within each single spot outline. Maxima are determined as those pixels which are brighter than the local background, by the prominence value initially defined by the user. This ensures detection of bright signals and avoids eventual noise.\n\n4. Once a local maximum is found, its position is marked as a new landmark. The corresponding landmark on the mask image is determined as the centroid of the selection that was used for searching the maximum.\n\nFollowing this procedure, a new pair of landmarks (one set for the blot, one set for the mask) is identified and the plug-in “Landmark Correspondences” is re-run, now with more landmarks.\n\nOnce the mask has been fully registered to the scanned blot, it can be used for creating the final spot selections: the centroids of each spot of the final transformed mask are extracted and used to draw a new selection of defined size. This ensures that each spot is measured with a selection of equal size. The size of selection is provided by the user at the beginning of the script. The user has the possibility to correct the position of the final selections.\n\nBefore doing the measurements, the background of the image is subtracted using the “Subtract Background” command4, based on the rolling ball algorithm. The radius of the rolling ball is set to three times the user-defined spot size, to ensure that only background but not signal is removed.\n\nAfter background subtraction the intensities of each spot are measured.\n\nThe final output of the macro is a result table that lists the following measurements for each spot:\n\n- Label of the spot\n\n- Area (for reporting, since it is the same area for all spots)\n\n- Mean intensity (“Mean”)\n\n- Minimum intensity (“Min”)\n\n- Maximum intensity (“Max”)\n\n- X- and Y-coordinate of the spot\n\n- Intensity density: the product of area (scaled units, if set) and mean intensity\n\n- RawIntDen: the sum of the pixel values within the spot\n\nSee the ImageJ documentation5 for more detailed information about the measurements.\n\nThe macro runs within the open-source and free software Fiji (https://fiji.sc/), which is supported on the following systems: Windows XP, Vista, 7, 8 and 10; Mac OS X 10.8 \"Mountain Lion\" or later; Linux on amd64 and x86 architectures.\n\n\nUse case\n\nThe macro and the data for testing are available for download at https://github.com/ahklemm/AnalyzeDotBlots. Download the material from GitHub by clicking on the green “code” button and choose “download ZIP”. Unzip the material and open DotBlot_Analysis.ijm by drag&drop to Fiji. For testing purposes, open blot.tif and mask.tif. Run the macro.\n\nFigure 1a shows as use case the scanned blot, blot.tif, and its mask, mask.tif. Four landmarks are marked in both the scanned blot and the mask (yellow arrows). Figure 1b shows the outcome after the first registration step. The blot has been inverted to create bright signals over dark background. While the spots already overlay the signals of the scanned dot blot quite well after this first registration, some selections (indicated with red arrow heads in Figure 1b) are offset with respect to the center of the actual signal. After the first registration the macro automatically identifies more landmarks, which are marked as yellow crosses in Figure 1c. The automatically identified landmarks are used for a second, finer registration step. The result of this second registration is visible in Figure 1d. Note that the center of the selections now overlays well with the center of the spots (red arrow heads, Figure 1d). Figure 1e shows the final result: the overlay of the final measurement selection over the background-corrected blot and the final results table.\n\na) User selected landmarks in the scanned dot blot and the mask. b) Overlay of the spots over the dot blot after the first registration step. Red arrowheads mark spots with major shifts in respect to the signal center. c) Yellow crosses show the automatically detected landmarks in the scanned blot image as well as on the mask. d) Overlay of the spots after the second registration step. The marked spots (red arrowheads) now match the signal center better. e) Final results of the workflow.\n\n\nAdaptations needed for different dot patterns\n\nThe use case shows the scanned blot and mask of a very specific dot blot pattern/array. Unless the user wants to evaluate blots of exactly that array one needs to provide a new labelled mask. Within the labelled mask the spot on the upper left has an ID/gray-value = 1 and IDs/gray values of spots increase from the left to the right and from the top to the bottom. In mask.tif there are 218 spots, so the spot on the lower right has the gray value of 218. The ID/grayvalue is important in order to identify the spot even after the rotation/scaling/translation of the two affine registration steps.\n\nMaskGenerator.ijm in the downloadable material helps to transform a typical vendor template6 into a labelled mask. Running MaskGenerator.ijm the user draws one selection around a spot of the template. The macro then identifies bright spots of the expected size and by roundness.\n\n\nSummary\n\nThe presented Fijj macro DotBlot_Analysis.ijm supports users to analyze images of dot blots. It requires minimal input of few landmarks to then automatically measure the intensities of single spots in dot blots.\n\nWhile scanning the dot blot one should avoid saturation of the signal. Signals should not reach a value of 0 but should always contain a minimum value of intensity. Reduce exposure times when imaging the film if needed.\n\nSupport for problems when using the script is provided via forum.image.sc – please link to the username of the author: @aklemm\n\n\nData availability\n\nSource code is available from: https://github.com/ahklemm/AnalyzeDotBlots\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.41342196\n\nLicense: BSD-3-Clause", "appendix": "Acknowledgements\n\nThe image of the dot blot was provided by Katja Eubler, Mayerhofer-lab, BMC, LMU Munich, Germany.\n\nThe author thanks Giovanni Cardone, MPI of Biochemistry, Munich for comments on the manuscript and the macro script and Andreas Thomae, CFBim, BMC, LMU Munich for discussions about the imaging of dot blots and analysis.\n\nThis publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology).\n\n\nFootnotes\n\n1 http://bigwww.epfl.ch/thevenaz/stackreg/\n\n2 https://imagej.net/Linear_Stack_Alignment_with_SIFT\n\n3 https://imagej.net/Landmark_Correspondences\n\n4 https://imagejdocu.tudor.lu/gui/process/subtract_background\n\n5 https://imagej.nih.gov/ij/docs/menus/analyze.html\n\n6 E.g. https://www.rndsystems.com/products/proteome-profiler-human-xl-cytokine-array-kit_ary022b\n\n\nReferences\n\nThévenaz P, Ruttimann UE, Unser M: A pyramid approach to subpixel registration based on intensity. IEEE Trans Image Process. 1998; 7(1): 27–41. PubMed Abstract \| Publisher Full Text\n\nLowe DG: Distinctive Image Features from Scale-Invariant Keypoints. Int J Comput Vis. 2004; 60(2): 91–110. Publisher Full Text\n\nOhgane K: Quantification of Gel Bands by an Image J Macro, Band/Peak Quantification Tool. 2019. Publisher Full Text\n\nProtein Array Analyzer for ImageJ - Gilles Carpentier Research Web Site: Computer Image Analysis. (accessed Oct. 01, 2020). Reference Source\n\nSchindelin J, Arganda-Carreras I, Frise Erwin, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nahklemm: ahklemm/AnalyzeDotBlots: 1. release AnalyzeDotBlots (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4134219" }	[ { "id": "75775", "date": "18 Dec 2020", "name": "Christopher Schmied", "expertise": [ "Reviewer Expertise Bioimage analysis", "computer vision", "data science" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Author introduces a workflow for the semi-automated analysis of dot blots using Fiji macros. To measure the intensity of the individual dots, a template is registered to the input blot using a two stage landmark based registration. First a registration is applied using manual landmarks specified by the user. This achieves a coarse alignment. The second registration step uses automatic landmarks, generated via local maxima detection. Affine transformation is used to match the template with the dot blot. Based on the template mask, the macro then extracts a number of useful measurements and presents a result table. The results as well as files for quality control are saved into the folder that contain the input files.\n\nI tested the workflow with the provided material. The download from the github repository provides the necessary scripts as well as test images for the blot and the mask. I was able to reproduce the main analysis on the first try. The controls of the macro are easy and intuitive, thus match with the described minimal user input. I like that the blot.tif test image provides already selections for the initial user input, so that I see how the author intended the macro to be used. I like that the necessary files for quality control are saved and easily accessible. I usually prefer to give the user an option to specify a different output directory, but this does not interfere with the usage of this macro. Two very small but important details of the article I like to highlight: the download of the material from github is well described, this is often a problem for people that never used github. I liked that the author referred to forum.image.sc and their user name there, as a way to find support for potential users.\n\nThe method is introduced and motivated well, although I have the feeling that references in the introduction are missing, when describing the method behind dot blots. The workflow logic is in general well described in its details and detailed components of the workflow are described sufficiently and also clearly. Unfortunately the structure of the article does not lend itself for an easy and quick understanding. Also details of how the input data needs to look like is not described clearly. Here are my points for revision:\n\nMajor: The article describes in detail how the macro works. However, I review this now also with the perspective of a user who wants to analyze my own blot and wants to get to the important bits fast. Here the article structure is confusing and not conducive for easy and quick adoption. Let me explain:\n\nMethods:\n\nRunning DotBlot_Analysis.ijm: The author first introduces the general method and usage of the workflow, no mention of where to find the script or an illustration of the workflow.\n\nImplementation: The author explains the detailed methods and the individual components in the section Implementation, this is good. Still it lacks the way to action on this or references to an illustration to easily understand the workflow.\n\nOperation: explains the macro uses Fiji and is OS independent.\n\nUse case:\n\nUse case data: introduces us finally to the location of the actual script and test files, something I would have liked to have at the beginning.\n\nUse Case: shows us a good schematic of the workflow and really illustrates well how to operate the workflow. Something that could have helped to understand the \"Methods\" and also the \"Implementation\".\n\nAdaptions needed for different dot patterns: we learn how to customize this workflow to a different dot blot. The author even provides a script for that which is great by the way. I just thought that a user, that presumably wants to analyze a different dot blot, would have needed that information earlier. I think this is also an important part of the workflow.\n\nSummary: is fine in the end.\n\nLimitations of the analysis: is an afterthought, although this is actually an important prerequisite.\n\nI think this can be amended by changing the structure without changing the content a lot. Here are my suggestions for that:\n\nMethods\n\nPrerequisite: content of \"Limitations of the analysis\" but more (see other Major point)\n\nDownload & Installation: Discuss download of script and example data (\"Use case data\")\n\nOperation\n\nCustom Mask: \"Adaptions needed for different dot patterns\"\n\nRunning DotBlot_Analysis.ijm fused with Use case\n\nImplementation\n\nCould stay the same but could use the Figure better.\n\nUse case Maybe could describe how this particular example blot has been generated and scanned. Could then discuss the results of this example blot and how to interpreted the results of the macro based on this blot briefly.\n\nSummary\n\nLimitations: could describe potential conditions were the macro fails to register properly.\n\nMajor: The Limitations of the analysis are very lightly touched upon and only at the end. Although they are, in the case of the discussed information, an important prerequisite for the analysis. I find them to be too brief and not clearly described for a user to understand immediately. Please explain clearer how a good blot should look like or describe briefly how one scans a good blot and/or refer to an article that does this in more detail.\n\nMajor: Figure1b&d: Unfortunately, the details in the examples images shown are very small also in the online version of the article. Figure 1b and Figure 1d are crucial in illustrating the results of the registration and why there needs to be two distinct registration steps in the first place. However, the overlay with the yellow ROIs are very hard to see and the images are too small. One can enlarge parts of the image as insets and show specifically the effect of the registration. One can also enlarge the yellow ROI outline or change the color of the ROI to make the ROIs more visible.\n\nMajor: Figure 1c: from the description there should be automatically detected landmarks. But these are not visible in the image. Again cropping and enlarging can help.\n\nMajor: Using the same overlapping numbers for citations and footnotes does not work! Super confusing.\n\nMinor: The Figure illustrates the workflow and the results of the individual processing steps for the DotBlotAnalysis.ijm. To reflect the entire complete workflow it would be good to set apart the input original blot and the generation of the original mask also visually.\n\nMinor: The content of the upper row of images is very hard to see when printed.\n\nMinor: it is great that the MaskGenerator.ijm is also included. Since I see this as a crucial part of the workflow. It would thus be great if an example input file for testing would also be provided for this script.\n\nMinor: All the material is nicely provided on the github repository. Please also include a description of the usage in the readme there.\n\nMinor: I think it is not obvious that the mask initially also has a different size than the blot and the adjustments are a result of the transformations applied during the registration. I think this needs to be noted in the usage or the figure legend.\n\nMinor: the DotBlotAnalysis.ijm on the github repo is refered to as DotBlot_Analysis.ijm in the text.\n\nMinor: I found this macro: https://www.optinav.info/MicroArray_Profile.htm a similar approach to Gilles Carpentier Protein Analyzer. Could be included in the references to regular patterned ROIs.\n\nMinor: no citations for the methods used to generate and scan dot blots are provided.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "75773", "date": "22 Dec 2020", "name": "Bram van den Broek", "expertise": [ "Reviewer Expertise BioImage Analysis", "Fluorescence Microscopy", "Cell Biophysics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary The author describes a tool to quantify dot blots or antibody arrays. These are membranes with antibodies absorbed to it in a certain pattern, used for (quantitative) detection of specific compounds in a solution. The presented ImageJ1 macro utilizes a well-established registration tool to map a template spot pattern (mask) to the scanned image of the dot blot, in a clever iterative way. The user needs only to provide a few matching point selections (corresponding landmarks) on both images. The macro then automatically optimizes the alignment process by finding more landmarks, leading to a good match for every spot. When the user is satisfied with the registration, intensities and positions of the dots are reported, as well as an overlay image for inspection.\nThe paper starts off by describing why other open source registration tools and dot blot analysis tools fail in some or most cases. Fully automatic registration is a utopia, because of the sparsity of positive spots in the dot blot, compared to the periodic spot alignment in the template. The author also makes a valid point by stating that ‘other tools require or assume full regular patterns, which is not always the case’. I cannot judge how often this actually happens, but the provided example blot is clearly not fully regular. The paper is well written, and the workflow steps are clearly described. Additionally, the author provides ample information on how to get the software to run on your computer, and where to go for support. I found the software tool easy to use, and rather robust. The clever procedure of iterative registration makes that the spots are correctly detected, even if the dot blot is somewhat distorted. The macro provides an output that is not overly complicated. Compared to another tool that is briefly mentioned, the Protein Array Analyzer by Gilles Carpentier, the functionality is in fact rather limited, but if you just need to quantify the intensities of the spots in a blot it will serve you well.\nThings that could be improved At the very end, the paper also briefly describes a second macro, on how to create a suitable template mask for a different dot blot pattern. For any user of the script, this part is of utmost importance, and I believe it needs a bit more attention. I tried to reproduce the same template mask by downloading the vendor mask in reference 6, but could only find it as a picture embedded in the datasheet PDF. The template image consists of black open circles on a white background. This is important information missing in the manuscript and/or the MaskGenerator macro, because the macro only functions with such a template image. Still, I could only get it to create a functional mask (from a screen capture of the template) after looking into the code. (See also remarks below).\nThe panels in figure1 are rather small, and the resolution is not great. The yellow crosses in figure 1c, for instance, can hardly be discerned. (Here, using a color more contrasting with gray may also help.) The same holds for Figure 1d. The three registered masks on the bottom all look the same; the landmarks are almost invisible. A larger image, perhaps with the first and second iteration of the registration mask overlaid in different colors may clarify things.\nFootnotes and references use the same numbering and typesetting. This is quite confusing and should be changed.\nI have many other, more technical remarks and suggestions that are mostly meant as improvements for the macro:\nI wonder why the background is subtracted after the registration process. This could in theory lead to the detection of false maxima (landmarks). Perhaps I’m missing something, but I do not see a disadvantage of first subtracting the background.\n\nWhen the dot blot image has light spots on a dark background, the macro does not work (no landmarks found). I recommend to include a choice (drop-down menu) in the script parameters, and then invert the image only if required.\n\nAlthough described in the text, ‘prominence’ is not very intuitive for users, nor is there feedback on whether the chosen prominence has been a good choice. It would be helpful to provide information on the number of landmarks found in e.g. the log window, and also the possibility to pause the macro at this step (again with a checkbox in the parameters dialog) to inspect the secondary landmarks in case the registration fails.\n\nThe Gaussian blur in line 75 should be a certain fraction of the spot diameter.\n\nThings go wrong if there are more than 255 spots in the mask and/or blot image. (Transformation2_Thres does not look as it should for spot number 256 and up, leading to the error message ‘incorrect number of ROIs detected’.) It can be fixed by allowing 16-bit spot template images, and changing the upper thresholds in line 65 and 146, from 255 to 65535.\n\nThe message ‘Incorrect number of ROIs detected, please check the ROI manager.’ isn’t very helpful. Please either explain what the user is supposed to do, if action is required.\n\nShow the labels of the ROIs in the manual modify step. This allows dragging the ROIs without the need to update the ROI in the ROI manager. Also, more information on how to do this will be helpful to the user.\n\nNice-to-have: a possibility to change the size of the spots in the manual modification step (e.g. with a (non-blocking) dialog instead of a waitForUser statement.)\n\nShow ROI labels in the flattened RGB overlay output image as well, so it is easy to check if the measured values make any sense. (Ideally, one would overlay the position in [column, row] format, but I realize that this would require a lot of work, and may be error-prone.)\n\nFile handling and output, some suggestions:\nLet the user choose a different output folder (or a subfolder) than the one containing the data. Do not close/modify the original images, i.e. duplicate before processing. Overlay the ROIs also on the original dot blot image, not only the processed inverted RGB.\n\nSome error catching:\nA message when there are less than 2 images open. A message/dialog when the prominence is set too high (now this leads to ‘not enough landmarks selected to find a transformation model’), perhaps even with the possibility to change it without rerunning the macro. The macro crashes if the image name does not contain a dot (e.g. if it is processed/duplicated with another name prior to running the macro).\n\nThe ID_Array() function is overly complicated. Use IJ.pad(n, length) to introduce leading zeros. The whole function can be replaced by\nID_Array = newArray(nSpots); for(i = 0; i < nSpots; i++) {\n\nID_Array[i] = IJ.pad(i, 3); }\n\nComments on MaskGenerator.ijm:\nThe message ‘Draw a circle around one of the Dots’ is somewhat misleading, because it seems to imply that the user should draw a bit larger than a single spot, in which case the macro often fails to recognize any round structures. In other words: 0.8DotArea is too strict.\n\nVery minor: Pre-select the circle selection tool before asking to draw a circle.\n\nWhen looking for test dot blots on the web, I found that many are in a fully regular pattern. In this case a mask could easily be generated without a vendor template. (It does not matter if in the real DotBlot Array some spots are missing, as long as the pattern is regular.) Below is the code I wrote for generating masks while testing several dot blots. It could be integrated in the macro:\n#@ Integer (label=\"Number of columns\" , value=12) cols #@ Integer (label=\"Number of rows\" , value=8) rows #@ Integer (label=\"Spot size\" , value=20) size\ndist = 8;\nnewImage(\"Mask_temp\", \"16-bit black\", cols(size+dist) + dist, rows(size+dist) + dist, 1); setForegroundColor(255,255,255); for (r = 0; r < rows; r++) {\n\nfor (c = 0; c < cols; c++) {\n\nmakeOval(dist + c(size+dist), dist + r*(size+dist), size, size);\n\nfill();\n\n} } run(\"Select None\"); setAutoThreshold(\"Default dark\"); run(\"Convert to Mask\"); run(\"Analyze Particles...\", \" show=[Count Masks]\"); run(\"glasbey_on_dark\"); rename(\"Mask\"); close(\"Mask_temp\");\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1385
https://f1000research.com/articles/9-1383/v1	30 Nov 20	{ "type": "Systematic Review", "title": "Exploring the inclusion of dental providers on interprofessional healthcare teams treating patients with chronic obstructive pulmonary disease: a rapid review", "authors": [ "Navia I. Novosel", "Greta J. Fratarcangeli", "Jasdip Randhawa", "Olivia M. Novosel", "Shannon L. Sibbald", "Navia I. Novosel", "Greta J. Fratarcangeli", "Jasdip Randhawa", "Olivia M. Novosel" ], "abstract": "Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world. Emerging concepts like One Health, integrated care models for COPD, and associations between oral and respiratory health are innovative ways to approach COPD treatment. This study explored contemporary evidence on the inclusion of dental providers on interprofessional healthcare teams treating patients with COPD. The first objective was to explore the current state of interprofessional care for COPD, and the second objective was to explore dentistry used in interprofessional care. A rapid review was conducted from January–June 2020 using Scopus and PubMed. Upon assessing for duplication and relevance, 85 articles were included for Objective 1, and 194 for Objective 2. The literature suggests that when dental providers are included on interprofessional healthcare teams, treatment outcomes for patients with multi-morbid, chronic disease such as COPD, are improved. The papers collected for review suggest that educational and clinical programs should implement interprofessional collaboration when treating chronic diseases. Healthcare teams can utilize the expertise of professionals outside the traditional medical field to better understand patients’ needs. Healthcare administration should consider a One Health approach when developing COPD treatment guidelines. We believe our results are transferable to the Canadian healthcare system. The collaborative nature and holistic philosophy of a One Health approach provides a novel way to develop policies and procedures that can effectively address the burden of COPD.", "keywords": [ "Chronic Obstructive Pulmonary Disease", "COPD", "dental providers", "interprofessional care", "One Health" ], "content": "Introduction\n\nChronic obstructive pulmonary disease (COPD) is a complex disease characterized by persistent adverse respiratory symptoms and airflow limitation due to airway and alveolar abnormalities1. COPD is often attributed to smoking, occupational exposures to particulate matter, and pre-existing asthma among other risk factors1. The possibility of acquiring comorbidities, such as cardiovascular disease, osteoporosis, and psychological distress, is very likely for patients with COPD2. COPD is currently the third leading cause of death in the world1. When analysing global health-related quality of life measures, patients with COPD often experience poorer physical, social, and psychological health attributed to increased emergency department visits and hospitalisations2,3. Given the complexity of COPD, patients often face challenges with accessing and receiving coordinated care, resulting in a fragmented healthcare experience4. Interprofessional team-based care for COPD exists, and is often quite successful; however, not all patients have access and there are many barriers to their implementation4. We believe, along with others in the scientific literature, that a larger focus on interprofessional team-based care is essential to improve the overall quality of care and quality of life for patients with COPD1–4.\n\n\nBackground\n\nPatients with COPD are often at risk of developing multi-organ complications; therefore, COPD has been classified as a systemic disease2. While a variety of healthcare professionals (respiratory therapists, family physicians, nurses, pulmonary technicians, etc.) are involved in caring for patients with COPD, dental providers are often overlooked5. Despite this gap in practise, the relationship between oral health and COPD has been explored in scientific, clinical, and educational domains, pointing to a vital need for integrating dental care into COPD treatment5.\n\nStudies have shown that the microbiome in both the lung and oral cavity is similar and connected through a bi-directional relationship5,6; a pathogen exchange between the lung and oral cavities occurs through saliva aspiration and hematogenous spread6. Infected teeth or gums act as a reservoir for anaerobic bacteria that provoke respiratory symptoms like wheezing, and the same bacteria can also be isolated in infected lungs5,6. The most common dental condition associated with COPD is periodontitis: a chronic inflammatory disease of the periodontal tissue, or gums, caused by bacterial infection5–7. If a patient has COPD or periodontitis, it is highly likely that the severity of disease progression for both conditions is exacerbated by the other6. Training dental students to better understand chronic medical disease processes (such as those pertaining to COPD) will better prepare them for working with other healthcare professionals in clinical team settings; therefore, improving quality of care for chronic disease patients8.\n\nPatients with COPD are often shown to have poorer oral health compared to healthy control patients, evidenced by limited dental visits and lack of dental hygiene habits (in particular, not flossing enough)5. Clinical practise guidelines advise dental providers on specific accommodations for their COPD patients during appointments, including: performing procedures with the dental chair upright, being cautious when irrigating and suctioning, having emergency oxygen supply nearby, conducting routine periodontal treatments, and providing education on proper oral and denture hygiene7. From a clinical perspective, healthcare teams should incorporate the expertise of dental providers when managing patients with COPD in order to acknowledge the oral-systemic health link and treat accordingly9.\n\nInterprofessional education (IPE) between medical, dental, and nursing students is becoming increasingly common both in professional degree programs and in on-site work training10. IPE prepares students to apply what they learned in the classroom to clinical care settings with other health professionals11. One study showed that the success of IPE programs depend on institutional infrastructures, inclusivity between programs, student workload, and faculty time and buy-in11. Studies surveying the attitudes of dental hygiene and dental students towards IPE have found that students believe it is useful because they can see the real-world benefit of applying themselves as collaborative practitioners12.\n\nAs a multifaceted, prevalent disease, COPD cannot be treated with conventional reductionist principles of medicine, but rather, with models emphasising collaboration between different health professionals13. Recognized as one of the ten goals for future Canadian medical education, interprofessional treatment approaches are essential for enhancing patient-centred care14. Interprofessional collaboration involves regular meetings between different health and social care professions to discuss how to solve complex care problems, like those involved in COPD, by integrating expertise from their respective fields15. This is different from interprofessional teamwork whereby those involved in treatment planning do not share a common identity15. Multidisciplinary teamwork occurs when members from disciplines outside of traditional healthcare (such as economics and the social sciences) work alongside each other in a parallel rather than an integrated fashion to solve complex cases15. Finally, transdisciplinary teamwork is defined when members in one professional group undertake the roles and responsibilities of another profession; although outside their usual scope, it is assumed that these members are competent enough to complete the presenting tasks15.\n\nOne Health is an emerging approach that extends beyond the ideas found in interprofessional care by stressing the connections between humans, animals, and their shared environment, as well as the need for transdisciplinary communication, or communication between experts in different fields inside and outside of medicine16. One Health is currently being endorsed by prominent organizations like the Centers for Disease Control and Prevention and the World Health Organization as a systems framework that considers the patient as part of a larger system with levels ranging from cellular to societal17. The etiology of several infectious diseases trace back to how humans interact with animal reservoirs and their surrounding environmental conditions; therefore, a One Health approach has proven to be very applicable to infectious disease16. One Health experts are also highlighting the benefits of applying this approach to prevalent non-infectious diseases like COPD16. Studying animal models or analysing environmental sources of COPD like biomass fuel burning in resource-poor countries presents new areas of research to better understand the pathophysiology of COPD1. Therefore, the patient-provider encounter can be enriched by One Health principles that encourage healthcare teams to include a variety of experts in medical, dental, and public health sectors to better understand their patients’ needs and history17. For the purpose of this review, we are selectively using the inclusive collaboration aspect of a One Health approach to strengthen and support interprofessional rather than multi- or trans-disciplinary care for COPD.\n\nThe burden of COPD on healthcare systems and global health, the relationship between COPD and oral health, and interprofessional interventions for chronic diseases highlight that effective COPD treatment demands a diverse, collaborative One Health-focused approach. The aim of this study was to explore contemporary evidence on the inclusion of dental providers on interprofessional healthcare teams treating patients with COPD. The objective was to explore the current state of interprofessional care for COPD treatment, and dentistry used in interprofessional care settings through a One Health lens.\n\n\nMethods\n\nA rapid literature review was conducted from January through June 2020. Rapid reviews are a synthesis of the most current evidence in the literature by exercising elements of systematic reviews in a timely fashion to inform and support public health decision-making18. Papers describing interprofessional care approaches to treat COPD were categorised as Objective 1, and interprofessional care approaches incorporating dentistry were categorised as Objective 2. We gathered papers based on each objective by following a sequence of steps: literature search, appraisal, synthesis, and analysis18. Papers collected at each step were screened by all authors and reviewed by the senior author (SLS) to assess for bias and accuracy. The articles were analysed through a One Health approach, which focuses on the importance of comprehensive, holistic care.\n\nA literature search for each objective was conducted on PubMed and Scopus. Inclusion criteria included primary and secondary studies and practise guidelines published between January 2015–May 2020 in either Canada or the United States. Given the nature of a rapid review and the relatively novel topic of this paper, any range shorter than five years may not have produced many results. Papers were excluded if they did not mention any keywords in the title or abstract, were not written in English, were not accessible electronically, were not available in full text, were classified as grey literature, and were not final publications. Search query strings and collected articles were documented on a Microsoft Excel document and Mendeley citation management software. Duplicate papers were removed. For the remaining articles, titles and abstracts were assessed for eligibility by the lead author (NIN), which included whether the paper aligned with the aim of this study or not. The literature search step is summarized in Figure 1. Search query strings for Objectives 1 and 2 are shown in Table 1. The last search was run on the databases on May 1st, 2020.\n\n(A) Objective 1; (B) Objective 2.\n\nAppraisal checklists specific to each article type were used from the Critical Appraisal Skills Program (CASP) developed by Oxford Centre for Triple Value Healthcare19. Examples include checklists for systematic reviews, cohort studies, and qualitative studies19. We used the CASP checklists to assess the quality of study methods to determine if the findings of the eligible articles are trustworthy and meaningful (Extended data20)18. Articles were examined in more detail during this step. As a result, 12 were removed for Objective 1 and 58 for Objective 2 because they did not align with inclusion or exclusion criteria, or the aim of this study. This step was completed by NIN, GJF, JR, and OMN.\n\nRelevant data from the appraised papers for each objective were extracted based on the aim(s), population/setting(s), methods, results, limitations, relevance to the aim, and applicability to the Canadian healthcare context18. The co-authors established conclusions and recommendations for each objective by evaluating the commonalities and differences across the studies, weighting the results by their methodological quality, and assessing their relevance to a One Health approach18. See Extended data for more information20.\n\nThese conclusions were then analysed for feasibility within the Canadian healthcare context using McMaster University’s Applicability and Transferability Tool21. The tool is designed to capture important issues related to proposed policy or program interventions within a given healthcare context21.\n\n\nResults\n\nOver 90% of papers for Objective 1 found that interprofessional healthcare teams are traditionally restricted to clinical practitioners, yet COPD exacerbations are experiences that, for most patients, extend beyond a physical biomedical episode13. Many patients with COPD struggle with social and psychological concerns related to loss of meaning, hope, and fear of death22. One study that investigated the correlation between anxiety and patient treatment interventions documented a 45% reduction in anxiety related to COPD diagnosis after completing a multidisciplinary pulmonary rehabilitation program23.\n\nFor Objective 2, the majority of articles support the addition of oral health practitioners within primary care teams. This interprofessional approach shifts care from curative, reductionist healthcare principles to preventive, patient-centered principles24–28. The literature suggests that dental providers should adopt a comprehensive approach that accounts for both the patient’s biomedical needs, along with physical and social29. Several articles suggest empowering patients to be contributing members of their treatment teams29. One study showed that when a patient’s role on the care team is ignored, or information is not properly communicated, recovery is negatively affected, and the perception of fragmented care can lead to confusion and distrust from the patient30. Effective communication is enhanced when a care team acknowledges the patient as a whole, including their cultural and linguistic preferences and socioeconomic status31,32. Studies have found that medical and dental providers agree that basic preventive oral health services should be incorporated into medical visits to reduce the burden of dental disease carried by patients in at-risk, low socioeconomic areas33,34. Several authors have demonstrated a need to incorporate the social determinants of health into dental education, modeled by interprofessional healthcare teams with diverse knowledge beyond a biomedical approach33,34.\n\nIn addition to the significance of interprofessional healthcare teams for patients with COPD, other papers assessed the practicality of these teams in clinical and educational settings35–41. After the implementation of a COPD interprofessional care program, one study reported the 30-day emergency department revisit rate declined from 49% to 30%. Another study reported a COPD 30-day readmission rate decrease from 22.7% to 14.7%35,42. In contrast, a study by Bhatt et al. did not report a reduction in 30-day all-cause readmissions from a comprehensive COPD multidisciplinary intervention43. Mansoor et al. propose that interdisciplinary care is beneficial only with well-trained team members, well-established team norms, funded resources, and schedule flexibility34. Another article demonstrated the challenge of miscommunication within healthcare systems, highlighting how a lack of coordination between veteran patients with COPD and community care providers resulted in delayed, missed, or inefficient care44. A possible explanation, proposed by Ly et al., is that healthcare professionals experience role changes as threats to their professional identity which may in turn cause resistance to collaboration45. Role clarification can improve the efficacy of interprofessional teams and provide valuable insight on how to optimize quality improvement and the performance of these teams in clinical practise45. A study that implemented an interprofessional population panel management curriculum targeted to diverse health profession trainees found trainees overwhelmingly reported an increased ability to identify patients who would benefit from interdisciplinary care or referral to another team member46.\n\nMany articles also stressed that collaboration between dental providers and other health professions within both clinical and educational domains is essential for improving health care outcomes for patients with complex medical and oral health needs, especially those with health inequities47,48. Research assessing the current status of interprofessional collaboration states that in order to improve patient outcomes there needs to be changes made in the type of personnel providing health care services, with more non-dental professionals providing select dental services and dental providers providing select medical services49. These changes require better integration of medical and dental education and patient care49,50. It is expected that this long-term goal will improve health outcomes, increase patient satisfaction, better overall health of populations, and reduce per capita costs of healthcare49,50. Primary healthcare teams in both rural and urban areas of Quebec have expressed their concerns on the absence of integrated oral health services for treatment purposes51. They suggest increased implementation of government policies, the prioritization of educational and management measures, and interprofessional collaboration toward innovative care models will facilitate the integration of these health services51.\n\nThere are challenges in incorporating IPE programs successfully at health professional schools largely due to the fact that the integration of these programs are still in their infancy48,52. One IPE activity for health professional students supported progress toward interprofessional socialization, but student learning was inconsistently demonstrated in teamwork products52. Incorporating IPE may be particularly challenging for dental hygiene programs, as their curricula primarily emphasises employment in private practise where providers deliver care intraprofessionally (collaboration only between other dental providers)48 and in relative isolation.\n\nThere is a high probability of being diagnosed with other comorbidities along with COPD53–57. Given this, interprofessional healthcare teams must approach treatment plans with a multi-morbidity (more than one comorbidity) outlook, which requires the inclusion of experts from several different specialties53–57. One systematic review estimated that ≥1 chronic conditions (such as cardiovascular disease) coexist in >85% of patients with COPD54. The authors stated that by utilizing an interprofessional approach, improvements could be made in the management of patients with COPD and other comorbidities54. Such models have been associated with a 66% decrease in hospitalisations and a reduction in the number of exacerbations COPD patients experience54. For COPD patients living with multimorbidities, the best care option is to change from disease-focused to patient-centred care, whereby providers look beyond the COPD diagnosis and instead see the patient as a whole to better understand the etiology of their comorbidities55. Since comorbidities are often associated with a COPD diagnosis, healthcare teams should also shift to a more palliative-focused structure through partnerships with doctors, palliative experts (mainly nurses), and administrative executives to optimise quality of life and mitigate chronic suffering as much as possible58.\n\nLike COPD, oral diseases are also often associated with comorbidities34,50,59–63. The inclusion of oral health into treatment plans has benefited patients across the lifespan who are living with chronic diseases34,50,59–63. One case-control study concerning the inclusion of oral healthcare for patients with hemophilia makes a comparison with healthy individuals and reveals that hemophilic patients had higher debris and calculus scores, suggestive of poor oro-dental health status59. The perception of childhood obesity as being a medical rather than dental issue, as well as dentists’ unwillingness to suggest interventions due to the fear of being offensive and judgemental, has led to many missed opportunities for screening childhood obesity at the dental office50. Patients with syndromic craniosynostosis also pose unique oral health conditions and dentofacial deformities that make it necessary to have oral healthcare providers as part of the interprofessional care team61. There is also a lack of knowledge surrounding sleep-disordered breathing among pediatric medical professionals; therefore, interprofessional care approaches are needed to benefit from the unique perspectives that dentists have on sleep-disordered breathing62. Including dentistry as part of interprofessional healthcare delivery directly addresses the comorbidities associated with dental diseases and can subsequently reduce hospital stays, advance patient well-being, and allow for fiscal savings60.\n\n\nDiscussion\n\nBased on the results of our review, we developed four key recommendations. Firstly, dental providers should be essential members on healthcare teams treating patients with COPD. The comorbidities associated with COPD are often other chronic diseases that have been treated and managed with dental expertise from dental providers; therefore, dental providers can offer valuable insight on some of the systemic conditions associated with COPD34,50,53–57,59–61,63. Secondly, dental providers can offer a valuable perspective and skill set within any interprofessional team. This is particularly important for educational and clinical purposes to enhance treatment outcomes for patients with COPD22,35–42,48,64–68. Thirdly, it is imperative that healthcare leaders and decision-makers embrace interprofessional principles in the development of policies and guidelines to manage complex, chronic diseases like COPD69. Lastly, our results align with the collaborative, multisectoral, and interprofessional aspect of the One Health approach which stresses that the patient is part of a larger system17. In order to fully embrace patient-centred care for patients with COPD, interprofessional teams are encouraged to follow One Health principles by extending beyond medicine, but into dentistry as well70,71. Most importantly, the patient themselves, as well as their families and caregivers, should feel they are valuable members of their healthcare teams30,72. By making interprofessional healthcare teams more holistic and inclusive of dental providers, they can better manage a patient’s experience with COPD physically, mentally, and emotionally, and ultimately, provide quality comprehensive care11,22,23,37,64,73–78.\n\nOur recommendations were analysed using the Applicability and Transferability Tool to better understand their applicability to the Canadian healthcare context (Table 2). We surmise there are important lessons from our review that are transferable to the Canadian healthcare system. There remain many complexities and system-level barriers that will challenge their implementation. Despite provincial and territorial reforms to enable primary interprofessional healthcare teams to be functional, barriers still exist such as ineffective communication and role clarity, lack of functional dental and medical equipment under one health care setting, and inability to share dental and medical electronic health records34,45,79. Changes at a system level are required to enable interprofessional teams to function to their full potential. One example would be to use redistributive policy, whereby transfers are made from one sector to another, to support more interprofessional care implementation80. Focusing on evidence-based implementation will also support transitioning research into policy and clinical practise81. Sustainability of interprofessional care models must consider both patient and provider satisfaction82. As discussed by Bodenheimer and Sinsky, healthcare workers frequently report burnout and dissatisfaction, especially when new interventions (such as interprofessional care teams) are frequently implemented82. Overall, this review can support the foundation of reliable evidence-based policy to ensure that research investments maximise healthcare value and public health efforts81.\n\nInformation was gathered from the rapid review results and paper by Sibbald (2013)35.\n\nBased on preliminary database searches conducted from September to December 2019, search queries were grouped together on the three main components of the study aim: ‘dental providers,’ ‘interprofessional care,’ and ‘COPD.’ This yielded one irrelevant result in PubMed and zero results in Scopus. Arguably a methodological limitation, it also presents as a gap in the literature; therefore, driving the motivation and strength of this study. Finding adequate, eligible papers to analyse was possible under two objectives that grouped together the concept of interprofessional care with COPD and then dentistry, having interprofessional care as the common search term (visualised in Figure 2). This may seem like a limitation as the rapid review methodology does not directly address the three main components of the aim. However, by conducting a literature search on two objectives that commonly share the theme of interprofessional care, the inclusion of dental providers on interprofessional healthcare teams for COPD can be adequately justified and explored in the databases.\n\nVisualizing how the objectives are a re-grouping of the three main components of the study aim.\n\nStudies harbouring significant results may have been eliminated during the Literature Search step based on the specificity of the search query strings for each objective or simply by gaps in the database algorithms. During the Appraisal step, papers classified as intervention evaluations or case reports did not have corresponding CASP checklists, resulting in possible inaccurate assessments of the study methods, further questioning the significance of the papers. In order to maintain the vigour and validity of the results, intervention evaluations were appraised with CASP economic evaluation checklists in accordance with the Workgroup for Intervention Development and Evaluation Research (WIDER) checklist83. Case reports were evaluated with CASP case-control checklists and consultation from a publication in the Journal of Medical Case Reports84. Non-specific literature reviews were also analysed with CASP systematic review checklists.\n\n\nConclusion\n\nThe landscape of COPD is complex and multilayered, making it an inherently difficult disease to manage; however, understanding COPD through the collaborative and multisectoral One Health approach is an innovative and promising solution because of its emphasis on collaborative and holistic principles71. Since this review explored an understudied topic in the literature, it is imperative that the results are used to guide current evidence-based healthcare decision-making. As stated by Mertz, author of the Dental-Medical Divide, “integrating oral health within broader health care systems promises to improve patient experiences and outcomes through better screening, referrals, and coordination of care, while decreasing overall costs (both dental and medical) through increased prevention and early treatment.”85 In order to alleviate the burden of chronic diseases like COPD, we must close the gap between medicine and dentistry which has historically been separated85. This review serves as a starting point for an evidence-based, collective movement of interprofessional practitioners striving to increase awareness around the advantages of including dental providers on healthcare teams treating and improving the lives of patients with COPD.\n\n\nData availability\n\nNo data is associated with this article.\n\nDryad: Exploring the inclusion of dental providers of interprofessional healthcare teams treating patients with chronic obstructive pulmonary disease: a rapid review, https://doi.org/10.5061/dryad.31zcrjdjj20.\n\nThis project includes the following extended data:\n\n- Appraisal Section of Rapid Review: Quality of the literature as assessed by CASP.\n\n- Synthesis Section of Rapid Review: Characteristics of the literature for which data were extracted\n\nDryad: PRISMA checklist for ‘Exploring the inclusion of dental providers of interprofessional healthcare teams treating patients with chronic obstructive pulmonary disease: a rapid review’, https://doi.org/10.5061/dryad.31zcrjdjj20.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors wish to acknowledge Dr. Francisco Olea Popelka for providing expertise on the foundational principles of One Health.\n\n\nReferences\n\nGlobal Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease. 2020; Accessed April 2, 2020. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "80586", "date": "12 Apr 2021", "name": "Maryam Tabrizi", "expertise": [ "Reviewer Expertise Geriatric oral health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well researched and timely for a chronic condition such as COPD, extremely relevant to the oral cavity's health condition. The topic is relevant and in line with Age-Friendly Health System and the 4Ms framework. An innovative health system allows oral health conditions to be recorded while taking the baseline health records in primary care and hospital settings. Further clinical studies are needed.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "6567", "date": "14 Apr 2021", "name": "Navia Novosel", "role": "Author Response", "response": "Dear Dr. Tabrizi, On behalf of the other authors and myself, we thank you for your insightful review and comments on our paper \"Exploring the inclusion of dental providers on interprofessional healthcare teams treating patients with chronic obstructive pulmonary disease\" for F1000 Research. Your expertise in geriatric oral health certainly makes your review extremely valuable and pertinent to our article. We would like to thank you again for your positive feedback. Sincerely, Navia I. Novosel" } ] }, { "id": "297747", "date": "22 Jul 2024", "name": "Sharla King", "expertise": [ "Reviewer Expertise interprofessional education", "health professional education" ], "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 appreciate the One Health perspective the authors took with the study and I think it is a strength of this work. I would like to see an extraction table with all of the reviewed studies listed, even as an appendix. My main concern is the date range for the review. I understand this was a rapid review with literature identified between January-June 2020. I am concerned that the information in the review is already outdated and not as contemporary as the authors state. The authors need to address this in the manuscript.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/9-1383
https://f1000research.com/articles/9-1375/v1	26 Nov 20	{ "type": "Research Article", "title": "Evidencing the need for psycho-socio-economic action to support the rural upskilled youth to cope with the COVID-19 health crisis: a state-wide audit", "authors": [ "M D Saju", "Lorane Scaria", "Natania Cheguvera", "Anuja Maria Benny", "Lizy P J", "Binoy Joseph", "Lorane Scaria", "Natania Cheguvera", "Anuja Maria Benny", "Lizy P J", "Binoy Joseph" ], "abstract": "Background: The impact of the COVID-19 pandemic extends beyond morbidity and mortality to social, psychological, and economic implications. This study aims to determine the grouping of modifiable impacts of COVID-19 among the rural poor youth working in unorganized sectors in Kerala, India. Methods: We conducted a state-wide telephonic survey, in the context of a COVID-19 national lockdown with 14430 youth, who had been trained through the Deen Dayal Upadhyaya Grameen Kaushalya Yojana (DDU-GKY), a skill development program of the Government of India, in the last year. Economic, health and health care, social and emotional issues, basic amenity needs, and interpersonal issues were explored in detail. We further prioritized the needs of vulnerable subgroups; pregnant women, people living alone, and those living with comorbid health conditions within this population. Results: All the participants were aged between 18-35 years and were economically poor rural residents. Only 28% had a permanent job and 6.8% of the participants were not working due to the COVID-19 related lockdown. Among the issues shared, the five domains with the highest frequency were financial toxicity, such as loss of income (32.99%), poor financial backup and debt (31.29%), concerns regarding the future job opportunities (23.92%) and fear of losing their current job (14.75%). 98% of the respondents expressed interest in following up with supportive engagements. Conclusion: This research aims to voice students’ needs to the concerned authorities to design a multi-sectoral, multi-disciplinary, and multi-systemic approach to reduce their distress in the context of pandemic outbreak.", "keywords": [ "COVID 19", "Upskilled youth", "psychological distress", "Kerala" ], "content": "Introduction\n\nThe impact of the COVID-19 pandemic, a public health emergency (WHO, 2020), is beyond morbidity and mortality, and there will certainly be massive social, psychological and economic ramifications for a large population around the world (Li et al., 2020; Wang et al., 2020; Xiao et al., 2020a; Xiao et al., 2020b). Pandemic-related actions such as social distancing and contact restrictions have hampered daily schedules, collapsed many industries and lowered GDP, which has further resulted in financial distress, job loss, job insecurity, temporary/permanent unemployment, unavailability of community services, social isolation, poor mental health outcomes and so on (Cao et al., 2020; Kar et al., 2020; Panchal et al., 2020; Pulla, 2020; Rubin & Wessely, 2020). It has also put a halt to emergency medical services and transportation facilities (The Indian Express, 2020), causing social disruptions worldwide. Concerns around the exacerbation of pre-existing mental health issues and greater difficulty in accessing health support and services have also been reported (The Academy of Medical Sciences, 2020). In addition, an increase in domestic abuse and violence against women is also widely prevalent (Qantarade, 2020).\n\nThe impact is much more disastrous for low and middle-income countries like India where more than 90% of the population depends on daily wage jobs or represent the informal job sector (Buheji et al., 2020). The economic loss imparted by pandemic related restrictions further imposes major mental health problems of depression, anxiety, and even suicidality for the people within the low-income group (Panchal et al., 2020). The quarantines and economic shutdowns speed up unemployment, leading to poverty and a minimum standard of living. A recent UN study pointed out that global poverty could increase for the first time since 1990, posing a major threat to the UN Sustainable Development Goal of ending poverty by 2030 (Sumner et al., 2020). To minimize the crisis, the world should aim to overcome the negative impacts of COVID-19 by focussing initially on the poorer communities (Buheji et al., 2020).\n\nThis study is conceptualized in the context of the Deen Dayal Upadhyaya Grameen Kaushalya Yojana (DDU-GKY) program of the Government of India (GoI). DDU-GKY is one of the clusters of initiatives of the Ministry of Rural Development (MoRD), spread across 28 states of India, with 1575 projects, implemented through 771 Project Implementing Agencies (PIAs), which seeks to promote rural livelihoods by upskilling the rural youth (aged between 15- 35), who are poor, and provide them jobs with regular monthly wages.\n\nThe current study is the first to describe the grouping of modifiable factors within the different domains of impact for youth belonging to impoverished backgrounds engaged in the unorganized work sector. The aim of DDU-GKY involves training unemployed youth from rural poor households with employable skills (DDUGKY, 2020). Measures are built into the guidelines to ensure exclusivity in the recruitment of the poor and the vulnerable for participation in the program. It is for the first time that the Participatory Identification of Poor (PIP) strategy has been used to identify vulnerable groups. Additionally, support strategies including placement services, placement counselling and job tracking for one year after course completion are mandated in this scheme (DDUGKY, 2020). This process is facilitated by PIA driven call centres. PIAs are further coordinated through state and district coordinating agencies of DDU-GKY. This audit is an attempt to optimize its ecosystem to achieve its well-defined goals. In addition, our study helps to identify the key issues faced by the participants in the context of pandemic, thus helping to make a positive difference in the lives of the youth and their families by reengaging this productive workforce into the economic system for their improved economic outcomes.\n\n\nMethods\n\nThe study was approved as an exploratory study by the Institutional Review Board of Rajagiri College of Social Sciences (Autonomous) with number RIRB201906. Obtaining written consent was impossible due to COVID-19 related lockdown and, realizing the urgency of the study, the Institutional Review Board permitted the researchers to obtain oral consent from the participants. Oral consent was obtained from participants and their consent was audio recorded. Participants were informed about the voluntary nature of participation prior to the data collection.\n\nThe aim of the study was to identify, clarify and quantify the issues and challenges faced due to the COVID-19 related lockdown and resulting economic toxicity in recently upskilled youth trained through DDU-GKY centres across Kerala. The issues were grouped under job related concerns and health domains. This multi-site cross-sectional audit included all 90 PIAs across the State of Kerala, India, and a total of 723 PIA staff members within 35 working days, from 4th May 2020 to 19th June 2020. Eligibility criteria for the recruitment of PIA staff members for data collection included a minimum educational qualification of graduation from university or above. Information about all pass out students upskilled through the DDU-GKY program from 1st April 2019 to 31st April 2020, aged above 18 years were retrieved from student records kept at various agencies. For this study, census method was used, where all 15200 students who passed out from these 90 PIAs during the specified period were contacted over telephone and an average of 30 minutes were spent on each participant. A total of 14430 respondents shared their experience, with 770 who did not consent.\n\nThe state coordinating agency organized a series of virtual training programs for the PIA staff members about the context, purpose, process and content of the project and details of what their individual participation would entail. Staff members were trained to use the audit tool for the purpose of data collection. Additional training was given to the PIA project leads and professional social workers (liaison team) working in these agencies, to address the questions and queries of the program implementing staff.\n\nIndividual audit sheets were collected from each agency after the completion of the audit. Data entry training was provided to staff members and staff were instructed to perform data entry on the day of the interview. The study data was collected and managed at the Research Centre of Rajagiri College of Social Sciences (Autonomous).\n\nA two-layer supervisory process was introduced to ensure data quality. In the first layer, the resource institute navigated the whole process through a direct link with the liaison team working in these PIAs. In the second level, the liaison team supervised their staff members. The liaison team allocated 20–25 students to each eligible staff member randomly using a systematic sampling technique. The staff members proactively engaged with their allotted students over telephone. Upon the completion of two interviews, a brief feedback session was conducted by the liaison team to clarify doubts and sort out any issues faced by the interviewers. This process helped in standardizing the process of completion of the study tool. Almost 10% of collected data was then randomly checked by the liaison team for any missing information, wrong entries or unrecorded data. For this purpose, systematic sampling technique was used, where every 6th person in the list was contacted to verify the accuracy of the data.\n\nThe audit tool (Saju et al., 2020) captured data across multiple domains including general physical health, mental health and emotional stressors, social issues, economic and job-related concerns, interpersonal conflicts and other practical concerns. The demographic profile included gender, marital status, working status and course attended under the DDU-GKY scheme. Health-related concerns were about health of family members, their own health, fear of accessing hospital care due to fear of infection, concerns about higher mortality of COVID -19 infection in chronically ill family members, fears of higher risk in old and very young family members, sedentary lifestyle and pregnancy-related worries. Financial concerns were related to loss of income, poor financial backup, future job opportunities, current job loss, debts, inability to access basic amenities like food and medicine, pay rent, financial resource crunches, pay-cuts and not being permitted by their family to undertake a job due to fear of COVID-19 spread. Situational stressors included concerns regarding community spread, geographical alienation from family members (due to lockdown and travel restrictions), and anxiety about family members working in COVID-19 hotspots. Psychological issues included feelings of loneliness, boredom, screen addiction, disrupted routine activities and disconnect from others. Issues with extended family members and other marital issues were elicited under this heading, interpersonal issues and attitudes to interstate migrant workers were also explored.\n\nStudy data was collected and managed using the Excel 2016 database hosted at Rajagiri College of Social Sciences (Autonomous). Data from all the 90 PIAs were aggregated and quantitatively analysed. A descriptive analysis of this aggregated data is detailed in this paper. All statistical analysis procedures were performed using Stata 14 and R version 3.6.3. Chi-square tests and t-tests were performed to test the significance of the study variables.\n\n\nResults\n\nThe current study explored the issues and concerns of 14430 rural upskilled youth from 90 PIAs of the DDU-GKY programme (Saju et al., 2020). More than half of the participants were males (55%) and the majority were unmarried (87%) (Table 1). Participants opted for a variety of courses, including food and beverage (12.8%), technician related courses (12.1%), courses related to banking and accounting (11.9), and hospitality (11.9%). Among the participants, 28% of them had permanent jobs, 6.8% were not working due to the COVID-19 related lockdown and 0.5% had permanently lost their job due to the pandemic situation. Out of the 77 respondents who permanently lost their job, 77% were males and 97% were unmarried. The highest percentage of people who had lost their job permanently were from the automobile industry (59.7%), followed by other technicians (16.9%).\n\naNot working includes all the participants who are students, participants who are unemployed, and participants who had lost their job due to COVID-19.\n\nA range of 28 different issues were shared with the staff (Table 2, Table 3 and Table 4).\n\nChi-square test was employed to find the statistical difference and a p value of <0.05 was considered significant.\n\nChi Square statistics was employed to find the statistical difference and a p value <0.05 was considered significant.\n\n*Chi-square test was employed to find the statistical difference and a p value of <0.05 was considered significant.\n\nThe six issues with the highest frequency were related to economic issues such as loss of income, poor financial backup (31.29%), concerns regarding future job opportunities (23.92%), and insecurity regarding current job (14.75%). Second level issues were related to health: fear of future (27.55%) and concerns regarding COVID-19 spread. 7.67% of the respondents had food scarcity. Interpersonal issues included problems with extended family members (1.37%) and marital issues (0.2%).\n\nMale participants generally expressed concerns regarding finance, employment, and basic needs, while the major concerns of female participants were related to health and interpersonal issues. It is to be noted that psychological issues and concerns were expressed more by males than females. Unmarried respondents had more concerns in all the domains studied when compared to married respondents.\n\nFigure 1 demonstrates major issues expressed by vulnerable populations among the respondents. Some of the major issues expressed by pregnant women were concerns regarding their health, concerns about the baby, potential COVID-19 infection during check-ups and other hospital engagements, poor financial background and loss of income due to the pandemic. The major issues expressed by participants who were living alone included loss of income, poor financial backup, fear of the future in the context of the epidemic, future job concerns and loneliness. Food scarcity was reported by 24.5% of the respondents who were living alone. People with comorbid health conditions had fears of the future, concerns regarding COVID-19 infection, loss of income, poor financial backup, conflict with family, future job concerns and job insecurity.\n\n\nDiscussion\n\nThis research has the potential to identify the felt needs of socially and economically vulnerable youth in the context of the COVID-19 pandemic. This inquiry was developed based on a resilience and strength perspective, which assumes that even though people have existing strengths and motivations, there are situations where they might need a helping hand in solving problems in their life. Recent research found that youth from rural areas, families without a steady income, and people living alone were associated with increased COVID-19 related impacts (Cao et al., 2020). Therefore, the information gathered from the current study on the needs of the vulnerable youth will guide service navigation, helping them to better cope with the pandemic situation.\n\nA total of 14430 upskilled youth from DDU-GKY centres across Kerala participated in this audit and shared their issues. A range of 28 major issues were identified. All these issues were further grouped under five different domains. Firstly, were issues were related to financial toxicity, such as loss of income (32.99%), poor financial backup and debt (31.29%), concerns regarding future job opportunities (23.92%) and fear of losing their current job (14.75%). The second group of issues were related to health, which included anxiety about health (27.55%) and concerns about the fast spread of COVID-19 (22.20%). The other three domains were the adjustment to pandemic situations, family and social relationships and psychological distress. These five domains and their subcategories demonstrate the complexity of the range of issues participants faced. These study findings are in agreement with the findings of recent studies conducted in similar areas, where almost 25% experienced psychological issues and perceived COVID-19 as a threat to their existence (Chakraborty & Chatterjee, 2020). The study highlights that unmarried male youths had higher levels of financial, employment, and psychological concerns, whereas young females were more concerned about their own and their family member’s health. These concerns included issues related to both physical health as well as difficulty in accessing different health care services. Previous studies also found that females experienced greater psychological impacts due to the pandemic (Varshney et al., 2020; Wang et al., 2020).\n\nFinancial crunches and concerns regarding future job opportunities and the security of current jobs were the major reasons for financial and employment-related worries among the participants, as most of these youth are working in sectors, such as aviation, travel, hospitality, retail, manufacturing, and automotive, which are most affected by the COVID-19 pandemic (Thomas, 2020; Wang et al., 2020). The economic domain was the most affected domain of the married participants. Consistent with our findings, Chakraborty & Chatterjee (2020) also revealed that the major reason for the concern is current as well as future job opportunities.\n\nIn agreement with other studies, the situational stressors like fear of infection and rampant spread of the virus were worrisome for many. Chakraborty & Chatterjee (2020) also found that the majority of participants were \"preoccupied with the idea of getting infected with COVID-19”. Additionally, those with physical comorbidities had concerns regarding their own health, resulting in increased distress. This coincides with previous findings (Fu et al., 2020; Varshney et al., 2020). In addition, Cao et al. (2020) reported that those with a relative or an acquaintance infected with COVID-19 were more likely to experience severe anxiety. Inadequate access to hospital care, as a result of the halt to emergency medical services and transportation facilities, was also found to be an area of worry (The Indian Express, 2020), which was found to be a cause of distress in all participants, but especially female participants, of this study.\n\nMass quarantine and nation-wide lockdown have wide consequences on mental health and well-being, especially for vulnerable populations. A lack of supplies of basic essentials, financial crunches, job related uncertainties and families needing separation (Brooks et al., 2020; Dubey et al. 2020; Hawryluck et al., 2004; Maunder et al., 2003) were some of the significant contributors of stress. Stressors like living alone, conflicts with extended family members, marital issues resulting in increased worry were also found in this study and it is consistent with previous findings that social support reduced psychological pressures during epidemics (Cao et al., 2020; Chen et al., 2020). These findings propose an ecosystem framework which acknowledges the socio-economic environment of the students (Green & McSermott, 2010), and consequently adds value to the DDU-GKY program. It advocates for positive change by initiating early economic and social engagements. This proactive empathetic engagement has twin benefits; this initiative gives this program a positive support service reputation, where the students and families maintain their long term trust in the institution and the program. Secondly, it helps the staff members to be sensitive to the needs of the students and become responsive to their needs through developing pathways of care. They become empowered to voice students’ needs to the concerned authorities and advocate for them in mobilizing internal resources as well as external community resources for achieving the goals of this program.\n\nOur study also has its limitations. The current study explored the various domains of issues from the perspective of the researcher. A qualitative study on the experience of the DDUGKY participant is necessary for a clearer understanding of the issues from the participant’s perspective.\n\n\nConclusion\n\nThis staff engagement facilitates speedier reengagement of students through the navigation of both formal and informal support systems, to address their psycho-socio- economic needs. This proactive engagement is a unique model that encourages the PIAs to engage with their alumni to offer support to sustain them in their jobs. This cost-effective support model can be replicated to similar programs to ensure their sustainability and to achieve stipulated goals and objectives.\n\n\nData availability\n\nFigshare: Evidencing the need for psycho-socio-economic action to support the rural upskilled youth to cope with the COVID-19 health crisis: a state-wide audit, https://doi.org/10.6084/m9.figshare.13258826.v4 (Saju et al., 2020).\n\nThis project contains the following underlying data:\n\n- DATA_FILE.xls (participant characteristics and responses)\n\nFigshare: Evidencing the need for psycho-socio-economic action to support the rural upskilled youth to cope with the COVID-19 health crisis: a state-wide audit, https://doi.org/10.6084/m9.figshare.13258826.v4 (Saju et al., 2020).\n\nThis project contains the following extended data:\n\n- Audit tool.docx (copy of audit tool)\n\n- CODE BOOK.docx (code book for underlying data)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication)", "appendix": "References\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuheji M, da Costa Cunha K, Beka G, et al.: The extent of covid-19 pandemic socio-economic impact on global poverty. a global integrative multidisciplinary review. Am J Econ. 2020; 10(4): 213–224. Publisher Full Text\n\nCao W, Fang Z, Hou G, et al.: The psychological impact of the COVID-19 epidemic on college students in China. Psychiatry Res. 2020; 287: 112934. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChakraborty K, Chatterjee M: Psychological impact of COVID-19 pandemic on general population in West Bengal: A cross-sectional study. Indian J Psychiatry. 2020; 62(3): 266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Q, Liang M, Li Y, et al.: Mental health care for medical staff in China during the COVID-19 outbreak. Lancet Psychiatry. 2020; 7(4): e15–e16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDDUGKY: Deen Dayal Upadhyaya Grameen Kaushalya Yojana. Retrieved from 2020. Reference Source\n\nDubey S, Biswas P, Ghosh R, et al.: Psychosocial impact of COVID-19. Diabetes Metab Syndr. 2020; 14(5): 779–788. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFu L, Wang B, Yuan T, et al.: Clinical characteristics of coronavirus disease 2019 (COVID-19) in China: A systematic review and meta-analysis. J Infect. 2020; 80(6): 656–665. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreen D, McDermott F: Social Work from Inside and Between Complex Systems: Perspectives on Person-in-Environment for Today’s Social Work. Br J Soc Work. 2010; 40(8): 2414–2430. Publisher Full Text\n\nHawryluck L, Gold WL, Robinson S, et al.: SARS control and psychological effects of quarantine, Toronto, Canada. Emerg Infect Dis. 2004; 10(7): 1206–1212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKar SK, Arafat SY, Kabir R, et al.: Coping with mental health challenges during COVID-19. In Coronavirus Disease 2019 (COVID-19). Singapore: Springer. 2020; 199–213. Publisher Full Text\n\nLi Z, Ge J, Yang M, et al.: Vicarious traumatization in the general public, members, and non-members of medical teams aiding in COVID-19 control. Brain Behav Immun. 2020; 88: 916–919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaunder R, Hunter J, Vincent L, et al.: The immediate psychological and occupational impact of the 2003 SARS outbreak in a teaching hospital. CMAJ. 2003; 168(10): 1245–1251. PubMed Abstract \| Free Full Text\n\nPanchal N, Kamal R, Orgera K, et al.: The implications of COVID-19 for mental health and substance use. Kaiser Family Foundation. 2020. Reference Source\n\nPulla P: Covid-19: India imposes lockdown for 21 days and cases rise. BMJ. 2020; 368: m1251. PubMed Abstract \| Publisher Full Text\n\nQantarade: Science versus covidiots – India's coronavirus challenge. 2020. Retrieved from Reference Source\n\nRubin GJ, Wessely S: The psychological effects of quarantining a city. BMJ. 2020; 368: m313. PubMed Abstract \| Publisher Full Text\n\nSaju MD, Scaria L, Cheguvera N, et al.: Evidencing the need for psycho-socio-economic action to support the rural upskilled youth to cope with the COVID-19 health crisis: a state-wide audit. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13258826.v4\n\nSumner A, Hoy C, Ortiz-Juarez E: Estimates of the impact of COVID-19 on global poverty. WIDER Working Paper 2020/43. Helsinki: UNU-WIDER. 2020. Reference Source\n\nThe Academy of Medical Sciences: Prepare now for a winter COVID-19 peak, warns Academy of Medical Sciences. 2020; Retrieved from Reference Source\n\nThe Indian Express: The Indian Express Lockdown woes: 70-year-old Kerala woman dies after Karnataka refuses entry to her ambulance at border. 2020; Retrieved from Reference Source\n\nThomas KS: Why upskilling will be important in post COVID-19 job scene. The Week. 2020; Retrieved from Reference Source\n\nVarshney M, Parel JT, Raizada N, et al.: Initial psychological impact of COVID-19 and its correlates in Indian Community: An online (FEEL-COVID) survey. PLoS One. 2020; 15(5): e0233874. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Pan R, Wan X, et al.: A longitudinal study on the mental health of general population during the COVID-19 epidemic in China. Brain Behav Immun. 2020; 87: 40–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Mental health and psychosocial considerations during the COVID-19 outbreak. 2020; Retrieved from Reference Source\n\nXiao H, Zhang Y, Kong D, et al.: The Effects of Social Support on Sleep Quality of Medical Staff Treating Patients with Coronavirus Disease 2019 (COVID-19) in January and February 2020 in China. Med Sci Monit. 2020a; 26: e923549. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiao H, Zhang Y, Kong D, et al.: Social Capital and Sleep Quality in Individuals Who Self-Isolated for 14 Days During the Coronavirus Disease 2019 (COVID-19) Outbreak in January 2020 in China. Med Sci Monit. 2020b; 26: e923921. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "87005", "date": "28 Jun 2021", "name": "Alena Novotná", "expertise": [ "Reviewer Expertise Social Work", "Counseling", "Supervision", "Psychotherapy", "Theories of social Work", "Social research in the fields of social work", "young people", "seniors", "poverty", "voluntary simplicity and others" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 contribution brings up-to-date results of the research evidencing the need for psycho-socio-economic action to support the rural upskilled youth to cope with the COVID-19 health crisis. It was carried out using a significant sample of such young people. I appreciate the appropriate methodology and design of the research. The research provides significant and even alarming results (lack of food, pregnant women’s concerns, 25% of the respondents having experienced psychological problems during the pandemic, fears caused by inadequate access to healthcare due to the standstill of medical services and transport facilities, Covid-19 pandemic being an existential threat, etc.). As the aim of the research was to obtain this data, the application section of the research is absent in the report. I believe that the authors, the involved institutions, and the government of India will all further process the outcome of the research and put them into practice to reduce the impact of the pandemic on the vulnerable groups of population.\nMinor comments:\nIt is not clear from the published research report whether and how the outcome of the research could have been affected by the fact that the respondents were participants of an educational program.\n\nSome of the interpretations could be broader. For instance, the connections resulting from the finding that the unmarried respondents are more concerned about all the examined domains compared to those married, are only formulated partially, as the factor of social support.\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? Partly\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/9-1375
https://f1000research.com/articles/9-1370/v1	26 Nov 20	{ "type": "Case Report", "title": "Case Report: Aortitis associated with rheumatoid arthritis: A challenging rheumatoid vasculitis presentation", "authors": [ "Zeineb Teyeb", "Mohamed Ben Salah", "Lobna Kharrat", "Imen Abdellali", "Taieb Jomni", "Mohamed Hedi Douggui", "Mohamed Ben Salah", "Lobna Kharrat", "Imen Abdellali", "Taieb Jomni", "Mohamed Hedi Douggui" ], "abstract": "Rheumatoid vasculitis (RV) is a rare but serious extra-articular manifestation of rheumatoid arthritis (RA). Its varied clinical presentation makes it hard to diagnose and treat. Hereby we describe a case of an aortitis revealing RV, which is a rare presentation of a rare complication of RA. A 56-year-old man with rheumatoid arthritis treated with methotrexate presented with fever, chest pain and arthritis. Blood tests revealed inflammatory syndrome associated with cholestasis. The diagnosis of pericarditis associated with aortitis was retained. Cholestasis was mostly due to methotrexate. The patient was treated with cyclophosphamide pulses and high doses of prednisolone. The patient was in complete remission of articular and extra-articular manifestations after two months of treatment.", "keywords": [ "rheumatoid arthritis", "rheumatoid vasculitis", "aortitis", "immunosuppressive therapy", "case report" ], "content": "Introduction\n\nRheumatoid arthritis (RA) is a connective tissue disease predominantly affecting the joints. Extra-articular manifestations develop in up to 40% of cases1, of which rheumatoid vasculitis (RV) is the most serious. The widespread vascular involvement, which effects not only the synovia but also other organs such as the skin, eye and nerves, can be life threatening. Mortality can reach up to 40% within five years of disease onset2. Fortunately, RV is a rare complication that occurs in 1-5% of RA patients3. It commonly affects small and medium blood vessels4. Large vessel vasculitis is unusual during RA. Hereby we describe a case of an aortitis revealing RV, which is a rare presentation of a rare complication of RA.\n\n\nCase description\n\nA 54-year-old man, a North African policeman with a personal history of smoking and pulmonary tuberculosis in 1991 was diagnosed in 2009 with seropositive and erosive RA associated with Sjögren’s syndrome. He was being treated with methotrexate (25mg/week). In November 2019, the patient presented to our hospital with fever, fatigue and chest pain that had started one week prior. Physical examination found a high temperature of 38.5°C. Systolic blood pressure was 100mmHg and diastolic blood pressure was 60mmHg in both arms. He had tachycardia, with a heart rate of 115 beats per minute. There were no signs of heart failure and respiratory rate was normal. Mobilization of the wrists, elbows and shoulders was painful, with a swollen right wrist. He had a dislocation of the right ulnar styloid. He had rheumatoid nodules on the outer side of both elbows. The rest of the physical examination was normal.\n\nAn electrocardiogram showed atrial fibrillation with a heart rate of 115 beats per minutes associated with diffuse ST-segment elevation with upward concavity.\n\nBlood tests showed hyper leukocytosis at 11000/mm3, C-reactive protein (CRP) levels of 117 mg/l, an erythrocyte sedimentation rate (ESR) of 118mm and cholestasis with increased gamma glutamyl transferase and phosphatase alkaline levels of 199UI/L (four times the normal rate) and 348UI/L (five times the normal rate), respectively. Transaminase levels were normal (ALAT level of 35UI/L and ASAT level of 25UI/L). A procalcitonin test was negative (<0.5 μg/L). Blood gas analysis was normal (pH level of 7.40, PaO2 level of 90mmHg, PaCO2 level of 42mmHg, HCO3- level of 25 mmol/l). A chest X-ray showed a flask-shaped enlarged cardiac silhouette. Transthoracic echocardiography confirmed a non-compressive large posterior pericardial effusion. Abdominal ultrasound was normal. A thoraco-abdominal computed tomography (CT) scan showed pericardial effusion with enhancement of the pericardium, compatible with pericarditis, and regular parietal hypodense circumferential thickening of the aortic arch and supra aortic arterial trunk root, confirming aortitis (Figure 1 and Figure 2). There was emphysema in the pulmonary parenchyma but no evidence of active tuberculosis on chest CT.\n\nA biopsy of the cardiac effusion was not possible due to its posterior location. The sputum test for Koch’s bacillus was negative. Viral hepatitis B and C and syphilis serology were negative. Antinuclear antibodies were positive at 1/1200. Anti-LKM1, anti-CCP and rheumatoid factor levels were 1/80, 40 IU/ml and 50 IU/ml, respectively. Immunoglobulin levels were normal (IgG level of 14.3 g/L, IgM level of 2.08 g/L and IgA level of 1.8g/L).\n\nLiver biopsy showed peliosis with no sign of auto-immune hepatitis nor auto-immune biliary cholangitis.\n\nThe diagnosis of RV with large vessel and cardiac involvement was retained. Disease activity was evaluated as moderate by the DAS-28 CRP score.\n\nThe patient was treated with a high dose of prednisone (60 mg/day) and IV pulses of 1000mg cyclophosphamide every month for six months. Treatment with methotrexate was stopped. Because of a personal history of pulmonary tuberculosis and the prescription of corticosteroids and cyclophosphamide in an epidemic country of tuberculosis, we administered prophylactic antitubercular therapy. The patient was also by treated with 200mg/day of amiodarone for the atrial fibrillation.\n\nThe patient was apyretic after day 2 and their heartbeat became normal. Chest pain and articular manifestations decreased and disappeared after one month of treatment. CRP levels decreased to 12mg/L after steroids and cyclophosphamide pulses. ESR became normal after two months of treatment. Cholestasis disappeared after one and a half months of methotrexate withdrawal.\n\nInvestigations of the cholestasis highlighted the iatrogenic involvement of methotrexate, confirmed by a report from the National Drug Safety Department in Charles Nicolle’s Hospital of Tunis.\n\nCardiac echography showed the disappearance of the cardiac effusion at two months. A chest CT scan showed a significant regression of the vasculitis.\n\n\nDiscussion\n\nThe epidemiology of RV is hard to define. Heterogeneous clinical presentation, paucity of specific data to confirm the diagnosis and lack of a unanimous definition of RV are some of the reasons why it is hard to define. Many authors claim that RV can be observed in up to 5% of RA patients5.\n\nOur patient was 54 years old. The mean age of patients at diagnosis of aortitis in a literature review was 56±15.2 years old6.\n\nRV commonly affects small and medium sized vessels. The skin is the most commonly damaged organ (90% of patients). The association of aortitis with RV is not widely recognized. However, many authors reported an association of RA with aortitis6. Our patient had a lesion typical of vasculitis of the aorta on his chest CT scan. Differential diagnoses were ruled out such as syphilis, tuberculosis, systemic erythematous lupus, Takayasu’s arteritis and Horton’s disease.\n\nAortitis was the only manifestation of RV in our patient. Other similar cases have been reported. In half of the cases, aortitis was isolated, with no other features of vasculitis6.\n\nIn our patient, RV was revealed after 11 years of RA onset while the patient was treated with methotrexate and a low dose of corticosteroids. In a review of the literature, RV appeared after a mean disease duration of six years. Rheumatoid nodules were observed in up to half of patients with RV6.\n\nOur patient had poor articular manifestations with disease activity evaluated as moderate by the DAS-28 score. The concept of ‘burnt out’ disease is described by many authors, consisting of the contrast between benign articular presentation and severe life-threatening RV4. This leads us to insist on actively screening this rare but fatal complication.\n\nRV typically occurs in long-standing seropositive and erosive RA, especially in males, smokers and patients with rheumatoid nodules or rheumatoid pericarditis1,5. Our patient had all these conditions.\n\nThere are no randomized controlled studies to guide the management of RV. However, treatment must be guided by the severity of organ involvement. High doses of corticosteroids and cyclophosphamide have been known to be the treatment of severe forms of RV such as aortitis1,7,6–9. Our patient had a good response to high doses of prednisolone and cyclophosphamide. Biotherapy such as TNF inhibitors, rituximab, abatacept and anakinra could be a good alternative4,10.\n\nAnother particularity of our patient is his liver injury. The patient had increased gamma glutamyl transferase and phosphatase alkaline levels with normal transaminases. This cholestasis is mostly due to methotrexate. This was confirmed by the complete normalization of liver enzyme levels after methotrexate withdrawal and the report from the drug safety department. However, our patient had autoimmune hepatitis (AIH) antibodies without any histological pattern of AIH and with normal levels of transaminase and immunoglobulins. There was not enough evidence to retain the diagnosis of AIH. Furthermore, surveillance of liver biology is recommended to assess the risk of developing AIH11–13.\n\n\nConclusion\n\nIn the past years, the incidence of RV has decreased. Early diagnosis of RA, treat-to-target treatment strategies and the large use of methotrexate and biological molecules has improved the quality of life of RA patients5. Better management of the disease has led to a diminishing incidence of RV. However, clinical presentation remains unchanged. The mortality rate remains high, making RV a life-threatening condition that must be screened and treated early and aggressively. In addition, liver injury in RA patients varies from infectious (hepatitis B or C), toxic (paracetamol, methotrexate) and autoimmune.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and images was obtained from the patient.", "appendix": "References\n\nMakol A, Crowson CS, Wetter DA, et al.: Vasculitis associated with rheumatoid arthritis: a case-control study. Rheumatology (Oxford). 2014; 53(5): 890–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuresson C, O'Fallon WM, Crowson CS, et al.: Occurrence of extraarticular disease manifestations is associated with excess mortality in a community based cohort of patients with rheumatoid arthritis. J Rheumatol. 2002; 29(1): 62–7. PubMed Abstract\n\nCojocaru M, Cohocaru IM, Chico B: New insight into the rheumatoid vasculitis. Rom J Intern Med. 2015; 53(2): 128–32. PubMed Abstract \| Publisher Full Text\n\nKishore S, Maher L, Majithia V, et al.: Rheumatoid Vasculitis: A Diminishing Yet Devastating Menace. Curr Rheumatol Rep. 2017; 19(7): 39. PubMed Abstract \| Publisher Full Text\n\nGenta MS, Genta RM, Gabay C: Systemic rheumatoid vasculitis: a review. Semin Arthritis Rheum. 2006; 36: 88–98. PubMed Abstract \| Publisher Full Text\n\nKaneko S, Yamashita H, Sugimori Y, et al.: Rheumatoid arthritis-associated aortitis: a case report and literature review. SpringerPlus. 2014; 3: 509. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMakol A, Matteson EL, Warrington KJ: Rheumatoid vasculitis: an update. Curr Opin Rheumatol. 2015; 27(1): 63–70. PubMed Abstract \| Publisher Full Text\n\nScott DG, Bacon PA: Intravenous cyclophosphamide plus methylprednisolone in treatment of systemic rheumatoid vasculitis. Am J Med. 1984; 76: 377–84. PubMed Abstract \| Publisher Full Text\n\nNanatsaki E, Mooney J, Scott DGI, et al.: Systemic rheumatoid vasculitis in the era of modern Immunosuppressive therapy. Rheumatology (Oxford). 2014; 53(1): 145–152. PubMed Abstract \| Publisher Full Text\n\nvan der Bijl AE, Allaart CF, Van Vugt J, et al.: Rheumatoid vasculitis treated with infliximab. J Rheumatol. 2005; 32(8): 1607–9. PubMed Abstract\n\nGatselis NK, Zachou K, Koukoulis GK, et al.: Autoimmune hepatitis, one disease with many faces: Etiopathogenetic, clinico-laboratory and histological characteristics. World J Gastroenterol. 2015; 21(1): 60–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeinblatt ME, Tesser JR, Gilliam JH 3rd: The liver in rheumatic diseases. Semin Arthritis Rheum. 1982; 11(4): 399–405. PubMed Abstract\n\nUtiyama SRR, Zenatti KB, Nóbrega HAJ, et al.: Rheumatic Disease Autoantibodies in Autoimmune Liver Diseases. Immunol Invest. 2016; 45(6): 566–73. PubMed Abstract \| Publisher Full Text" }	[ { "id": "75567", "date": "16 Dec 2020", "name": "Dionicio Ángel Galarza-Delgado", "expertise": [ "Reviewer Expertise Rheumatology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have read this article with interest.\nThe title of the article is presented as the association of aortitis with rheumatoid arthritis, it should be noted that there would also be an association between the patient's history of tuberculosis with aortitis, perhaps the title could be modified.\n\nOther causes of infectious aortitis are not discussed in your article.\nPlease see: Journeau L, de la Chapelle M, Guimard T, Ferfar Y, Saadoun D, Mahé I, Castier Y, Montravers P, Lescure X, Van Gysel D, Asseray N, Lascarrou JB, Ngohou C, Vandamme YM, Connault J, Cepoy PD, Brochard J, Goueffic Y, Pistorius MA, Boutoille D, Espitia O. A strobe multicenter descriptive study of 55 infectious aortitis. Medicine (Baltimore). 2020 Oct 2;99(40):e224221..\nFor example, among rare infectious causes is a infection by C burnetii which usually occurs in men aged over 50 and lived in rural environment.\n\nYou do not mention whether fungal causes were ruled out as rare causes of aortitis.\n\nIt is noteworthy that there were no or no other common manifestations of rheumatoid vasculitis such as skin ulcerations, erythema nodosum, and only “vasculitis” of large vessels was found in his patient.\n\nIt is not mentioned whether there was evidence of atherosclerosis or aneurysm given the age of the patient. Please clarify.\n\nIt is not mentioned whether positron emission tomography (PET) -CT was done to rule out other sites of vascular involvement. Please clarify.\n\nYou could specify in your article if the aortic vascular affection was concentric or localized, if there were any Sacciform aneurysm.\n\nAlso determine if in the imaging studies there was presence of gas bubbles in or around the aorta wall or mural thrombus. As well as whether blood cultures were taken to detect rare and sometimes indolent infectious causes such as Salmonella infections of the aorta in patients with atherosclerosis\nPlease see. Looi JL, Cheung L, Lee AP. Salmonella mycotic aneurysm: a rare cause of fever and back pain in elderly. Int J Cardiovasc Imaging. 2013 Mar;29(3):529-312.\nFinally, you say in your article that giant cell arteritis was excluded, but it is not mentioned how it was excluded. Please expand the discussion on other causes of non-infectious aortitis.\nPlease see. Loricera J, Blanco R, Hernández JL, Carril JM, Martínez-Rodríguez I, Canga A, Peiró E, Alonso-Gutiérrez J, Calvo-Río V, Ortiz-Sanjuán F, Mata C, Pina T, González-Vela MC, Martínez-Amador N, González-Gay MA. Non-infectious aortitis: a report of 32 cases from a single tertiary centre in a 4-year period and literature review. Clin Exp Rheumatol. 2015 Mar-Apr;33(2 Suppl 89):S-19-31.3.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "80665", "date": "29 Mar 2021", "name": "Shunta Kaneko", "expertise": [ "Reviewer Expertise Rheumatology" ], "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\nTeyeb et al. reported a rare case of aortitis in patients with rheumatoid arthritis. While the treatment of rheumatoid arthritis has made remarkable progress, this paper is interesting and important especially because aortitis is a life-threatening condition. I have several minor comments:\nDiagnostic assessment: The RF titer of this patient was low for rheumatoid vasculitis. Does the patient have any findings suggestive of rheumatoid vasculitis such as interstitial pneumonia, skin ulcers, and low complement except for rheumatoid nodules? Without these findings, the possibility of Takayasu's arteritis and extracranial giant cell arteritis can’t be ruled out.\n\nFollow-up and outcomes: Do you have follow-up CT images? You should show an improvement in the thickening of the aortic wall by CT.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1370
https://f1000research.com/articles/9-1369/v1	25 Nov 20	{ "type": "Research Article", "title": "The effect of halogen bulb and light-emitting diode light curing units on temperature increase and fibroblast viability", "authors": [ "Georgia Memari Trava", "Juliane Almeida Santos", "Lucas Paula Ramos", "Pamela Beatriz Rosário Estevam dos Santos", "Amjad Abu Hasna", "Karen Cristina Yui", "Adriano Bressane", "Luciane Dias de Oliveira", "Marianne Spalding", "Georgia Memari Trava", "Juliane Almeida Santos", "Pamela Beatriz Rosário Estevam dos Santos", "Amjad Abu Hasna", "Karen Cristina Yui", "Adriano Bressane", "Luciane Dias de Oliveira", "Marianne Spalding" ], "abstract": "Background: This study aimed to compare the temperature increase produced by halogen bulb (HAL) and light-emitting diode (LED) light curing units (LCUs) by irradiating dentin discs (0.5 mm and 1 mm thickness), and to evaluate their cytotoxic effects on fibroblast culture in the presence of dentin discs due to the increasing demand on resin composite restorations and teeth bleaching for esthetic purposes. Methods: A total of 20 bovine incisors were used to obtain dentin discs and divided into four experimental groups (n=10): HAL0.5: irradiation with halogen-tungsten bulb Curing Light XL 3000 at an intensity of 470 mW/cm2 over a dentin disc of 0.5 mm; LED0.5: irradiation with LED Optilight Max (GNATUS- Ribeirão Preto, SP, Brazil) at an intensity of 1200 mW/cm2 over a dentin disc of 0.5 mm; HAL1: irradiation as in HAL0.5 but over a dentin disc of 1 mm; LED1: irradiation as in LED0.5 but over a dentin disc of 1 mm. The temperature increase was measured using a digital thermometer and the cytotoxicity was evaluated using an MTT assay with a mouse fibroblast cell line (L929). Parametric Data were analyzed by ANOVA and Tukey and non-parametric data were analyzed by Kruskal Wallis with Conover-Iman for non-parametric data (all with α=0.05). Results: A significant statistical difference was found between the groups HAL0.5 and HAL1 and both were different of LED0.5 and LED1 which presented higher temperature. All the experimental groups were different of the control group (without irradiation), and promoted reduction of cellular viability. Conclusions: HAL LCU promoted a lower temperature change in the dentin compared to LED, regardless of the dentin thickness (0.5-1 mm). Both HAL and LED LCUs decreased fibroblast viability; however, LED promoted more significant cytotoxic effects.", "keywords": [ "Halogen light", "Light-emiting-diode", "mouse fibroblasts", "temperature increase" ], "content": "Introduction\n\nDentistry patients are looking for esthetically pleasing smiles and increasingly demanding resin composite restorations and teeth bleaching (Al Otaibi et al., 2020; Sebold et al., 2020). These processes require photopolymerization and photoactivation by halogen bulb (HAL) and light-emitting diode (LED) light curing units (LCUs) (Gallinari et al., 2020; Pieniak et al., 2014). However, these LCUs can increase the temperature and induce thermal transfer, depending on the light source intensity and type (Armellin et al., 2016; Kim et al., 2017).\n\nThe elevated temperature resulting from daily clinical procedures can cause an increase in pulp temperature, with the subsequent development of symptoms such as hyperalgesia, dentin hypersensitivity, and spontaneous typical pain of acute pulpitis (Vinagre et al., 2019). The thermal change causes heat-induced bone tissue injury (Eriksson & Albrektsson, 1983) and pulp tissue necrosis, pathology or alteration (Matalon et al., 2010; Nyborg & Brännström, 1968).\n\nAdditionally, some thermal injuries may affect the surrounding tissue cells (Baldissara et al., 1997) and form lesions in the odontoblastic layer, which leads to its degeneration, protein coagulation and fluid expansion in the dentinal tubules (Vinagre et al., 2019). Some contributing factors affect the extent of injury, such as the remaining dentin thickness, the type of the LCU, the type of ultrasonic device, or the type of water spray used (Kwon et al., 2013).\n\nTherefore, controlling the temperature increase during the emission of LCUs is an important factor in the use of photopolymerizers. The objective of this study was to compare the temperature increase produced by HAL and LED LCUs by irradiating dentin discs (0.5 mm and 1 mm thickness), and to evaluate their cytotoxic effects on fibroblast culture in the presence of dentin discs.\n\n\nMethods\n\nA total of 20 bovine incisors were used in this study, the crowns were separated from the roots 2 mm below the level of cemento-enamel junction and then embedded in self-curing acrylic resin (TDV, Santa Catarina, Brazil) in a prefabricated PVC mold. Later, longitudinal dentin discs (without enamel) were obtained (10 discs of 0.5 mm and 10 discs of 1 mm) by sectioning the crowns using diamond disc (0.3 mm thickness) and an EXTEC cutting machine (Labpol 8-12, Extec Corp®, Enfield, Connecticut, USA) (Figure 1).\n\n(A) the bovine tooth was cross-cut on the level of cemento-enamel junction of the lateral surface and the enamel was removed totally with sand paper. (B) the crown was fixed in self-curing acrylic resin JET (fabrication) in a PVC prefabricated mold. (C) Longitudinal discs were obtained (10 discs of 0.5 mm and 10 discs of 1 mm).\n\nA digital thermometer (MT-507 Minipa, São Paulo) was used to measure the temperature variation during light irradiation with HAL and LED LCUs (Table 1). The base of the specimen was covered with an insulating thermal paste (Implastec, Votorantim Ind. Brasileira, São Paulo, SP, Brazil), and the tip of the thermocouple surrounded by paste was placed in contact with the lower wall of each dentin disc. The specimens were irradiated for 20 s and the temperature was measured one time for each specimen obtaining 10 measurements for each experimental group (n= 10).\n\nMouse fibroblast cells (L929) (Rio de Janeiro Cell Bank, APABCAM, RJ, Brazil) were grown in cell culture flasks (TPP, Switzerland) containing Dulbecco’s modified Eagle medium (DMEM) (LGC Biotecnologia, Cotia, Brazil) and supplemented with 10% fetal bovine serum (Invitrogen, New York, USA) at 37°C and 5% CO2 with atmospheric humidity. Next, 2×104 cells/mL were cultivated in 96-well microplates (TPP, Trasadingen, Switzerland) in the same medium for 24 hours for cell adhesion. DMEM was used as a control group (0 mg/mL).\n\nThe treatment was carried-out for the groups (n= 10), in which each dentin disc was positioned over a well containing 100 µL of cell suspension in DMEM and light irradiation performed (Table 1). This positioning was to simulate the clinical situation when the LCU irradiates the dentin and this irradiation may affect the fibroblast in the adjacent soft tissues (Figure 2).\n\nNext, MTT solution (100 µL/well) was added to the 96-well plate and the plates were incubated at 37°C with 5% CO2 for 1 h. Then, the MTT solution was discarded and 100 µL/well of dimethylsulfoxide (DMSO; Sigma, Missouri, USA) was added and the plates were incubated again for 10 min and shaken for 10 min. The absorbance of the wells was measured using a spectrophotometer at 570 nm and data generated were converted to cell viability percentage using the formula: = OD of each group × 100 / OD of control group (OD = optical density).\n\nAfter normality testing, data were analyzed by one-way ANOVA with Tukey’s post hoc test for parametric data or Kruskal-Wallis with post hoc Conover-Iman test for non-parametric data (α=0.05) using GraphPad Prism 6 (La Jolla, CA, USA).\n\n\nResults\n\nA significant statistical difference was found between the groups HAL0.5 and HAL1 and both were significantly different to LED0.5 and LED1, which presented higher temperatures. However, no significant difference was observed between the two LED groups (Figure 3).\n\nDifferent letters indicate statistically significant differences among the experimental groups with (P ≤ 0.05).\n\nAll groups were significantly different to the control group, and promoted reduction of cellular viability. There was no significant difference between the groups HAL0.5 (cell viability 43.5%) and HAL1 (cell viability 41.1%), and between the groups LED0.5 (cell viability 17.1%) and LED1 (cell viability 17.3%). However, both HAL0.5 and LED0.5 were significantly different to HAL1 and LED1 (Figure 4).\n\nDifferent letters indicate statistically significant differences among the experimental groups with (P ≤ 0.05).\n\nRaw cytotoxicity and temperature results are available as Underlying data (Paula Ramos, 2020).\n\n\nDiscussion\n\nThis paper investigated the heating generated by HAL and LED when irradiating dentin discs of thickness 0.5 mm and 1 mm of for 20 seconds. Hannig & Bott (1999) obtained different readings were obtained (2.9 to 7.9°C) when they evaluated six LCUs, including HAL, for 40, 10 and 5 s, finding that significantly higher pulp chamber temperatures were obtained when compared to conventional LCUs like Heliolux II. Uhl et al. (2003) evaluated the heating generated after resin composite photopolymeriztion and founded that LED LCUs represent a viable alternative to HAL LCUs for dental composite photopolymerization due lower temperature increases within the composite. Different results were obtained in the present study, as HAL generated lower temperature increases than LED in dentin. Conversely, another study showed no difference between HAL and LED LCUs in generating heat (Drost et al., 2019). These different results in the literature may be related to the kind of temperature sensor and the methodology used (Jiang et al., 2019).\n\nBoth LED and HAL LCUs negatively influence cellular viability (Passarelli et al., 2020); however, there is insufficient evidence that they cause pulp inflammation/cytotoxicity (Benetti et al., 2018). In the present study, it was verified that LED was more cytotoxic than HAL LCUs; however, Gonçalves et al. (2016) found that LED had minimal cytotoxicity. This result may be influenced by the dentin thickness, as Daronch et al. (2007) found that the increase in pulp temperature was directly related to the remaining dentin thickness. In the present study, the application of LED light to the thickest dentin disc (1.0 mm) was less cytotoxic than the thinnest dentin disc (0.5 mm).\n\nThe divergence observed in the present work in relation to the dentin thickness, light source and the possible greater protection that it can confer to the pulp could be related to the wavelength that the devices emit. The HAL LCU used emits a wavelength of 400–500 nm, and the dental structure is capable of absorbing light in a spectrum from 350–400 nm, meaning it can thus exhibit fluorescence at 410-500 nm. Therefore, the HAL LCU employed herein emits light at an absorbable wavelength for the dentin disc. Thus, the greater the dentin thickness, the greater the absorbance of light and the higher the concentration of photons, thus explaining the increase in the temperature of the disc (Neumann et al., 2005).\n\nThis study found that HAL LCU promoted a lower temperature change in the dentin compared to LED, regardless of the dentin thickness (0.5–1 mm). HAL and LED LCUs decreased fibroblast viability; however, LED resulted in greater cytotoxicity.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Dataset. https://doi.org/10.7910/DVN/M4FYVV (Paula Ramos, 2020).\n\nFile ‘Dataset.tab’ contains raw data for cell viability and temperature generated in the present study.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAl Otaibi FL, Althumairy AF, Al Ahmadi BT, et al.: Patients’ preferences on different types of esthetic treatment in saudi arabia. J Contemp Dent Pract. 2020; 21(1): 62–67. PubMed Abstract \| Publisher Full Text\n\nArmellin E, Bovesecchi G, Coppa P, et al.: LED curing lights and temperature changes in different tooth sites. Biomed Res Int. 2016; 2016: 1894672. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaldissara P, Catapano S, Scotti R: Clinical and histological evaluation of thermal injury thresholds in human teeth: a preliminary study. J Oral Rehabil. 1997; 24(11): 791–801. PubMed Abstract \| Publisher Full Text\n\nBenetti F, Lemos CAA, de Oliveira Gallinari M, et al.: Influence of different types of light on the response of the pulp tissue in dental bleaching: a systematic review. Clin Oral Investig. 2018; 22(4): 1825–1837. PubMed Abstract \| Publisher Full Text\n\nDaronch M, Rueggeberg FA, Hall G, et al.: Effect of composite temperature on in vitro intrapulpal temperature rise. Dent Mater. 2007; 23(10): 1283–1288. PubMed Abstract \| Publisher Full Text\n\nDrost T, Reimann S, Frentzen M, et al.: Effectiveness of photopolymerization in composite resins using a novel 445-nm diode laser in comparison to LED and halogen bulb technology. Lasers Med Sci. 2019; 34(4): 729–736. PubMed Abstract \| Publisher Full Text\n\nEriksson AR, Albrektsson T: Temperature threshold levels for heat-induced bone tissue injury: a vital-microscopic study in the rabbit. J Prosthet Dent. 1983; 50(1): 101–107. PubMed Abstract \| Publisher Full Text\n\nGallinari M, de O, Cintra LTA, et al.: Evaluation of the color change and tooth sensitivity in treatments that associate violet LED with carbamide peroxide 10 %: A randomized clinical trial of a split-mouth design. Photodiagnosis Photodyn Ther. 2020; 30: 101679. PubMed Abstract \| Publisher Full Text\n\nGonçalves RS, Costa CAS, Soares DGS, et al.: Effect of different light sources and enamel preconditioning on color change, H2O2 penetration, and cytotoxicity in bleached teeth. Oper Dent. 2016; 41(1): 83–92. PubMed Abstract \| Publisher Full Text\n\nHannig M, Bott B: In-vitro pulp chamber temperature rise during composite resin polymerization with various light-curing sources. Dent Mater. 1999; 15(4): 275–281. PubMed Abstract \| Publisher Full Text\n\nJiang X, Lin C, Huang Y, et al.: Hybrid Fiber Optic Sensor, Based on the Fabry-Perot Interference, Assisted with Fluorescent Material for the Simultaneous Measurement of Temperature and Pressure. Sensors (Basel). 2019; 19(5): 1097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim MJ, Kim RJY, Ferracane J, et al.: Thermographic analysis of the effect of composite type, layering method, and curing light on the temperature rise of photo-cured composites in tooth cavities. Dent Mater. 2017; 33(10): e373–e383. PubMed Abstract \| Publisher Full Text\n\nKwon SJ, Park YJ, Jun SH, et al.: Thermal irritation of teeth during dental treatment procedures. Restor Dent Endod. 2013; 38(3): 105–112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaula Ramos L: Replication Data for: Dataset. Harvard Dataverse, V3. 2020. http://www.doi.org/10.7910/DVN/M4FYVV\n\nMatalon S, Slutzky H, Wassersprung N, et al.: Temperature rises beneath resin composite restorations during curing. Am J Dent. 2010; 23(4): 223–226. PubMed Abstract\n\nNeumann MG, Miranda WG, Schmitt CC, et al.: Molar extinction coefficients and the photon absorption efficiency of dental photoinitiators and light curing units. J Dent. 2005; 33(6): 525–532. PubMed Abstract \| Publisher Full Text\n\nNyborg H, Brännström M: Pulp reaction to heat. J Prosthet Dent. 1968; 19(6): 605–612. PubMed Abstract \| Publisher Full Text\n\nPassarelli PC, Saccomanno S, De Angelis P, et al.: Study of cellular toxicity in vitro of two resins for orthodontic use. Eur Rev Med Pharmacol Sci. 2020; 24(2): 930–934. PubMed Abstract \| Publisher Full Text\n\nPieniak D, Niewczas AM, Walczak M, et al.: Influence of photopolymerization parameters on the mechanical properties of polymer-ceramic composites applied in the conservative dentistry. Acta Bioeng Biomech. 2014; 16(3): 29–35. PubMed Abstract \| Publisher Full Text\n\nSebold M, Lins RBE, André CB, et al.: Flowable and Regular Bulk-Fill Composites: A Comprehensive Report on Restorative Treatment. Int J Periodontics Restorative Dent. 2020; 40(2): 293–300. PubMed Abstract \| Publisher Full Text\n\nUhl A, Mills RW, Jandt KD: Polymerization and light-induced heat of dental composites cured with LED and halogen technology. Biomaterials. 2003; 24(10): 1809–1820. PubMed Abstract \| Publisher Full Text\n\nVinagre A, Ramos JC, Rebelo C, et al.: Pulp Temperature Rise Induced by Light-Emitting Diode Light-Curing Units Using an Ex Vivo Model. Materials (Basel). 2019; 12(3): 411. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "75399", "date": "07 Dec 2020", "name": "Talal Al-Nahlawi", "expertise": [ "Reviewer Expertise Endodontics and Operative Dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aimed to evaluate a relevant topic in operative dentistry nowadays because of the increased demand and need for light curing units in many procedures including photo-polymerization of resin composite restorations and for teeth bleaching despite the heavy discussion about the light curing efficacy on improving the bleaching.\n\nIntroduction:\n\nThe authors were able to successfully approach these topics in the introduction section. The objective defined at the end of this section was clearly reported.\n\nMaterial and methods:\nThis study protocol is well described and standardized. The light irradiation and temperature measuring method were described in the literature in other studies. I think it would be more appropriate to cite the original studies, however, it is not of great relevance as a number of these studies were cited in the discussion section. The same should be followed in the MTT assay, it is not a unique test of this study, I think in the future, all the original studies of these tests should be cited.\n\nI should congratulate the authors for the schematic illustration of MTT assay, this part of the test of applying the dentin discs over the 96-well plate is innovative, or at least, to the best of my knowledge, it was not used in any other study.\n\nResults and Discussion:\n\nEven I don't agree with the results. However, they were well described and illustrated. And the fact the LED light curing was more cytotoxic and generated more thermal changes than the Halogen light curing unit may be explained by the LED unit intensity used in the study (1200 mW/cm2) which relatively 3 times the intensity of the halogen light curing unit used in this study 470 mW/cm2). Why did the authors use light curing units with great intensity difference?\n\nWhy did the authors not evaluate the laser as well? What about laser-induced photopolymerization?\n\nThere are divergent results in the literature about the thermal changes caused by LED and halogen light curing units, however, the common concept is that the LED generates less thermal changes (Mahant RH et al. 2016).1 Still, the results of this study answered its objective, and the conclusion is supported by the findings.\n\nI think this study has the potential to be indexed justly to alert the importance of more studies about this topic of great relevance.\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": [] }, { "id": "80646", "date": "22 Mar 2021", "name": "Afnan Al-Zain", "expertise": [ "Reviewer Expertise Dental light-curing units", "resin-based composites", "dental adhesives." ], "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 the effect of heat generated from a quartz-tungsten-halogen and a light-emitting-diode curing unit on the temperature increase and fibroblast cell viability by irradiating 0.5-mm and 1-mm dentin slices. The article topic is important and interesting. A few points need to be justified, and sections of the methods need to be clarified.\nTitle\nAuthors should use scientific terminology and abbreviations. According to the literature, the scientific term for “Halogen bulb” light is “quartz-tungsten-halogen” and the correct abbreviation is “QTH”. Authors need to kindly correct this by using the scientific term and abbreviation throughout the article.\n\nIntroduction\nIn the last sentence of the first paragraph, the authors mentioned that \"… LCUs can increase temperature and induce thermal transfer depending on the light source and intensity and type\"; nevertheless, the LCUs used in this study had approximately a 60% difference in the light irradiance. Therefore, it was not clear why authors used units with such vast differences in irradiance values. This point will be further addressed in the methods.\n\nThe authors used the term “intensity”. However, this term should no longer be used. The updated term is “irradiance”. A Glossary of Terms for Light Curing was published in 2014. Authors may kindly find these terms in several publications, and the following research article is one of them: Jeffrey A Platt, Richard B Price; Light Curing Explored in Halifax. Oper Dent November 1 2014; 39 (6): 561–563. DOI: https://doi.org/10.2341/1559-2863-39.6.5611\n\nMaterials and Methods\nI appreciate the authors' novelty in the methods, but it does not simulate in vivo or clinical situations. The authors placed a dentin slice over the well-plate, which resulted in a distance between the slice and cells. It seems that this design would test the amount of light transmission through the dentin slices rather than simulating clinical situations. Did the authors consider using a larger well-plate and placing the dentin slice inside the well? This may better simulate the clinical situation where the dentin is closer to the cells since the soft tissue surrounds the tooth structure. Therefore, when light curing, the emitted light hits the tooth and the surrounding tissue simultaneously. So, it is more clinically relevant if a larger well-plate is used and the dentin slice is placed inside the well. Furthermore, authors could use different slice thicknesses as they did in this study. Authors may consider this for future research. More details, justification, or the thinking process behind the methodology would be appropriate for the readers to relate to clinical settings.\n\nAuthors need to kindly clarify a few things to the readers based on the methodology; how does dentin's thickness relate to temperature increase? Does 0.5- and 1-mm slice represent curing? i.e., which class does it represent curing? a class II or a class III resin-based composite restoration or another clinical situation? What is the degree of insulating vs. presence of composite layer on top of dentin? The authors mentioned a few of these points. However, it is suggested for authors to mention more details and address these points.\n\nIt is not clear why the authors obtained one section per tooth? Since teeth have variations among them, it would have been more relevant for authors to consider collecting both slices (0.5 and 1 mm) from the same tooth or collect more slices per tooth and account for within-sample variation during the statistical analysis.\n\nIt is not clear why authors obtained vertical sections instead of horizontal if the sections were placed flat on top of the well-plate. Also, the dentin histology would differ from the top to the bottom of the specimens. Did the authors consider that during the study design? How would the authors justify their sections? Authors need to add justifications in the discussion section.\n\nIt is not clear how the specimens were stored and tested. The authors need to add details. In what medium were the dentin slices stored? For how long were they stored? were the dentin slices hydrated or dehydrated before testing? These details may impact the light transmission, and it is important to mention them in the methods.\n\nThe authors had a control group without irradiation. However, it would be relevant to include an additional control group with irradiated cells without the dentin slice's presence.\n\nIt is not clear why the authors selected 20 seconds to light cure the dentin slices. The authors needed to justify this point.\n\nQTH Irradiance values are approximately half that of the LED. Therefore, it would be more relevant to double or triple the curing time when light curing with the QTH or use different units with relatively similar irradiance values. Although the literature is conflicting regarding applying the law of reciprocity on light-curing resin-based composites, the concept is relatively valid, according to some publications. The radiant exposure is the amount of energy the restoration receives over time [radiant exposure (J/cm2) =irradiance x time]; therefore, we would expect the amount of radiant exposure the dentin slices received using the LED is double or more than the QTH unit.\n\nIn the cytotoxicity analysis, the authors needed to consider having an additional control group without the disks, as mentioned previously.\n\nThe authors did not mention the average dimensions of the dentin slices. What was the diameter of the slices relative to the diameter of each well in the 96-well plate?\n\nResults\nFigure 3: the control group is missing. Also, the authors need to consider adding another control group with irradiated cells with no dentin slice over the well. However, it may be challenging at this point but may be considered in future studies.\n\nFigure 4: there is a space without a bar present between the control bar and the LED 1-mm bar. This space is best to be removed. The control here is cells without light irradiation; as mentioned in Figure 3 comment, adding a control group of irradiated cells without dentin slices would be relevant.\n\nFigures 3 and 4: it is best to place the letter \"A\" on the bar with the highest significant bar, followed by \"B\", and then \"C\" on the lowest significant bar. It would be easier for the reader to follow.\n\nIt would have been nice for authors to show cell morphology images for the different groups. The cell morphology images may be considered in future studies.\n\nThe authors would expect significant differences between the QTH and LED groups due to the significant differences in irradiance values between units.\n\nDiscussion\nAuthors should discuss the results of their study first before discussing other research articles. Readers would want to know the justification of their study first.\n\nJustifications for the mentioned comments would best be added in the discussion section.\n\nConclusion\nThe conclusion is accurate to the results and aligned with the aim.\n\nOverall, the research is valuable, and considering the suggested comments would be beneficial for future research.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1369
https://f1000research.com/articles/9-1367/v1	24 Nov 20	{ "type": "Research Article", "title": "Analysis of environmental migrants and their mental health in strengthening health systems", "authors": [ "Keren Herrán", "Dawn Biehler", "Dawn Biehler" ], "abstract": "Background: It is predicted that by 2050 more than 250 million people will have to relocate as a result of climate-related change to their home environment. The existential threat posed by anthropogenic phenomena such as forest fires, floods, sea level rise, drought, and intensified weather events (e.g. tropical storms) has caused a new type of migrant to emerge. Within academic literature, these migrants are referred to as climate migrants, environmental migrants, or eco migrants, among other terms. These individuals’ migration journey and this journey’s impact on their mental health is currently an understudied research area. This paper summarizes the mental health challenges climate migrants face via a narrative review. Methods: Google Scholar was used as the main search database throughout May, 2020 until authors determined data saturation had been reached. Grey literature was also included. Sources were included if they focused directly on evaluating environmental migrants and their mental health experiences. Academic sources must have been peer-reviewed and published within the past 10 years. Information was coded and evaluated according to the three migration journey stages of before, after, and during relocation. Results: Main findings include that the slow or sudden degradation of one’s surroundings can cause the onset of mental health disorders that are later exacerbated by challenges faced when migrating, such as lack of access to health services. Mental health challenges faced upon reaching destination communities consist mainly of social marginalization and disruption of social ties. Lastly, action items for health systems are outlined and the need for more research on the mental wellbeing of climate migrants throughout their migration journey is stressed. Conclusions: This review is an urgent call to policymakers, health professionals, and researchers to strengthen health systems by making them more climate resilient and inclusive towards environmental migrants.", "keywords": [ "Mental health", "migration", "climate migrant", "environmental migrant", "eco migrant" ], "content": "Introduction\n\nRelevant human geography statistics. Scientists studying the nexus of climate change and human population movement predict that by 2050, more than 250 million people will have migrated from their homelands due to climate-related change (Heaney & Winter, 2016). Within Sub-Saharan Africa alone, it is estimated that there will be 86 million internal climate migrants by 2050 (Negev et al., 2019).\n\nOur era’s new migrants: Who are they? Escalation in anthropogenic greenhouse gases is increasing both the frequency and the intensity of extreme weather conditions and natural disasters (Schwerdtle et al., 2018). Those who are displaced by these changes or by their implications are often identified as climate migrants, environmental migrants, or eco migrants (International Organization for Migration, n.d.). Since climate change is not viewed as a force that acts alone in driving population displacement, these labels and their definitions are highly contested (Schwerdtle et al., 2018). It is also worth noting that these migrants are not recognized as refugees by international regulations, and thus labeling them as such is a misnomer (International Organization for Migration, n.d.).\n\nThe silent understudied health component of climate migration. Scholars have noticed a positive growth in governmental attention directed toward addressing the physical health consequences of climate change and climate migration, such as injuries and infectious disease (Torres & Casey, 2017). However, researchers also increasingly stress that there is a dire need for discussion and study of the mental health consequences endured by eco migrants (Acharibasam & Anuga, 2018; McMichael et al., 2012; Schwerdtle et al., 2018; Schwerdtle et al., 2019; Torres & Casey, 2017). For instance, child and adolescent health specialists have been adamant in their concern and demand for studies on the state of mental health among environmental migrants (Myles et al., 2018). Children have been particularly vulnerable to post-disaster trauma given that their cognitive development does not prepare them for the witnessing of mass quantities of dead bodies (Hasan et al., 2020). Unfortunately, these mental scars are usually only exacerbated by the psychological stressors of climate migration (Schwerdtle et al., 2018). Despite pleas from professionals to invest in mental health evaluations and resources for environmental migrant populations, leaders and officials are often preoccupied by fighting for the provision of basic needs to these vulnerable communities and neglect addressing psychological concerns (Hasan et al., 2020; Nahar et al., 2014). This is especially unfortunate since there is evidence that environmental migrants themselves view their mental health as a valuable part of their overall health. In Tanzania, when asked what it meant to be healthy, “[climate] migrants placed more importance on mental health and non-migrants on physical health” (Heaney & Winter, 2016, p.645).\n\nDespite the advancing onset of climate change (and thus climate-driven migration), research on the health experiences of climate migrants is limited in regards to mental health knowledge. Therefore, this paper endeavors to answer the following question: What does the synthesis of available environmental migrant mental health studies reveal regarding environmental migrants’ mental health and how health systems can be strengthened to best serve their psychological well-being?\n\n\nMethods\n\nThe first author searched for the following phrases within Google Scholar: mental health of environmental migrants, environmental migrant mental health, climate migrant mental health, and eco migrant mental health, in order to find pertinent literature. For each phrase, search result pages one through six were reviewed to identify papers that met the following inclusion criteria: 1) The paper centered on environmental migrants as the population of focus for the study; 2) The manuscript directly discussed mental health experiences faced by climate migrants; 3) Published information was peer-reviewed and thus reliable; 4) Article had been released within the last 10 years and was thereby relatively recent.\n\nRegarding reasons for exclusion, literature that focused on migrants in a general light and did not specifically analyze the experiences of eco migrants were not chosen for this narrative review. Papers that concentrated on physical health and mentioned mental health impacts briefly and only in passing were also omitted. In addition to direct searching, the technique of snowball searching was also utilized to identify scholarly information that relate to this analysis’ research question and met inclusion criteria. Searching occurred during May of 2020 and stopped once data saturation was reached on May 23, 2020. Ultimately, 15 scientific studies were identified for review. Full text reading of manuscripts involved coding and categorizing information according to their relationship to the three distinct stages of migration: before, during, and after relocation. The authors also chose to supplement the narrative review with grey literature from the World Health Organization, news sources such as The Guardian and Gizmodo, and documentary films on climate migrants in Pacific Island countries. Inclusion criteria for grey literature were the same as the aforementioned criteria minus the requirement of peer-review validation. Grey literature was deemed reliable if affiliated with a larger recognized organization that collects primary data. All materials have been included in the reference list of this paper.\n\n\nPre-existing mental health challenges among eco migrants\n\nThis section will examine what mental health disorders environmental migrants develop in the course of their experiences with climate change. It is important to comprehend their prior state of mental health since the stress of migrating to and entering into a new community can exacerbate or resurface previous psychological struggles. Anthropologist Dr. Lawrence Palinkas (2019) has stated that eco migrants are often, “able to bring little with them apart from the mental health problems created by the environmental changes [that caused them to move]” (p.2). According to the World Health Organization (WHO), up to half of all earthquake and tsunami survivors in South East Asia coastal lands suffer from post-disaster mental health issues that range from moderate to severe levels of distress (WHO, 2016). A population-based survey specific to south India reported that five years after the 2004 tsunami, around 78% of survivors continued to express symptoms of mental health morbidity (Hasan et al., 2020).\n\nThe main acute mental health disorders that result from witnessing sudden climate change disasters include post-traumatic stress disorder (PTSD), insomnia, depression, hopelessness and suicidal ideation, generalized anxiety disorder (GAD), and mood disorders (Acharibasam & Anuga, 2018; Hasan et al., 2020; Palinkas, 2019). Past studies have concluded that even the anticipation of forced migration due to environmental changes significantly increases a community's level of depression (Torres & Cassey, 2017). In communities where a climate change disaster has progressed gradually, such as extreme drought in the Central American Dry Corridor, mental health consequences such as stress and substance abuse are chronic (Acharibasam & Anuga, 2018; Lakhani, 2019). The accumulation of years of unaddressed mental health imbalances in an individual can be distinctly detrimental since it interrupts their ability to accumulate human capital assets such as education (Palinkas, 2019).\n\nNew specific mental health diagnoses have also emerged as a result of anthropogenic environmental transformations. These new clinical illness categories include terms such as eco-anxiety and solastalgia (Palinkas, 2019; Tschakert et al., 2013). Whereas general anxiety is an unshakeable sense of concern and unease, eco-anxiety includes these sentiments in addition to the dread of existential threat due to climate change (Nugent, 2019). Solastalgia is the distress and isolation felt by no longer being able to find solace in the present condition of one’s home environment (Palinkas, 2019).\n\nMany of those who are vulnerable to climate migration reside in countries where mental health care access is limited; thus, existing mental health issues often go unaddressed before migration (DW Documentary, 2017; Hanna & Oliva, 2016; Hasan et al., 2020; Lakhani, 2019; McIver et al., 2016; Nahar et al., 2014; Torres & Casey, 2017; Tschakert et al., 2013; Vorobyov, 2020). For instance, while Bangladesh ranks first worldwide in natural disaster mortality, the country only has one mental health hospital for its entire population (Nahar et al., 2014). This is a disservice to the mental health needs of natural disaster survivors in need of counsel and support after suffering from the trauma and loss of their loved ones or homes. With few resources for managing their illness appropriately, some individuals may turn to physical and sexual abuse of romantic partners or children as a coping mechanism (Hanna & Oliva, 2016; Nahar et al., 2014). Such actions only further deteriorate the mental health of fellow victims of climate change.\n\nWhat happens when those affected by climate change decide to migrate without support for recuperating their mental and emotional wellbeing? Will these populations receive support for their mental health along their journey, or have these needs overlooked, as in their home countries? These questions will be explored further in the subsequent section.\n\n\nMental health risks along the climate migration journey\n\nBased on investigations in Sub-Saharan Africa, Israeli public health investigators inferred that migration shapes health by disrupting access to health services and social networks of support (Negev et al., 2019). The researchers suggested that mobile phones and telemedicine technology ought to be optimized in order to provide care along migration routes. They also noted that health clinics should be established at key areas of repose along migration paths. Lack of access to physical health supports can be connected with poor mental health outcomes. If health services are not available for climate migrants along their route of travel, then treatment for other noncommunicable diseases, such as diabetes, is interrupted (Schwerdtle et al., 2019). Not having access to medication if one becomes ill or injured and not being able to receive needed medical treatment for noncommunicable diseases along migration routes can deteriorate a travelers’ physical health. Consequently, worsened physical health can complicate one’s ability to migrate, potentially causing a state of despair and decline in a migrant’s mental health (Schwerdtle et al., 2018). Furthermore, since migrants may transit through regions with cultures and languages different than their own, this unfamiliarity with their surroundings and lack of protection places migrants at a, “greater risk of sexual exploitation, human trafficking, and sexual and gender-based violence” (Piguet et al., 2011, p.34; Palinkas, 2019). For migrants traveling with already delicate mental health states, undergoing such violations of their human rights only worsens their psychological and emotional wellbeing.\n\nSome may reason that migration is not actually detrimental to climate migrants’ mental health because their mental health is not unstable in the first place. This argument is based on the theory of immigrant self-selection. Economists explain immigrant self-selection as the conclusion that the, “healthiest and wealthiest individuals are the people most likely to migrate” (Kennedy et al., 2006, p.3). Under this mindset, the journey eco migrants undertake is not seen as an exacerbation of their prior mental health concerns because their mental health must by default have been steady beforehand for them to have been strong enough to assume the risks of migrating.\n\nIn contrast, some mental health researchers argue that, in certain circumstances, individuals do not fund their own displacement. Hence, the assumption that they must have robust health and come from a strong economic standing due to a privileged level in society that allows them to migrate, is false (Scaramutti et al., 2019). For example, after Hurricane Maria tore through Puerto Rico in 2017, the Federal Emergency Management Agency (FEMA) funded thousands of Puerto Ricans’ displacement to Florida. Hence, those that left Puerto Rico were probably, “those who suffered the greatest personal and property losses during and after the storm” (Scaramutti et al., 2019, p.3). As the degree of duress on migrants increases, there may be a tendency for more unhealthy individuals to migrate. Diverse push and pull factors combined with different kinds of health conditions may lead to various levels of migration inhibition. In communities where climate change causes severe drought and famine, or results in an increase in armed conflict over limited resources, environmental migrants are essentially cornered. Just as staying in their hometowns would be a death sentence, deciding to leave and undertake the difficulties of traveling without guaranteed food, water, shelter, and protection from crime can also be mortal (Lakhani, 2019).\n\n\nMental health challenges faced upon reaching destination community\n\nDue to differences in cultural background, language, and legal status, receiving communities may treat eco migrants as inferior members of society (Heaney & Winter, 2016; McMichael et al., 2012; Torres & Casey, 2017; Vorobyov, 2020). This social stigma and discrimination can cause eco migrants to suffer from sentiments of isolation and depression. As reasoned by a citizen from the Republic of Kiribati (a country of islands that is disappearing due to sea level rise), “moving away to somewhere that does not belong to you, you will always become a second class person - in your heart you know you don’t belong there” (DW Documentary, 2017, 27:45). The documentary, There Once Was an Island, portrays similar strains for people being displaced from the Takuu islands to Bougainville. Both Takuu and Bougainville islands are currently part of Papua New Guinea but have acutely distinct local communities and cultures (March & Collie, 2010).\n\nRecently in Brazil, farmers from the Northern region have had to relocate to cities in the South due to crop failure from excess heat and drought (Piguet et al., 2011). Given these migrants have limited funds as a result of unsuccessful harvest seasons, they often move to cheap housing in the slums, or “favelas,” that are already overcrowded. In Brazil and other developing countries, rural to urban migration strains already overstretched resources of major cities (DW Documentary, 2017; McMichael et al., 2012). Brazil’s current president, President Bolsonaro, has derided what he sees as a disorderly migration influx into favelas, limiting government investment and support. His harsh rhetoric towards favela dwellers, characterizing them as criminals, fails to set an example for how others should treat and welcome climate migrants with warmth in support of their mental health. Many have labeled these former agricultors as uneducated delinquents. These prejudices escalate migrants’ likelihood of developing mental health illnesses (Vorobyov, 2020).\n\nHost communities’ hostility towards outsiders not only degrades the mental health of environmental migrants, but it can also prevent them from accessing mental health resources. Given that eco migrants already face extensive prejudice and discrimination, fear of further social stigmatization may deter them from seeking treatment for their mental health conditions (Heaney & Winter, 2016). Furthermore, approaching public hospitals may be worrisome for eco migrants who lack legal status in the countries where they have relocated. Public health analysts have phrased this dilemma as a disadvantage that is, “exacerbated every time they must decide whether to interact with a system that may deport them” (Negev et al., 2019, p.1).\n\nFurthermore, an environmental migrant’s relocation to another community can adversely impact their mental health due to migration’s tendency to disrupt social ties (Heaney & Winter, 2016; McMichael et al., 2012; Schwerdtle et al., 2018; Torres & Casey, 2017). Friends and family members that migrants leave behind constitute an important support network that migrants were previously able to count on for emotional support, guidance, and resources (Torres & Casey, 2017). Stripped of these systems of resilience and surrounded by a new group of people that may not be supportive of their presence, eco migrants can find it difficult to maintain optimal mental health. Although technology bridges this gap to a certain extent, studies have shown that when individuals are separated, the mental health of those who stay in situ is as equally aggravated as the mental health of those who migrate (Schwerdtle et al., 2018). Therefore, since the mental health of those who are left may also suffer after community members migrate, their ability to provide substantial encouragement remotely via technology is questionable. In fact, if the well-being of those in situ amid environmental disaster is poor, this may exacerbate the mental health of those who did migrate. Furthermore, climate migrants’ responsibility to send remittance obligations to financial dependents back home can drain their mental health since it is yet another burden they must bear in their stressful transition and adaptation to a new location (Heaney & Winter, 2016; Torres & Casey, 2017).\n\nWhen climate change directly or indirectly forces one to leave their home in search of a better quality of life, it does not always lead one to a destination with fewer mental health threats. In fact, the end of a climate migrant’s journey may be a city with more job opportunities at the expense of exposure to even more environmental threats than before (McMichael et al., 2012; Piguet et al., 2011). For example, environmental migrants arriving to favelas in Brazil often lack electricity, water, hygienic living spaces, and basic services as a result of their rural to urban migration (Vorobyov, 2020). Apart from the fact that climate migrants’ mental health may be further aggravated by their new community, not living on their land of origin and changing livelihoods can also deteriorate their sense of identity (Acharibasam & Anuga, 2018; March & Collie, 2010; McMichael et al., 2012; Tschakert et al., 2013). Geographers and anthropologists have noted that, “there is an intimate relationship between people and their everyday landscapes,” and that hence, losing one’s sense of place can cause collateral psychological damage (Tschakert et al., 2013, p.14).\n\n\nConclusion\n\nIt would be an injustice to collect, organize, and scrutinize as much information as possible about the mental health consequences of environmental migration without shedding light on what can be done to mitigate the phenomenon. From a medical sociology standpoint it is indeed, “a matter of social justice to invest in the mental health of these populations in motion” (Torres & Casey, 2017, p.8). Climate change repeatedly victimizes the poor since their economic levels limit their housing options to environmentally risky areas that lack protective infrastructure (Hanna & Oliva, 2016; Lakhani, 2019; McIver et al., 2016; Nahar et al., 2014; Scaramutti et al., 2019; Torres & Casey, 2017; Tschakert et al., 2013). Environmental transformations and destructions ironically impact developing countries the most, contributing to a cycle of disempowerment and vulnerability despite the fact that developing nations are the smallest contributors to climate change (McIver et al., 2016; Piguet et al., 2011; Vorobyov, 2020). It is also important to recognize that females’ mental health is all the more endangered by eco migration because they are exposed to additional traumatic events such as lack of autonomy upon widowhood, gender based violence, and sexual exploitation (Hasan et al., 2020; McMichael et al., 2012; Nahar et al., 2014; Palinkas, 2019; Piguet et al., 2011, p.34). Taking into consideration the synthesis of information presented thus far and the unique vulnerabilities that add to its complexity, the remaining end of this paper will proceed to outline action items health systems ought to take.\n\nTo clarify, addressing the mental health of climate migrants will demand support and collaboration at multiple levels worldwide. Given the multidimensional nature of the issue, a truly sustainable and effective solution will likewise require interdisciplinary investment (Nahar et al., 2014; McMichael et al., 2012; Torres & Casey, 2017). It will necessitate expertise from international development workers, gender and women's studies experts, policymakers, nations’ leading economists, public health strategists, and technology innovators, among other professionals. Thus assigning recommendations specific to health systems should be seen as suggestions only worthwhile if part of a larger global plan of intervention. Such a plan will undoubtedly also entail further research on the crisis.\n\nFirst and foremost, across publications, there is widespread agreement that health systems should endeavor to fight structural barriers that impede climate migrant’s access to health services (Hasan et al., 2020; Negev et al., 2019; Schwerdtle et al., 2019). For instance, as previously stated, concerns over uncovering personal legal status may dissuade climate migrants from seeking treatment for their mental health (both along their journey and once at their destination). Therefore, health systems along migration routes and within countries that receive migrants should brainstorm how they can intake patients without asking for information that patients do not wish to disclose due to fear of deportation (Negev et al., 2019). Furthermore, since this subdivision of migrants is often economically disadvantaged, healthy systems need to implement pro bono/free/non-compensated mental health counseling services options that remove any financial barriers. An exemplary model of successful allocation of funds toward complementary mental health services would be the healthcare facilities in Arusha, Tanzania that, “provide free psychiatric and psychological counseling to underprivileged populations,” given it is an area where many environmental migrants resettle (Negev et al., 2019, p. 312). Social policy experts and the WHO also increasingly stress that healthcare professionals train community health workers and non-professionals in psychological first aid interventions to improve their range of outreach (Nahar et al., 2014; Palinkas, 2019). This may resolve access barrier issues that relate to lacking transportation to health services.\n\nHowever, regardless of the preceding fundamental action items, eco migrants are unaware of even the current mental health services they have available (Heaney & Winter, 2016). Health systems must recognize that development and expansion of programming is futile if these efforts are not effectively promoted so that eco migrants know what is available to them. Hence, healthcare strategists should also reflect on information dissemination methods and how to reduce stigma surrounding the notion of seeking assistance for mental health.\n\nIn conclusion, although natural disasters and drastic changes in weather can be unpredictable, climate change “loads the dice” in favor of increasing incidence of natural disasters (Hansen et al., 2012). By reflecting on current mental health information related to climate migrants, efforts to assist these migrants in their healing and restoration of mental wellbeing can and should be achieved while conducting further research in this area.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nThe author would like to thank her mentor Dr. Dawn Biehler for encouraging her to research this topic and for contributing to the author’s academic and professional development.\n\n\nReferences\n\nAcharibasam JW, Anuga SW: Psychological distance of climate change and mental health risks assessment of smallholder farmers in Northern Ghana: Is habituation a threat to climate change? Clim Risk Manag. 2018; 21: 16–25. Publisher Full Text\n\nDW Documentary: Kiribati: a drowning paradise in the South Pacific. [Video file]. 2017. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTschakert P, Tutu R, Alcaro A: Embodied experiences of environmental and climatic changes in landscapes of everyday life in Ghana. Emot Space Soc. 2013; 7: 13–25. Publisher Full Text\n\nVorobyov N: Brazil is Cracking Down on Climate Migrants While Worsening the Climate Crisis. 2020. Reference Source\n\nWorld Health Organization: Disasters and Mental Health. 2016. Reference Source" }	[ { "id": "75979", "date": "01 Feb 2021", "name": "Pablo Daniel Estrella Porter", "expertise": [ "Reviewer Expertise Public Health", "Medical Education", "General Medicine", "and Global Health." ], "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 global health migration crisis has become one of the issues that are threatening human society worldwide. This research article has raised an important approach to the migration crisis, centering on the mental health area. The analysis presents an overview of the mental health situation on the three stages of migration: before, during, and after the relocation. The different disorders of the mental health illnesses were identified, as well as the baseline situation of many migrants, and the challenges faced upon arrival. The authors acknowledged the need to involve different stakeholders and decision-making actors, to generate positive changes on environmental migration.\nTo make this research more impactful, the methodology could have been strengthened. For example, using a qualitative-systematic review, where research evidence could be collected, analyzed, and presented in a framework where is easier to replicate and summarize. The manuscript could have benefited from presenting the total scientific studies found before using the inclusion and exclusion criteria or using a simplified version of a PRISMA diagram to present the results. Furthermore, including a table with a summary of the main findings of each paper that went to full-text reading, would have increased the relevance of the results and conclusions of this analysis.\n\nAlthough the document presents a highly relevant topic, the structure of the article could be improved to give a better understanding. In such a way that the information collected from the selected scientific studies is presented more sequentially and the subsequent analysis is done afterward.\n\nNonetheless, the results obtained from this research are important to show the need for greater intervention and action in mental health in this emerging group of environmental migrants that is growing every day.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "77714", "date": "23 Feb 2021", "name": "Mithila Faruque", "expertise": [ "Reviewer Expertise My field of research interest is noncommunicable diseases like epidemiology and risk factors of hypertension", "cardiovascular disease", "diabetes", "chronic respiratory diseases", "cancer and their prevention management. Also strengthening primary health care system for the prevention and control of NCDs." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author tried to highlight a very important issue regarding the eco-migrants mental health which should have been emphasized a long before the era. Definitely the health system of the country where this type of environmental or climate change impacts are prominent should have a specific intervention strategy at the policy level to address the mental health needs of the migrants.\n\nBut regrding methodology, I would like to mention that the author should include a flow chart with the reviewed literature serching pattern and numbers with and without inclusion criteria (before and after), so that the readers have a clear idea about the literature searching process. At present, the method is clumsy and review process is not understandable.\nAlso need a clear visualization of the findings analysed from the reviewed literature. So the author is requested to add a table with highlighting the summary of findings obtained from the literature with reference list.\n\nConclusion is too big, I suggest to concise the conclusion with including a part of recommendations from the obtained findings form the review.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1367
https://f1000research.com/articles/9-1109/v1	09 Sep 20	{ "type": "Opinion Article", "title": "Adaptive platform trials using multi-arm, multi-stage protocols: getting fast answers in pandemic settings", "authors": [ "Nurulamin M. Noor", "Sarah L. Pett", "Hanif Esmail", "Angela M. Crook", "Claire L. Vale", "Matthew R. Sydes", "Mahesh K.B. Parmar", "Nurulamin M. Noor", "Sarah L. Pett", "Hanif Esmail", "Angela M. Crook", "Claire L. Vale", "Matthew R. Sydes" ], "abstract": "Global health pandemics, such as coronavirus disease 2019 (COVID-19), require efficient and well-conducted trials to determine effective interventions, such as treatments and vaccinations. Early work focused on rapid sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), subsequent in-vitro and in-silico work, along with greater understanding of the different clinical phases of the infection, have helped identify a catalogue of potential therapeutic agents requiring assessment. In a pandemic, there is a need to quickly identify efficacious treatments, and reject those that are non-beneficial or even harmful, using randomised clinical trials. Whilst each potential treatment could be investigated across multiple, separate, competing two-arm trials, this is a very inefficient process. Despite the very large numbers of interventional trials for COVID-19, the vast majority have not used efficient trial designs. Well conducted, adaptive platform trials utilising a multi-arm multi-stage (MAMS) approach provide a solution to overcome limitations of traditional designs. The multi-arm element allows multiple different treatments to be investigated simultaneously against a shared, standard-of-care control arm. The multi-stage element uses interim analyses to assess accumulating data from the trial and ensure that only treatments showing promise continue to recruitment during the next stage of the trial. The ability to test many treatments at once and drop insufficiently active interventions significantly speeds up the rate at which answers can be achieved. This article provides an overview of the benefits of MAMS designs and successes of trials, which have used this approach to COVID-19. We also discuss international collaboration between trial teams, including prospective agreement to synthesise trial results, and identify the most effective interventions. We believe that international collaboration will help provide faster answers for patients, clinicians, and health care systems around the world, including for future waves of COVID-19, and enable preparedness for future global health pandemics.", "keywords": [ "multi-arm", "multi-stage", "MAMS", "adaptive", "platform", "trials", "efficient", "conduct", "epidemic", "pandemic", "SARS-CoV-2", "COVID-19", "FAME", "IPD", "meta-analysis" ], "content": "Introduction\n\nGlobal health pandemics pose many challenges, which require rapid clinical answers. There have been a growing number of global health emergencies caused by infectious diseases, including the novel coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1, and recent outbreaks of Ebola and Zika2. These have all been associated with significant morbidity and mortality, and demanded urgent evaluation for treatments and development of vaccines. With increasing globalisation, similar challenges are likely to be encountered with further waves of these diseases, and further novel infectious pathogens entering the human population. There is therefore a need for more efficient, well-conducted, and collaborative clinical trials to provide answers for such pandemic settings.\n\nFrom the outset of the coronavirus disease 2019 (COVID-19) pandemic, there has been an astounding pace of discovery to characterise this condition. Within months after the first widely-reported cases, the genomic structure of SARS-CoV-2 causing COVID-19 was identified and work initiated to explore downstream biology3. Accordingly, there are multiple potential interventions with plausible biological mechanisms that have been proposed and are under investigation for treatment or prevention of COVID-194,5.\n\n\nUrgent need for clinical trial efficiency in a pandemic setting\n\nSARS-CoV-2 exemplifies the typical challenges of highly infectious pathogens entering the human population for the first time, given that there were initially no approved treatments nor any available vaccines. There was a need for answers even more quickly from clinical trials. Both a need to identify effective treatments for those most vulnerable to COVID-19 and, equally important, to identify ineffective interventions early, in order to direct resources to the most promising interventions.\n\nResearchers are generally aware of the potential biases from non-randomised trials and small, underpowered randomised controlled trials (RCTs)6, as well as the difficulties of interpreting non-controlled cohort studies7. However in a pandemic setting, particularly for a condition with high risks of morbidity and mortality, there is typically demand for early access to treatments with potential benefit - even in the absence of any compelling RCT data8.\n\nA review by the National Academy of Science, Engineering and Medicine from the United States of America (USA), on the international response to the 2014 Ebola outbreak in West Africa, highlighted many apparent errors and lessons to be learned9. A key criticism was that small, underpowered clinical trials did not provide answers to help direct clinical care, nor did “compassionate use” trial programmes, since even if treatments were provided to large numbers of individuals, little sense of relative efficacy could be made in the absence of a comparative control arm10. Indeed, there was a widespread recognition of the need for randomisation to determine if interventions are effective or not in global health pandemics.\n\n\nEfficiencies of MAMS platform designs to investigate multiple interventions\n\nEach potential intervention for COVID-19 could be investigated across separate traditional two-arm, RCTs. This is, however, an inefficient process, particularly if, as expected, a large number of these interventions turn out to be clinically ineffective.\n\nUnfortunately, the lessons from previous epidemics have not been widely applied to COVID-19. The use of efficient trial approaches have been the exception rather than the rule, with over 2,500 separate clinical trials registered for COVID-19 by 25 August 2020 on the World Health Organisation (WHO) International Clinical Trials Registry Platform (ICTRP)11. A large number of these registrations are for small, underpowered trials using traditional non-adaptive designs, and this inefficiency has been compounded by many overlapping and competing trials seeking to answer the same clinical question12.\n\nMoreover, the inefficiencies of using multiple, classical, two-arm RCTs have been felt even more keenly during the COVID-19 pandemic, given the ability of such an infectious pathogen to rapidly overwhelm health service capacity. First, each two-arm trial has a separate control group, which is hugely inefficient given the large overall numbers of patients required; second, the choice of interventions to be tested is often based on very limited information, which will inevitably lead to a large number of ‘negative’ trials. Finally, as information emerges, further interventions demand evaluation immediately and setting up a separate, new trial is again hugely inefficient and time consuming.\n\nThere has been a growing recognition of the need to adopt efficient RCT approaches in pandemic settings13. Indeed, irrespective of the medical condition, whenever there are multiple interventions meriting further assessment, there has increasingly been a cultural shift away from two-arm RCTs, towards multi-arm designs14. A multi-arm, multi-stage (MAMS) platform design, provides a solution to many of the problems described and could be considered for evaluation of treatments or preventative strategies.\n\nThe multi-arm element allows multiple different interventions (comparisons) to be investigated simultaneously against a shared, standard-of-care control arm15. The shared control arm approach means that fewer patients are required overall compared with numerous, separate trials and allows for greater assignment of participants to receive comparison interventions.\n\nThe multi-stage element of a MAMS design uses interim analyses to assess accumulating data from the trial and ensure that only treatments showing promise continue to recruit new participants during the next stage of the trial. Whilst therapies showing a lack of sufficient benefit or, indeed, signals of real harm, have recruitment halted.\n\nThe ability to test many interventions at once and move recruitment away from insufficiently effective interventions, significantly speeds up the rate at which answers can be achieved16. The design also ensures resource and funding are allocated to the most promising arms. Importantly, the MAMS platform allows new interventions to be rapidly added for assessment at any time, via an approved protocol amendment rather than by launching a new or competing trial. The selection of new intervention arms is based on a range of information, including promising data from laboratory, animal or human studies, as well as consideration of factors such as cost and wider availability of the intervention16.\n\n\nAdvantages of MAMS platform design in non-pandemic settings\n\nThe MAMS platform has been successfully implemented in non-pandemic settings. Whilst adaptive, platform approaches have increasingly been used across early-phase trials for drug screening programmes in industry17, uptake in the late-phase setting has been more gradual18.\n\nThe MAMS approach was perhaps first designed and applied in the late-phase setting to ovarian cancer in the ICON5 trial (NCT00011986)19,20, where many design features were implemented for the first time, and numerous lessons learned were incorporated into the STAMPEDE trial (NCT00268476)21. STAMPEDE has utilised a MAMS platform approach to investigate potential treatments for prostate cancer. This trial has been running since 2005 and will address at least ten randomised treatment comparisons over 20 years (by 2025). In the past 15 years, results from STAMPEDE have contributed to three changes in standard-of-care for patients with prostate cancer22. Separate parallel-group, two-arm trials would have taken many decades to produce such results.\n\nThe MAMs platform approach changes the standard research question from a two-arm RCT of ‘does this intervention improve outcomes?’, to the more informative question ‘do any of these interventions, and any further new interventions identified, which need to be tested, improve outcomes?’. Given the need for speed and the large proportion of RCTs that do not show a new intervention is better than the control, we believe that this is a more relevant research question to ask – and potentially offers great utility for infectious diseases in pandemic settings.\n\n\nUse of a MAMS platform approach for infectious disease pandemics\n\nIn recent years, it has been shown that the MAMS approach can also be applied in the fast-moving context of global health emergencies, and equally within resource-limited healthcare settings.\n\nIn the 2016 Ebola outbreak in West Africa, four potential treatments for Ebola were simultaneously investigated in the PALM trial (NCT03719586)23, with two treatments being “dropped” following interim analysis and the two more effective treatments proceeding to a further next stage of recruitment23. The PALM consortium demonstrated the ability of such modern RCTs to be rigorously conducted during infectious disease outbreaks and importantly to deliver clinical answers in a timely fashion.\n\nMany treatments and trials have been proposed and initiated for COVID-19, and the extensive, ongoing screening projects of new and re-purposed agents will likely lead to many more potential interventions that require evaluation in the near future. Commendable private-public collaborations, such as the Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV), programme have been setup to develop and test interventions at much greater speed than typical development programmes24. Using a MAMS platform design offers a very efficient way to assess these interventions in RCTs, in order to make progress as rapidly as possible25.\n\nOnly a small number of trials have utilised a MAMS platform approach for COVID-19. However, the benefits of a co-ordinated and collaborative approach using an efficient platform design, are well illustrated by the RECOVERY trial (NCT04381936). RECOVERY, being co-ordinated from the United Kingdom (UK), initially started as a four-arm trial, with multiple further comparisons added to date. Recruitment was able to start within nine days of the protocol being finalised26, and remarkably three research questions were answered - with over 12,000 participants recruited - within a period of just over 100 days. Most importantly this trial has provided convincing results to influence changes in clinical practice27,28.\n\nThere are a number of further notable trials using a MAMS platform approach for patients with COVID-19. The SOLIDARITY master protocol (ISRCTN83971151) advocated and co-ordinated by the WHO and adopted across many individual countries. The ACTT-3 trial (NCT04492475) -- an international trial co-ordinated from the National Institute of Allergy and Infectious Diseases in the USA, initially starting as a three-arm trial. This builds on successes of the adaptive two-arm ACTT-1 trial (NCT04280705), which demonstrated improved time to recovery with remdesivir, an antiviral medication, when compared with placebo29. The TACTIC-R (NCT04390464) and TACTIC-E (NCT04393246) trials are investigating repurposed and experimental immunomodulatory medications, respectively, for COVID-19 across the UK. In addition, the PRINCIPLE trial (ISRCTN86534580) has demonstrated that the MAMS platform can also be adopted for assessment of potential interventions in a primary care setting during a global health pandemic.\n\nWhilst this article has focused on the MAMS platform approach, additional and complementary multi-arm approaches can be used, including multi-arm, multi-factorial designs - such as the REMAP-CAP trial (NCT02735707). Notably, factorial approaches can also be combined with MAMS designs. A multi-arm, multi-factorial design may offer particular benefits when assessing multiple combinations of interventions, as is typically the case for patients being treated in an intensive care environment.\n\n\nChallenges of a MAMS platform approach for pandemic trials\n\nAn often highlighted challenge, especially for longer-lasting platform protocols, is the potential for standard-of-care to change during the course of the trial and implications of this for a MAMS design30. Indeed, in the context of a fast-moving infectious disease pandemic, it is likely that usual or standard care will change based on interim analysis of comparison arms (and reporting) from the current trial, or from emerging data and findings of other research studies. However, there are now well-reported methods to overcome the challenges from changing standards of care in a MAMS design31. These solutions principally ensure comparison of interventions to participants recruited to a contemporary standard-of-care treatment arm, rather than comparison with participants receiving historical standard-of-care treatment.\n\nSetting up and undertaking multi-national trials of any design in non-pandemic settings can offer considerable challenges32, and the difficulties may be an order of magnitude greater in a pandemic - given the speed needed to setup these trials. Challenges to consider include ensuring adequate funding, staffing, alignment of a single trial protocol to enable appropriate regulation, monitoring and oversight in each country, and logistical considerations, such as implementation of protocol amendments simultaneously across participating countries and sites. An additional challenge, which can be very difficult to predict, will be which countries are most likely to be affected by a pandemic and at what time they are most likely to be affected. The relative national incidence of a disease, and fluctuations in these numbers, will have a critical bearing on the ability to recruit into any pandemic trial. Moreover, different nations will have variable infrastructures in place to allow rapid setup of sites and ability to deliver RCTs efficiently, including crucial aspects such as rapid distribution of medications.\n\nIt is sobering to reflect that a likely major contributor to the speed of setup for RECOVERY was that this trial was taking place in one country and within one healthcare system - without the need to overcome multiple regulatory and administrative hurdles across multiple countries. The WHO SOLIDARITY trial approach likely offers the best practical solution around some of these challenges for multi-national trials – using a single overarching master protocol, with individual and separate trial registrations and delivery in each respective country.\n\n\nFuture horizons\n\nAn important future consideration is the need to collate and synthesise information from RCTs in order to derive maximum benefit for patients worldwide. Accordingly, there is an urgent need for collaboration between trial teams, to ensure the most accurate and rapid reporting of findings.\n\nThis collaboration could be achieved using the prospective framework for adaptive meta-analysis (FAME), which has been successfully used to pool data across ongoing trials of prostate cancer33, and helped facilitate the identification of patient subgroups for whom individual treatments may be most effective34. In the COVID 19 setting, meta-analysis using individual participant data (IPD) may be key to delineating which treatment strategies are most effective in individual patients35. Accordingly, extending the FAME approach, to include prospective agreement to share IPD from relevant trials, could reduce any potential delay to IPD meta-analysis. Irrespective of which method is used, these collaborative approaches enable rapid synthesis and reporting of all the evidence, providing a clear message to patients, clinicians and the wider public.\n\nIt is also clear that there is a need to be prepared for both further waves of COVID-19 and future potential global health pandemics. A key aspect of preparedness will be for trial organisations to pre-prepare MAMS protocols ready to implement at speed, ideally using a seamless phase two/three approach. Given the multiple potential vaccines under development for COVID-1936, appropriate consideration should be given to the application of MAMS designs to preventative vaccine trials in the near future. Similarly to treatment trials, the WHO is commendably supporting global efforts for evaluation of multiple vaccines in a SOLIDARITY vaccine trial protocol37.\n\n\nConclusion\n\nThere is an urgent need for reliable evidence in pandemic settings, as illustrated by the COVID-19 pandemic. Well conducted, adaptive platform trials utilising a MAMS approach, offer substantial advantages over multiple, separate, two-arm trials. Indeed, trials using efficient approaches have provided some of the rapid answers needed in the COVID-19 pandemic. However, these efficient trial approaches have been the exception rather than the rule. In this respect, an important consideration for the future will be for funders, regulators and other key stakeholders to prioritise more efficient trial designs. Focusing efforts on MAMS protocols in particular, with international collaboration between co-ordinating trial teams, and prospectively planned meta-analyses of emerging data, should lead to faster identification of effective therapies and vaccines. This should also contribute to faster answers for patients, clinicians, and health care systems around the world, including for future waves of COVID-19 and enable preparedness for future global health pandemics.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nPetersen E, Koopmans M, Go U, et al.: Comparing SARS-CoV-2 with SARS-CoV and influenza pandemics. Lancet Infect Dis. 2020; S1473-3099(20)30648-4. PubMed Abstract \| Publisher Full Text\n\nKhubchandani J, Jordan TR, Yang YT: Ebola, zika, corona…what is next for our world? Int J Environ Res Public Health. 2020; 17(9): 3171. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVale CL, Fisher DJ, White IR, et al.: What is the optimal systemic treatment of men with metastatic, hormone-naive prostate cancer? A STOPCAP systematic review and network meta-analysis. Ann Oncol. 2018; 29(5): 1249–57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTierney JF, Fisher DJ, Burdett S, et al.: Comparison of aggregate and individual participant data approaches to meta-analysis of randomised trials: An observational study. PLoS Med. 2020; 17(1): e1003019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThanh Le T, Andreadakis Z, Kumar A, et al.: The COVID-19 vaccine development landscape. Nat Rev Drug Discov. 2020; 19(5): 305–6. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization (WHO): An international randomised trial of candidate vaccines against COVID-19. World Health Organization. 2020; [cited 2020 Aug 25]. Reference Source" }	[ { "id": "71099", "date": "08 Oct 2020", "name": "Cyrus Mehta", "expertise": [ "Reviewer Expertise I am a biostatistician with expertise in design of adaptive clinical trials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Comments This paper highlights the many benefits of designing a randomized clinical trial so that several treatment arms are compared in a pairwise fashion to a common control arm, with interim analyses to identify effective treatments and drop ineffective ones from further consideration. The considerable efficiency that such a design offers relative to the more conventional two-arm clinical trial arises in three ways. First, since many treatment arms share the same control arm, there is a saving of sample size. Second, the efficacy and/or futility data for each of the treatment interventions is obtained much earlier, and more reliably, than it would be were the interventions to be studied in separate two-arm trials that are conducted in sequence and with smaller sample sizes. Thirdly the operational infrastructure associated with setting up the multi-arm trial need only be created once, and can be used repeatedly, with minor adjustments, as current treatment arm exit the platform and new arms replace them. The references to on-going and completed trials, many of which are pandemic related, are a special strength of this paper.\n\nThe authors have also pointed some of the challenges of the MAMS platform approach. Much of this discussion relates to aligning the special requirements of participants from different countries and sites, with different medical needs, different treatment interventions and different funding sources and onto a single protocol. This is a vast problem that cannot be discussed adequately in a short survey paper. Perhaps it is worthwhile to mention in this context the EU-PEARL Consortium (eu-pearl.eu).\n\nIssues in Need of Further Discussion:\nChanging the Control Arm. How does one handle the problem of comparability if one of the interventions attains statistical and clinical significance at an interim analysis and thereby becomes the new control arm? Suppose, for example, that while subjects are still being followed, one of the arms attains statistical significance by crossing a group sequential boundary. Would you continue with the current SOC until the pre-specified follow-up of the remaining arms was completed? Would that be ethical? On the other hand, if you were to immediately replace the SOC with the new intervention, how would you interpret the results of a comparison to a control arm that was replaced in mid-stream?\n\nConcurrent Controls. If one or more arms are dropped for futility and are replaced by new arms, what are your thoughts on using all the control-arm data in the comparison with the new arms, versus using only the concurrent control-arm data? Some Bayesian trials have used the former approach.\n\nControl of Multiplicity. Multiplicity issues can arise if one performs repeated significance tests over time on a specific treatment arm to determine if it is efficacious. This source of multiplicity can be controlled by use of group-sequential efficacy boundaries. A different source of multiplicity arises because multiple treatments are being tested in the same protocol. Therefore one must ask whether these treatments are members of one family or whether there is no relationship whatsoever between them, and they have merely been placed in the same protocol for the convenience of sharing a common control arm. In the former case the family-wise error rate must be controlled while in the latter case, only the pair-wise error rate must be controlled. Which type of control is more appropriate in the Covid setting? These are important conceptual issues that should be discussed.\n\nAdaptive Sample Size Re-assessment. What are the authors' views on estimating the treatment effect at an interim analysis and adjusting the sample size based on the new evidence from the trial itself? Appropriate statistical methods are available to control the error rate in this situation. In a Covid setting, where very little is known about the efficacy of the treatment interventions, would it not be desirable to commit to a final sample size only after having observed a reasonable amount of data from the trial?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6124", "date": "23 Nov 2020", "name": "Nurulamin Noor", "role": "Author Response", "response": "We are grateful to both reviewers for their careful assessment of this manuscript, and their constructive comments. We have provided a point-by-point response to each of the reviewers’ comments below, added in citations to support the additional text, and where applicable updated current citations. The changes to the manuscript are described and, where applicable, added changes are highlighted as tracked changes in the revised manuscript. Best wishes, Reviewer: 1 Comment: This paper highlights the many benefits of designing a randomized clinical trial so that several treatment arms are compared in a pairwise fashion to a common control arm, with interim analyses to identify effective treatments and drop ineffective ones from further consideration. The considerable efficiency that such a design offers relative to the more conventional two-arm clinical trial arises in three ways. First, since many treatment arms share the same control arm, there is a saving of sample size. Second, the efficacy and/or futility data for each of the treatment interventions is obtained much earlier, and more reliably, than it would be were the interventions to be studied in separate two-arm trials that are conducted in sequence and with smaller sample sizes. Thirdly the operational infrastructure associated with setting up the multi-arm trial need only be created once, and can be used repeatedly, with minor adjustments, as current treatment arm exit the platform and new arms replace them. The references to on-going and completed trials, many of which are pandemic related, are a special strength of this paper. Response: Thank you for your positive comments and for your careful reading of this manuscript. Comment: The authors have also pointed some of the challenges of the MAMS platform approach. Much of this discussion relates to aligning the special requirements of participants from different countries and sites, with different medical needs, different treatment interventions and different funding sources and onto a single protocol. This is a vast problem that cannot be discussed adequately in a short survey paper. Perhaps it is worthwhile to mention in this context the EU-PEARL Consortium (eu-pearl.eu). Response: We thank the reviewer for this suggestion and agree that these are important points that will likely be best considered by international collaborative efforts. We have now included reference to important international efforts in this regard, including mention of both the EU-PEARL Consortium and to the Clinical Trials Transformation Initiative (CTTI). Comment: Changing the Control Arm. How does one handle the problem of comparability if one of the interventions attains statistical and clinical significance at an interim analysis and thereby becomes the new control arm? Suppose, for example, that while subjects are still being followed, one of the arms attains statistical significance by crossing a group sequential boundary. Would you continue with the current SOC until the pre-specified follow-up of the remaining arms was completed? Would that be ethical? On the other hand, if you were to immediately replace the SOC with the new intervention, how would you interpret the results of a comparison to a control arm that was replaced in mid-stream? Response: Thank you for this question about change in standard of care for control arms. We certainly agree that in the context of a fast-moving infectious disease pandemic, it is likely that usual or standard care will change based on interim analysis (and reporting) of comparison arms. We agree that it would be unethical to continue treating new patients with a historical standard of care in light of an evidence-based and available better standard of care, and have now made this clear in the discussion. We have also included about the need to compare comparison/intervention arms in a pairwise manner with the participants in the standard of care arm, at the time of enrolment. For ongoing patients in the trial when convincing results emerge, there would need to be careful consideration for each participant – the trial results apply to patients who meet the entry criteria and many ongoing participants may no longer be in this same state; this is more likely in trials and disease courses that run over a longer period. Comment: Concurrent Controls. If one or more arms are dropped for futility and are replaced by new arms, what are your thoughts on using all the control-arm data in the comparison with the new arms, versus using only the concurrent control-arm data? Some Bayesian trials have used the former approach. Response: Thank you for this challenging question on use of control data from trials. We agree that there has been variable practice across platform trials. Whilst some trials do use historical control group data from the entirety of a platform trial, we encourage applying caution, particularly in the late-phase trial setting, and when there a changes in standard of care for the control arm group within the course of a trial. Staying with the contemporaneously randomised patients is likely the better course of action in most instances. Comment: Control of Multiplicity. Multiplicity issues can arise if one performs repeated significance tests over time on a specific treatment arm to determine if it is efficacious. This source of multiplicity can be controlled by use of group-sequential efficacy boundaries. A different source of multiplicity arises because multiple treatments are being tested in the same protocol. Therefore one must ask whether these treatments are members of one family or whether there is no relationship whatsoever between them, and they have merely been placed in the same protocol for the convenience of sharing a common control arm. In the former case the family-wise error rate must be controlled while in the latter case, only the pair-wise error rate must be controlled. Which type of control is more appropriate in the Covid setting? These are important conceptual issues that should be discussed. Response: Thank you for this question about multiplicity, which is of interest to many of the authors. We have now included a short discussion about multiplicity and control of error rates when adding intervention arms in the context of a pandemic platform and added in a recent citation to support this text (Choodari-Oskooei et al. Clinical Trials, 2020). Comment: Adaptive Sample Size Re-assessment. What are the authors' views on estimating the treatment effect at an interim analysis and adjusting the sample size based on the new evidence from the trial itself? Appropriate statistical methods are available to control the error rate in this situation. In a Covid setting, where very little is known about the efficacy of the treatment interventions, would it not be desirable to commit to a final sample size only after having observed a reasonable amount of data from the trial? Response: Carefully planned and explained adaptive sample size re-assessment could also be incorporated into a MAMS platform protocol. However, this is a controversial topic. We do advise an element of caution as with all adaptations made following interim analysis in terms of clarifying which individuals/committees have access to unblinded interim outcome data and pre-specifying how data will be used to inform decision-making. We note with interest that the RECOVERY trial - used as one of the exemplar trials in this manuscript - did not have an initial sample size calculation, and used data from early periods within the trial to allow subsequent sample size calculations. We have now accommodated this in the discussion section of the manuscript." } ] }, { "id": "73671", "date": "29 Oct 2020", "name": "Dmitri Nepogodiev", "expertise": [ "Reviewer Expertise Public health", "epidemiology", "surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 make a clear case for the benefits of adaptive platform trials in the context of the COVID-19 pandemic. The RECOVERY trial has demonstrated that MAMS design can significantly accelerate the rate at which urgent research questions can be robustly answered. However, even with the increased efficiency that MAMS offers, such platform trials still require large numbers of patients to be recruited. For example, RECOVERY has now recruited 15,000 patients but this has required concerted recruitment across most (n=176) UK hospitals. Realistically, during a pandemic it is only feasible for available local research infrastructure to support a limited number of such large-scale trials. This raises issues about how a consensus is reached on what trials are supported and what treatments they test.\nWhat research questions should be addressed? There are a large number of research questions relating to SARS-CoV-2 across different domains (prophylaxis, treatment, public health measures etc), different patient groups, and different settings (primary care, acute hospitals, nursing homes etc). It is not feasible to support a large number of trials so research questions must be prioritised. Who should do this and on what basis?\n\nWho should lead such trials? This requires multi-disciplinary (multi-institutional) teams with a track record of delivering large-scale studies. A pandemic is probably not the best time for novice trialists to get started.\n\nFor a treatment trial (e.g. RECOVERY) how should treatments be selected? Although a large number of potential repurposed treatments were proposed, there was relatively little early phase research to indicate which should go to phase III trials. So who should make these decisions and how?\n\nNew treatments are now being developed for COVID-19. How should a pipeline be set up linking phase II studies to the phase III platform trial to ensure that promising treatments seamlessly and efficiently progress?\n\nOnce the results for a trial arm have been analysed and final results are available how should the results be disseminated? Should this be through the traditional peer review, pre-print servers, press release? How can research findings be rapidly linked in to clinical guidelines?\nAlthough these points are beyond the specifics of the MAMS design, they are important to the overall aim of rapidly delivering impactful research during the pandemic and they are likely to be issues faced by anyone setting up a MAMS trial in this context. Perhaps a few brief comments on this might be interesting?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6125", "date": "23 Nov 2020", "name": "Nurulamin Noor", "role": "Author Response", "response": "We are grateful to both reviewers for their careful assessment of this manuscript, and their constructive comments. We have provided a point-by-point response to each of the reviewers’ comments below, added in citations to support the additional text, and where applicable updated current citations. The changes to the manuscript are described and, where applicable, added changes are highlighted as tracked changes in the revised manuscript. Best wishes, Reviewer: 2 Comment: The authors make a clear case for the benefits of adaptive platform trials in the context of the COVID-19 pandemic. The RECOVERY trial has demonstrated that MAMS design can significantly accelerate the rate at which urgent research questions can be robustly answered. Response: Thank you for your positive comments and for your careful consideration of this manuscript. Comment: However, even with the increased efficiency that MAMS offers, such platform trials still require large numbers of patients to be recruited. For example, RECOVERY has now recruited 15,000 patients but this has required concerted recruitment across most (n=176) UK hospitals. Realistically, during a pandemic it is only feasible for available local research infrastructure to support a limited number of such large-scale trials. This raises issues about how a consensus is reached on what trials are supported and what treatments they test. Response: Thank you for this question about prioritisation of trials in a pandemic. We agree that one of the benefits from using the MAMS platform protocol approach is the reduced competition between trials and greater efficiencies overall. We also agree that another source of efficiency will be from approaches where there are wide-scale support for fewer platforms rather than many overlapping and competing platforms. Ultimately, in a pandemic setting given the need for speed for answers, we believe the platforms, which can be setup at speed to start answering research questions are most likely to provide the answers needed. We have now included these above points in our discussion. Comment: What research questions should be addressed? There are a large number of research questions relating to SARS-CoV-2 across different domains (prophylaxis, treatment, public health measures etc), different patient groups, and different settings (primary care, acute hospitals, nursing homes etc). It is not feasible to support a large number of trials so research questions must be prioritised. Who should do this and on what basis? Comment: For a treatment trial (e.g. RECOVERY) how should treatments be selected? Although a large number of potential repurposed treatments were proposed, there was relatively little early phase research to indicate which should go to phase III trials. So who should make these decisions and how? Comment: New treatments are now being developed for COVID-19. How should a pipeline be set up linking phase II studies to the phase III platform trial to ensure that promising treatments seamlessly and efficiently progress? Response: Thank you for these three linked questions about selection of research questions and interventions within a MAMS platform protocol. We entirely agree that the MAMS platform can be used to evaluate a number of different interventions. To date, the majority of interventions that have been assessed have been investigational medicinal products (IMPs) for treatment. However, we strongly believe that non-IMP interventions also require assessment in a pandemic setting. The MAMS platform offers many benefits for evaluating prophylactic, treatment or preventative interventions. Ultimately the decision for which interventions should be studied within a trial platform including future intervention arms, rest with the trial team itself. We have now included this in the discussion and about factors for trial teams to consider when adding in new intervention arms and assessing their progress. Comment: Who should lead such trials? This requires multi-disciplinary (multi-institutional) teams with a track record of delivering large-scale studies. A pandemic is probably not the best time for novice trialists to get started. Response: We thank the reviewer for this question and agree that given the complexities and workload associated with delivering MAMS platform trials, especially in the late-phase trial setting, these trials would likely be best delivered by multi-disciplinary organisations and teams with experience and track record in the area. We have included this in our discussion section and cited three citations further exploring MAMS platform trials from: a trial management perspective (Schiavone et al. Trials, 2019), a data management perspective (Hague et al. Trials, 2019), and from a trial team perspective for working within and delivering large MAMS platform trials in the late-phase trial setting (Morrell L et al. Trials, 2019). Comment: Once the results for a trial arm have been analysed and final results are available how should the results be disseminated? Should this be through the traditional peer review, pre-print servers, press release? How can research findings be rapidly linked in to clinical guidelines? Although these points are beyond the specifics of the MAMS design, they are important to the overall aim of rapidly delivering impactful research during the pandemic and they are likely to be issues faced by anyone setting up a MAMS trial in this context. Perhaps a few brief comments on this might be interesting? Response: Thank you for this question about the important topic of when and how to report findings from comparison intervention arms during the course of a MAMS platform. The challenge of rapid and appropriate dissemination is pertinent to all trials. In a MAMS platform trial where comparison arms will have primary results available at different times during the course of the platform, we strongly believe that primary outcome results from intervention arms should be reported as soon as these results have been analysed and are available - and that it would be unethical to delay reporting these findings until all comparison arms have primary results available. We support the process of traditional peer review but given the length of time that this can take, we also support appropriate use for earlier dissemination of headline findings such as through the use of open-access, pre-print reports. We have now included a section on this topic in the challenges section of our manuscript." } ] } ]	1	https://f1000research.com/articles/9-1109
https://f1000research.com/articles/9-1355/v1	20 Nov 20	{ "type": "Case Report", "title": "Case Report: Unusual presentation of concomitant gastric outlet obstruction and jaundice complicating giant hepatic angioma", "authors": [ "Nour Elleuch", "Aya Hammami", "Amira Hassine", "Wafa Ben Ameur", "Wafa Dahmani", "Ben Slama Aida", "Ksiaa Mehdi", "Braham Ahlem", "Ajmi Salem", "Hanen Jaziri", "Jmaa Ali", "Nour Elleuch", "Amira Hassine", "Wafa Ben Ameur", "Wafa Dahmani", "Ben Slama Aida", "Ksiaa Mehdi", "Braham Ahlem", "Ajmi Salem", "Hanen Jaziri", "Jmaa Ali" ], "abstract": "Hemangioma is the most common benign tumor affecting the liver. The vast majority of liver hemangiomas (LH) are less than 30 mm in diameter, asymptomatic, and are most often identified incidentally during radiological investigations for other reasons. Giant LH greater than 50 mm can lead to the development of symptoms and complications that require prompt surgical intervention. Herein, we report the case of a 58-year-old man who presented with gastric outlet obstruction and obstructive jaundice as a result of a giant hepatic hemangioma that was complicated with fatal spontaneous rupture.", "keywords": [ "Giant hemangioma", "Obstructive jaundice", "gastric outlet obstruction", "Acute spontaneous rupture" ], "content": "Introduction\n\nHemangiomas are the most common primary benign liver tumours with an incidence estimated up to 20%1. Most of the lesions are less than 3 cm2. They are typically asymptomatic and are most often discovered incidentally during radiological investigations for other reasons. Although there is no general consensus on size, the most authors agree that hemangiomas >4 cm are described as giant3,4. Symptoms depend on the location and the size of the haemangioma. Concomitant gastric outlet obstruction (GOO) and obstructive jaundice is an extremely rare presentation of liver hemangiomas (LH)2.\n\nWe report the case of giant LH developing in the left lobe of the liver responsible for GOO and obstructive jaundice with fatal outcomes secondary to spontaneous rupture.\n\n\nCase report\n\nA 58-year-old male Tunisian patient was admitted to our department owing to a 3-week history of worsening postprandial vomiting, associated to abdominal pain in the right upper quadrant and jaundice. His past medical history was unremarkable.\n\nThe physical examination revealed a dehydrated patient with jaundice and moderately reduced general condition. His vital signs were stable, body temperature was 37.2°C, pulse rate was 72 beats/minute, and blood pressure was 120/75 mmHg. Abdominal palpation identified a large firm abdominal mass in the right upper quadrant extending to the epigastrum and the umbilicus.\n\nLaboratory tests performed at admission showed hemoglobin levels of 12.6 g/dL, platelet count of 251,000/mL, international normalized ratio (INR) of prothrombin time of 1.12, albumin of 32 g/L (normal range: 36–48 g/L), aspartate aminotransferase of 45 UI/L (normal range up to 50 UI/L), alanine aminotransferase of 65 UI/L (normal range up to 40 UI/L), alkaline phosphatase of 360 U/L (normal range: 32–91 U/L), gamma glutamyl transpeptidase of 310 UI/L (normal range up to 50 UI/L) and notable hyper bilirubinemia (total bilirubin of 279 µmol/L (normal range up to 17 µmol/L) and direct bilirubin 90 of µmol/L (normal range up to 7 µmol/L). Renal function tests, electrolytes and c-reactive protein level were all within normal limits.\n\nAs the patient presented with symptoms related to gastric outlet obstruction, upper digestive endoscopy was performed one week after admission, following fluid resuscitation and nasogastric decompression. It showed that the stomach was full of fluid and food particles with evident extrinsic compression of the prepyloric region and the duodenum. The abdominal CT-scan revealed a 24 x 18 x 32 cm lobulated mass with a dense contrast uptake. It caused compression of the stomach, the duodenum, the common bile duct, and the hepatic pedicle (Figure 1). A diagnosis of compressive giant LH was made on the basis of typical radiological aspects.\n\nA giant cavernous hemangioma, filling the left lobe of the liver, showing peripheral intermittent nodular contrast enhancement. with exophytic extension, compressing adjacent structures.\n\nAlthough risky, surgery (partial liver resection) was decided upon because of the pressure being put on both the gastrointestinal system and the biliary tract. Unfortunately, before the intervention, the patient developed severe right upper quadrant abdominal pain, and became hemodynamically unstable. On palpation, the upper abdomen showed diffuse tenderness and fullness. Acute spontaneous rupture of the LH was strongly suspected and the patient died despite resuscitation attempts, three weeks after his admission.\n\n\nDiscussion\n\nHemangioma is a frequent benign hepatic tumor with an estimated incidence of 0.4 to 7.3% in the general population5,6. Unfortunately, there are no national statistics for incidence in Tunisia. Regarding sex distribution, it seems that women are more frequently affected. The pathogenesis of this tumor is not yet completely understood. Female-specific hormones have been is involved and it has been suggested that they stimulate the enlargement of haemangiomas7.\n\nA diagnosis of LH can be made easily through imaging techniques including ultrasonography, CT, magnetic resonance imaging, either alone or in combination, with a high sensitivity greater than 90%. Occasionally positron emission tomography (PET) or angiography can be used8. On ultrasound, small hemangioma appears as a uniform hyperechoic mass with relatively well- defined margins. Larger lesions, can appear hypoechoic or inhomogeneous, because of possible necrosis, hemorrhage or fibrosis9.\n\nLH occurs more frequently in the right lobe of the liver10. Although there is no general agreement on size, LH are considered giant when they exceed 40 mm in diameter3. However, other sizes have been subsequently specified to categorize a LH as giant (4, 8 and 10 cm in diameter)11. Most lesions are less than 30 mm1, usually asymptomatic, and most often diagnosed incidentally on imaging tests for other reasons, laparotomy or autopsy.\n\nGiant hemangiomas are symptomatic in 90% of cases, with various signs and findings depending on the location and size of the lesion12,13. The most frequently seen manifestation is abdominal pain resulting from the stretching of the Glisson capsule or necrosis of the lesion. In addition, giant LH may cause haematological abnormalities such as thrombocytopenia, anemia and leukopenia. Kasabach-Merritt syndrome is a life-threatening complication that may occur in 1.7% of patients with LH. It includes thrombocytopenia, consumptive coagulopathy and microangiopathic hemolytic anemia14. In rare cases, LH located in the left lobe, close to the hepatic hilum, may cause obstructive jaundice by biliary obstruction. GOO is also a rare manifestation of giant LH typically located in the left lobe of the liver, resulting from pressure on the stomach and the duodenum.\n\nSpontaneous rupture of LH is an extremely rare and life-threatening event that is more likely to occur in lesions with a diameter ≥40 mm that are near the surface of the liver or with an exophytic growth15. It is associated with severe abdominal pain, peritonitis, and shock. The management of complications is challenging as the mortality rate ranges between 60 and 75%. Only a few cases have been reported16,17. Our patient had an extremely giant hemangioma exceeding 20 cm. To the best of our knowledge, this is the first case reported in literature to have LH with concomitant obstructive jaundice, GOO, and spontaneous rupture.\n\nTreatment is only indicated when symptoms occur14,18.\n\nAs most liver hemangiomas do not increase in size over time, it has been suggested that asymptomatic patients with hemangiomas less than 5 cm require no intervention or observation. Additionally, giant LH with no or few symptoms requires no treatment since there is little risk of developing complications.\n\nSymptomatic LH is conventionally treated surgically. Enucleation and resection are the most commonly used techniques and they are both safe. If feasible, enucleation is generally the preferred approach19. The choice of approach depends on the experience of the surgeon and the location and size of the tumor. Other options, such as hepatic artery ligation, transcatheter arterial embolization or in rare cases liver transplantation, have also been proposed. Surgical management of extremely giant LH >20 cm remains challenging18. In the case of our patient, we considered that hepatic resection was the most suitable therapeutic option. Unfortunately, the patient died before surgery because of the occurrence of a fatal spontaneous LH rupture.\n\nTo conclude, giant LH does not necessarily require any treatment unless symptoms appear. Lesions close to the hepatic hilum may cause compression of the bile duct and cause obstructive jaundice, those close to the stomach and duodenum may cause GOO. The exact risk of spontaneous rupture remains unknown. The association of several complications such as the case of our patient is a very uncommon clinical presentation that requires prompt surgical intervention.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the family of the patient.", "appendix": "References\n\nOzdemir E, Akbulut S, Kutluturk K, et al.: Giant hepatic hemangioma: An unusual cause of gastric compression. Turk J Gastroenterol. 2019; 30(10): 930–1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAydin C, Akbulut S, Kutluturk K, et al.: Giant hepatic hemangioma presenting as gastric outlet obstruction. Int Surg. 2013; 98(1): 19–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoszka AJ, Ferreira FG, de Aquino CG, et al.: Resection of a rapid-growing 40-cm giant liver hemangioma. World J Hepatol. 2010; 2(7): 292–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUetama T, Yoshida H, Hirakata A, et al.: A symptomatic giant hepatic hemangioma treated with hepatectomy. J Nippon Med Sch. 2011; 78(1): 34–9. PubMed Abstract \| Publisher Full Text\n\nIshak KG, Rabin L: Benign tumors of the liver. The Medical clinics of North America. 1975; 59(4): 995–1013.\n\nOchsner JL, Halpert B: Cavernous hemangioma of the liver. Surgery. 1958; 43(4): 577–82.\n\nvan Malenstein H, Maleux G, Monbaliu D, et al.: Giant liver hemangioma: the role of female sex hormones and treatment. Eur J Gastroenterol Hepatol. 2011; 23(5): 438–43. PubMed Abstract \| Publisher Full Text\n\nCorigliano N, Mercantini P, Amodio PM, et al.: Hemoperitoneum from a spontaneous rupture of a giant hemangioma of the liver: report of a case. Surg today. 2003; 33(6): 459–63. PubMed Abstract \| Publisher Full Text\n\nHo HY, Wu TH, Yu MC, et al.: Surgical management of giant hepatic hemangiomas: complications and review of the literature. Chang Gung Med J. 2012; 35(1): 70–8. PubMed Abstract \| Publisher Full Text\n\nZerpa R, Abdelghani EH, Iliescu G, et al.: Enormous haemangioma of the liver. BMJ case rep. 2019; 12(3): e226983. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdam YG, Huvos AG, Fortner JG: Giant hemangiomas of the liver. Annals of surgery. 1970; 172(2): 239–45.\n\nVagefi PA, Klein I, Gelb B, et al.: Emergent orthotopic liver transplantation for hemorrhage from a giant cavernous hepatic hemangioma: case report and review. J Gastrointest Surg. 2011; 15(1): 209–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChoi J, Lee YJ, Hwang DW, et al.: Surgical treatment of giant hepatic hemangiomas: technical point of view. Am Surg. 2011; 77(1): 48–54. PubMed Abstract\n\nYoon SS, Charny CK, Fong Y, et al.: Diagnosis, management, and outcomes of 115 patients with hepatic hemangioma. J Am Coll Surg. 2003; 197(3): 392–402. PubMed Abstract \| Publisher Full Text\n\nZhao W, Guo X, Dong J: Spontaneous rupture of hepatic hemangioma: a case report and literature review. Int J Clin Exp Pathol. 2015; 8(10): 13426–8. PubMed Abstract \| Free Full Text\n\nSantos Rodrigues AL, Silva Santana AC, Carvalho Araujo K, et al.: Spontaneous rupture of giant hepatic hemangioma: a rare source of hemoperitoneum. G Chir. 2010; 31(3): 83–5. PubMed Abstract\n\nGoidescu OC, Patrascu T: Ruptured liver cavernous hemangioma - rare cause of hemoperitoneum. J Med Life. 2015; 8(1): 73–4. PubMed Abstract \| Free Full Text\n\nLiu X, Yang Z, Tan H, et al.: Characteristics and operative treatment of extremely giant liver hemangioma >20 cm. Surgery. 2017; 161(6): 1514–24. PubMed Abstract \| Publisher Full Text\n\nVeerankutty FH, Rather SA, Yeldho V, et al.: Totally Laparoscopic Resection of an Extremely Giant Hepatic Hemangioma. Surg J (N Y). 2019; 5(3): e110–e2. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "84819", "date": "17 May 2021", "name": "Phillipe Abreu", "expertise": [ "Reviewer Expertise Abdominal Transplant Surgery", "Liver and Pancreas Surgery" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very superficial case report from a patient who presented with a large hepatic hemangioma and was poorly managed, resulting in a fatal outcome.\n\nAuthors should better describe full information regarding the patient's clinical history and progression of the disease. Even in the course of the admission, it fails to describe symptoms and clinical presentation.\n\nThere is no discussion about the potential use of endovascular approach pre-surgical management of such large lesions, or any discussion about advanced endoscopic procedures that could be made to downsize the lesion prior to an exploratory surgical intervention was even considered (see attached citation as an example).\n\nThe current case as presented is a catastrophic example of how not to manage patients with liver hemangiomas, his clinical course is not even well described, rather only simple information as \"died despite resuscitation attempts, three weeks after his admission\". What resuscitation attempts? Why an emergency surgery was not performed? How come this patient remained in the hospital for 3 weeks and was not operated on before? Why no endovascular procedure was attempted?\nAuthors should better review this case in detail and present it in another way, considering alternatives of treatment and better justifications of the management adopted to clarify why the state of the art medicine was not the choice for the current patient.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "83691", "date": "05 Jul 2021", "name": "Shariful Islam", "expertise": [ "Reviewer Expertise All aspects of General and Laparoscopic and Breast Surgery" ], "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 unusual presentation of giant hepatic hemangioma.\nThe Title: use the commonly used term Hepatic hemangioma rather hepatic angioma.\n\nAbstract: Needs further revision. The authors should start by saying how rare the giant hemangioma is, the difficulties in the management and outcomes if not treated adequately or promptly - rather simply describing an hepatic hemangioma- which has been already described in the literature.\nIntroduction: Should also include brief but updated treatment options.\nCase report: Not written well. Proper terminology was not used in many places. Needs further revision\n\nAuthors should explain the reasons for delayed treatment. Why an emergency surgery was not performed? and also why not early endovascular management was not offered to this patient.\nDiscussion: Is too short, it should include the role of advanced endoscopic/endovascular option for a giant hemangioma\nConclusion: Also requires further revision. In this section the authors need to highlight the importance of this case report to the literature. Also to state what is learnt from this case report.\nDecision: I my opinion, it is an important finding and can add new information to the literature, however this article needs major revision, addressing all above concerns before indexing.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1355
https://f1000research.com/articles/9-226/v1	02 Apr 20	{ "type": "Research Article", "title": "Quality of reporting acupuncture interventions for chronic obstructive pulmonary disease: Review of adherence to the STRICTA statement", "authors": [ "Carles Fernández-Jané", "Mireia Solà-Madurell", "Mingkun Yu", "Changhao Liang", "Yutong Fei", "Mercè Sitjà-Rabert", "Gerard Úrrutia", "Mireia Solà-Madurell", "Mingkun Yu", "Changhao Liang", "Yutong Fei", "Mercè Sitjà-Rabert", "Gerard Úrrutia" ], "abstract": "Background: The quality of reporting of acupuncture interventions is critical to ensure the applicability and reproducibility of acupuncture clinical trials. In the past, different publications have evaluated the quality of reporting of acupuncture interventions for different clinical situations, such as knee osteoarthritis, neurological diseases or cancer. However, this has not been done for acupuncture trials for chronic obstructive pulmonary disease (COPD). Objective: To assess the quality of reporting of acupuncture interventions in trials for COPD. Methods: A total of 11 English and Chinese databases were screened up until May 2019 for randomised or quasi-randomised control trials of acupuncture for COPD. The STRICTA checklist was used to determine the quality of the reporting of acupuncture interventions. Results: A total of 28 trials were included in our review. Out of the 16 STRICTA checklist subitems analysed, only 3 were considered appropriately reported in more than 70% of the trials, while 7 were correctly reported in less than 40%. Conclusion: The adherence to STRICTA guidelines of acupuncture trials for COPD is suboptimal, and future efforts need to be addressed to improve the quality of reporting.", "keywords": [ "Quality of Reporting", "Acupuncture", "COPD" ], "content": "Introduction\n\nRecent systematic reviews have assessed acupuncture’s efficacy for chronic obstructive pulmonary disease (COPD)1–3. These reviews have concluded that, even though acupuncture could have some benefits, the risk of bias of the included trials is too high to draw any strong conclusion. Risk of bias is certainly a critical aspect in randomised control trials; however, the quality of reporting is also a key point.\n\nComplete and clear information regarding clinical trial methodology is not only essential to adequately assess health research but also for its applicability and reproducibility. This is especially important in complex interventions, such as acupuncture, that can be practiced in many different styles and variations. Aspects such as point selection, depth of the insertion, stimulation method and response sought, which may be very different between practitioners, could have an impact on the therapeutic effect4. Therefore, to be able to replicate an acupuncture intervention in clinical practice or reproduce it in another trial, it is necessary to fully describe how it is applied.\n\nThe STandards for Reporting Interventions in Controlled Trials of Acupuncture (STRICTA) guidelines were created to improve the quality of reporting in acupuncture trials and facilitate transparency in published reports5. These guidelines were updated in 2010 as an extension of the CONsolidated Standards Of Reporting Trials (CONSORT) guidelines6 and consist of 6 key items and 17 subitems addressing aspects such as acupuncture rationale, needling details, treatment regime, other components of the treatment, practitioner background and details about the control or comparator.\n\nAlthough there is evidence that STRICTA guidelines have helped to improve acupuncture’s reporting, there is still a lot of room for improvement7. This has also been seen in recent publications for some specific conditions such as neurological diseases8–10 or cancer11, concluding that reporting is still suboptimal in these conditions. However, to our knowledge, there is currently no publications regarding the quality of reporting of acupuncture interventions in COPD trials.\n\nEvaluating the adherence of acupuncture clinical trials for COPD to SRICTA guidelines is crucial to detect underreported subitems and therefore highlight current deficiencies. This will help to improve the reporting of future trials and facilitate their applicability in clinical practice and the reproducibility of future research.\n\nThe aim of this study is to comprehensibly evaluate the adherence to STRICTA guidelines of trials using acupuncture for COPD.\n\n\nMethods\n\nIn this study, we used the results of our previous systematic review, which included randomised or quasi-randomised trials using filiform needle acupuncture for COPD2. Published studies were comprehensively searched in the following databases from their inception to May 2019: Cochrane Central Register of Controlled Trials (CENTRAL), Medline, Embase, CINAHL, AMED (Ovid), PEDro, PsycINFO, CNKI, VIP, Wanfang, and Sino-Med. Detailed descriptions of the inclusion criteria, information sources, search strategies and study selections are published elsewhere2 and the protocol is available at http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42014015074.\n\nReviewers (CFJ, MSM, MY and CHL) worked in pairs and independently extracted the data using a standardised data extraction form, and disagreements were solved by consensus.\n\nFor data collection and STRICTA assessment, a specific extraction table with instructions of how to assess each of the 16 STRICTA subitems was created by the authors (Extended data12). This extraction table was tested with pilot data to ensure its usability.\n\nOur aim was to assess the acupuncture interventions, and therefore, the 17th STRICTA subitem “precise description of the control or comparator” was not considered. However, we did include the 16th STRICTA subitem “rationale of the chosen comparator”, since it is critical to justify which component of the acupuncture treatment is being assessed.\n\nSince some STRICTA subitems refer to multiple aspects (e.g., “names of points used” subitem refers not only to the name or location of points but also to if they are used unilaterally or bilaterally), besides considering items just as reported or not reported, we also considered partially reported items and recorded the reasons for there being. This was done to provide more detailed information regarding the aspects that should be improved in the reporting of future trials. Partial reporting was also considered when the authors reported information in other sections, such as the introduction or the discussion. Although some subitems were considered that could potentially be “not applicable” (NA) for some pragmatic designs, none of the trials required this.\n\nA descriptive analysis was used to summarise the results using percentages and absolute numbers.\n\n\nResults\n\nIn our systematic review, we screened all 5030 unduplicated titles and abstracts retrieved, and obtained 166 full text articles. Finally, we included 28 trials using filiform needles for COPD (Figure 1). Of those, only 6 were published in English-language journals, 1 in a German-language journal and 21 in Chinese-language journals. Details regarding the study characteristics and inclusion process have been published elsewhere2.\n\nRatings for STRICTA domains are summarized in Figure 2. Details for each trial are shown in Table 1, including reasons for considering partial reporting.\n\nSTRICTA, STandards for Reporting Interventions in Clinical Trials of Acupuncture.\n\nAcupuncture rationale. Acupuncture rationale was considered adequately reported in 20 trials (71%) regarding acupuncture style, 18 trials (64%) regarding reasoning of the treatment and 24 trials (85%) regarding treatment variation. Trials classified with partial reporting mentioned acupuncture style (3 trials) and reasoning (4 trials) in the introduction section but not in the methods section.\n\nDetails of needling. Adequate reporting of needling details was very heterogenous along all 4 subdomains. Best reported subdomains were “needle retention time” (21 trials, 75%) and “needle stimulation” (19 trials, 67%). Worse reported items were “name of the points” (10 trials, 35%), “depth of insertion” (8 trials, 28%) and “needle type” (6 trials, 21%). Partial reporting was observed in great proportion in “name of acupuncture points” (16 trials, 57%) being the main reason not describing if points were used unilaterally or bilaterally. Regarding the 46% of the trials classified with a partial reporting in the “needle type” item, there was missing information about the needle manufacturer (32.1%, 9 trials), material (25%, 7 trials) and length and diameter (10.7%, 3 trials).\n\nTreatment regime. While “frequency and duration of the treatment sessions” was considered adequately reported for all trials (100%), the “number of treatment sessions” was considered completely reported in none of them (0%). Although this might seem a contradiction, since the number of treatment sessions can be calculated from the treatment regime, in STRICTA, the number of treatment sessions does not only include the planned number of sessions but also the actual number of treatments received. This information was missing or considered not clear in all trials.\n\nOther components of treatment. Other components of treatment were one of the poorer described items. “Details of other interventions administrated” was only reported in 8 trials (28%) and “settings and context of treatments”, which refers to “instructions to practitioners that might modify their normal practice, for example, prescribing or proscribing explanations to patients about their diagnosis”, were only reported in 2 trials (7.1%).\n\nPractitioner background. This item was only addressed completely in 1 trial (3.5%), and only 2 more trials (7.1%) partially reported it without stating practitioner’s years of experience.\n\nControl or comparator. The “rational of the chosen comparator” was only correctly described in 2 trials (7.1%), while in 2 more trials (7.1%), this was mentioned in the introduction but not in the methods section.\n\n\nDiscussion\n\nWe found important limitations in the quality of reporting of acupuncture interventions in trials for COPD, especially regarding “depth of insertion”, “needle type”, “number of treatment sessions”, “details of other interventions administered”, “setting and context of treatments”, “description of acupuncturist” and “rationale for the control or comparator”.\n\nRecently, several similar studies have been published. Lu et al.11 and Hughes et al.13 used STRICTA to evaluate trials with cancer patients, and Wei et al. used STRICTA to evaluate trials with stroke patients8. Although they all concluded that reporting should be improved, there were also some differences. While Hughes et al. found better reporting regarding details of needling and treatment regimen, other reviews found lower reporting on these subitems, especially regarding number of needles per session and depth of insertion. Poor reporting on “details of other interventions administered”, “description of acupuncturist” and “rationale for the control or comparator” subitems was found in all studies. The differences between studies could be due to several reasons.\n\nFirst, Lu’s and Weis’s reviews, as well as our own, included Chinese-language trials, while Hugs’ study included only English-language trials. Trials published in English-language journals seem to have greater quality of reporting than those in Chinese-language journals, according to Lu’s review. However, Wei et al. found better reporting of the subitems “treatment reasoning” and “response sought” in Chinese journals and better reporting on “practitioner’s background” in English journals.\n\nSecond, since STRICTA does not clearly specify how items should be judged, authors might have used different criteria. For example, regarding the subitem “number of treatment sessions”, the STRICTA statement says that “the actual number of treatments received by participants should be reported in the Results section” not only the planned ones. Whereas in our review, we did not consider that this subitem was fully reported unless this information was explicitly stated; others might have been more permissive. Also, the criteria to consider proper reporting on “other components of treatment” might vary widely between reviewers.\n\nThird, sometimes information might have been reported in sections different from the ones they should appear in. While some authors might not have given much importance to this, we decided to take it into consideration.\n\nTo our knowledge, this is the first study to assess the quality of reporting of acupuncture interventions for COPD. We included all acupuncture trials for COPD published until May 2019 with no language restriction, which is important since we only found 6 acupuncture trials published in English. Also, we did not only assess if STRICTA subitems were adequately reported or not but also analysed partial reporting and its reasons, which might be more helpful for authors to realise what specific information is currently missing.\n\nLimitations of this study include that the STRICTA guidelines are not a rating scale; therefore, there are no clear indications of how to judge each subitem and when to consider it fully reported. To minimise this problem, each item was assessed by two reviewers independently, and a standardised extraction form was developed to unify reviewers’ criteria. Also, it would have been interesting to compare the quality of reporting depending on the language of publication, so we could explore differences in journal standards. However, due to the low number of non-Chinese-language publications found (1 in German and 6 in English), we decided not to do so. Finally, STRICTA guidelines are specific for the filiform needle acupuncture technique and are not suitable to assess other interventions. Therefore, we did not include trials using only moxibustion, acupressure or transcutaneous electrical nerve stimulation.\n\nThe quality of reporting of acupuncture interventions in COPD trials according to STRICTA guidelines is suboptimal. Greater attention needs to be paid to include all relevant information necessary to adequately assess the results of the trials by researchers and journals.\n\n\nData availability\n\nFigshare: Raw data file, https://doi.org/10.6084/m9.figshare.11999970.v114.\n\nFigshare: Extended data 1 Extraction form, https://doi.org/10.6084/m9.figshare.11999994.v112.\n\nData is available under a Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank the students from the Beijing University of Chinese Medicine for participating in the project and helping to define the suitability of the data extraction form. The corresponding author is a PhD candidate at the Universitat Autònoma de Barcelona, Spain.\n\n\nReferences\n\nCoyle ME, Shergis JL, Huang ET, et al.: Acupuncture therapies for chronic obstructive pulmonary disease: a systematic review of randomized, controlled trials. Altern Ther Health Med. 2014; 20(6): 10–23. PubMed Abstract\n\nFernández-Jané C, Vilaró J, Fei Y, et al.: Filiform needle acupuncture for copd: A systematic review and meta-analysis. Complement Ther Med. 2019; 47: 102182. PubMed Abstract \| Publisher Full Text\n\nHsieh PC, Yang MC, Wu YK, et al.: Acupuncture therapy improves health-related quality of life in patients with chronic obstructive pulmonary disease: A systematic review and meta-analysis. Complement Ther Clin Pract. 2019; 35: 208–218. PubMed Abstract \| Publisher Full Text\n\nShi GX, Yang XM, Liu CZ, et al.: Factors contributing to therapeutic effects evaluated in acupuncture clinical trials. Trials. 2012; 13: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacPherson H, Altman DG, Hammerschlag R, et al.: Revised STandards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA): Extending the CONSORT statement. J Evid Based Med. 2010; 3(3): 140–55. PubMed Abstract \| Publisher Full Text\n\nSchulz KF, Altman DG, Moher D: CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010; 340: c332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSvenkerud S, MacPherson H: The impact of STRICTA and CONSORT on reporting of randomised control trials of acupuncture: a systematic methodological evaluation. Acupunct Med. 2018; 36(6): 349–357. PubMed Abstract\n\nWei JJ, Yang WT, Yin SB, et al.: The quality of reporting of randomized controlled trials of electroacupuncture for stroke. BMC Complement Altern Med. 2016; 16(1): 512. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo G, Lu L, Zeng J: Quality of reporting of randomised controlled trials of acupuncture for neurological diseases conducted in China. Acupunct Med. 2014; 32(5): 411–7. PubMed Abstract \| Publisher Full Text\n\nZeng J, Lin G, Li L, et al.: Assessment of reporting quality in randomised controlled trials of acupuncture for post-stroke rehabilitation using the CONSORT statement and STRICTA guidelines. Acupunct Med. 2017; 35(2): 100–106. PubMed Abstract \| Publisher Full Text\n\nLu L, Liao M, Zeng J, et al.: Quality of reporting and its correlates among randomized controlled trials on acupuncture for cancer pain: application of the CONSORT 2010 Statement and STRICTA. Expert Rev Anticancer Ther. 2013; 13(4): 489–98. PubMed Abstract \| Publisher Full Text\n\nFernández C: Extended data 1 Extraction form. figshare. Dataset. 2020. https://doi.org/10.6084/m9.figshare.11999994.v1\n\nHughes JG, Lewith G, MacPherson H, et al.: Assessment of the quality of reporting in studies of acupuncture for patients with cancer using the STRICTA guidelines. Acupunct Med. 2019; 37(4): 223–227. PubMed Abstract\n\nFernández Carles: Raw data file.figshare. Dataset. 2020; https://doi.org/10.6084/m9.figshare.11999970.v1" }	[ { "id": "71710", "date": "06 Oct 2020", "name": "Erik Cobo", "expertise": [ "Reviewer Expertise RCTs", "reporting guidelines", "causal modeling", "meta-researcch" ], "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 read this paper and I think it could be accepted provided authors correct my three major suggestions and address my minor suggestions.\nMajor suggestions:\nPlease revise the numbers in your flow diagram and add explanations (maybe in a legend) about your more important labels, such us ineligible population/comparison/… Regarding the numbers, please note that the box “records screened” has more records than the previous one. Or than 62 >< 28+7+13+7+6+1+1=63. Please also note that studies are not the same unit as articles. Please, unify.\n\nPlease consider to re-write your conclusions clarifying which recommendations could potentially improve quality in the future. I mean, recommendations to STRICTA authors; recommendations to journals; recommendations to reviewers, recommendations to authors; and so on.\n\nPlease, allow access to your data to other scientists. Please, consider a public repository.\n\nMinor suggestions:\nAs you stated, “the STRICTA guidelines are not a rating scale”. However, you are repeating through the text that you are attempting to “assess the quality of reporting”. I wonder if you should also highlight the need to further develop reporting guidelines to provide such a tool to assess the quality of reporting. Should you speak about the completeness of reporting? I definitively like much more your statement “evaluate the adherence to Stricta guidelines”.\n\nIn the introduction, I wonder if you should clarify that “risk of bias” applies to the study; and “quality of reporting” to the paper.\n\nAlso in the introduction, you use the terms \"replication\" and \"reproducibility\". I would like you to clarify your meanings, perhaps with some references. For example, are you following the definition from Goodman, Fannelly & Ioanidis in “What does research reproducibility mean?”1\n\nPlease, further specify how reviewers “worked in pairs”? Did they split the sample? Did both pairs of reviewers analyze some papers?\n\nPlease, consider to explain the pilot process and how did you “ensure” its usability.\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? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6026", "date": "21 Oct 2020", "name": "Carles Fernández", "role": "Author Response", "response": "We thank you for your time and comments on the manuscript. Please find below a point by point response to them: Major suggestions:Please revise the numbers in your flow diagram and add explanations (maybe in a legend) about your more important labels, such us ineligible population/comparison/… Regarding the numbers, please note that the box “records screened” has more records than the previous one. Or than 62 >< 28+7+13+7+6+1+1=63. Please also note that studies are not the same unit as articles. Please, unify.As it is already explained in the figure, one trial included two interventions, that is why the total number of studies included is 62 but the summation of all the interventions is 63.Studies has been changed for articles in the diagram Please consider to re-write your conclusions clarifying which recommendations could potentially improve quality in the future. I mean, recommendations to STRICTA authors; recommendations to journals; recommendations to reviewers, recommendations to authors; and so on.Conclusions have been re-written to include the mentioned recommendations“The quality of reporting of acupuncture interventions in COPD trials according to STRICTA guidelines is suboptimal. Strategies for improving the understanding of the guides for authors, reviewers and journal editors are needed, as well as to improve its implementation.” Please, allow access to your data to other scientists. Please, consider a public repository.The data is already public and available in the “data availability” section.Minor suggestions:As you stated, “the STRICTA guidelines are not a rating scale”. However, you are repeating through the text that you are attempting to “assess the quality of reporting”. I wonder if you should also highlight the need to further develop reporting guidelines to provide such a tool to assess the quality of reporting. Should you speak about the completeness of reporting? I definitively like much more your statement “evaluate the adherence to Stricta guidelines”.We agree on highlighting the need of developing a reliable tool for assessing the quality of reporting and this has been added in the discussion section.We would prefer not to change the nomenclature as the real the objective of the study is to assess the quality of reporting, and using the adherence to current reporting guidelines is the method to do so. We believe that introducing the term “completeness of the reporting” may confuse reader. In the introduction, I wonder if you should clarify that “risk of bias” applies to the study; and “quality of reporting” to the paper.This has been clarified in the introduction section“Risk of bias of bias of the studies is certainly a critical aspect in randomised control trials; however, the quality of reporting of the published papers is also a key point.” Also in the introduction, you use the terms \"replication\" and \"reproducibility\". I would like you to clarify your meanings, perhaps with some references. For example, are you following the definition from Goodman, Fannelly & Ioanidis in “What does research reproducibility mean?”1Here replication and reproducibility are used in a common language sense and the meaning is clarified at the end of the introduction section:“Therefore, to be able to replicate an acupuncture intervention in clinical practice or reproduce it in another trial, it is necessary to fully describe how it is applied.”We believe that using the terms “methods reproducibility”, “Results reproducibility” or “inferential reproducibility” might not clarify but confuse the reader. Please, further specify how reviewers “worked in pairs”? Did they split the sample? Did both pairs of reviewers analyze some papers?Every paper was analysed by two reviewers, this has been now clarified in the methods section:“Data from each trail was extracted independently by two reviewers (CFJ, MSM, MY and CHL) using a standardised data extraction form, and disagreements were solved by consensus.” Please, consider to explain the pilot process and how did you “ensure” its usability.This has been now clarified in the “data collection process” section.“This extraction table was tested with pilot data of 3 papers to solve disagreements on its understanding and ensure its usability.”" } ] } ]	1	https://f1000research.com/articles/9-226
https://f1000research.com/articles/9-1354/v1	20 Nov 20	{ "type": "Brief Report", "title": "Dried blood spots are an efficient blood sampling method for the detection of SARS-CoV-2 antibodies", "authors": [ "Juan Carlos Cassano", "Michael Reut", "Wolfgang Korte", "Michael Reut", "Wolfgang Korte" ], "abstract": "A novel coronavirus termed SARS-CoV-2 caused an outbreak in December of 2019 which has led to pandemic. Currently several serological diagnostic assays exist for the detection of SARS-CoV-2, which require the collecting of whole blood that brings about problems including the invasive nature of venepuncture, poor acceptance by patients and their storage and transportation. A more fast, efficient and less tedious method that allows mass blood sampling is necessary during a pandemic to quickly diagnose disease and obtain population serological data. Dried blood spot (DBS) sampling has been used for several decades for the accurate detection of viral specific antibodies and remains one the most convenient methods for obtaining serological data on exposed patients. Here we evaluate the use of DBS sampling on current viral serological assays including SARS-CoV-2. DBS samples were collected from six patients (five control and one positive for SARS-CoV-2 infection) and patient serum was extracted and tested blindly using commercially available antibody test kits for Coxiella burnetti, parvovirus B19 and SARS-CoV-2. The results demonstrate that antibodies recovered from DBS after elution are comparable to those found in serum, indicating that serological tests can be adapted to test DBS samples from patients using our modified protocol. Because DBS sampling is a much faster and cheaper method of sampling blood, this modification could therefore allow for potential nationwide testing for epidemiological studies.", "keywords": [ "SARS-CoV-2", "Coronavirus", "Dried Blood Spot", "Blood", "Patient", "Serology", "Sampling", "Antibody" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) was first reported in December, 2019 in Wuhan, China and on March 11 2020 it was declared a pandemic by the World Health Organization. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which shares up to 82% genome identity with SARS-CoV, which first caused an outbreak in 20031. It is characterized by fever, cough, malaise, shortness of breath and in severe cases pneumonia2.\n\nReliable serological data are in urgent need to guide treatment, infection control, epidemiological measures and vaccination. Whilst several serological assays are now available for antibody detection in the form of anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) kits, most of these require the collection of serum from whole blood. Whole blood collection can pose several problems in their collection, storage and transportation. The collection of whole blood requires venepuncture, which is an invasive procedure and can be poorly accepted by patients. Equipment requirements for the isolation of serum include syringes, tubes, centrifuges, refrigerators and freezers. After collection, whole blood requires maintenance at the site of collection with refrigerated storage and transportation from distant locations3.\n\nDried blood spot (DBS) sampling offers practical, clinical, and financial advantages as compared to conventional blood testing. Compared with standard venepuncture, the fingerstick or heelpricking sampling of capillary blood is easy to perform and relatively painless. It can be carried out by patients at home without a trained health care worker or medical laboratory, which is of particular advantage in settings with limited infrastructure. Utilizing capillary blood requires less sampling volume, negates the use of centrifuges or even basic laboratory equipment, and transportation is facilitated as it does not require refrigeration or high skill preventing contamination4.\n\nDBS has been used for the detection of antibodies to several viral disease including rubella, HIV, measles and hepatitis and has shown good correlation to serum samples when assayed with commercially available kits5–9. Further, DBS sampling allows for the detection of different immunoglobulin isotypes IgG, IgA, IgE and IgM5–8,10. For this reason, we decided to evaluate the use of DBS sampling on current viral serological assays for SARS-CoV-2. DBS samples were collected from six patients (five control and one positive for SARS-CoV-2 infection) and patient serum was eluted and tested blindly using commercially available antibody test kits for Coxiella burnetti, parvovirus B19 and SARS-CoV-2.\n\nOur results demonstrate that antibodies recovered from DBS after elution are comparable to those found in serum, indicating that commercially available serological assays can be adapted to test DBS samples from patients using our modified protocol. Adapting ELISA assays to DBS sampling may facilitate potential nationwide testing for epidemiological studies into SARS-CoV-2.\n\n\nMethods\n\nAccording to Swiss legislation, the Human Research Act is applicable to research on human diseases and on the structure and function of the human body. As this project is a quality control on the blood samples of six participants, it does not fall under the scope of research under Article 3 HFG and an assessment by an ethics committee was therefore deemed unnecessary by the Ethics Committee Of Eastern Switzerland. All patients gave both oral and written consent to participate in this study.\n\nPotential participants were identified within our institute and selected based on their medical history. DBS samples were collected by a medical laboratory technician from six adult patients (five control and one SARS-CoV-2 positive, as confirmed by PCR and antibody testing). Blood was collected through skin puncture twice over two separate weeks. Briefly, the skin was cleaned on the palmar side of the 4th finger on the left hand with 70% isopropyl alcohol. The skin was punctured using a single-use safety BD Microtainer contact activated lancet (Becton Dickinson, Dublin, Ireland) and the first drop of blood was wiped off. Blood was collected on to a Whatman 903 Protein Saver Card (GE Healthcare, UK). DBS were allowed to dry completely overnight at room temperature. Using a single-use 6mm biopsy punch (GSK Consumer Healthcare, UK), one spot was punched out from each DBS and transferred into a well of a 96-well plate using a single-use disposable tweezer (Megro, Wesel, Germany). This process was repeated for all punched spots. To elute serum, 250 µl elution buffer (1 X PBS, 0.05% Tween 20, 0.1% BSA) was placed in each well and the 96-well plate was placed on a rotator plate overnight at RT. Eluates were then collected and stored at 4°C for ELISA.\n\nDBS eluates were subjected to the following commercial immunoassay kits: Parvovirus B19 IgG (Kit number ESR122G, Serion Diagnostic, Germany), Coxiella burnetii Phase 2 IgG (Kit number ESR1312G, Serion Diagnostics), Anti-SARS-CoV-2-ELISA IgG and Anti-SARS-CoV-2-ELISA IgA (Kit numbers EI 2606-9601 G and EI 2606-9601 A, Euroimmun, Lübeck, Germany) and the EDI™ Novel Coronavirus COVID-19 IgG ELISA kit (Kit number KT-1032, Epitope Diagnostics, San Diego, USA) to detect the presence of antibodies. Because seroprevalence is high for parvovirus11 and low for Coxiella burnetii in Europe12,13, the parvovirus B19 IgG and Coxiella burnetti Phase 2 IgG ELISAs acted as positive and negative controls, respectively. All the listed commercial immunoassays in our study require a minimum of 10 µl serum sample to be diluted in a 1:100 dilution ratio with sample diluent at a final volume of 1000 µl. It has been reported that a 6mm punched spot contains approximately 5 µl of serum5. Therefore, sample preparation for the commercial ELISA required 2 x 250 µl patient sample eluates to be pooled (500 µl), to which 500 µl of sample diluent was added to make a final volume of 1000 µl.\n\nThe commercial ELISAs were run on a DSX® Automated ELISA Processing System (Dynex Technolgies, Denkendorf, Germany) according to the manufacturer’s instructions. A total of two DBS samples per patient was run for each commercial ELISA over two separate weeks. OD values for each DBS sample were determined at λ = 405 nm (λ = 405 nm reference wavelength) for Coxiella burnetii, λ =405 nm (λ = 650 nm reference wavelength) for parvovirus B19, λ = 450 nm (λ = 620 nm reference wavelength) for both anti-SARS-CoV-2 IgG and IgA, and λ = 450 nm (λ = 450nm reference wavelength) for EDI™ Novel Coronavirus COVID-19 IgG.\n\nELISA assays were performed on the DYNEX DSX, and analysed on DSXLab software (version 640.2, DYNEX Technologies GmbH, Denkendorf, Germany). OD values for all DBS samples were corrected using the average control serum sample giving a ratio, as described by the manufacturer. For the Coxiella burnetti ELISA, DBS samples registering a ratio of >1.1 were considered positive results, whilst ratios of <0.9 indicated negative results. For the parvovirus B19 ELISA, DBS samples registering a ratio of 5.01 indicated positive results whilst those registering a ratio of <2.99 were considered negative results. For the commercial anti-SARS-CoV-2 IgG and IgA ELISA kits from Euroimmun, DBS samples registering ratios of >1.1 indicated positive results, ratios of <0.8 indicated negative results, whilst ratios between 0.8 and 1.1 indicated borderline results. For the commercial EDI™ Novel Coronavirus COVID-19 IgG ELISA kit, DBS samples that resulted in ratios >1.1 indicated positive results, ratios of <0.9 indicated negative results, whilst ratios between 0.9 and 1.1 indicated borderline results.\n\n\nResults\n\nThe elution buffer (1 X PBS, 0.05% Tween 20, 0.1% BSA) was found to be effective at eluting serum from DBS samples. Because each 6mm punched spot contains approximately 5 µl of serum, and elution with 250 µl represented a 1:50 dilution of the DBS sample.\n\nAll six patient (A-F) DBS samples were tested for antibodies using the commercial ELISA kits parvovirus B19 (IgG), Coxiella burnetii Phase 2 (IgG), anti-SARS-CoV-2-ELISA (IgG and IgA) and the EDI™ novel coronavirus COVID-19 (IgG) ELISA kit. All DBS samples were positive for parvovirus B19 IgG antibodies, whilst negative for Coxiella burnetti IgG as expected (Table 1). DBS samples assayed on both the SARS-CoV-2 IgG from Euroimmun and Epitope Diagnostics were negative in control patients (A-E), whilst positive for the single patient (F) with a previous history of SARS-CoV-2 infection (Table 2, Table 3).\n\nCalculated ratios were used to determine serological status. For the Coxiella burnetti ELISA, positive results (pos) indicate a ratio >1.1, negative results (neg) indicate a ratio <0.9. For the parvovirus B19 ELISA, positive results (pos) indicate a ratio >5.01, negative results (neg) indicate a ratio <2.99. For each patient, DBS samples were taken and measured over two separate weeks.\n\nCalculated ratios were used to determine serological status. For both the SARS-CoV-2 IgG and IgA ELISA, positive results (pos) indicate a ratio >1.1, negative results (neg) indicate a ratio <0.8. Borderline results (gw) indicate a ratio between 0.8 and 1.1. For each patient, DBS samples were taken and measured over two separate weeks.\n\nCalculated ratios were used to determine serological status. Positive results (pos) indicate a ratio >1.1, negative results (neg) indicate a ratio <0.9. Borderline results (gw) indicate a ratio between 0.9 and 1.1. For each patient, DBS samples were taken and measured over two separate weeks.\n\nThe ELISA for IgA antibodies from Euroimmun indicated that the DBS sample from the SARS-CoV-2 patient (F) was positive for specific IgA antibodies (Table 2). Interestingly, several DBS samples from control patients displayed borderline positive results, whilst one DBS sample from one patient (E) with no apparent history of exposure to SARS-CoV-2 showed the presence of IgA antibodies (Table 2).\n\n\nDiscussion\n\nThe results that we have obtained serve to confirm the effectiveness of using DBS samples for the serological detection of antibodies, whilst extending the applicability of current commercial immunoassays for the detection of SARS-CoV-2. Our results are in accordance with the expected seroprevalence of Coxiella burnetii and parvovirus in Europe11–13. Of importance is that we were able to detect both IgG and IgA antibodies in the patient with a history of SARS-CoV-2 infection using the commercially available kits. This indicates that DBS sampling may be as reliable as serum sampling via venepuncture for the detection of antibodies to SARS-CoV-2. Although IgA was detected as both borderline and positive results in asymptomatic control patients which were thought to be negative for exposure to SARS-CoV-2, this observation may be due to several reasons. Borderline cross-reactivity has been reported for the anti-SARS-CoV-2-ELISA (IgA) from Euroimmun14. Samples negative for IgG and borderline or positive for IgA may also indicate prior exposure to SARS-CoV-2.\n\nThe advantages of adapting commercial immunoassays to DBS are many. Rapid diagnostic testing is necessary in limited-resource settings. DBS offers an affordable alternative to venepuncture. Because little training is required to sample blood using the DBS method, a greater number of the population can be sampled for epidemiological studies. DBS samples can be dried and stored at room temperature for several weeks, they require little storage space and can even be sent to testing sites via mail. Such sampling may be especially suitable in serological screening programs in developing countries, where SARS-CoV-2 is having its greatest impact. DBS sampling may further facilitate widespread sampling of capillary blood, enabling analysis with high-throughput laboratory-based immunoassays, which would allow rapid nationwide serological screening that is of absolute necessity for disease control during a pandemic.\n\nThe main disadvantage of DBS sampling is that most of the existing commercial immunoassays have not been validated or received the necessary regulatory approval for DBS sampling to be implemented. Despite this drawback, DBS sampling has shown promise, in that it has been successfully used to detect the presence of antibodies to several disease-causing viral pathogens including rubella, HIV, measles and hepatitis5–9. A meta-analysis on the diagnostic accuracy of HIV-antibody and HBV- surface antigen from DBS samples compared to venous blood samples was associated with excellent diagnostic accuracy9. However, the study found that the lack of standardization of sampling, handling, processing, storage and transportation processes limits their use. The use of DBS sampling for routine or high-throughput serological assays would therefore require a standardized method for the pre-analytical treatment of specimens and processing of the sample and subsequent validation with commercial assays.\n\nSeveral commercially available automated biopsy punch instruments exist that would facilitate the processing of DBS samples for high-throughput immunoassay application. Coupled with automated immunoassays platforms, automated biopsy punch instruments could facilitate the standardization of DBS technology and bring us one step closer to adapting DBS sampling to routine immunoassays. What remains a challenge is the standardization of the sampling protocol, the storage and transportation to a facility that would combine such a set-up.\n\n\nConclusions\n\nDBS sampling offers a promising alternative to serum sampling via venepuncture and can accurately detect antibodies to SARS-CoV-2. DBS sampling could therefore be adapted for future serological screening of large populations. Lack of standardization of sampling and testing limits the wider application of DBS.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nChan JFW, Kok KH, Zhu Z, et al.: Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect. 2020; 9(1): 221–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellini WJ, Helfand RF: The challenges and strategies for laboratory diagnosis of measles in an international setting. J Infect Dis. 2020; 187(Suppl.1): S283–S290. PubMed Abstract \| Publisher Full Text\n\nSnijdewind IJM, van Kampen JJA, Fraaij PLA, et al.: Current and future applications of dried blood spots in viral disease management. Antiviral Res. 2012; 93(3): 309–21. PubMed Abstract \| Publisher Full Text\n\nLillo F, Varnier OE, Mantia E, et al.: Detection of HIV-1 antibodies in blood specimens spotted on filter-paper. Bull World Health Organ. 1992; 70(3): 323–326. PubMed Abstract \| Free Full Text\n\nRiddell MA, Byrnes GB, Leydon JA, et al.: Dried venous blood samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay. Bull World Health Organ. 2003; 81(10): 701–7. PubMed Abstract \| Free Full Text\n\nMercader S, Featherstone D, Bellini WJ: Comparison of available methods to elute serum from dried blood spot samples for measles serology. J Virol Methods. 2006; 137(1): 140–9. PubMed Abstract \| Publisher Full Text\n\nHelfand RF, Cabezas C, Abernathy E, et al.: Dried blood spots versus sera for detection of rubella virus-specific immunoglobin M (IgM) and IgG in samples collected during a rubella outbreak in Peru. Clin Vaccine Immunl. 2007; 14(11): 1522–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLange B, Cohn J, Roberts T, et al.: Diagnostic accuracy of serological diagnosis of hepatitis C and B using dried blood spot samples (DBS): two systematic reviews and meta-analyses. BMC Infect Dis. 2017; 17(Suppl 1): 700. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndersen NJ, Mondal TK, Preissler MT, et al.: Detection of immunoglobulin isotypes from dried blood spots. J Immunol Methods. 2014; 404: 24–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMossong J, Hens N, Friedrichs V, et al.: Parvovirus B19 infection in five european countires; seroepidemiology, force of infection and maternal risk of infection. Epidemiol Infect. 2008; 136(8): 1059–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTobudic S, Nedomansky K, Poeppl W, et al.: Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucell abortus and Brucell Melitensis in Austrian adults: A cross- sectional survey among military personnel and civilians. Ticks Tick Borne Dis. 2014; 5(3): 315–7. PubMed Abstract \| Publisher Full Text\n\nvan Roeden SE, Holsboer EW, Oosterheert JJ, et al.: Seroprevalence of Coxiella burnetii antibodies and chronic Q fever among post-mortal and living donors of tissues and cells from 2010 to 2015 in the netherlands. Euro Surveill. 2018; 23(9): 17–00384. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeavis KG, Matushek SM, Abeleda APF, et al.: Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA assay for detection of IgA and IgG antibodies. J Clin Virol. 2020; 129: 104468. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "75137", "date": "15 Jan 2021", "name": "Livia Melo Villar", "expertise": [ "Reviewer Expertise Virology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes a method for detecting SARS CoV-2 antibodies in dried blood spot samples.\nThere are some issues that is not clear:\nWhy include Parvovirus and Coxiella testing in the paper?\n\nAuthors informed that there are 5ul of serum in each 6mm of paper disc, however the paper cited as reference indicates that 5mm of paper disc contains 10ul of serum. In addition, variation could happen according hematocrit.\n\nThe major issue is the few number of samples included for analysis since just one individual presented IgG anti-SARS CoV-2, for example. More samples should be tested to confirm the effectiveness of using DBS samples for the serological detection of SARS CoV-2.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "90319", "date": "11 Aug 2021", "name": "Davor Brinc", "expertise": [ "Reviewer Expertise Clinical chemistry" ], "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\nGeneral comments The manuscript reports a study of antibody detection against SARS-CoV-2 using DBS samples. Use of DBS for testing different analytes in blood, including testing of infectious diseases and serology, is an important and developing field. The topic is therefore relevant and would be of wide interest.\nThe main issue with reported study is very low n value; there is only one patient positive for SARS-CoV-2 which makes it difficult to draw conclusions about the performance of the particular methodology used in the study.\nSpecific comments: Method section: Patient selection is described as based on PCR and antibody testing. More details need to be given; which PCR test was used (which specific test, manufacturer, Ct/viral load if possible) on which sample type; which antibody testing (isotype, manufacturer, antigen) on which sample type. Also days since symptom onset relative to PCR and antibody testing as well as symptom description (mild/severe/hospitalized) are needed.\nFor DBS sampling, days since symptom onset and days since PCR and antibody testing sample collection are needed. More details about DBS elution would be helpful, for example, how many circles on the filter paper were spotted, how many total punches were made and then transferred to 96 well plate. Was 250 uL elution buffer used for one 6 mm punch, this seems to be suggested in the Results section, but perhaps could be described more clearly in the Methods section. For the cutoffs described for ELISAs, are the cutoffs the same as for plasma/serum as suggested by the manufacturer? Source of cutoffs should be clearly given.\nResults section For SARS-CoV-2 DBS results, comparison of signals obtained from corresponding matched plasma/serum using the same assays would be helpful.\nFor control samples showing IgA positivity, knowing the corresponding result from plasma/serum would be helpful.\nDiscussion section There are several published studies of DBS serology for SARS-CoV-2 using in-house developed ELISA, commercial ELISA, and commercial automated analyzers, including Euroimmun. Euroimmun also has specific dried blood spot application.\nClinical performance of DBS for SARS-CoV2 detection (e.g. sensitivity, specificity) reported by such studies should be discussed.\nDetection of IgA in control samples should be discussed in more detail based on previous Euroimmun assay performance. Specificity of Euroimmun assay (both IgG and IgA) based on published studies should also be discussed as any limited specificity could affect data interpretation.\nIn conclusion, while DBS results from the investigated COVID19+ patient does show likely ability of the proposed DBS methodology to detect positive serology, and this is in agreement with other DBS studies, it is difficult to make more general comments about the performance due to low n value.\n\nAt this stage, the study is basically a case report.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\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? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1354
https://f1000research.com/articles/9-349/v1	11 May 20	{ "type": "Opinion Article", "title": "Physical activity promotion in the age of climate change", "authors": [ "Karim Abu-Omar", "Peter Gelius", "Sven Messing", "Peter Gelius", "Sven Messing" ], "abstract": "The importance of the global climate crisis requires linking physical activity promotion and climate action. This article provides a first overview of interconnections between physical activity promotion and climate action, potential synergies and discrepancies, aiming to stimulate further discussion about this topic. The analysis is based on the World Health Organization’s Global Action Plan on Physical Activity 2018-2030 (GAPPA). The GAPPA covers five perspectives that are of particular relevance with respect to potential links with climate policy: (1) Infrastructures supporting active transport, (2) green spaces and recreational/exercise facilities, (3) exercise programs, (4) mass communication campaigns and mass participation events, and (5) training of professionals. Our analysis demonstrates a considerable alignment between strategies for physical activity promotion and efforts for the reduction of greenhouse gas emissions. However, in some of the areas, this alignment could still be improved. Additionally, more climate-conscious policies, research and surveillance need to be developed in the field of physical activity promotion.", "keywords": [ "physical activity", "sport", "active transport", "exercise", "training", "recreation", "climate change", "climate action" ], "content": "Introduction\n\nIn recent years, there has been a new wave of recognition regarding the urgency of the global climate crisis. The Intergovernmental Panel on Climate Change (IPCC) has clearly stated that the next ten years will be crucial for limiting global warming to less than 1.5 degrees Celsius1. Studies by the World Bank predict the devastating impact global warming will have on development of poor countries2 and consequently on migration3. The IPCC has also reported on the dramatic implications of climate change on land use4. Nonetheless, the United Nations have vividly described the acute failure to curb global greenhouse gas emissions5. In light of these sobering prospects, a number of American public health organizations have called on governments, business leaders and civil society to treat climate change as a “health emergency”6. Also, there have been calls from scientists to engage in civil disobedience as a mean to put pressure on world leaders to step up efforts to combat the climate crisis7.\n\nLikewise, physical activity (PA) promotion has evolved significantly in the past decades and has asserted itself as a stand-alone public health issue8. This development was triggered by a robust understanding of the role of PA for the prevention of non-communicable diseases9 on the one hand and stubbornly high rates of physical inactivity in adolescents10 and adults11 on the other. PA rates in children are higher but still leave room for improvement12. The policy response has resulted, among others, in the PA Strategy for the World Health Organization (WHO) European Region 2016–2025 (EuroPAS;13), the Global Action Plan on PA 2018–2030 (GAPPA;14), the EU Council Recommendation on Health-Enhancing PA across Sectors15, as well as numerous strategies, action plans, and recommendations at the national level16–18.\n\nBut are efforts to promote PA in any way linked to the climate crisis, and if so, what are these links? Will the climate crisis dampen efforts for PA promotion or, to the contrary, are there potential synergies, with PA promotion potentially supporting efforts to combat climate change? What is the carbon footprint of different strategies currently employed or suggested to promote PA? What are potential upcoming research priorities in our field that relate to the climate crisis? These questions are highly relevant given the urgency of climate change and the need to prioritize limited resources. The global-level GAPPA acknowledges this by including a link to the United Nation’s Sustainable Development Goals (SDGs), including SDG 13 on climate action. However, the link between PA promotion and climate change plays a rather minor role in the GAPPA and, in our opinion, still needs to be further explored. This article is an attempt to provide a first overview of the available information and stimulate further discussion about these topics.\n\n\nInterconnections between physical activity promotion and the climate crisis\n\nThe recent strategies developed by WHO to support national-level PA promotion provide a good overview of the most common action areas currently proposed. For example, the PA Strategy for the European Region13 employs a life-course approach with four major action areas (leadership and coordination; children and adolescents; adults; older people; monitoring, surveillance, evaluation and research) and 14 specific objectives. The global-level GAPPA14 builds extensively on this earlier strategy and has very similar key messages, but it uses a slightly different structure with four strategic objectives (active societies; active environments; active people; active systems) and 20 specific policy actions. The overview provided in the next sections is based on this most recent WHO policy document, taking a closer look at its different policy actions using five perspectives that bear particular relevance for climate change.\n\nSeveral actions recommended by the GAPPA14 in the strategic objective “Create active environments” stress the need for governments to develop highly connected mixed-land-use neighborhoods (Action 2.1), create infrastructures to increase active transport by walking and cycling as a means of PA promotion (Action 2.2), and improve road safety (Action 2.3).\n\nIt will be evident to most readers that these recommendations line up extremely well with actions to promote sustainable mobility proposed elsewhere. For example, the UN Economic Commission for Europe has stated that in many nations, more than 30% of final energy (i.e. energy consumed by end users such as households, industry and agriculture) is consumed in transport19. The report concludes that cities striving to become carbon neutral should develop a comprehensive cycling/walking infrastructure and better integrate working, shopping and entertainment opportunities19. Also, the global “C40 Cities Climate Leadership Group” showcases cities around the world that strive to become carbon neutral by supporting active travel20. Cities such as Copenhagen have demonstrated that it is possible to increase the modal share of walking and cycling (currently 45% of all trips) beyond the share of car use (34%)20.\n\nIn order to do so, however, more concerted action involving public health policymakers, practitioners and researchers on the one side and urban/transport planners on the other would be needed. Environmental and climate activists are another important asset in taking a strong stance against motorized transport. The GAPPA recommends to use and further develop the WHO Health Economic Assessment Tool for Cycling and Walking (HEAT)21 to enable an assessment of the health, climate and environment-related benefits of transport and urban design policies (Appendix 2 of GAPPA). On top of that, we would recommend promotion of good practice examples of cities that are already shifting from motorized traffic to active travel such as Copenhagen or Amsterdam. The direct public health burden of motorized transport may serve as an important supporting argument: Globally, there are 1.36 million deaths due to road fatalities22 and 3.2 million deaths related to ambient air pollution every year23.\n\nAnother area of PA promotion with obvious climate connections relates to green spaces and recreational/exercise facilities. The GAPPA addresses these in the context of the strategic objective “Create active environments” (Actions 2.4, and 2.5).\n\nBesides binding carbon dioxide, parks and green spaces play an important role in climate-proofing cities by mitigating urban heat islands24. It has been shown that, in some cities, an additional square kilometer of green space per 1,000 people might prevent up to 7.4 deaths caused by heat25 and that green spaces can directly and positively influence people’s health26. In these areas, public health and PA promotion objectives to create more green spaces align very closely with objectives for counteracting climate change. Some cities have already taken steps to expand green spaces by planting trees, such as Manchester27, or by increasing inner-city vegetation as an important part of climate adaptation, such as Copenhagen28.\n\nBy contrast, the “climate case” for other types of recreational and exercise facilities is far less clear. Our knowledge about the carbon footprints of such sites is currently limited. One study by Boussabaine et al.29 has shown that heated indoor-pools have a much higher energy consumption per square meter and year (1,250–1,750 kWh) than indoor gyms (210–350 kWh). Likewise, the use of fertilizers on the turf grass surfaces needed for many outdoor sports (e.g. football and cricket) causes high nitrogen dioxide emissions when compared to regular pastures, making these compounds a substantial contributor to land consumption30.\n\nIn order to better align efforts for PA promotion in this area with those for limiting global warming, the GAPPA recommends implementing assessments of public and green open spaces and natural spaces to evaluate health, climate and environmental benefits of urban ecosystems (Appendix 2 of GAPPA). We suggest that, additionally, experts should engage with the urban planning and transport sectors to ensure that parks and green spaces are built in close proximity to people’s homes and are easily accessible by active and public transport. Regarding recreational and exercise facilities, land-use and carbon footprint need to be considered in the planning of new facilities and the decision to maintain existing ones. One might also need to reconsider whether new outdoor facilities for sports that require vast land use (e.g. golf, baseball) should be build. Generally, facilities that require extensive heating or cooling are likely to have a higher carbon footprint, i.e. indoor facilities will tend to be less climate-friendly than outdoor facilities. Therefore, newly built indoor infrastructures should preferably be multi-purpose and/or be able to contribute to energy production, e.g. via roof-mounted solar panels.\n\nWhile many health promoters and researchers are likely to be aware of the potential links between active transport, PA facilities and climate change, other areas of PA promotion may have rarely been considered with respect to their effects on speeding up or slowing down global warming. Importantly, this applies to GAPPA’s strategic objective “Create active people”: Actions 3.3, 3.4, 3.5, and 3.6 recommend the implementation of PA programs and services tailored to different target groups, across different settings, and supported in co-development by all stakeholders and grassroots initiatives.\n\nTo our knowledge, there has been very little research on the carbon footprint of different sport and exercise programs. A recent study by Wicker31 has investigated the travel behavior (e.g. for training, league games, day trips) for German athletes in 20 different sports. The results indicate average carbon emissions of 844 kg per person and year, with stark variation across different sports. Most individual sports, such as climbing/bouldering (1,156 kg CO2) and surfing (2,074 kg) have higher emissions than team sports such as soccer (337 kg). However, there are also some individual sports (e.g. fitness/gym with 228 kg) with a comparably small carbon footprint. The study found some nature sports, such as alpine skiing, to have a particularly high carbon footprint31. The environmental impact of these sports has also been pointed out by previous studies32,33.\n\nFuture efforts for PA promotion through services and programs should consider carbon emissions. Partly, these emissions are caused by motorized travel to and from such offers, and partly also by the energy consumption of the facilities they take place in. To minimize motorized travel, offers and programs should be easily reachable by active and public transport. Offers should be attractive to multiple target groups, which will help increase local participation and, as a consequence, shorten the distances that teams need to travel for away matches.\n\nTo minimize emissions, activities using facilities with comparably low carbon emissions should be promoted with priority (e.g. outdoor Zumba rather than water aerobics in a heated indoor pool). Respecting the seasonality of activities (e.g. winter sports) will certainly help in this regard. If (recreational) league play is involved, strong efforts should be made to limit (motorized) travel. This might be achieved by encouraging tournaments involving multiple teams in a single location rather than round-robin series of individual home/away matches. Where possible, events should be scheduled during daylight hours to avoid the need for artificial lighting.\n\nAnother core element of many policy recommendations to promote PA, including GAPPA’s strategic objective “Create active societies”, are campaigns and events. Actions 1.1 and 1.2 recommend communication campaigns to inform the public about the multiple benefits of PA, and action 1.3 suggests community mass events for PA.\n\nMass communication campaigns that stress the environmental benefits of active transport line up extremely well with objectives for the reduction of carbon emissions. Potentially, future campaigns should place even more emphasis on promoting walking and cycling as an important means for health and environmental benefits. Additionally, they should also (where appropriate) include advice on being physically active in hot weather. There is evidence, particularly for vulnerable groups such as older people, that the weather influences PA patterns34 and that PA in hot weather can have detrimental health effects35. Campaigns should also highlight sporting activities that have a comparably low carbon footprint. Potentially, due to the substantial environmental impact of sports apparel production36, campaign messages could also emphasize that the latest sportswear is no prerequisite for becoming physically active.\n\nBy contrast, using mass events as a means of PA promotion might be rather a double-edged sword from an environmental perspective. The high carbon footprint of professional events such as the Football World Cup has already been described37, while their presumed ‘festival effect’ on PA behavior seems to be very limited38. Comparable research on amateur mass events or those in which recreational athletes compete alongside professionals is scarce, but there is reason to believe that events such as major marathons also come with considerable carbon emissions. Most of these emissions will be caused by participants’ (air) travel to the venue – for example, around 47% of finishers of the New York Marathon in the 2010s were international participants39.\n\nTaking this into consideration, mass events at the community level should be within easy reach of public and active transport in order to limit the climate impact. Organizers of such events might also need to balance the number of participants from other regions or nations and potentially even consider downsizing. Additionally, events would need to be organized in a way that limits detrimental environmental effects, e.g. by avoiding plastic-bottled water, reducing overall waste, offering vegetarian/vegan food options, and limiting free giveaways with high carbon footprints such as t-shirts. As a source of inspiration, the Canadian Sport Tourism Alliance has issued guidelines on how to host sustainable sport events40.\n\nIn its strategic objectives “Create active societies” and “Create active people”, the GAPPA advises countries to invest in the training of professionals for PA (Action 1.4), in particular but not limited to (physical education, PE) teachers (Action 3.1) and health professionals (Action 3.2).\n\nResearch indicates that professionals’ training on PA in general leaves much room for improvement41, so that cross-references between PA and climate in curricula can be expected to be even less frequent. A look at current training standards for exercise prescription, such as the ACSM Guidelines42, shows that health professionals are provided with information on how to advise patients on PA in hot environments. However, patient advice regarding the environmental benefits of walking and cycling are currently not covered, not to mention guidance regarding activities that come with a comparably low carbon footprint. Implementing such information into future training of health professionals will more closely align PA promotion with objectives for the reduction of greenhouse gas emissions.\n\nBy and large, the same can be said to hold true for the training of PE teachers. In many countries, the climate impact of different exercises and sport tourism might not be explicitly covered. For example, the PE curriculum for secondary schools in the German state of Bavaria requires all pupils to learn winter sports43, which is consequently an integral part of the university curriculum for PE teacher education44. Both schools and universities often teach the required competences through practical alpine or cross-country skiing courses in the Alps. Studies predict the closure of many ski resorts in the Alps due to lack of snow unless massive amounts of artificial snow are used on a regular basis45, thus causing high carbon emissions33. Consequently, re-considering the types of sports and exercise that are taught as part of PE in schools (and by extension in PE teacher training) might be warranted. Preferably, these should be sports/exercises that can be performed locally or regionally, have a low carbon footprint, and require little land-use.\n\n\nImplications for future PA promotion\n\nOur cursory analysis has demonstrated a considerable potential for alignment between strategies for PA promotion and efforts for the reduction of greenhouse gas emissions. The GAPPA acknowledges the importance of this topic by referring to the SDG on climate action. However, the link between climate action and PA promoting policies/interventions remains unspecific as it is limited to a few sentences. Our analysis shows that these links are already widely acknowledged in some action areas of the GAPPA, most notably in the field of active transport (strategic objective “create active societies”) and regarding green spaces and (at least in part) recreational/exercise facilities (strategic objective “create active environments”). In other areas, such as media campaigns, mass events, and professional training, however, the alignment of PA promotion with efforts to reduce carbon dioxide emissions could still be improved.\n\nIn order to actually give PA promotion a new, more climate-conscious outlook, changes will be required in PA policymaking, research and surveillance As policy actions, the GAPPA proposes health economic assessments of health, climate and environmental benefits in the areas of active transport and urban design (Action 2.1) and public and green open spaces and natural spaces (Action 2.4). However, these recommendations are just mentioned in the appendix and are not an adequate substitute for a more systematic look at climate-conscious policy development, research and surveillance. GAPPA’s strategic objective “Create Active Systems” that targets the governance of PA promotion (without linking it to climate change) served as a basis for our following recommendations (Actions 4.1-4.5).\n\nFor one, it will be important to raise awareness for the potential benefits and hazards of sport and PA for the climate among policymakers across all levels of government, the media, the private sector, and community leaders (Action 4.4). This does not only pertain to the fields of health and sport but also to other relevant sectors such as transport, environment, urban design, tourism, and social care. The transport sector, in particular, has a huge potential for devising policies that combine increased health/quality of life with a reduction in carbon dioxide emissions. Environmental protection groups and climate initiatives should also be considered as potential allies for PA promotion.\n\nAs also advocated by the GAPPA (Action 4.1), a logical next step is to increase the integration of the two issues in policy frameworks, leadership structures and governance systems, e.g. via multisectoral coordination mechanisms. Historically, both the health and the environmental sector have called for mainstreaming their concerns into all sectors of government – one in the form of “Health in all policies”46, the other under the moniker of “Environmental policy integration”47. In practice, however, there still seems to be room for improvement: data on the implementation of the EU Council Recommendation for Health-Enhancing PA across Sectors indicate that the transport and environment sector are integrated into cross-sectoral PA coordination mechanisms in only 17 out of 27 participating EU Member States48.\n\nAnother important action area (Action 4.5) in this context are financing mechanisms. In the future, it will be important to further integrate health and environment-related funding lines to ensure sustainable financial support for activities that promote both the climate and population-level PA. Yet again, data from the EU indicate that it is difficult for governments to even collect information on investments made in sectors other than health that may also help promote PA, be it as an intended or unintended side effect, let alone to act towards a better coordination of sectoral funding49.\n\nResearch and surveillance (Actions 4.2 and 4.3) are covered as important cornerstones for future PA policy in the GAPPA. However, with respect to climate change, there are a number of key issues in these areas that should be urgently tackled, including the following:\n\n• Understanding the environmental impact of various forms of sport and exercise in order to tailor promotion efforts towards those with a low carbon footprint.\n\n• Identifying ways to limit the carbon footprint of sport tourism.\n\n• Developing curricula to integrate knowledge on the interconnections of PA promotion and climate protection in the training of health and other professionals.\n\n• Increasing our understanding of how to succeed in transitioning neighborhoods and communities towards high rates of active transport.\n\nImportantly, current PA surveillance systems should strive to integrate indicators that have a high relevance for climate change. This could mean that physical activity questionnaires should include dedicated measures for walking and biking, thus enabling governments to monitor changes in active transport behavior more accurately. Currently-utilized questionnaires often either only assess walking (such as the International Physical Activity Questionnaire IPAQ,50) or walking and cycling in a combined indicator (Global Physical Activity Questionnaire GPAQ51). A positive exception is the relatively new European Health Interview Survey Physical Activity Questionnaire EHIS52, which does assess walking and biking via separate indicators.\n\nThe same holds true for PA policy monitoring. As mentioned above, the regular joint EU/WHO surveys on the implementation of the EU Council Recommendation for Health-Enhancing PA across Sectors48,53 already provide information on a set of highly useful indicators such as the level of cycling and walking, and other supplementary WHO tools (such as the HEPA Policy Audit Tool54, and the HEAT Tool for the health economic assessment of cycling and walking55) may help countries gather additional data. In the years ahead, efforts to strengthen PA policy monitoring and to potentially integrate it with similar efforts in the field of transport, environmental and climate policy should be stepped up.\n\n\nConclusions\n\nThere are several interconnections between PA promotion and climate action. While they are most recognizable with regards to active transport, green spaces and – partially – recreational or exercise facilities, these links could be strengthened in other areas (media campaigns, mass events, professional training). Climate-conscious policy development, research and surveillance are needed in the field of PA. Recognizing the close alignment between PA promotion and climate action is an important message for public health professionals and policymakers.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nRogelj J, Shindell D, Jiang K, et al.: Mitigation Pathways Compatible with 1.5°C in the Context of Sustainable Development. 2018. Reference Source\n\nWorld Bank: World development Report 2010. Development and climate change. 2010. Reference Source\n\nWorld Bank: Groundswell. Preparing for internal climate migration. 2018. Reference Source\n\nIPCC: Climate Change and Land: an IPCC special report on climate change, desertification, land degradation, sustainable land management, food security, and greenhouse gas fluxes in terrestrial ecosystems. [P. R. Shukla, J. Skea, E. Calvo Buendia, V. Masson-Delmotte, H.-O. Pörtner, D. C. Roberts, P. Zhai, R. 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Publisher Full Text\n\nBreda J, Jakovljevic J, Rathmes G, et al.: Promoting health-enhancing physical activity in Europe: Current state of surveillance, policy development and implementation. Health Policy. 2018; 122(5): 519–527. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGelius P, Rütten A, Goodwin L, et al.: Study on the implementation of the European Physical Activity Guidelines. Luxembourg: Publications Office of the European Union; 2016. Reference Source\n\nIPAQ: International Physical Activity Questionnaire. 2020. Reference Source\n\nWHO: Global Physical Activity Surveillance. 2020. Reference Source\n\nFinger JD, Tafforeau J, Gisle L, et al.: Development of the European Health Interview Survey - Physical Activity Questionnaire (EHIS-PAQ) to Monitor Physical Activity in the European Union. Arch Public Health. 2015; 73: 59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Physical activity country factsheets 2018. 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Reference Source" }	[ { "id": "63281", "date": "21 May 2020", "name": "Kevin Lanza", "expertise": [ "Reviewer Expertise climate change", "physical activity", "health equity", "urban heat island adaptation" ], "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 timely and clearly written article, the authors describe the relation between physical activity promotion and climate change, and do so through the World Health Organization’s Global Action Plan on Physical Activity 2018-2030 (GAPPA). From the four strategic objectives (i.e., create active societies, environments, people, and systems) of GAPPA and each objective’s proposed policy actions, the authors offer five perspectives that connect physical activity to climate change: 1) development of infrastructures to enable active transport, 2) green spaces and recreational/exercise facilities to promote PA, 3) physical activity and exercise programs, 4) mass communication campaigns and mass participation events, and 5) training of professionals. The authors conclude with the implications of their overview for future PA promotion, in particular on climate-conscious 1) policy development for PA promotion and 2) PA research and surveillance.\nI commend the authors for this article because the link between physical activity and climate change has not been adequately addressed in the literature, and there is potential for public health and environmental sectors in local government to partner on the topic. Before sharing my thoughts on specific sections of the article, I have a few overall comments. The article would benefit from a table that includes each of the GAPPA objectives and a summary of each of the policy actions. This way, readers can refer to GAPPA while the authors share their five perspectives. Second, there are several impacts of greenhouse gas emissions on our climate (e.g., rising temperatures, increase in extreme weather events, change in precipitation patterns, and sea-level rise), but the authors seem to address only rising temperatures and its relation to physical activity promotion. Similarly, the authors focus their perspectives on physical activity and climate change mitigation (i.e., opportunities for the reduction of greenhouse gas emissions), yet less attention is given to climate change adaptation and resilience. Lastly, the authors should review their article for minor issues with grammar (e.g., inconsistent use of the Oxford comma, “the GAPPA” versus “GAPPA,” and “build” versus “built”) and language choice (e.g., “extremely well” and “less climate-friendly”).\nPlease see below for my comments specific to select sections.\nIntroduction The authors state that there are “… stubbornly high rates of physical inactivity in adolescents and adults…” and “PA rates in children are higher but still leave room for improvement.” Both of these statements can be refined. There is a difference between physical inactivity (i.e., sedentary behavior) and not being sufficiently active (i.e., failing to reach recommended levels of physical activity for health benefits). This distinction is important, because although children may have higher PA rates than adults (which depends on method of surveillance and individual-level factors), children in the US are recommended to engage in more PA each week than adults. The authors can be more precise here, and include values.\nDevelopment of infrastructures to enable active transport The authors focus their overview of active transport on the reduction of greenhouse gas emissions and the benefits to human health. The authors conclude this section with a statement on the health burden of motorized transport—I am wondering if the authors can dig deeper into the literature here. Can the authors tease out how many road fatalities involve a non-motor vehicle (e.g., pedestrian and bicycle)? Similarly, can the authors cite research that has modeled the amount of lives saved, injuries prevented, and pollution reduced from switching from vehicular transport to alternative modes?\nGreen spaces and recreational/exercise facilities to promote PA The authors focus their overview of green spaces on carbon sequestration, urban heat island mitigation, and climate adaptation; and their overview of recreational/exercise facilities on energy and land consumption. I think this section can be improved by providing further detail on each of the above points. Regarding green spaces, I would mention the amount of cooling offered by parks, which has been found to be 0.94°C, on average:\nBowler, D. E., Buyung-Ali, L., Knight, T. M., & Pullin, A. S. (2010). Urban greening to cool towns and cities: A systematic review of the empirical evidence. Landscape and urban planning, 97(3), 147-1551.\nThe specific vegetation is also worth mentioning, in relation to how trees and non-tree vegetation have different effects on physical activity levels of different populations; and how climate change is impacting what trees are able to be planted in an area with continued global warming:\nLanza, K., Stone Jr, B., & Haardörfer, R. (2019). How race, ethnicity, and income moderate the relationship between urban vegetation and physical activity in the United States. Preventive medicine, 121, 55-612.\nLanza, K., & Stone Jr, B. (2016). Climate adaptation in cities: What trees are suitable for urban heat management?. Landscape and Urban Planning, 153, 74-823. For recreational/exercise facilities, the authors can mention outdoor fitness classes/groups/meet-ups, which do not have the energy use requirements (e.g., air conditioning and lighting) of indoor facilities.\nPhysical activity and exercise programs The authors focus their overview of exercise programs on the reduction of greenhouse gas emissions and the benefits to human health. The authors state “…outdoor facilities with comparably low carbon emissions should be promoted with priority (e.g. outdoor Zumba rather than water aerobics in a heated indoor pool).” This recommendation also relates to the authors’ previous perspective on recreational/exercise facilities. In organizing the article’s content, the authors may want to consider green spaces as a separate perspective, and recreational/exercise facilities and exercise programs as another perspective.\nMass communication campaigns and mass participation events The authors focus their overview of mass communication and participation on the reduction of greenhouse gas emissions and the benefits to human health. The authors raise the pertinent point that weather conditions may influence physical activity patterns. However, most studies show temperature to have a positive correlation with physical activity, with individuals being more active during summer over other seasons:\nChan, C. B., & Ryan, D. A. (2009). Assessing the effects of weather conditions on physical activity participation using objective measures. International journal of environmental research and public health, 6(10), 2639-26544.\nIn recent work, US adults have been found to not modify their physical activity behavior on days with high temperatures, which suggests cities in hot climates may be placing adults at increased risk of exertional heat illness. The authors recommend incorporating the risk of exertional heat illness in health impact assessments and deploying heat management strategies (i.e., tree planting and installation of reflective building materials):\nLanza, K., Stone, B., Chakalian, P. M., Gronlund, C. J., Hondula, D. M., Larsen, L., ... & Haardörfer, R. (2020). Physical Activity in the Summer Heat: How Hot Weather Moderates the Relationship Between Built Environment Features and Outdoor Physical Activity of Adults. Journal of Physical Activity and Health, 1(aop), 1-95.\nThe authors point about the environmental impact of sports apparel production seems tangential to physical activity promotion and climate change, and therefore may be removed. Instead, I believe this article section would benefit from including content on the increased risk of exertional heat illness from projected temperature increases. The authors could also include content on how weather service providers, such as the National Weather Service in the United States, can incorporate alerts about unsafe physical activity conditions within its heat products (e.g., heat advisory and excessive heat warning).\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6098", "date": "20 Nov 2020", "name": "Sven Messing", "role": "Author Response", "response": "We would like to thank the reviewer for his valuable feedback. We have addressed his comments by modifying our article in the respective sections as described below. Q1: In this timely and clearly written article, the authors describe the relation between physical activity promotion and climate change, and do so through the World Health Organization’s Global Action Plan on Physical Activity 2018-2030 (GAPPA). From the four strategic objectives (i.e., create active societies, environments, people, and systems) of GAPPA and each objective’s proposed policy actions, the authors offer five perspectives that connect physical activity to climate change: 1) development of infrastructures to enable active transport, 2) green spaces and recreational/exercise facilities to promote PA, 3) physical activity and exercise programs, 4) mass communication campaigns and mass participation events, and 5) training of professionals. The authors conclude with the implications of their overview for future PA promotion, in particular on climate-conscious 1) policy development for PA promotion and 2) PA research and surveillance. I commend the authors for this article because the link between physical activity and climate change has not been adequately addressed in the literature, and there is potential for public health and environmental sectors in local government to partner on the topic. Before sharing my thoughts on specific sections of the article, I have a few overall comments. The article would benefit from a table that includes each of the GAPPA objectives and a summary of each of the policy actions. This way, readers can refer to GAPPA while the authors share their five perspectives. Second, there are several impacts of greenhouse gas emissions on our climate (e.g., rising temperatures, increase in extreme weather events, change in precipitation patterns, and sea-level rise), but the authors seem to address only rising temperatures and its relation to physical activity promotion. Similarly, the authors focus their perspectives on physical activity and climate change mitigation (i.e., opportunities for the reduction of greenhouse gas emissions), yet less attention is given to climate change adaptation and resilience. Lastly, the authors should review their article for minor issues with grammar (e.g., inconsistent use of the Oxford comma, “the GAPPA” versus “GAPPA,” and “build” versus “built”) and language choice (e.g., “extremely well” and “less climate-friendly”). Please see below for my comments specific to select sections. A1: Thank you very much for your positive comment. The link between physical activity and climate change has indeed a high potential for joint action of the public health and environmental sector. As suggested, we added a table that provides an overview about all GAPPA objectives and policy actions (table 1). Furthermore, we added a sentence at the end of the introduction to clarify that the article focuses on climate change mitigation and the relation of rising temperatures and physical activity promotion. Additionally, we described this focus as a limitation in the discussion section and recommended future research to address these aspects. The minor issues with grammar and language choice were addressed as well. Introduction Q2: The authors state that there are “… stubbornly high rates of physical inactivity in adolescents and adults…” and “PA rates in children are higher but still leave room for improvement.” Both of these statements can be refined. There is a difference between physical inactivity (i.e., sedentary behavior) and not being sufficiently active (i.e., failing to reach recommended levels of physical activity for health benefits). This distinction is important, because although children may have higher PA rates than adults (which depends on method of surveillance and individual-level factors), children in the US are recommended to engage in more PA each week than adults. The authors can be more precise here, and include values. A2: Thank you very much for this helpful comment. We have refined the first statement and included values. We state now that there are “… stubbornly high rates of insufficient physical activity in adolescents (81,0%) and adults (27,5%). …”. We removed the second statement. Development of infrastructures to enable active transport Q3: The authors focus their overview of active transport on the reduction of greenhouse gas emissions and the benefits to human health. The authors conclude this section with a statement on the health burden of motorized transport—I am wondering if the authors can dig deeper into the literature here. Can the authors tease out how many road fatalities involve a non-motor vehicle (e.g., pedestrian and bicycle)? Similarly, can the authors cite research that has modelled the amount of lives saved, injuries prevented, and pollution reduced from switching from vehicular transport to alternative modes? A3: We agree with this suggestion and added additional information on the number of deaths per year due to road fatalities among pedestrians and cyclists (26% of 1.36 million) and on the health benefits of shifting from car to bicycling (1,300 Euro per person and year for a commute of 5 km per day, effects of pollution and accidents are much smaller). Green spaces and recreational/exercise facilities to promote PA Q4: The authors focus their overview of green spaces on carbon sequestration, urban heat island mitigation, and climate adaptation; and their overview of recreational/exercise facilities on energy and land consumption. I think this section can be improved by providing further detail on each of the above points. Regarding green spaces, I would mention the amount of cooling offered by parks, which has been found to be 0.94°C, on average: Bowler, D. E., Buyung-Ali, L., Knight, T. M., & Pullin, A. S. (2010). Urban greening to cool towns and cities: A systematic review of the empirical evidence. Landscape and urban planning, 97(3), 147-1551. The specific vegetation is also worth mentioning, in relation to how trees and non-tree vegetation have different effects on physical activity levels of different populations; and how climate change is impacting what trees are able to be planted in an area with continued global warming: Lanza, K., Stone Jr, B., & Haardörfer, R. (2019). How race, ethnicity, and income moderate the relationship between urban vegetation and physical activity in the United States. Preventive medicine, 121, 55-612. Lanza, K., & Stone Jr, B. (2016). Climate adaptation in cities: What trees are suitable for urban heat management?. Landscape and Urban Planning, 153, 74-823. For recreational/exercise facilities, the authors can mention outdoor fitness classes/groups/meet-ups, which do not have the energy use requirements (e.g., air conditioning and lighting) of indoor facilities. A4: We added additional information regarding the cooling effect of parks (Bowler et al 2010) and the influence of trees on physical activity levels (Lanza et al 2019). This evidence strengthens the argument and is highly appreciated. We did not, however, add the study that analyses what trees are suitable for urban heat management because we felt that it is not closely related to our line of argument. Outdoor fitness classes are mentioned in the next section (physical activity and exercise programs). Physical activity and exercise programs Q5: The authors focus their overview of exercise programs on the reduction of greenhouse gas emissions and the benefits to human health. The authors state “…outdoor facilities with comparably low carbon emissions should be promoted with priority (e.g. outdoor Zumba rather than water aerobics in a heated indoor pool).” This recommendation also relates to the authors’ previous perspective on recreational/exercise facilities. In organizing the article’s content, the authors may want to consider green spaces as a separate perspective, and recreational/exercise facilities and exercise programs as another perspective. A5: We added the following sentence to the first paragraph of this section: “This aspect is strongly related to the previous one, as the type of PA and exercise programs that can be offered depends on the availability of green spaces and recreational/exercise facilities.” We decided not to reorganize the article’s overarching structure because we believe that this would not have solved the issue completely: Physical activity and exercise programs usually take place either in green spaces or in recreational/exercise facilities. There is an overlap between the GAPPA’s strategic objectives “create active environments” and “create active people”. Mass communication campaigns and mass participation events Q6: The authors focus their overview of mass communication and participation on the reduction of greenhouse gas emissions and the benefits to human health. The authors raise the pertinent point that weather conditions may influence physical activity patterns. However, most studies show temperature to have a positive correlation with physical activity, with individuals being more active during summer over other seasons: Chan, C. B., & Ryan, D. A. (2009). Assessing the effects of weather conditions on physical activity participation using objective measures. International journal of environmental research and public health, 6(10), 2639-26544. In recent work, US adults have been found to not modify their physical activity behavior on days with high temperatures, which suggests cities in hot climates may be placing adults at increased risk of exertional heat illness. The authors recommend incorporating the risk of exertional heat illness in health impact assessments and deploying heat management strategies (i.e., tree planting and installation of reflective building materials): Lanza, K., Stone, B., Chakalian, P. M., Gronlund, C. J., Hondula, D. M., Larsen, L., ... & Haardörfer, R. (2020). Physical Activity in the Summer Heat: How Hot Weather Moderates the Relationship Between Built Environment Features and Outdoor Physical Activity of Adults. Journal of Physical Activity and Health, 1(aop), 1-95. A6: We clarified this section and added both references: “Even though most studies indicate that temperature has a positive correlation with the PA behavior of children and adults, and adults have been found not to modify their PA behaviour on days with high temperatures, this topic is of particular relevance for vulnerable groups. There is evidence for older people, that the weather influences PA patterns and that PA in hot weather can have detrimental health effects”. Q7: The authors point about the environmental impact of sports apparel production seems tangential to physical activity promotion and climate change, and therefore may be removed. Instead, I believe this article section would benefit from including content on the increased risk of exertional heat illness from projected temperature increases. The authors could also include content on how weather service providers, such as the National Weather Service in the United States, can incorporate alerts about unsafe physical activity conditions within its heat products (e.g., heat advisory and excessive heat warning). A7: We deleted the sentence about the environmental impact of sports apparel production as it is not closely related to our topic. As suggested, we added a sentence about the incorporation of alerts about unsafe PA conditions into the products of weather service providers." } ] }, { "id": "70762", "date": "16 Oct 2020", "name": "Stephanie Levy", "expertise": [ "Reviewer Expertise Biological anthropology", "human energetics", "cardio-metabolic health", "climate change" ], "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 argue that many of action points outlined by the recent Global Action Plan on Physical Activity (GAPPA) can be leveraged to also mitigate carbon emissions and global climate change. In particular, the authors highlight how efforts to promote physical activity by establishing active transport and green spaces can also serve to limit carbon emissions. The article is well researched and the argument is clearly presented. However, the paper would benefit from a discussion of the economic, political, and cultural factors that will constrain stakeholders’ ability to carry out these synergistic policies. For instance, in middle-income countries there is a simultaneous increase in carbon emissions and growing prevalence of sedentary lifestyles; however, the capacity to implement climate-minded GAPPA policies might be more limited due to financial constraints.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "6099", "date": "20 Nov 2020", "name": "Sven Messing", "role": "Author Response", "response": "We would like to thank the reviewer for her valuable feedback. We have addressed her comments by adding a paragraph to the discussion section. A1: The authors argue that many of action points outlined by the recent Global Action Plan on Physical Activity (GAPPA) can be leveraged to also mitigate carbon emissions and global climate change. In particular, the authors highlight how efforts to promote physical activity by establishing active transport and green spaces can also serve to limit carbon emissions. The article is well researched and the argument is clearly presented. However, the paper would benefit from a discussion of the economic, political, and cultural factors that will constrain stakeholders’ ability to carry out these synergistic policies. For instance, in middle-income countries there is a simultaneous increase in carbon emissions and growing prevalence of sedentary lifestyles; however, the capacity to implement climate-minded GAPPA policies might be more limited due to financial constraints. Q1: Thank you very much for pointing out this important aspect. We have added it to the discussion by including the following paragraph at the end of the section: “Climate-conscious policy development for PA promotion” that refers to this issue: “However, the implementation of climate-conscious policies for PA promotion also depends on economic, political and cultural factors. For example, health spending per capita is lower in low- and middle-income countries. These financial constraints might severely impact the capacity of these countries to implement policies for climate change mitigation in general and for health promotion in particular.”" } ] } ]	1	https://f1000research.com/articles/9-349
https://f1000research.com/articles/9-586/v1	10 Jun 20	{ "type": "Software Tool Article", "title": "iMutSig: a web application to identify the most similar mutational signature using shiny", "authors": [ "Zhi Yang", "Priyatama Pandey", "Paul Marjoram", "Kimberly D. Siegmund", "Priyatama Pandey", "Paul Marjoram", "Kimberly D. Siegmund" ], "abstract": "There are two frameworks for characterizing mutational signatures which are commonly used to describe the nucleotide patterns that arise from mutational processes. Estimated mutational signatures from fitting these two methods in human cancer can be found online, in the Catalogue Of Somatic Mutations In Cancer (COSMIC) website or a GitHub repository. The two frameworks make differing assumptions regarding independence of base pairs and for that reason may produce different results. Consequently, there is a need to compare and contrast the results of the two methods, but no such tool currently exists. In this paper, we provide a simple and intuitive interface that allows such comparisons to be easily performed. When using our software, the user may download published mutational signatures of either type. Mutational signatures from the pmsignature data source are expanded to probabilistic vectors of 96-possible mutation types, the same model specification used by COSMIC, and then compared to COSMIC signatures. Cosine similarity measures the extent of signature similarity. iMutSig provides a simple and user-friendly web application allowing researchers to compare signatures from COSMIC to those from pmsignature, and vice versa. Furthermore, iMutSig allows users to input a self-defined mutational signature and examine its similarity to published signatures from both data sources. iMutSig is accessible online and source code is available for download on GitHub.", "keywords": [ "Mutational Signatures", "pmsignature", "COSMIC", "Web interface", "Shiny", "R" ], "content": "Introduction\n\nEach human is subject to a variety of mutational processes throughout their lifetime, which result in the formation of a catalog of somatic mutations as his/her unique mutational profile1. A mutational signature captures the pattern of the mutations and contexts in which those mutations occur (i.e., the neighboring bases). Examples of important mutational processes with distinct mutational signatures include aging and ultraviolet (UV) radiation. Additionally, many research groups are performing analysis to discover de novo mutational signatures in cancer1–4.\n\nCurrently, there are two frameworks used to characterize and visualize mutational signatures5,6. The first, proposed by Alexandrov et al., used a vector of 96 probabilities to capture the possible composition of the six nucleotide substitutions (C>A, C>T, C>G, T>A, T>C, T>G) and the neighboring base immediately on each of the 5′ and 3′ side of the mutated base1. A list of published mutational signatures can be downloaded from the Catalogue Of Somatic Mutations In Cancer (COSMIC) website7 (version 2, v2). Later, Alexandrov et al. published an expanded set of mutational signatures, which was referred to as version 3 (v3)8. The 67 COSMIC v3 Single Base Substitution (SBS) signatures include 30 v2 signatures. Based on the signature concept, but using different model assumptions, Shiraishi et al. proposed a mixed-membership model, pmsignature, which substantially reduced the number of parameters needed to characterize a signature9. They achieved this by assuming independence across bases, thereby reducing the number of parameters from 644-1 = 95 to (6-1)+(4-1)+(4-1) = 119. The reduction in the number of parameters is greater if more flanking bases are included. All Shiraishi’s signatures can be downloaded from their GitHub repository9. In this paper, we will refer to signatures resulting from these two methods as “COSMIC signatures” with version numbers (for those resulting from Alexandrov et al.’s method) and “PM signatures” (for those resulting from Shiraishi et al.’s method).\n\nA large number of researchers have published scientific findings resulting from the COSMIC signature-based method10–12, which was defined as the “gold standard\" in the field by Baez-Ortega et al.6. Meanwhile, an increasing number of researchers are using the pmsignature-based method for samples with lower numbers of somatic variants due to it requiring fewer parameters9,13,14. Given that both methods are widely used, investigators need the ability to compare results from their analysis with those reported in earlier databases, which may have been produced using the alternate method. For example, researchers have adopted both tools for gastric cancer and tried to compare and integrate the information from two data sources in a somewhat ad hoc manner15. No rigorous tool exists for this task. In this paper we present iMutSig, an easy-to-use tool that allows users to identify the most similar mutational signatures across methods and to compare the information characterizing those signatures using simple point-and-click navigation.\n\n\nMethods\n\nIn order to measure the similarity between mutational signatures across two databases, we need to represent PM signatures in a way that is comparable with those from COSMIC. To do this we convert the PM signature into a probabilistic vector with the same length as the COSMIC signature, i.e., 96. To calculate each of 96 resulting probabilities in the vector, we take the constituent components that make up the COSMIC signature - which refer to the nucleotide substitution and two flanking bases at the -1 and +1 position - calculate the probability of each component for the given PM signature, and then multiply those probabilities using pm-signature’s assumption of independence. For example, to calculate the probability of the COSMIC signature C[C >A]T we multiply three PM signature’s probabilities: P(C at pos -1), P(C >A), and P(T at pos +1). This example is shown in Table 1 and Equation 1.\n\nNow that we have represented both forms of signature using probabilistic vectors of length n = 96, P and C say, we can directly compare the two signature types. In order to measure the similarity between them we use cosine similarity, CS, defined as shown in Equation 2:\n\nIntuitively speaking, cosine similarity is the cosine of the angle between the two vectors. As such, cosine similarity ranges from 0 to 1 (inclusive). In our context, if two mutational signatures have a cosine similarity of 1, they must be identical, i.e., the angle between them is 0°; in contrast, if two mutational signatures have a cosine similarity of 0, they are maximally dissimilar (i.e., orthogonal). Computing the cosine similarity between the input signature and each of the candidate signatures, and then sorting the similarities from highest to lowest value, we identify the candidate signature with the highest cosine similarity as the most similar mutational signature.\n\niMutSig is built in R with its key features depending on the R package, pmsignature9. As shown in Figure 1, the Shiny app currently supports three possible workflows for users to choose from, depending on the type of signatures they have already obtained: 1) starting with a COSMIC signature; 2) starting with a PM signature; 3) starting with a self-defined signature that could follow either the COSMIC or PM format.\n\nThe first two tabs allow users to finding the most similar PM signature to an input COSMIC signature (highlighted in green) and vice versa (highlighted in orange). In addition, users can identify the most similar signatures from both data sources to an input signature (highlighted in blue).\n\nThe first tab in the Shiny app window, “COSMIC to pmsignature\", allows users to select an input COSMIC signature via a drop-down list and returns the best-matched PM signature. The returned results are divided and organized separately in the top and the bottom portion of the page. The top half tab summarizes background information regarding the input signature by presenting: 1) visualized plots of the input signature and its membership among all cancer types, i.e., in which kind of cancers the mutational signatures has been found; 2) a table showing the cosine similarity between this signature and all PM signatures, sorted in decreasing order, along with a visualization of a similarity heatmap with color and intensity proportional to assessed similarity. The bottom half tab presents plots and descriptions of the input COSMIC signature, the most similar PM signature, and a second PM signature that the user can select. Thus, users can easily access all the vital information and results regarding these signatures rather than having to manually gather and organize information from publications. The top half of the tab will be automatically updated via a control panel in the middle section of the tab, which enables users to select a signature to start with and also highlights information about the currently selected signature, the most-similar signature from the alternate model framework, and the cosine similarity.\n\nThe second tab was designed in a similar manner to the first tab, but for the case in which we are starting with a PM signature and looking for the most similar COSMIC signature. For the first two tabs, users can choose which version of COSMIC signatures to input from the sub-menus, i.e., v2 or v3.\n\nUnlike the first two tabs, the third tab enables users to enter a user-supplied signature, which can be in either PM or COSMIC format, and then identify the most similar signature from each online database. The user will be requested to enter a sub-menu based on the type of the input signature and to upload a comma-separated values (CSV) file containing a single signature. A sample CSV file is provided for download to give the user a better sense of the format of the input file. Then, the tab will be updated to display three tables, one from each data source (COSMIC v2, v3 and PM), listing the signatures from that data source and the cosine similarity of each signature with the user-uploaded signature. The tables are ordered from most similar to least similar signature. In addition, the user is able to view figures of the best-matched signatures (i.e., those with highest cosine similarity) from each data source, allowing users to observe any similarities and dissimilarities. Below, users will see a list of cancer types that contain the best-matched signature.\n\n\nUse cases\n\nWe use iMutSig to identify the most similar signature for a given PM/COSMIC signature or a user-supplied signature. Figure 2 shows the input panel after inputting COSMIC v3 signature SBS1 and Figure 3 shows the input panel after inputting PM signature P1. If users provide a user-supplied signature of either COSMIC-kind or PM-kind, the results can be seen in Figure 4 and Figure 5. Consider the example shown in Figure 4, where we input COSMIC v2 signature C1. iMutSig returned the most similar signatures COSMIC v2 signature C1, COSMIC v3 signature SBS1, and PM signature P7 (similarity = 1.000, 0.947, and 0.948, respectively) along with the names of its associated cancer types. When providing PM signature P1, iMutSig returned COSMIC v2 signature C10, v3 signature C10a and PM signature P1 (similarity = 0.816, 0.957, 1.0, respectively).\n\n\nConclusions\n\niMutSig is a user-friendly interactive browser-based application that allows users who have a signature that they have discovered in an analysis of their own data to identify the best-matched existing mutational signature from the COSMIC and PM databases. It also allows users to directly compare signatures between the two databases. It does this in an interactive way, and also allows straightforward visualization of results. iMutSig enables researchers to easily identify the most similar mutational signature and to easily access characteristic information from both data sources without additional software installation and programming of their own.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSoftware available from: https://zhiyang.shinyapps.io/iMutSig/\n\nSource code available from: http://www.github.com/USCbiostats/iMutSig\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.387388816\n\nLicense: MIT", "appendix": "References\n\nAlexandrov LB, Nik-Zainal S, Wedge DC, et al.: Signatures of mutational processes in human cancer. Nature. 2013; 500(7463): 415–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang Z, Pandey P, Shibata D, et al.: HiLDA: a statistical approach to investigate differences in mutational signatures. bioRxiv. 2019; 577452. Publisher Full Text\n\nGröschel S, Hübschmann D, Raimondi F, et al.: Defective homologous recombination DNA repair as therapeutic target in advanced chordoma. Nat Commun. 2019; 10(1): 1635. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÜlgen E, Can C, Bilguvar K, et al.: Whole exome sequencing–based analysis to identify DNA damage repair deficiency as a major contributor to gliomagenesis in adult diffuse gliomas. J Neurosurg. 2019; 1(aop): 1–12. PubMed Abstract \| Publisher Full Text\n\nOmichessan H, Severi G, Perduca V: Computational tools to detect signatures of mutational processes in DNA from tumours: a review and empirical comparison of performance. bioRxiv. 2018. Publisher Full Text\n\nBaez-Ortega A, Gori KComputational approaches for discovery of mutational signatures in cancer. Brief Bioinform. 2017; 20(1): 77–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForbes SA, Beare D, Boutselakis H, et al.: COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 2016; 45(D1): D777–D783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlexandrov LB, Kim J, Haradhvala NJ, et al.: The repertoire of mutational signatures in human cancer. Nature. 2020; 578(7793): 94–101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShiraishi Y, Tremmel G, Miyano S, et al.: A simple model-based approach to inferring and visualizing cancer mutation signatures. PLoS Genet. 2015; 11(12): e1005657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNik-Zainal S, Loo PV, Wedge DC, et al.: The life history of 21 breast cancers. Cell. 2012; 149(5): 994–1007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchulze K, Imbeaud S, Letouzé E, et al.: Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets. Nat Genet. 2015; 47(5): 505–511. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHelleday T, Eshtad S, Nik-Zainal S: Mechanisms underlying mutational signatures in human cancers. Nat Rev Genet. 2014; 15(9): 585–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYokoyama A, Kakiuchi N, Yoshizato T, et al.: Age-related remodelling of oesophageal epithelia by mutated cancer drivers. Nature. 2019; 565(7739): 312–317. PubMed Abstract \| Publisher Full Text\n\nGuo J, Huang J, Zhou Y, et al.: Germline and somatic variations influence the somatic mutational signatures of esophageal squamous cell carcinomas in a chinese population. BMC Genomics. 2018; 19(1): 538. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSahasrabudhe R, Lott P, Bohorquez M, et al.: Germline mutations in PALB2, BRCA1, and RAD51C, which regulate DNA recombination repair, in patients with gastric cancer. Gastroenterology. 2017; 152(5): 983–986.e6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang Z: Uscbiostats/imutsig v1.0. 2020. http://www.doi.org/10.5281/zenodo.3873888" }	[ { "id": "64568", "date": "22 Jun 2020", "name": "Adrian Baez-Ortega", "expertise": [ "Reviewer Expertise Computational biology", "Bioinformatics." ], "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\nYang et al. present an interactive software tool, iMutSig, which allows comparison between two alternative mathematical representations of mutational signatures. Both of these representations are widely used, but are remarkably different in their visual aspect, making intuitive comparisons difficult. To my knowledge, this is the first openly available method for comparison between signatures expressed in these two alternative representations.\nThe methods implemented for conversion between signature representations and for comparison between signatures are straightforward and based on a widely used similarity measure; although the main formula in the paper is incorrect, this mistake does not appear to extend to the implementation. Instead of simply reporting the most similar signature to the chosen signature, the tool provides information about the similarity of the chosen signature to all the signatures available in the alternative representation, allowing better assessment of the results. The user interface is thoughtfully and tastefully designed, making the tool both easy and pleasant to use. All the offered functionalities appear to work correctly and the platform runs smoothly. The authors provide their tool as an interactive website, as well as current and archived versions of the source code. However, there is a lack of information about how to install and run the software locally as an R package, which would increase the long-term usability of the tool.\nBelow I provide comments regarding major and minor issues in the article and online tool. I also provide a few optional suggestions that may be safely ignored, but which I think would enhance the functionality of the tool.\nMAJOR COMMENTS\n1. Implementation, paragraph 2: The formula for the cosine similarity defined in Equation 2 is not correct. While it is true that CS(P,C) = (P·C) / (\|\|P\|\|·\|\|C\|\|), it is not true that CS(P,C) = sum(P_i * C_i) / (sum(P_i) * sum(C_i)).\nThe correct formula for the third part of Equation 2 would be: CS(P,C) = sum(P_i * C_i) / (sqrt(sum(P_i^2)) * sqrt(sum(C_i^2))) (see here).\nPlease ammend this formula, and make sure that it gives the same values as the formula you have defined in your code (function getCosDistance).\nMINOR COMMENTS\n2. Introduction, paragraph 1: This is somewhat inaccurate, in the sense that it is the cells in an organism's tissues that are exposed to mutational processes throughout the organism's life, and each cell or tissue develops its own mutational profile. The existence of a \"unique mutational profile\" thus may be better described as a property of a tissue, organ or tumour: it is not really accurate to say that each human has \"his/her unique mutational profile\", as this varies widely across tissues (e.g. cells in the blood, skin, liver, and colon have very different mutational spectra), and the differences among individuals also tend to be tissue-specific.\n3. Introduction, paragraph 3: in the sentence where the motivation for PM signatures is mentioned (\"due to it requiring fewer parameters\"), it might be appropriate to add a brief note that the assumption of independence between substitution type and flanking bases also limits the representation of patterns where these features are dependent, although this is seen in relatively few COSMIC signatures (e.g. SBS8, SBS25, SBS35).\n4. Implementation, paragraph 1: it would be useful to complement the example in Table 1 and Equation 1 with a figure that shows the original PM signature in Table 1, and the equivalent COSMIC signature that results from applying Equation 1 to each of the 96 substitution types. This would help the reader to understand the conversion between both graphical representations, which is shown in later figures.\n5. Note that reference 5 has an updated version: Omichessan, Severi & Perduca (2019)1.\n6. Note that reference 6 is missing a colon between the author list and title.\n7. The \"About iMutSig\" web page states that \"On the Github page, you can: - install the iMutSig R pacakge and run it locally.\" However, on the GitHub page I found no instructions on how the package can be installed and run locally using R and Shiny. While I understand that the main purpose of the platform is to be an online tool, some users may be interested in having a local copy. For example, the availability of the tool seemed somewhat variable: I was able to access the website (https://zhiyang.shinyapps.io/imutsig/) on 17 June, but not on 18 June. Although this might be a rare issue, it highlights the advantage of providing users with an alternative way of accessing the tool in the long term. For example, some simple steps for installation and running could be added as a README.md file on the GitHub repository.\n8. Note that, in the platform interface, some of the signature names read \"COSIMIC\" instead of \"COSMIC\".\nOPTIONAL SUGGESTIONS\n9. I found it strange that in the tabs \"COSMIC to pmsignature\" and \"pmsignature to COSMIC\", the drop-down menus for choosing the COSMIC and PM signatures to compare are located in the middle of the interface, below the top panels that show the chosen signature. It seems to me that it would be more intuitive to place the selection menus at the top of the page, although I might be wrong.\n10. The authors might consider extending the user-supplied signature mode to allow the user to input a set of signatures, and then select the signature to compare using a drop-down menu (as in the other comparison modes), as it is likely that users will be interested in analysing sets of signatures, rather than single signatures. However, I understand this might not be straightforward to implement.\n11. Another potential extension could be an additional mode in which comparison could be performed between two sets of user-supplied signatures (each of which could be in COSMIC or PM format), in order to find the best one-to-one match between the signatures.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6096", "date": "19 Nov 2020", "name": "Zhi Yang", "role": "Author Response", "response": "We are very grateful to the reviewers for their comments and suggestions. We give detailed responses to each of those comments below. MAJOR COMMENTS 1. Implementation, paragraph 2: The formula for the cosine similarity defined in Equation 2 is not correct. While it is true that CS(P,C) = (P·C) / (\|\|P\|\|·\|\|C\|\|), it is not true that CS(P,C) = sum(P_i * C_i) / (sum(P_i) * sum(C_i)). The correct formula for the third part of Equation 2 would be: CS(P,C) = sum(P_i * C_i) / (sqrt(sum(P_i^2)) * sqrt(sum(C_i^2))) (see here). Please amend this formula, and make sure that it gives the same values as the formula you have defined in your code (function getCosDistance). Response: The formula has been corrected in this new version of the manuscript. We have also verified that the formula was correctly implemented in the getCosDistance function in the software (see below). getCosDistance <- function(F_1, F_2) { if(length(F_1)!=length(F_2)){ geterrmessage(\"Two signatures have different number of bases!\") } cos <- sum(F_1F_2)/(sqrt(sum(F_1^2))sqrt(sum(F_2^2))) return(cos) } MINOR COMMENTS 2. Introduction, paragraph 1: This is somewhat inaccurate, in the sense that it is the cells in an organism's tissues that are exposed to mutational processes throughout the organism's life, and each cell or tissue develops its own mutational profile. The existence of a \"unique mutational profile\" thus may be better described as a property of a tissue, organ or tumour: it is not really accurate to say that each human has \"his/her unique mutational profile\", as this varies widely across tissues (e.g. cells in the blood, skin, liver, and colon have very different mutational spectra), and the differences among individuals also tend to be tissue-specific. Response: We agree that our phrasing was poorly chosen here. We have altered the text to reflect this comment. The text now says “These processes result in a catalog of somatic mutations in the tissue creating…”. 3. Introduction, paragraph 3: in the sentence where the motivation for PM signatures is mentioned (\"due to it requiring fewer parameters\"), it might be appropriate to add a brief note that the assumption of independence between substitution type and flanking bases also limits the representation of patterns where these features are dependent, although this is seen in relatively few COSMIC signatures (e.g. SBS8, SBS25, SBS35). Response: We have added a sentence addressing this disadvantage of using the independence assumption. “However, the independence assumption might prevent signatures with dependent bases from being discovered, thereby resulting in fewer signatures.” 4. Implementation, paragraph 1: it would be useful to complement the example in Table 1 and Equation 1 with a figure that shows the original PM signature in Table 1, and the equivalent COSMIC signature that results from applying Equation 1 to each of the 96 substitution types. This would help the reader to understand the conversion between both graphical representations, which is shown in later figures. Response: : As requested, we have added a new Figure 1 that shows the signature in Table 1 and the ‘expanded’ signature in COSMIC format to help illustrate Equation 1. 5. Note that reference 5 has an updated version: Omichessan, Severi & Perduca (2019)1. Response: Reference 5 is now updated. 6. Note that reference 6 is missing a colon between the author list and title. Response: : The colon has been added to reference 6. 7. The \"About iMutSig\" web page states that \"On the GitHub page, you can: - install the iMutSig R pacakge and run it locally.\" However, on the GitHub page I found no instructions on how the package can be installed and run locally using R and Shiny. While I understand that the main purpose of the platform is to be an online tool, some users may be interested in having a local copy. For example, the availability of the tool seemed somewhat variable: I was able to access the website (https://zhiyang.shinyapps.io/imutsig/) on 17 June, but not on 18 June. Although this might be a rare issue, it highlights the advantage of providing users with an alternative way of accessing the tool in the long term. For example, some simple steps for installation and running could be added as a README.md file on the GitHub repository. Response: Thank you for the suggestion. A new README.md file was added to the GitHub repository (https://github.com/USCbiostats/iMutSig) providing instructions on how to install the necessary packages and host the app locally. The website failure on June 18th resulted from a scheduled automatic downloading procedure which exceeded the server response time. Subsequently, we found that the COSMIC website posted new versions of signatures (v3.1) under a different website, allowing us to remove the automatic download procedure and avoid such an issue in the future. 8. Note that, in the platform interface, some of the signature names read \"COSIMIC\" instead of \"COSMIC\". Response: Those typos have been corrected. OPTIONAL SUGGESTIONS 9. I found it strange that in the tabs \"COSMIC to pmsignature\" and \"pmsignature to COSMIC\", the drop-down menus for choosing the COSMIC and PM signatures to compare are located in the middle of the interface, below the top panels that show the chosen signature. It seems to me that it would be more intuitive to place the selection menus at the top of the page, although I might be wrong. Response: The dropdown menu for selection is now at the top of the webpage. 10. The authors might consider extending the user-supplied signature mode to allow the user to input a set of signatures, and then select the signature to compare using a drop-down menu (as in the other comparison modes), as it is likely that users will be interested in analysing sets of signatures, rather than single signatures. However, I understand this might not be straightforward to implement. Response: Thank you for the suggestion. Indeed, allowing multiple signature inputs will require introducing a drop-down menu in order to maintain the current layout. Although not incorporated at this time, we plan to add this feature in the future. 11. Another potential extension could be an additional mode in which comparison could be performed between two sets of user-supplied signatures (each of which could be in COSMIC or PM format), in order to find the best one-to-one match between the signatures. Response: Thank you for this suggestion as well. In the future, we also plan to add this feature by adding a tab allowing the comparison between user-supplied signatures." } ] }, { "id": "64570", "date": "15 Jul 2020", "name": "Vittorio Perduca", "expertise": [ "Reviewer Expertise Applied statistics", "biostatistics." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents an original online tool for comparing mutational signatures represented according to two alternative formats, namely COSMIC vectors with the relative frequencies of the 96 types of substitutions on one side1, and lower dimensional \"pmsignature\" vectors on the other side2. The article is well written and the method behind the tool is clearly explained. The interactive tool runs smoothly and has the potential to provide useful support to researchers running mutational signature analyses using alternative frameworks.\nMy comments:\nOne important point worth stressing is that the proposed solution for comparing \"PM signatures\" and \"COSMIC signatures\" is to represent the former lower dimensional probabilistic vectors in the larger space of the latter vectors. This is clearly explained in the methods, but I believe it is worth mentioning explicitly that this happens even when the input is of the COSMIC type.\n\nMy major concern is that the described method relies on the pmsignature assumption of independence between the mutation features. Could the authors comment about the possible limitations entailed by this assumption?\n\nIn my understanding, another possibility would have been to represent \"COSMIC signatures\" as 11-dimensional \"PM signatures\" by eliminating through summation two features out of three. For instance, one could compute the \"PM signature\" component representing the probability of the substitution S=[C>A] as P(S = [C>A]) = sum_{l, r} P(L=l, S=[C>A], R=r), where L and R denote the -1 and +1 flanking bases, and P(L=l, S=[C>A], R=r) is one of the 96 probabilities in the input \"COSMIC signature\". This approach does not rely on the pmsignature assumption of independence. (This solution does not make it possible to consider \"PM signatures\" with more than two flanking bases, but in any case the information about such extra bases is lost when converting \"PM signatures\" to \"COSMIC signatures\" using the method described in the paper). Have the authors explored this other method? A comment on this point could possibly help clarifying the reason why the authors have decided to rely on the independence assumption.\n\nI suggest to add a figure with the heatmap showing the cosine similarity between the original 27 \"PM signatures\" and the 30 v2 \"COSMIC signatures\". This would help understanding the type of correspondence between the two databases.\nMinor point:\n\"Implementation\" paragraph, line 6: pm-signature -> pmsignature.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6097", "date": "19 Nov 2020", "name": "Zhi Yang", "role": "Author Response", "response": "This paper presents an original online tool for comparing mutational signatures represented according to two alternative formats, namely COSMIC vectors with the relative frequencies of the 96 types of substitutions on one side1, and lower dimensional \"pmsignature\" vectors on the other side2. The article is well written and the method behind the tool is clearly explained. The interactive tool runs smoothly and has the potential to provide useful support to researchers running mutational signature analyses using alternative frameworks. My comments: One important point worth stressing is that the proposed solution for comparing \"PM signatures\" and \"COSMIC signatures\" is to represent the former lower dimensional probabilistic vectors in the larger space of the latter vectors. This is clearly explained in the methods, but I believe it is worth mentioning explicitly that this happens even when the input is of the COSMIC type. Response: We now summarize explicitly the tasks the software performs at the end of the introduction: “iMutSig, an easy-to-use tool that allows users to 1) input a new mutational signature, 2) compare it using Cosine similarity to all published signatures from both the COSMIC and PM-signature databases, 3) identify the most similar signatures previously reported, and 4) to assemble the information characterizing those signatures using simple point-and-click navigation.” My major concern is that the described method relies on the pmsignature assumption of independence between the mutation features. Could the authors comment about the possible limitations entailed by this assumption? Response: We are not recommending one method over another, but are providing a means for comparing signatures that are estimated under the different modeling assumptions. One can transform the format of either signature type to the format used by the other signature type (PM signature to COSMIC or COSMIC to PM signature). In the original submission we only considered expanding the PM signature to the COSMIC format. In this revision we have added the capability of collapsing the COSMIC signature to the PM signature format (see response to next comment). These are not symmetric activities and can lead to differences in the identification of most similar signature from the opposite model. The new method of ‘collapsing’ the COSMIC signature is described in the section: “Methods/Implementation”. The consequences for identifying the most similar signature of the opposite type are described in the last paragraph under “Methods/Operation”. In my understanding, another possibility would have been to represent \"COSMIC signatures\" as 11-dimensional \"PM signatures\" by eliminating through summation two features out of three. For instance, one could compute the \"PM signature\" component representing the probability of the substitution S=[C>A] as P(S = [C>A]) = sum_{l, r} P(L=l, S=[C>A], R=r), where L and R denote the -1 and +1 flanking bases, and P(L=l, S=[C>A], R=r) is one of the 96 probabilities in the input \"COSMIC signature\". This approach does not rely on the pmsignature assumption of independence. (This solution does not make it possible to consider \"PM signatures\" with more than two flanking bases, but in any case the information about such extra bases is lost when converting \"PM signatures\" to \"COSMIC signatures\" using the method described in the paper). Have the authors explored this other method? A comment on this point could possibly help clarifying the reason why the authors have decided to rely on the independence assumption. Response: Thank you for this suggestion. As we noted in our response to your last point, we have added this additional comparison to the app using a function implemented in the decompTumor2Sig package. Interestingly, the cosine similarity values tend to be higher using the above method. We interpret this in the paper as follows: “When a COSMIC signature is collapsed to the PM signature format the independence assumption is imposed on both signature types. However, when a PM signature is expanded to the COSMIC signature format, the PM signature probability vector still represents the fit under feature independence whereas the COSMIC signature does not. This difference in model assumptions results in lower estimates of cosine similarity.” (section Methods/Operation). I suggest to add a figure with the heatmap showing the cosine similarity between the original 27 \"PM signatures\" and the 30 v2 \"COSMIC signatures\". This would help understanding the type of correspondence between the two databases. Response: The heatmap is now added to the Shiny app and the manuscript. Minor point: \"Implementation\" paragraph, line 6: pm-signature -> pmsignature. Response: We have corrected this typo in this version of the manuscript." } ] } ]	1	https://f1000research.com/articles/9-586
https://f1000research.com/articles/9-1348/v1	19 Nov 20	{ "type": "Brief Report", "title": "Effect of herbicides on soil respiration: a case study conducted at Debrecen-Látókép Plant Cultivation Experimental Station", "authors": [ "Zsolt Sándor", "Ida Kincses", "Magdolna Tállai", "Daniel A. Lowy", "Jesus R. Melendez", "Nelly Ivonne Guananga Diaz", "Luis Elias Guevara Iñiguez", "Gerardo Cuenca Nevarez", "Viviana Talledo Solórzano", "János Kátai", "Ida Kincses", "Magdolna Tállai", "Daniel A. Lowy", "Jesus R. Melendez", "Nelly Ivonne Guananga Diaz", "Luis Elias Guevara Iñiguez", "Gerardo Cuenca Nevarez", "Viviana Talledo Solórzano", "János Kátai" ], "abstract": "Measuring the effect of herbicides on the natural environment is essential to secure sustainable agriculture practices. Amount of carbon dioxide released by soil microorganisms (soil respiration) is one of the most important soil health indicators, known so far. In this paper we present a comprehensive quantifying study, in which we measured the effect of 14 herbicides on soil respiration over 16 years, from 1991 to 2017, at Debrecen-Látókép Plant Cultivation Experimental Station. Investigated herbicides contained different active ingredients and were applied in various doses. It was found that 11 out of the examined 14 herbicides had a detrimental effect on soil respiration.", "keywords": [ "CO2 emission", "Chernozem", "Herbicides", "Látókép", "Debrecen", "soil respiration" ], "content": "Introduction\n\nCarbon dioxide (CO2) is an important greenhouse gas, which affects significantly global warming and climate change (Rastogi et al., 2002). Approximately 30% of the total CO2 emissions are released by agricultural activities. It is notable that agricultural CO2 emissions increased by 27% over two decades, from 1970 to 1990 (Lal, 2004).\n\nPrimary sources of soil CO2 emissions are root respiration and degrading of organics by soil microorganisms. Soil microbial activity mainly depends on soil properties, including soil temperature, organic matter and soil moisture content (Smith et al., 2003). Increasing scientific attention is focused on understanding the role of the soil microbial community (Bautista et al., 2017; Cho-Tiedje, 2000; Mátyás et al., 2018; Mátyás et al., 2020) and nutrient cycles (Jakab, 2020; Sándor et al., 2020). It has been documented that different cultivation technologies significantly impact soil microbiological activity (Sándor et al., 2020).\n\nDifferent chemicals (such as fertilizers and/or herbicides) are utilized in agricultural technologies. Use of herbicides constitutes an integral part of crop production, and one should be aware that they cause a “secondary effect” on both soil life and so called “non-target” organisms (Kecskés, 1976). Sensitive organisms are killed after using herbicides, and their remains are easily decomposed by the surviving microorganisms (Cervelli et al., 1978). At present, the selection criteria for allowed chemicals is more rigorous and stricter than over past decades, and they are restricted to smaller concentrations (Inui et al., 2001). Soil microbes play a major role in maintaining soil quality (Mendes et al., 2018; Wang et al., 2008).\n\nIn this paper, we discuss carbon dioxide emission levels of chernozem soil at the Debrecen-Látókép Plant Cultivation Experimental Station, where herbicides were applied to control the weeds. We compare results of carbon dioxide production in treated plots to untreated control parcels.\n\n\nMethods\n\nFirst, we conducted a literature review on types and doses (L-ha-1 or kg-ha-1) of herbicides (Molnár & Ocskó, 2000; Ocskó, 1991; Ocskó et al., 2017) that had been applied from 1991 to 2017 at Debrecen-Látókép Plant Cultivation Experimental Station (47°33’ 55.36” N; 21°28’ 12.27” E). The type of soil is calcareous chernozem; according to the International Classification (WRB) it is designated as Calcic Endofluvic Chernozen (Endosceletic). Prior absolute control soil was measured; control soil did not receive any treatment or fertilizers.\n\nSoil CO2 was measured in triplicate by NaOH absorption. Experiments were performed between 1991 and 2017. In 1991, 2000, 2008 and 2017 soil samples were obtained two weeks after the herbicide(s) was applied. For incubation, 10 g of soil was weighed and placed in a polyester bag (0.1 mm ø holes), from where CO2 could escape. One took 500 mL laboratory glassware in which 10 cm3 of 0.1 M NaOH solution (Sigma-Aldrich, USA) was introduced to absorb the released carbon dioxide. Soil samples were hung above the NaOH solution, and the glass containers were sealed tightly. Since CO2 has a greater density than air, it sunk in the container, and was absorbed by the alkaline solution. After an incubation period of 7 days, the remaining alkali solution was back titrated with 0.1 M HCl (Sigma-Aldrich, USA), in the presence of phenolphthalein, and then with methyl orange indicator. From the volume of equivalence one can calculate the amount of CO2 formed during soil respiration, according to Equation 1.\n\nmg (CO2)· 10 g−1 · 7 day−1 = (C-S) · f(NaOH) · f(HCl) · 2.2 * dm (1)\n\nwhere, C: 0.1 M/ dm3 HCl loss for methyl orange indicator (Sigma-Aldrich, USA); S: 0.1 M dm3 HCl loss for phenolphthalein indicator (Sigma-Aldrich, USA); f: 0.1 mol dm-3 HCl and a 0.10 mol dm-3 NaOH factor; 2.2: titer (1 mL 0,1 mol dm-3 HCl equivalent 2.2 mg CO2); dm: multiplication factor for dry soil.\n\nData analysis was performed using Microsoft Excel 2003 (mean values and standard deviation). Two-factor variance analysis was performed to obtain the significant effect on measured parameters. Significant differences were accepted at the level 1%, but the evaluation was calculated by LSD 5% values, as widely accepted in agricultural research.\n\n\nResults and discussion\n\nIn 1991, three herbicides were applied, and even the basic doses were high. Results were compared to the control; CO2 production was significantly reduced at single doses and a further decrease was experienced at 2–3 times greater doses. Consequently, CO2 production declined gradually with increasing doses of herbicides. The smallest production was obtained at 3 times the dose of Anelda Plus 80 EC, its value being only 59% of the control (Table 1).\n\n* kg ha-1 (quantity given in different units)\n\nIn 2000, six different active ingredients were used, and their effect examined. Much lower doses were applied, half and one third of the ones used in 1991. As compared to the control, soil respiration decreased significantly in all treated plots, after laboratory incubation. The lowest results were obtained with Acenit 880 EC; when 3x dose was used, only the 64% of the control being achieved.\n\nIn 2008, a significant decrease was found for the treated soil relative to the control. In the treatment with triple dose, only 74% of the control was measured. The herbicides used in 2008 are no longer authorized, as they were withdrawn from the market.\n\nIn 2017, three herbicides were examined. Out of them, Figaro TF, which contained glyphosate agent, was no longer authorized. When this herbicide was applied, CO2 production decreased significantly. Carbon dioxide production did not change considerably in Andengo and Capreno treatments; there was a slight increase in treatments with Andengo and decrease in treatments with Capreno.\n\n\nConclusions\n\nWe can conclude that CO2 production decreased significantly in the soil for 11 out of the 14 herbicides. With two herbicides, Merlin SC (izoxaflutol) and Capreno (Isoxadifen-ethyl, tembotrione), there was no significant change of treated soils relative to the untreated soil, and there was only one herbicide Adengo (Bayer, Germany), which increased soil respiration slightly, but not significantly. The main sources of CO2-emissions from soil is the respiration of plant roots and of the microbial community. Therefore, a significant decrease of CO2 emission indicates a change in these parameters. One can recommend for use those chemicals, which do not cause major changes in the microbial community and do not affect life conditions of other live organisms.\n\n\nData availability\n\nFigshare: Supporting data CO2 soil respiration, https://doi.org/10.6084/m9.figshare.13125290.v1 (Dama Research Center Limited, 2020).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBautista G, Mátyás B, Carpio I, et al.: Unexpected results in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity. F1000Res. 2017; 6: 1950. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCervelli S, Mannipieri P, Sequi P: Interaction between agrochemicals and soil enzymes. In: Soil Enzymes, (Ed. BURNS) Acad. Press, London, 1978; 252–293.\n\nCho JC, Tiedje JM: Biogeography and degree of endemicity of fluorescent Pseudomonas strains in soil. Appl Environ Microbiol. 2000; 66(12): 5448–5456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDama Research Center limited: Supporting data CO2 soil respiration. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13125290.v1\n\nInui H, Shiota N, Motoi Y, et al.: Metabolism of herbicides and other chemicals in human cytochrome P450 species and in transgenic potato plants co-expressing human CYP1A1, CYP2B6 and CYP2C19. J Pest Sci. 2001; 26(1): 28–40. Publisher Full Text\n\nKecskés M: Xenogén anyagok, mikroorganizmusok és magasabb rendű növények közötti kölcsönhatások talajmikrobiológiai értékelése. Ph.D. Dissertation, Hungarian Academy of Science (MTA), Budapest, 1976; 1–225.\n\nOcskó Z: II. and III. marketed authorized plant protection products, <in original language: Milyen szert használjunk? - II. és III. forgalmi kategóriájú engedélyezett növényvédő szerek> MEZŐGAZDASÁGI KIADÓ. Budapest, ISBN: 9632344502, 1991.\n\nOcskó Z, Erdős G, Haller G, et al.: Plant protection products, yield-increasing substances I-II. 2017. <in original language: Növényvédő szerek, termésnövelő anyagok I-II. 2017> AGRINEX BT, Budapest, ISBN: 2050000066504, 2017.\n\nJakab A: The ammonium lactate soluble potassium and phosphorus content of the soils of north-east Hungary region: a quantifying study. DRC Sustainable Future. 2020; 1(1): 7–13. Publisher Full Text\n\nLal R: Soil carbon sequestration to mitigate climate change. Geoderma. 2004; 123(1-2): 1–22. Publisher Full Text\n\nMátyás B, Andrade MEC, Chida NCY, et al.: Comparing organic versus conventional soil management on soil respiration. F1000Res. 2018; 7: 258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMátyás B, Lowy DA, Singla A, et al.: Comparison of effects exerted by bio-fertilizers, NPK fertilizers, and cultivation methods on soil respiration in Chernozem soil. La Granja: Revista de Ciencias de la Vida. 2020; 32(2): 7–17. Publisher Full Text\n\nMendes KF, Collegari SA, Pimpinato RF, et al.: Glucose mineralization in soils of contrasting texture under application of metolachlor, terbuthylazine, and mesotrione, alone and in mixture Bragantia Campinas. 2018; 77(1): 152–159. Publisher Full Text\n\nMolnár J, Ocskó Z: Plant protection products, crop enhancers 2000. <in original language: Növényvédő szerek, termésnövelő anyagok 2000. I-II.> AGRINEX BT, Budapest, 2000.\n\nRastogi M, Singh S, Pathak H: Emission of carbon dioxide from soil. Current Science. 2002; 82(5): 510–517. Reference Source\n\nSándor Z, Tállai M, Kincses I, et al.: Effect of various soil cultivation methods on some microbial soil properties. DRC Sustainable Future. 2020; 1(1): 14–20. Publisher Full Text\n\nSmith KA, Ball T, Conen F, et al.: Exchange of greenhouse gases between soil and atmosphere: interaction of soil physical factors and biological processes. European J Soil Sci. 2018; 69(1): 10–20. Publisher Full Text\n\nWang MC, Liu YH, Wang Q, et al.: Impacts of methamidophos on the biochemical, catabolic, and genetic characteristics of soil microbial communities. Soil Biol Biochem. 2008; 40(3): 778–788. Publisher Full Text" }	[ { "id": "75105", "date": "01 Dec 2020", "name": "Sahar El-Nahrawy", "expertise": [ "Reviewer Expertise Soil microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 provided greater clarity on the controls for their study, which measured the effect of 14 herbicides on soil respiration over 16 years from 1991 to 2017, and this study is highly interesting and readers will be intrigued by these results.\nI have some minor comment that are:\nCreating a map with the sites for sampling to be studied with a mention of the crops grown during the study period;\n\nThe number of doses should be indicated in the methods used;\n\nWhat about the standard deviation and LSD in Table 1.\nIn the future, I would like to use a phospholipid fatty acids (PLFAs) technique to study the biomass of the microbial community such as bacteria, fungi….etc.\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? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "75106", "date": "07 Jan 2021", "name": "Fa-Jun Chen", "expertise": [ "Reviewer Expertise Plant Protection", "Insect Ecology", "Soil Ecology" ], "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, the authors carried out experiments to study the effect of herbicides on soil respiration. In this study, they measured the effect of 14 herbicides on soil respiration over 16 years, from 1991 to 2017, at Debrecen-Látókép Plant Cultivation Experimental Station.This is an interesting and novel topics for the readers. There were some errors and shortcomings, which were following as:\n\nQ1: Title: In this title, it is no necessary to give the testing site. So it should be changed as “Effect of herbicides on soil respiration: a case study”.\n\nQ2: Abstract: Give more results of the measured indexes, not just the description of detrimental effects!\n\nQ3: M&M: Soil CO2 was measured in triplicate by NaOH absorption. Experiments were performed between 1991 and 2017. Why was just soil samples were obtained two weeks after the herbicide(s) in 1991, 2000, 2008 and 2017? What about the results of the other measuring years? And what were the two factors in the two-favtor ANOVAs? Sampling years and herbicide treatment? This should be given in the data analysis.\n\nQ4: Results: Why were just the results in 1991, 2000, 2008 and 2017 given in this study? What about the other years' results from 1991 to 2017?\n\nQ5: Table 1: In this stable, it shows confused and not clear to say \"Herbicide dose\", \"Initial herbicide dose\" (L ha-1), and 1x, 2x and3x!\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "76811", "date": "26 Feb 2021", "name": "Istvan Fekete", "expertise": [ "Reviewer Expertise Soil ecology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"Primary sources of soil CO2 emissions are root respiration and degrading of organics by soil microorganisms.\"\n\nthe above sentence needs some citatioms: e.g Béni et al. 2017 Beni Áron; Kate, Lajtha ; János, Kozma ; István, Fekete, (2017), Application of a Stir Bar Sorptive Extraction sample preparation method with HPLC for soil fungal biomass determination in soils from a detrital manipulation study, JOURNAL OF MICROBIOLOGICAL METHODS 136 pp. 1-5.1 PMID: 28238755, DOI: 10.1016/j.mimet.2017.02.009\n\n\"Soil microbial activity mainly depends on soil properties, including soil temperature, organic matter and soil moisture content Smith et al., 2003\"\nA more recent citation would also be helpful. e. g. Fekete et al. 2021 (Fekete, I ; Berki, I ; Lajtha, K ; Trumbore, S ; Francioso, O ; Gioacchini, P ; Montecchio, D ; Várbíró, G ; Béni, Á ; Makádi, M et al. (2021) How will a drier climate change carbon sequestration in soils of the deciduous forests of Central Europe?, BIOGEOCHEMISTRY 152 pp. 13-32. , 20 p.), https://doi.org/10.1007/s10533-020-00740-02\nKotroczó, Zsolt ; Koncz, Gábor ; Halász, L Judit ; Fekete, István ; Krakomperger, Zsolt ; Tóth, D Márta ; Balázsy, Sándor ; Tóth, János Attila, (2009) Litter decomposition intensity and soil organic matter accumulation in síkfőkút dirt site, ACTA MICROBIOLOGICA ET IMMUNOLOGICA HUNGARICA 56 pp. 53-54. , 2 p., DOI: 10.1556/AMicr.56.2009.Suppl.13\nHow many repetitions did the measurements take, when the soil respiration values in Table 1 were determined? If average values were used then standard error or standard deviation values should also be included.\nIt would also be useful to indicate in the table which values differed significantly from the control.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1348
https://f1000research.com/articles/9-1345/v1	18 Nov 20	{ "type": "Case Report", "title": "Case Report: Prostatitis tuberculosis mimicking prostate cancer", "authors": [ "Syah Mirsya Warli", "Dhirajaya Dharma Kadar", "Muhammad Haritsyah Warli", "Dhirajaya Dharma Kadar", "Muhammad Haritsyah Warli" ], "abstract": "A 56-year-old male who presented with recurrent gross hematuria and moderate lower urinary tract symptoms. After taking the patient’s medical history and performing a physical exam, suspicion of prostate cancer arose with prostate enlargement during rectal examination without tenderness and palpable nodes. Ultrasound showed estimated prostate volume of 95 gr and prostate specific antigen of 5.5. Transurethral resection of the prostate was performed and prostate tissue was checked by a pathologist, with histopathological examination showing active tuberculosis lesions. An interferon gamma release assay test was performed, which showed a positive result. The patient was diagnosed with prostatitis tuberculosis and treated with standard nine-month regimen for extra-pulmonary tuberculosis. Prostate enlargement due to tuberculosis is rare and its presence may mimic prostate cancer presentation; however, due to its rarity, careful examination is needed in order to provide optimal management.", "keywords": [ "Prostatitis", "Tuberculosis", "Prostate Cancer", "Genitourinary Tuberculosis", "Prostate Enlargement" ], "content": "Introduction\n\nTuberculosis (TB) is a chronic condition that is still endemic in Indonesia, with more than 800,000 new cases in 2018. Most cases are primary lung TB (88%); however, the genitourinary tract is one of the most common sites of hematogenous spread of extra-pulmonary TB. Genitourinary TB (GUTB), first introduced in 1937 by Wildbolz, is the second most common form of extra-pulmonary TB in endemic countries1. While male genital TB incidence are relatively low, in men, TB may affect the epididymis and the prostate (most common), followed by the seminal vesicles and the testicles1–3.\n\nWe present the case of a 56-year-old male who presented with recurrent gross hematuria and moderate lower urinary tract symptoms.\n\n\nCase presentation\n\nA 56-years-old male presented to the urology clinic with gross hematuria, which had been experienced for the past three months. Hematuria was intermittent and recurrent, with no history of urinary retention or previous trauma. The patient was also experiencing moderate lower urinary tract symptoms (LUTS) with daytime urinary frequency, poor urinary stream, nocturia up to three times a night, straining on bladder emptying and a sensation of incomplete emptying. His International Prostate Symptom Score was 18/35, indicating moderate symptoms, with 4/6 for the quality-of-life component, indicating mostly dissatisfied due to the symptoms. Physical examination was unremarkable, with no other comorbidities and no history of previous surgery. Digital rectal examination was performed, revealing a normal anal tone, normal perianal sensation, but an enlargement of the prostate with a firm consistency and smooth surface. Tenderness and palpable nodes were not found.\n\nWorkup was performed, revealing that blood markers were unremarkable with normal renal function, and a prostate specific antigen (PSA) value of 5.5 ng/dL (normal range, <4 ng/dL). Ultrasound and CT scan revealed prostate enlargement (Figure 1), with estimated volume of 95 gr. Cystoscopy and transurethral resection of the prostate (TUR-P) were performed for diagnosis as well as therapy to reduce prostate volume. During cystoscopy, the bladder was found to be unremarkable; the bladder wall was normal, with no mass or stone found. The prostate was found enlarged with protrusion to the bladder cavity, and a kissing lobe were found. Resection of the prostate was performed systematically and resected tissue was sent to pathology.\n\nPathology result showed a typical tuberculosis specimen with tubercles, consisting of epithelioid cells, central necrosis mass accompanied by chronic inflammatory lymphocyte cells, and presence of Langhans type of multinucleated giant cells (Figure 2). Interferon gamma release assay (IGRA) test was then performed to confirm TB diagnosis, which was positive.\n\nThe patient was given standard regimen treatment (combination of isoniazid, rifampicin, ethambutol and pyrazinamide for 2 months and isoniazid and rifampicin for the next 7 months) for TB for nine months, with evaluation of symptoms every three months. Evaluation and follow up consisted of history and physical examination, urinalysis and urine cytology and ultrasound of bladder and prostate. After 9 months of oral therapy, an additional CT scan was performed resulting normal prostate image.\n\n\nDiscussion\n\nTB is a long-term health issue in Indonesia. Current challenges with TB are increased incidence due to drug-resistant cases and HIV spread1. One of the most common sites of extra-pulmonary TB is the genitourinary tract, as it is the primary target of hematogenous infection. The kidney is the most common site of infection in the genitourinary tract. Prostatitis TB is a rare and uncommon presentation of extra-pulmonary tuberculosis. As a type of GUTB, diagnosis of this disease are challenging, as signs and symptoms, such as LUTS and hematuria, may mimic other disorders, such as urinary tract infection, benign prostate hyperplasia, and prostate cancer. Most of the time, diagnosis of prostate TB are incidental, for example made by the pathologist while performing examination of prostate tissue specimen taken from biopsy or prostate resection1,2,4. TB in the male genitalia is commonly found in the prostate or epididymal. Some of the most common symptoms are frequent urination, nocturia, dysuria, hematuria, urgency, and hematospermia. On physical examination indurations and nodules on the prostate can be found, which makes it difficult to distinguish from another malignancy; indeed it might be concurrent with other malignancies2,3. However, TB in the prostate is often asymptomatic, and has been shown to be found in about 10–14% of autopsy cases2.\n\nInvestigation for suspected prostate TB can be done by examining leukocytes on a three glass urine test, prostate massage secretions, ultrasound examination, and anatomical and cultural pathology examination. The gold standard is biopsy, and confirmation can be done by PCR test3. In our case, the patient had moderate LUTS symptoms and hematuria for the last three months. After examination, an enlargement of the prostate with high PSA value was found. After confirming the enlargement with ultrasound and CT scan, a TUR-P for biopsy was done which returned the result of TB. The positive IGRA test was used for confirmation. The IGRA test cannot be used alone because it will most likely always be positive in an endemic environment for TB, such as Indonesia. An alternative for TB confirmation is PCR.\n\nManagement of prostate TB is the same as other extra-pulmonary TB; administration of isoniazid, rifampicin, pyrazinamide and ethambutol or streptomycin every day for two or three months, followed by administration of isoniazid and rifampicin every day for six to seven months (for a total of nine months treatment). In our case, we used a nine month TB drug treatment, with follow-up of the symptoms every three months. Extra-pulmonary tuberculosis including genitourinary TB has a low mortality rate3.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nWorld Health Organization: Global Tuberculosis Report. World Health Organization. 2018. Reference Source\n\nLessnau KD: Tuberculosis of the Genitourinary System. Drug & Disease Urology. 2019. Reference Source\n\nPringgodigdo, Wahyudi I, Rasyid N: Tuberkulosis Urogenital. Tuberkulosis ekstra paru: Departemen Ilmu Penyakti Dalam FKUI. 2018; 329–42.\n\nSusilawati TN, Larasati R: A recent update of the diagnostic methods for tuberculosis and their applicability in Indonesia: a narrative review. Med J Indonesia. 2019; 28: 284–91. Publisher Full Text" }	[ { "id": "78265", "date": "18 Feb 2021", "name": "Dahril Dahril", "expertise": [ "Reviewer Expertise Clinical and basic of the Urology" ], "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\nPulmonary tuberculosis is the most common and most contagious form of tuberculosis, so if extrapulmonary TB is found, a comprehensive evaluation of the patient suspected of having TB should be done, including clinical history, physical exploration, chest x-ray/CT, and microbiological culture. This may also include a tuberculin test (TST) and serological testing.\n\nExtrapulmonary TB (ETB) remains a challenging diagnosis for both clinicians and microbiologists (7, 8). Signs and symptoms are most often nonspecific. Obtaining material for culture often requires invasive procedures that cannot be easily repeated. Because of the paucibacillary nature of extrapulmonary specimens and the irregular distribution of bacilli that tend to clump together, the sensitivity of smear microscopy is very low. Cases of ETB are more often culture-negative than cases of pulmonary TB, and when culture is positive, growth on a solid medium may require as long as 8 weeks. Moreover, histopathological findings are not always conclusive.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "82989", "date": "26 Apr 2021", "name": "Zhuo Tony Su", "expertise": [ "Reviewer Expertise Urology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report is well and concisely written and the topic is of interest.\n\nLimited description is available of the patient's past medical, surgical and social history and occupational/environmental exposure.\n\nIn the abstract, the authors mentioned that the DRE finding of an enlarged prostate and elevated BPH prompted a clinical suspicion for prostate cancer, but in the case report, there is no mention of further workup for prostate cancer including prostate biopsy was made before the patient underwent TURP. Please elaborate on the clinical reasoning on the decision to bypass biopsy to TURP.\n\nPlease also discuss/elaborate on the following:\n\nWhat were the patient's urinary symptoms are his post-op follow-up checks? Did any of the urinary symptoms persist and/or recur?\n\nDid the patient experience any systemic symptoms of TB at any point?\n\nLastly, some epidemiology data on prevalence/incidence of TB in Indonesia and any data on drug resistance in the region would be helpful.\n\nThanks for the opportunity to review this report.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1345
https://f1000research.com/articles/9-1344/v1	18 Nov 20	{ "type": "Case Report", "title": "Case Report: Painless obstructive jaundice caused by IgG4 autoimmune pancreatitis; the role of endoscopic ultrasound in diagnosis", "authors": [ "Sofia Voidila", "Panagiotis Sideris", "Constantinos Letsas", "Elias Anastasopoulos", "Ioanna Oikonomou", "Panagiotis Sideris", "Constantinos Letsas", "Elias Anastasopoulos", "Ioanna Oikonomou" ], "abstract": "We report the case of a 60-year-old woman, presenting with painless obstructive jaundice of unknown etiology, who was finally found to suffer from type I autoimmune pancreatitis (AIP). This case emphasizes AIP as a rare cause in the differential diagnosis of obstructive jaundice and the role of endoscopic ultrasound (EUS) in final diagnosis, which is difficult to establish. According to diagnostic criteria, we combined the results from serologic, imaging and histological features (specifically lgG4 levels, computed tomography, magnetic resonance imaging/magnetic resonance cholangiopancreatography and EUS) with cytological results, leading to a final diagnosis. Our patient’s response to corticosteroids was impressive, confirming the diagnosis, leading to complete remission of the disease. Whilst diagnosis of AIP is challenging, the application of diagnostic criteria can lead to correct diagnosis. Therapy is corticosteroid based, with very satisfying outcomes.", "keywords": [ "autoimmune pancreatitis", "obstructive jaundice", "IgG4-related disease", "endoscopic ultrasound", "corticosteroids." ], "content": "Introduction\n\nAutoimmune pancreatitis (AIP) is a rare disease, meaning that clinicians have little experience in diagnosis1. Clinical suspicion is the first step for correct diagnosis, arising from the patient’s demographics and clinical presentation. This case presented here emphasizes the major role of endoscopic ultrasound (EUS) and biopsy in the final diagnosis of AIP, when other diagnostic procedures fail to offer an accurate answer.\n\n\nCase description\n\nA female patient, 60-years-old, was admitted to the emergency department with painless jaundice. The patient did not have any history of alcohol consumption, drug abuse, or previous liver and biliary or hematologic diseases.\n\nOn clinical examination, the patient’s eyes and skin were yellow. Murphy and Giordano signs were negative. Blood tests showed serum lipase and amylase within normal limits, total bilirubin at 11.2mg/dl [normal range: 0.3–1.5mg/dL] (direct fraction 5.9 mg/dL, indirect fraction 5.3 mg/dL), hematocrit within normal limits, white blood cell count of 5.5 K/μL [normal range: 4.5–11 K/μL], and a normal fasting blood glucose level. Patient’s liver enzymes were elevated (aspartate aminotransferase 109 U/L [normal range 7–40U/L]; alkaline aminotransferase 230U/L [normal range: 5–45U/L]; alkaline phosphatase 238U/L [normal range: 40–150U/L]). Initial ultrasound examination revealed enlargement of the pancreas, with low echogenicity and dilatation of the biliary tree. The diameter of the pancreatic duct was normal (Figure 1).\n\nAnterioposterior diameter of pancreatic head was 48.9mm and body up to 23.6mm. Diameter of bile duct was 12.9mm.\n\nDynamic computed tomography revealed free peripancreatic fat with no other signs, indicating acute pancreatitis and mild enlargement of the pancreas with homogeneous density and enhancement. There were no signs indicating neoplasm.\n\nFor better evaluation of the pancreaticobiliary tree, an magnetic resonance imaging/magnetic resonance cholangiopancreatography (MRI/MPCP) was performed, showing mild pancreatic enlargement. There was no obvious dilatation of pancreatic duct, with stenosis of the final part of common bile duct (Figure 2).\n\nSince MRCP has inferior resolution in the imaging of pancreatic duct, an endoscopic retrograde cholangiopancreatography (ERCP) was performed, which confirmed the MRCP findings, showing no significant prestenotic dilatation of the common bile duct. During the procedure, a dilatation of common bile duct with a balloon-catheter and a plastic stent placement was performed to alleviate the patient from jaundice. Furthermore, tissue-sampling was conducted from the stenosis, with results indicating inflammatory process of the duct wall. The patient was transferred to a tertiary centre, where, in order to obtain a more precise and direct imaging of the pancreas, EUS and EUS elastography was performed, showing a diffusely enlarged gland with hypoechoic, patchy, heterogeneous appearing parenchyma, with regions of stiffness, suggesting AIP. In addition, the presence of a hypoechoic mass in the final part of the intrapancreatic portion of common bile duct was found, which increased the suspicion of cholangiocarcinoma, deteriorating the diagnostic procedure (Figure 3). EUS with fine needle aspiration of the mass, using a 25-gauge needle, was performed. Several inflammatory cells, fibroblasts and traces of fibrous tissue were found in the samples. lgG4 levels were elevated, measuring 250mg/dl [normal range: <140mg/dL]. The combination of clinical, imaging and cytological findings pointed to the diagnosis of AIP.\n\nHypoechoic mass in the distal part of intrapancreatic portion of common bile duct can be seen and diffusely enlarged gland with hypoechoic, patchy, heterogenous appearing parenchyma, with regions of stiffness.\n\nPrednisolone 40mg daily for a month was given as therapy, leading to clinical and radiological disease remission. MRl one month following treatment revealed almost a normal pancreas (Figure 4), with subsequent normalization of lgG4 levels in serum. Nine months later, during dosage reduction with a taper of 5mg/week, the patient attended the emergency room once again, presenting with painless jaundice. Ultrasound indicated pancreas enlargement, with elevation of IgG4 in serum and [200mg/dL] the relapse of the disease. “Rebound phenomenon” was the most possible scenario. Re-induction/increased dose of steroid therapy to 40mg/daily for a two month period caused the remission of the disease. Α dose reduction rate of 5mg every 1–2 weeks followed, until prednisolone dose reached 15 mg/day. Then the reduction rate was decreased to 2.5mg every 2 weeks, until a dose of 3.0mg/day was reached.\n\nMaintenance therapy for three years was decided to prevent disease relapse, since most relapses occur in the first three years after diagnosis2. At the moment, three years later, the patient stays in remission, receiving 3.0mg/day of prednisolone.\n\n\nDiscussion\n\nAutoimmune pancreatitis (AIP) consists of an uncommon type of chronic pancreatitis, accounting for 2–11% of all cases of chronic pancreatitis3. Two subtypes of the disease, defined by their histopathology, are clearly recognized since 20104: lymphoplasmacytic sclerosing pancreatitis, known as Type 1; and idiopathic duct-centric pancreatitis (ICDC), Type 2. Both types are steroid responsive. Type 1 is the pancreatic manifestation of lgG4-related disease. There are many extrapancreatic organs that may be involved, such as the biliary tree and gallbladder, kidneys, retro-peritoneum, prostate, the mesentery, blood vessels, gastrointestinal tract, thyroid, lacrimal glands and orbits, salivary glands, lymph nodes, and lungs5. The biliary tract is the most commonly involved extrapancreatic site, hence the painless jaundice in AIP patients, as in the present case5. The clinical features of AIP depend on the phase,acute or subacute. In the acute phase, the most common clinical presentation for both subtypes of AIP is obstructive jaundice, usually painless. In the subacute phase, AIP imitates chronic pancreatitis due to pancreatic atrophy, leading to steatorrhea. The incidence of diabetes mellitus in patients with AIP is up to 50%6. There are three established patterns of autoimmune pancreatitis depending on pancreatic appearance macroscopically: diffuse, as described in the current case, focal, and multifocal.\n\nDiagnosis of AIP is challenging, and many clinical, imaging and biochemical features overlap with other conditions, such as idiopathic pancreatitis, primary sclerosing cholangitis and malignancies, mainly pancreatic cancer and cholangiocarcinoma. A plethora of diagnostic criteria have been proposed by different groups around the world. The first was proposed by the Japan Pancreas Society in 20027, combining cardinal features, concerning histology, imaging, serology, other organ involvement, and steroid effect3 (Table 1). ICDC is the first universally accepted criterion of AIP because it considers national differences and establishes two types8.\n\nMPD, main duct dilation; This table was adapted from Cai and Tan9 under a Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nExclusion of malignancy is crucial for the clinician to choose either conservative or surgical treatment. Focal pattern is most difficult to distinguish from cancer, especially ductal adenocarcinoma (PDAC)10, for which there is no pathognomonic biomarker, including lgG4 levels and CA 19-92,11. The mistaken diagnosis of AIP as PDAC, or vice versa, can result in unnecessary or life-threatening surgery or delayed treatment. Among diagnostic procedures (Table 2), EUS-FNA consists of a sensitive method for detecting/excluding pancreatic cancer, as in our case12.\n\nThis table was adapted from Cai and Tan9 under a Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.\n\nThe prognosis of AIP is generally good and complications rare. Our patient remains free from symptoms three years after diagnosis with a successful treatment of relapse. However, the role of AIP as a predisposing factor for pancreatic cancer needs to be investigated. Internists should keep in mind AIP as a rare entity in the diagnostic spectrum of pancreatic diseases and when high clinical suspicion exists, based on clinical presentation, demographics and patient’s history, should consider this diagnosis. Diagnosis can be made according to ICDC 20118. Clinicians, pathologists and radiologists have to be aware of this recently recognized entity13, to suspect and timely diagnose it, to give patient the appropriate steroid therapy, avoiding inappropriate pancreatic resections, which lead to increased morbidity and mortality.\n\nAlthough diagnosis of AIP is challenging, respect to the established diagnostic criteria helps to have a definite and patient-safe result. When a clinical/radiological or other suspicion exists, clinicians should follow diagnostic algorithms to rule out other pathologies, especially pancreatic and billiard cancer. In our case, based on clinical presentation, the preferable diagnosis was malignancy, but imaging features of CT, ERCP and MRI/MRCP were not indicating this. EUS imaging deteriorated the diagnostic procedure, as it showed a hypoechoic mass, which is highly suspicious for malignancy, specifically cholangiocarcinoma. Finally, the combination of histological results, obtained after EUS biopsy, and elevated IgG4 levels established AIP diagnosis. In the current literature, the important role of EUS in the diagnosis of AIP has been highlighted. Our case emphasizes the role of EUS/elastography and EUS biopsy in diagnosis of the disease.\n\nExclusion of malignancy is crucial for the clinician to choose either conservative or surgical treatment. The misdiagnosis of AIP, especially focal pattern, as pancreatic duct adenocarcinoma, or the reverse, can result in medical malpractice. Sometimes biopsy is necessary, as in the present case and EUS is an ideal method for tissue sampling.\n\nGlucocorticoids are the cornerstone in the therapeutic approach of AIP and effectiveness of steroid treatment consists of a major diagnostic criterion. Therapeutic goal of AIP is to achieve clinical, serologic and radiological remission of the disease.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nSarles H, Sarles JC, Muratore R, et al.: Chronic inflammatory sclerosis of the pancreas--an autonomous pancreatic disease? Am J Dig Dis. 1961; 6: 688–98. PubMed Abstract \| Publisher Full Text\n\nNgwa T, Law R, Hart P, et al.: Serum IgG4 elevation in pancreatic cancer: diagnostic and prognostic significance and association with autoimmune pancreatitis. Pancreas. 2015; 44(4): 557–560. PubMed Abstract \| Publisher Full Text\n\nChari ST: Diagnosis of autoimmune pancreatitis using its five cardinal features: introducing the Mayo Clinic's HISORt criteria. J Gastroenterol. 2007; 42 Suppl 18: 39–41. PubMed Abstract \| Publisher Full Text\n\nChari ST, Kloeppel G, Zhang L, et al.: Histopathologic and Clinical Subtypes of Autoimmune Pancreatitis: The Honolulu Consensus Document. Pancreas. 2010; 39(5): 549–554. PubMed Abstract \| Publisher Full Text\n\nKamisawa T, Egawa N, Nakajima H: Autoimmune pancreatitis is a systemic autoimmune disease. Am J Gastroenterol. 2003; 98(12): 2811–12. PubMed Abstract\n\nKamisawa T, Shimosegawa T, Okazaki K, et al.: Standard steroid treatment for autoimmune pancreatitis. Gut. 2009; 58(11): 1504–1507. PubMed Abstract \| Publisher Full Text\n\nJapan Pancreas Society: Diagnostic Criteria for autoimmune pancreatitis 2002. J Japan Pancreas Soc. 2002; 17: 585–7.\n\nShimosegawa T, Chari ST, Frulloni L, et al.: International consensus diagnostic criteria for autoimmune pancreatitis: guidelines of the International Association of Pancreatology. Pancreas. 2011; 40(3): 352–8. PubMed Abstract \| Publisher Full Text\n\nCai O, Tan S: From Pathogenesis, Clinical Manifestation, and Diagnosis to Treatment: An Overview on Autoimmune Pancreatitis. Gastroenterol Res Pract. 2017; 2017: 3246459. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRen S, Chen X, Cui W, et al.: Differentiation of chronic mass-forming pancreatitis from pancreatic ductal adenocarcinoma using contrast-enhanced computed tomography. Cancer Manag Res. 2019; 11: 7857–7866. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Heerde MJ, Buijs J, Hansen BE, et al.: Serum level of ca 19-9 increases ability of IgG4 test to distinguish patients with autoimmune pancreatitis from those with pancreatic carcinoma. Dig Dis Sci. 2014; 59(6): 1322–1329. PubMed Abstract \| Publisher Full Text\n\nBuscarini E, Lisi SD, Arcidiacono PG, et al.: Endoscopic ultrasonography findings in autoimmune pancreatitis. World J Gastroenterol. 2011; 17(16): 2080–2085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshida K, Toki F, Takeuchi T, et al.: Chronic pancreatitis caused by an autoimmune abnormality. Proposal of the concept of autoimmune pancreatitis. Dig Dis Sci. 1995; 40(7): 1561–68. PubMed Abstract \| Publisher Full Text" }	[ { "id": "80531", "date": "08 Mar 2021", "name": "Shin Miura", "expertise": [ "Reviewer Expertise My specialty is biliary pancreatic disease. I also have a lot of clinical experience with autoimmune pancreatitis" ], "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 is a case report of autoimmune pancreatitis (AIP). AIP is a disease for which diagnostic criteria have been established and is a common disease for specialists in our area. Many papers on autoimmune pancreatitis have already been reported. This case is a typical case as AIP and is not novel. In addition, you do not provide proper images or patient information. You should submit a clear image of abdominal echo, endoscopic ultrasonography. The position of the body mark on the echo is also incorrect. Is the image you diagnosed with a bile duct tumor in Figure 3 appropriate? The tumor cannot be pointed out on MRI images. Did you misidentify the intestinal tract? If you insist on the importance of EUS, please provide the appropriate image that the reader will be satisfied with. The contents of Table 1 are not accurate. The latest guidelines for Japan were published in 2013. You state that the Japanese guidelines for other organ involvements and steroid effect for diagnostic items are not included. This is a mistake. You are providing incorrect information. There is no novel or useful information about Table 2. This paper does not contain any useful information for the reader and I disagree.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? No", "responses": [] }, { "id": "80533", "date": "23 Mar 2021", "name": "Zaheer Nabi", "expertise": [ "Reviewer Expertise Pancreatitis (acute and chronic)", "EUS", "drainage of pancreatic collections", "Third space endoscopy" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a case report entitled “Painless obstructive jaundice caused by IgG4 autoimmune pancreatitis; the role of endoscopic ultrasound in diagnosis”. The authors reported a classic case of autoimmune pancreatitis (AIP) presenting as obstructive jaundice. I have the following comments to make:\nThe report presents a typical case of AIP with no new information. Why do the authors feel it is important to highlight this case? Since, multiple studies have reported the clinical and histopathological features as well as the role of steroids in cases with AIP an atypical presentation or use of a new treatment approach would have been a welcome addition.\n\nThe quality of images need a great deal of improvement. Imaging forms the core of diagnosis in AIP and compromise in their quality may not be acceptable. The MRI image (Figure 4) shows several dates which should be otherwise masked.\n\nWhat was the follow-up duration in this case? The date on MRI (Fig 4) suggests it to be around six years in contrast to 3-years as mentioned in the manuscript.\n\nI would suggest the authors to include histology images. In case the number of figures exceeds the requirements of the journal, ultrasound images may be omitted.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1344
https://f1000research.com/articles/9-1343/v1	18 Nov 20	{ "type": "Data Note", "title": "Complete mitochondrial genome of the endangered species Brycon nattereri (Characiformes, Characidae)", "authors": [ "Snaydia Viegas Resende", "Rubens Pasa", "Fabiano Bezerra Menegídio", "John Seymour (Pat) Heslop-Harrison", "Trude Schwarzacher", "Karine Frehner Kavalco", "Rubens Pasa", "Fabiano Bezerra Menegídio", "John Seymour (Pat) Heslop-Harrison", "Trude Schwarzacher", "Karine Frehner Kavalco" ], "abstract": "Brycon nattereri is a Brazilian fish species of the order Characiformes (Bryconidae). Like others in the genus, B. nattereri is classified as \"vulnerable\" on the red list of endangered species. For this work, we collected a sample of B. nattereri from the Upper Paraná and São Francisco river basins, identified it and registered in an ichthyology collection. Whole genome sequencing was performed by Illumina. The raw reads were assembled with Novoplasty and the sequence annotated with MitoAnnotator. This is the third complete mitochondrial genome described for the genus and is available on GenBank: MT428073.1 and MT428074.1.", "keywords": [ "WGS", "Genomics", "mitDNA", "mitogenome" ], "content": "Introduction\n\nBrycon Müller & Troschel 1844 has 44 valid species and belongs to the order Characiformes. Species vary between medium and large size and are found in the hydrographic basins of South America and the Caribbean (Lima, 2017). Brycon, Chilobrycon, Henochilus and Salminus belong to the family Bryconidae (Abe et al., 2014). They occur in rivers of clean, flowing water, with a rock bed and high levels of oxygen (Botero-Botero & Ramírez-Castro,, 2011). Dependent on the riparian forest, for the most part they feed on allochthonous material, such as fruits, seeds and insects (Albrecht et al., 2009). Seven species of the genus are threatened, including Brycon nattereri, and the main reasons are fishing and environmental changes (Pavanelli et al., 2018). B. nattereri occurs in the Tocantins, Paraná and São Francisco river basins. Chromosome, molecular and population studies with specimens from tributaries of the last two river basins showed significant differences between individuals from different basins (Resende et al., 2020). B. nattereri would be the third complete mitochondrial genome sequenced in the genus, after B. orbignyanus and B. henni.\n\n\nMethods\n\nThe two B. nattereri specimens used in this work was collected in the Salto Stream (19°27’54.9” S; 46°13’57.2” W) and Espinha Stream (19°02’32.8”S; 46°01’12.9”W) tributaries belonging to the Paraná and São Francisco River basins, respectively, from Minas Gerais State, Brazil.\n\nAfter sampling, we brought the living specimens to the laboratory, euthanized them according to the technical standards of CONCEA (National Council for Control of Animal Experimentation of Brazil and CEUA/UFV) and the Animal Use Ethics Committee/Federal University of Viçosa (883/2019). We performed the sampling with licenses provided by SISBIO (Biodiversity Authorization and Information System; license number: 1938128) and SISGEN (National System for the Management of Genetic Heritage and Associated Traditional Knowledge; license number: A9FE946). We identified and deposited the specimens in the ichthyological collection of the Laboratory of Ecological and Evolutionary Genetics of the Federal University of Viçosa, Rio Paranaíba campus (voucher number: LaGEEvo-3928 and LaGEEvo-3847).\n\nTotal genomic DNA was extracted from liver and heart samples, as per the instructions of Invitrogen’s DNA extraction and purification kit (catalog number: K182002). After quality checking using Nanodrop (Lite Spectrophotometer ND-LITE-PR), Whole Genome Sequencing was performed by Illumina sequencing (San Diego, CA) at Novogene company, UK. The 2x150 raw reads were uploaded to Galaxy (Afgan et al., 2018), and trimmed to remove the adaptors and low-quality sequences using Trimmomatic Galaxy Version 0.38.0 (Bolger et al., 2014).\n\nWe assembled the mitogenome from raw reads on NOVOPlasty v3.7 (Dierckxsens et al., 2017) in a parallel cluster computer (64 Gb RAM) using the mitogenome of Brycon henni (GenBank KP027535.1) as seed. The mitogenome of Brycon from the Parana river basin was circularized on NOVOPlasty, but not from the São Francisco basin because of repeats on the D-loop region. Therefore, we uploaded the aligned reads to Galaxy and assembled de novo using SPAdes v3.13.0 (Bankevich et al., 2012). We blasted the sequences using megaBLAST implemented at NCBI obtained to be sure we had mitochondrial sequences (Altschul et al., 1990). We annotated the mitochondrial sequences on MitoAnnotator (Iwasaki et al., 2013) at MitoFish v. 3.60. We mapped as mitochondrial sequences in relation to the raw reads (after quality filtering) with Bowtie2 v. 2.4.1 (Langmead & Salzberg, 2012).\n\nTo validate our data, we aligned the CDS sequences of the two individuals of B. nattereri, B. henni (GenBank NC_026873.1), B. orbignyanus (GenBank NC_024938.1), Salminus brasiliensis (GenBank NC_024941.1), and Astyanax paranae (GenBank NC_031380.1) (outgroup) using MUSCLE (Edgar, 2004), followed by maximum likelihood analysis using MEGA X software (Kumar et al., 2018), with GTR model + gamma + invariant sites and 1000 bootstrap.\n\n\nDataset validation\n\nThe mitochondrial genome of B. nattereri from Paraná and São Francisco river basin has the expected size for vertebrates, 16,837 bp (GenBank MT428073) and 16,752 bp (GenBank MT428074 access), respectively. Both specimens have a total of 37 functional genes (Figure 1), 13 DNA coding regions (CDS), two ribosomal RNAs and 22 transporter RNAs.\n\nIllustrative diagram of the complete mitochondrial genome of Brycon nattereri from São Francisco (a) and Paraná (b) river basins, including genes encoding proteins, ribosomes and transporters, as well as their locations on the H and L strands.\n\nThe maximum likelihood dendrogram generated with our data and other complete GenBank mitochondrial genomes (Figure 2) rescued phylogenetic relationships according to literature (Abe et al., 2014; Hilsdorf et al., 2008).\n\nThe evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model. The tree with the highest log likelihood (-45932.69) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 3.3372)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 43.41% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved six nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. There was a total of 11549 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.\n\n\nData availability\n\nBrycon nattereri voucher LAGEEVO_3928 mitochondrion, complete genome, Accession number MT428073.1: https://www.ncbi.nlm.nih.gov/nuccore/MT428073.1\n\nBrycon nattereri voucher LAGEEVO_3847 mitochondrion, complete genome, Accession number MT428074.1: https://www.ncbi.nlm.nih.gov/nuccore/MT428074.1", "appendix": "Acknowledgements\n\nAuthors thank Henrique Peluzio for his support on the UFV cluster. S.V.R. thanks to CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the D.Sc. fellowship.\n\n\nReferences\n\nAbe KT, Mariguela TC, Avelino GS, et al.: Systematic and historical biogeography of the Bryconidae (Ostariophysi: Characiformes) suggesting a new rearrangement of its genera and an old origin of Mesoamerican ichthyofauna. BMC Evol Biol. 2014; 14: 152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfgan E, Baker D, Batut B, et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018; 46(W1): W537–W544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbrecht MP, Caramaschi É P, Horn MH: Population responses of two omnivorous fish species to impoundment of a Brazilian tropical river. Hydrobiol. 2009; 627: 181–193. Publisher Full Text\n\nAltschul SF, Gish W, Miller W, et al.: Basic local alignment search tool. J Mol Biol. 1990; 215(3): 403–410. PubMed Abstract \| Publisher Full Text\n\nBankevich A, Nurk S, Antipov D, et al.: SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19(5): 455–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBotero-Botero A, Ramírez-Castro H: Ecologia trofica de la sabaleta Brycon henni (Pisces: Characidae) en el rio Portugal de Piedras, Alto Cauca, Colombia. Rev MVZ Córdoba. 2011; 16(1): 2349–2355. Publisher Full Text\n\nDierckxsens N, Mardulyn P, Smits G: NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res. 2017; 45(4): e18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004; 32(5): 1792–1797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHilsdorf S, Oliveira C, Lima FCTD, et al.: A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA. Genet Mol Biol. 2008; 31(1): 366–371. Publisher Full Text\n\nIwasaki W, Fukunaga T, Isagozawa R, et al.: MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol Biol Evol. 2013; 30(11): 2531–2540. Publisher Full Text\n\nKumar S, Stecher G, Li M, et al.: MEGA X molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6): 1547–9. 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. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLima FC: A revision of the cis-andean species of the genus Brycon Müller & Troschel (Characiformes: Characidae). Zootaxa. 2017; 4222(1): zootaxa.4222.1.1. PubMed Abstract \| Publisher Full Text\n\nPavanelli CS, Bock CL, Oliveira C, et al.: “Brycon nattereri Günther, 1864”. In Instituto Chico Mendes de Conservação da Biodiversidade (Org.). Livro Vermelho da Fauna Brasileira Ameaçada de Extinção: Volume VI - Peixes. Brasília: ICMBio. 2018; 6: 85–88.\n\nResende SV, Silva IB, Pasa R, et al.: Hidden evolutionary units and its implications on conservation in a vulnerable species of a freshwater fish. Zebrafish. 2020; In Press." }	[ { "id": "75435", "date": "23 Dec 2020", "name": "Yong Zhang", "expertise": [ "Reviewer Expertise Genetics and molecular biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Resende et al. sequenced the mitochondrial genome of the Brazilian fish species Brycon nattereri, which is endangered. This is the third sequenced mitochondrial genome for this genus. The data showed in this work is pretty straightforward and clear. However, the rationale for this study can be better explained. Main discoveries of the mitochondrial genome that differ this species from others should also be mentioned in the manuscript.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "8584", "date": "30 Sep 2022", "name": "Snaydia Resende", "role": "Author Response", "response": "Thank you Yong Zhang for reviewing our manuscript. Brycon nattereri is listed as an endangered species and genetic studies are important for management and conservation programs. Our dataset provides specific mitochondrial markers that could be used in future projects with the species. The main difference between the two mitochondrial genomes described here are correspond to the intergenic regions and the size of the D-Loop region, composed of 1197 bp (from 15640 to 16837) in B. nattereri from Paraná and 1112 bp in B. nattereri from São Francisco. I would like to know what other details I can provide in the methodology to facilitate the replication of the work." } ] }, { "id": "96881", "date": "01 Nov 2021", "name": "Dora Davies", "expertise": [ "Reviewer Expertise Morphological and molecular studies of parasitic helminths of freshwater fish and invertebrates (Davies) and Molecular and phylogenetic studies on parasites of clinical and ecological relevance (Lauthier)." ], "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 sequenced the mitochondrial genome of 2 specimens of Brycon nattereri, one of 7 threatened species out of 44 that the genus encompasses. The species is distributed in the basins of the Paraná and San Francisco rivers, in Brazilian territory. The specimens from tributaries of both basins show chromosomal, molecular and population differences. The genome size is that expected for vertebrates and shares 100% similarity between both samples.\nKnowledge of the mitochondrial genome of specimens identified as Brycon nattereri is important taking into account its status as a threatened species of the Neotropical Region; however, the data shown in this work should be better explained, there’s no further explanation about these discoveries and how they differ from other related organisms; the authors fail to relate these results to morphological and population data, considering that variations are known between individuals from different populations. For example, did the 2 individuals studied present morphological or chromosomal differences?\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "8585", "date": "30 Sep 2022", "name": "Snaydia Resende", "role": "Author Response", "response": "Dora Davies and Juan Jo´se, thank you for reviewing our manuscript. Brycon nattereri is listed as an endangered species and genetic studies are important for management and conservation programs. Our dataset provides specific mitochondrial markers that could be used in future projects with the species. The main difference between the two mitochondrial genomes described here are correspond to the intergenic regions and the size of the D-Loop region, composed of 1197 bp (from 15640 to 16837) in B. nattereri from Paraná and 1112 bp in B. nattereri from São Francisco. In our maximum likelihood tree, we identified that there are phylogenetic differences between the two evolutionary units, as well as corroborating the hypothesis that the genus Brycon is not monophyletic. In another work we had previously published with populations from the same localities (https://doi.org/10.1089/zeb.2020.1916), we identified chromosomal differences (the two evolutionary units show differences in karyotypic formulas, despite their conserved diploid number) and morphometric, where differences are also found in the body configuration of the two ESUs. I would like to know what other details I can provide in the methodology to facilitate the replication of the work." } ] } ]	1	https://f1000research.com/articles/9-1343
https://f1000research.com/articles/9-709/v1	15 Jul 20	{ "type": "Software Tool Article", "title": "gprofiler2 -- an R package for gene list functional enrichment analysis and namespace conversion toolset g:Profiler", "authors": [ "Liis Kolberg", "Uku Raudvere", "Ivan Kuzmin", "Jaak Vilo", "Hedi Peterson", "Liis Kolberg", "Uku Raudvere", "Ivan Kuzmin", "Jaak Vilo" ], "abstract": "g:Profiler (https://biit.cs.ut.ee/gprofiler) is a widely used gene list functional profiling and namespace conversion toolset that has been contributing to reproducible biological data analysis already since 2007. Here we introduce the accompanying R package, gprofiler2, developed to facilitate programmatic access to g:Profiler computations and databases via REST API. The gprofiler2 package provides an easy-to-use functionality that enables researchers to incorporate functional enrichment analysis into automated analysis pipelines written in R. The package also implements interactive visualisation methods to help to interpret the enrichment results and to illustrate them for publications. In addition, gprofiler2 gives access to the versatile gene/protein identifier conversion functionality in g:Profiler enabling to map between hundreds of different identifier types or orthologous species. The gprofiler2 package is freely available at the CRAN repository.", "keywords": [ "g:Profiler", "R package", "functional enrichment analysis", "identifier mapping", "Gene Ontology", "pathways" ], "content": "Introduction\n\nInterpretation of gene lists is a key step in numerous biological data analysis workflows, such as differential gene expression analysis and co-expression clustering of RNA-seq or microarray data. Usually this involves associating these gene lists with previous knowledge from well curated data sources of biological processes and pathways. However, as the knowledge-bases are constantly changing, keeping the associations up to date requires careful data management. Handling numerous databases, especially when using different gene identifier types, can be a very time-consuming process for researchers.\n\ng:Profiler (https://biit.cs.ut.ee/gprofiler) is a popular web toolset that helps to handle gene lists from various biological and biomedical studies of more than 600 species and strains, including vertebrates, plants, fungi, insects and parasites1,2. g:Profiler’s best known functionality is the over-representation analysis to identify significantly enriched biological functions and pathways obtained from well established data sources which include, among others, Gene Ontology (GO)3, KEGG4 and Reactome5. The information about genes, identifier types and GO term associations in g:Profiler is mostly based on Ensembl databases6 including data from Ensembl Genomes, fungi, plants and metazoa specific versions of Ensembl. g:Profiler follows Ensembl’s quarterly update cycle while keeping the access to previous data versions as archives for reproducibility. The parasite specific data is included from the WormBase7.\n\nProviding users with fast and easy access has been the main goal of g:Profiler developers. Since 2007, g:Profiler has been in constant development and with the recent update in 2019 a new accompanying R package, gprofiler2, was developed8. The R package relies on the g:Profiler REST API requests providing an easy programmatic access to the same functionalities as in the web tool without performing heavy computations and mappings in R. While there are other popular R packages for functional enrichment analysis, such as topGO9 and clusterProfiler10, gprofiler2, unlike the others, provides access to numerous annotation data sources with a single query without requiring to download any of these sources to a local computer. Furthermore, the mapping between different gene identifiers is automatic and the input can be a mixed list of identifiers. g:Profiler’s continuous development and flexibility of usage has been recognised by the European Life Science Infrastructure ELIXIR, which has selected it as one of its Recommended Interoperability Resources.\n\ng:Profiler development team encourages and supports external tools and packages to use either gprofiler2 package or the public API to be part of their workflows. For example, RCAS Bioconductor package11 includes gprofiler2 for functional analysis of transcriptomic regions detected by different high-throughput experiments. Single-cell mapper package (scMappR)12 analyses cell-type specific gene lists with gprofiler2. OmnipathR13 suggests using gprofiler2 for enrichment analysis of protein complexes. Gene Co-expression Network analysis pipeline (GWENA) uses gprofiler2 in their pipeline for functional enrichment of co-expressed gene modules. A Nextflow differential gene expression analysis pipeline includes gprofiler2 for pathway analysis.\n\nHere we demonstrate how to conveniently incorporate the gprofiler2 R package into bioinformatics analysis pipelines using differential gene expression analysis as an example.\n\n\nMethods\n\nInherently, gprofiler28 is a collection of wrapper functions in R that simplify sending POST requests to the g:Profiler REST API using the RCurl package14. This means that all the annotation data sources and computations are centralised in a single well-maintained server and therefore the results from both the web tool and R package are guaranteed to be identical. Relying on the central API also simplifies the maintenance of the g:Profiler interfaces and enables the R users to get access to the most up-to-date data without having to download the heavy annotation data files to their own devices.\n\nThere are four main API wrapper functions in gprofiler2:\n\ngost for functional enrichment analysis\n\ngconvert for mapping gene identifiers between different namespaces\n\ngorth for mapping orthologous genes across species\n\ngsnpense for mapping SNP rs-IDs to chromosome positions, genes and variant effects.\n\nIn addition to fetching the results from the API, gprofiler2 uses the packages ggplot215 and plotly16 to provide visualisations for enrichment results that are similar to the web tool ones. Using ggplot2 allows users to customise the visualisations by adding or removing graphical layers, and to adjust the quality of images for publication.\n\nThis article was written using R version 3.6.1 (2019-07-05) and gprofiler2 version 0.1.9.\n\nThe gprofiler2 R package is available from CRAN and works on R versions 3.5 and above. The package also includes a detailed vignette.\n\nThe package can be installed from CRAN:\n\n\n\nThe most popular functionality of g:Profiler is functional enrichment analysis provided by the g:GOSt tool that performs over-representation analysis using hypergeometric test. This functionality is available in gprofiler2 under the function gost. The required inputs for this function are a vector of gene identifiers, query, and the name of the corresponding organism which is constructed by concatenating the first letter of the genus name and the specific epithet, e.g. hsapiens for human genes. The full list of supported species and strains, 641 in total, is available on the g:Profiler web page.\n\nThe query vector can include mixed types of gene/protein identifiers, SNP rs-IDs, chromosomal intervals or term IDs. Accepting a mixture of IDs is a unique feature that skips time-consuming manual steps of converting between different identifier types required by other functional enrichment tools. However, in case of analysing numeric identifiers (e.g. Entrez IDs) the user should specify the namespace using the numeric_ns parameter. The same description of input query and organism holds for the three other functions in gprofiler2.\n\n\n\nSeveral additional parameters in the gost function help to perform the analysis according to specific needs, including custom statistical options such as background definition, statistical significance threshold, method for multiple testing correction and testing for under-representation. Also, additional information like GO evidence codes and genes belonging to the intersection between the input list and the functional term is available.\n\ng:Profiler’s in-house database includes only reliable annotation data sources that are regularly updated such as Gene Ontology (GO)3, KEGG4, Reactome5, WikiPathways17, miRTarBase18, TRANSFAC19, Human Protein Atlas20, protein complexes from CORUM21 and Human Phenotype Ontology22. By default, all the data sources in g:Profiler database are used for the analysis in gprofiler2, but a specific selection can be made with the sources parameter of the gost function. In order to enable more flexibility, users can also use their own annotation data. The custom source can be uploaded in a Gene Matrix Transposed (GMT) file format. This feature is further described in the next section.\n\n\nUse case\n\nDifferential gene expression analysis determines lists of genes that show changes in expression between different conditions, cell types, time points, etc. Functional enrichment analysis using the gprofiler2 package8 helps to interpret these gene lists.\n\nHere we demonstrate the main functionality of gprofiler2 by following an analysis example from the existing RNA-seq Bioconductor workflow23 that uses the popular DESeq2 package24 for differential analysis. The example RNA-seq data are obtained from the airway package25 that comprises data from experiment where airway smooth muscle cells were treated with dexamethasone.\n\n\n\nFirst, we will detect the list of genes that are differentially regulated when stimulated with dexamethasone and then we will use the function gost from gprofiler2 to find the biological functions and pathways that are significantly enriched in this gene set.\n\n\n\n\n\nThe output of the gost function is a named list where the element result includes a data frame with the enriched functions and related statistics; and the element meta includes relevant metadata for reproducing these results.\n\n\n\n\n\nFor cases where the list of interesting genes can be ranked by some biologically meaningful measure, such as P-value or fold change in differential analysis, g:Profiler provides an ordered query option that takes the ranking into account when performing enrichment tests. The testing is then performed iteratively, starting from the first gene and sequentially adding genes one by one. For every term, the smallest enrichment P-value is reported along with the corresponding gene list size. Consequently, for different terms the query size can vary, especially as the broader terms can be enriched for larger lists only. This option is very similar to the idea of the GSEA analysis method26.\n\nFor example, to perform ordered query using gprofiler2 we first rearrange the list of up-regulated genes based on the log2 fold change values so that the first gene in the list has the highest value. Next we use this ordered list as a query in the gost function and set the parameter ordered_query = TRUE.\n\n\n\n\n\nThe resulting data frame is in the same format as shown previously. Only the size of the query in the table can vary as the algorithm detects the most significant cutting point from the input gene list considering every function separately.\n\nDifferent visualisations are useful to summarise and interpret functional enrichment results. With the recent update, g:Profiler introduced an alternative way for visualising functional terms, a Manhattan plot. On this plot, the x-axis shows the terms and y-axis shows the enrichment P-values on − log10 scale. Each circle on this plot corresponds to a single term. The circles are colored according to the annotation source and size-scaled according to the total number of genes annotated to the corresponding term. The locations on the x-axis are always fixed and ordered in a way that the terms from the same GO subtree are located closer to each other. This helps to highlight different enriched GO sub-branches as they form peaks in the Manhattan plot and makes plots from different queries easily comparable. For the same reason, by default the values on the y-axis are capped to a maximum value of 16 that corresponds to P-value less than 10−16 . The same default threshold is also used in the statistical tests in R. This selection can be switched off to show the P-values in a wider scale range.\n\nInteractive graphs are common in web tools and therefore the Manhattan plot in g:Profiler web interface also provides several interactive features to facilitate data exploration and enables to export the visualisations as high-quality image files. Mimicking the g:Profiler web interface, the Manhattan plot in gprofiler2 is implemented in the function gostplot that uses the resulting object from the gost function as an input. As a unique feature, compared to other similar packages, the parameter interactive enables to switch between interactive plotly graph for browsing or static ggplot graph for saving as an image file. The parameter capped enables to turn off the upper limit of y-axis.\n\n\n\nAfter exploring the interactive graph and deciding on the story to tell about the results, the user can compose a publishable figure that highlights the most important terms using the function publish_gostplot and defining the relevant terms in the parameter highlight_terms. The chosen terms are indicated with numbers on the plot and corresponding statistics are shown in the table below the Manhattan plot. For example, the enrichment results for up-regulated genes are shown in Figure 1. The Manhattan plot can be saved as an image file (PNG, PDF, JPEG, etc) specified by the filename parameter.\n\n\n\nAs the resulting plot is a standard ggplot object, it is easy to further customise the graphs by adding graphical layers or textual annotations.\n\nAbove we were analysing the up- and down-regulated gene lists separately, but the gost function also works with a (named) list of multiple gene vectors that enables to keep all the results in a single object and to easily compare different groups.\n\n\n\nIn this case, the resultant data frame is in a so-called “long format” where the column query includes the names of corresponding input vectors to differentiate between them. Alternative option is to set multi_query = TRUE which, in case of multiple gene lists, returns results as a comparison table in a “wide format”. That is, the rows are concatenated by terms and query statistics are shown in cells as vectors, e.g. the p_values column includes a vector of corresponding P-values from all the input queries, even the insignificant ones.\n\nResults from multiple gene lists can also be used for plotting. The function gostplot detects the case of multiple queries and plots the Manhattan plots under each other for comparison. The example enrichment results are shown in Figure 2.\n\n\n\nThe same enrichment results can also be viewed in the g:Profiler web tool. The user can generate a dedicated short-link by setting the parameter as_short_link = TRUE in the gost function which then returns the short-link to g:Profiler web tool instead of a data frame. This is a useful feature for sharing the results quickly with colleagues or to accompany a publication without the peers having to run the full analysis code in R.\n\n\n\nIn this case, the variable multi_gp_link is a character string that corresponds to a stable short-link to these enrichment results: https://biit.cs.ut.ee/gplink/l/0wgtcERnQT.\n\nAnother common but tedious task in handling gene lists is mapping between different identifiers. The function gconvert helps to easily solve this issue and translates the given input identifiers to some other user defined namespace together with gene names and descriptions. The function is able to map between at least 30 different namespaces for more than 190 species. All available namespaces for different organisms are listed on the g:Profiler page.\n\nAs an example we will convert the Ensembl IDs in our differential expression results to numeric Entrez IDs with gconvert. The function takes a vector of gene identifiers as an input and returns a data frame that includes a column with target identifiers together with the names and descriptions for the input genes.\n\n\n\n\n\nThe users can add this information to the differential expression results data frame and save it to a tab separated text file to include as a supplementary file in their article, for example.\n\n\n\nWhile g:Profiler enables to analyse genes from numerous organisms using high-quality annotation databases, there is still a need for custom data functionality for researchers interested in non-model organisms, that are not annotated in the Ensembl database, or in some specific, not so widespread annotation resource. In g:Profiler, this is solved by enabling users to upload custom annotation files in the GMT file format, which is essentially a tab delimited text file where every row describes a function by its identifier, description, and the genes annotated in this function. Here it is important to note that in case of custom annotation files, all the identifiers not present in the GMT file will be ignored in the analysis.\n\nFor example, to use the gene-disease association data from the DisGeNET database27 for enrichment analysis, the user can upload the GMT file in R using the upload_GMT_file function that returns a unique token for the file which can then be used as a value for the organism argument in the gost function.\n\nFirst, we use R utility function download.file to download an annotation GMT file from DisGeNET into a file in the working directory and name it “DisGeNET.gmt”.\n\n\n\nNow, when we have the file in our local environment, we can upload it to g:Profiler with the upload_GMT_file function.\n\n\n\nThe result of this upload is a unique token (in this case \"gp_goJy_Ej2J_rPc\") which should be saved by the user for future use. In order to find the enriched diseases in our gene list, we will use the token as a value for the organism in the gost function. As the DisGeNET database file includes gene symbols and not Ensembl identifiers, we first use gconvert to map our Ensembl IDs to gene names and use these as the input for the enrichment analysis.\n\n\n\nThe custom data source results can also be plotted using the Manhattan plots (Figure 3).\n\n\n\nAs the gprofiler2 R package and the web tool are in sync, this token will also work for the analysis in the web tool and can be inserted under the section “Bring your own data (Custom GMT)”. And vice versa, the token obtained from the web tool will work in the R package without uploading the data again. Thus, in order to analyse multiple gene lists with the same data source, the user needs to upload the file only once and can use the given token from then on. Furthermore, analysing multiple custom sources at once is enabled with the upload of a ZIP archive that includes multiple GMT files. GMT file names are used as the names for the data sources in the results and colored independently in the Manhattan plot.\n\nSometimes, in order to further investigate the interesting set of differential genes in human, researchers need to perform additional experiments on model organisms such as mice. This requires finding the corresponding orthologs of these interesting genes from other species. Another use for orthologous genes is the possibility to transfer the extensive knowledge from well studied organisms to less studied species.\n\nMapping orthologous genes between species in g:Profiler is enabled by the g:Orth tool and in the gprofiler2 package the access is wrapped into the function gorth. The function works very similarly to gconvert, only in this case the user has to define corresponding source_organism and target_organism. For example, the following code maps the detected up-regulated gene identifiers to corresponding mice genes.\n\n\n\n\n\nThis function returns a data frame that includes the input and target identifiers, and also the ortholog names and descriptions.\n\nPlots from enrichplot. Since the output of the gost function is stored in a standard data frame format, it is easy to alter it for custom visualisations using ggplot2, enrichplot28, clusterProfiler10 or any other similar package. Here we demonstrate how to convert the results from multiple gene lists into enrichResult and compareClusterResult objects required by the visualisations methods implemented in the enrichplot package. Similar approach also works for a single query.\n\n\n\n\n\nAfter creating an instance of the enrichResult or compareClusterResult (for multiple gene lists) class from the gost result, this object can be used as an input for the visualisation functions from enrichplot and clusterProfiler that are suitable for over-representation analysis such as dotplot, barplot, cnetplot, upsetplot, emapplot, etc. Figure 4 shows the dot plot for results in a compareClusterResult object.\n\n\n\nAs these plots are ggplot objects, using ggplot2 layers allows further customisation of the visualisations as shown in Figure 5.\n\n\n\nIn order to use the browseKEGG function to open KEGG pathway browser, the pathway IDs should be transformed according to the organism. In case of human pathways, the prefix KEGG should be replaced with hsa. The full list of organisms and their prefixes is available from the KEGG home page.\n\n\n\n\n\nThis command will open the KEGG browser page for the pathway Inflammatory mediator regulation of TRP channels.\n\nThe functional enrichment results from the gost function can be modified in order to save them into a Generic Enrichment Map (GEM) file format that is compatible with the EnrichmentMap application in Cytoscape29. This app helps to visualise enrichment results as a highly customisable network where nodes represent enriched terms and edges represent their mutual overlap.\n\nIn case of a single query, the GEM file can be generated with the following lines of code. The parameter value evcodes = TRUE is important for obtaining the intersection column with corresponding gene IDs in the query that are annotated to the term.\n\n\n\n\n\n\n\nIn the EnrichmentMap the user can set the “Analysis Type” parameter as Generic/gProfiler and upload the required files: GEM file with enrichment results (input field “Enrichments”) and GMT file that defines the annotations (input field “GMT”). Both of these files have to include gene identifiers from the same namespace for the EnrichmentMap to work.\n\nThe GMT files used by g:Profiler are downloadable from the web page under the “Data sources” section. Only the GMT files of KEGG and Transfac are not available as the sharing is restricted by data source licenses.\n\n\n\nThe demand for better reproducibility of computational analyses is constantly growing30. In bioinformatics analysis, many different tools and databases are combined in order to detect relevant findings. This adds an extra layer of complexity which often leads to reproducibility issues. Because of this, since 2011 all the past releases of g:Profiler are maintained and kept usable to ensure reproducibility and transparency of enrichment analysis results. The users can cite the exact extract of the annotation database and the state of the implementation by stating the version number in their research. In gprofiler2, this is available, along with other query information, from the metadata of gost enrichment results:\n\n\n\n\n\nThe version number notes that the results were obtained using the state of the database that includes data from Ensembl release 99, Ensembl Genomes release 46 and WormBase ParaSite release 14, and the g:Profiler codebase with the Git revision number f929183. The version number together with the details of applied parameters (available from multi_gp$meta$query_metadata) is enough to reproduce the results.\n\nIn order to reproduce the results obtained with a specific version, one can change the data version using the function set_base_url:\n\n\n\nAll the past versions and their URLs are available at https://biit.cs.ut.ee/gprofiler/page/archives. gprofiler2 works with versions e94_eg41_p11 and higher, earlier versions are still accessible using the deprecated R package gProfileR.\n\nFunction set_base_url also gives access to the most recent developments and data updates of g:Profiler available at the Beta version:\n\n\n\nIn order to determine the current g:Profiler URL used for the analysis one can use the function get_base_url:\n\n\n\n\n\n\nConclusion\n\nWe presented the gprofiler2 R package8 that is one of the programmatic access points to the widely used g:Profiler web toolset for gene list functional enrichment analysis and identifier conversion. This package enables effective integration of g:Profiler functionalities in various bioinformatics pipelines and tools written in R without the need of searching and downloading several data files. The suite of functions in gprofiler2 are implemented with the importance of analysis reproducibility and interoperability with other tools in mind. In addition, the package provides a way to easily create or customise the enrichment plots using the existing visualisation packages in R. For the researchers who prefer to perform their computational analysis pipelines through the web we have wrapped the gprofiler2 package as a tool for the Galaxy platform31.\n\nIt is important to note that using gprofiler2 for functional enrichment analysis is not limited to the use case of differential gene expression analysis. The package is useful whenever there is a set of genes/proteins/SNPs the user wants to characterise with biological functions or to convert to another namespace.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nR package gprofiler2 is available from CRAN: https://cran.r-project.org/package=gprofiler2.\n\nSource code available from: https://gl.cs.ut.ee/biit/r-gprofiler2.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.39197958.\n\nLicense: GNU General Public License v2.0.", "appendix": "Author information\n\nLK, UR and IK implemented the package. LK and HP wrote the article. HP and JV supervised the development.\n\n\nAcknowledgments\n\nWe would like to thank Kaur Alasoo for critically reading the manuscript.\n\n\nReferences\n\nReimand J, Kull M, Peterson H, et al.: g:profiler - a web-based toolset for functional profiling of gene lists from large-scale experiments. Nucleic Acids Res. 2007; 35(suppl_ 2): W193–W200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRaudvere U, Kolberg L, Kuzmin I, et al.: g:profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res. 2019; 47(W1): W191–W198. PubMed Abstract \| Publisher Full Text \| Free 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): 25–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Sato Y, Furumichi M, et al.: New approach for understanding genome variations in kegg. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHimes BE, Jiang X, Wagner P, et al.: RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells. PLoS One. 2014; 9(6): e99625. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubramanian A, Tamayo P, Mootha VK, et al.: Gene set enrichment analysis: a knowledge- based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43): 15545–15550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiñero J, Ramírez-Anguita JM, Saüch-Pitarch J, et al.: The disgenet knowledge platform for disease genomics: 2019 update. Nucleic Acids Res. 2020; 48(D1): D845–D855. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu S: enrichplot: Visualization of Functional Enrichment Result. 2019. Reference Source\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\nKulkarni N, Alessandrì L, Panero R, et al.: Reproducible bioinformatics project: a community for reproducible bioinformatics analysis pipelines. BMC Bioinformatics. 2018; 19(Suppl 10): 349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfgan E, Baker D, Batut B, et al.: The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018; 46(W1): W537–W544. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "67235", "date": "01 Sep 2020", "name": "Egon L. Willighagen", "expertise": [ "Reviewer Expertise R packages", "identifier mapping", "biological pathway resources", "metabolomics", "cheminformatics" ], "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 an R package (gprofiler2) that wraps around the REST interface of a g:Profiler web service. The paper outlines the method used to access the API, and explains that providing an R API makes it easier to integrate with the functionality of other R packages. The description of the software is sufficient and complementary to the source code repository (and backed by a Zenodo archive). The examples given nicely demonstrate the functionality and I tried many of them in rstudio (some comments below). The visualisation, as is most functionality, is inherited from the website, and as such benefits from a larger user base.\nThe choice of the combination of having the code base under version control, archiving on Zenodo, and distribution via CRAN shows high-quality standards. A test suite, unfortunately, is missing, and adding it is recommended. Furthermore, it could be interesting to explore curl as a replacement of RCurl, as I had trouble with SSL certificates with the RCurl package on Windows (but not essential).\nI can build the package from source. When applying a regular CRAN check on the package I get a couple of warnings and notes, but none that are affecting this paper. I left two source code suggestions: https://github.com/egonw/r-gprofiler2/pull/1.\nI also like to note that I found out that the interactive plot does not seem to work in RStudio: I only get an empty window.\nRegarding the content of the article, I have the following questions. First, the argument that the computation is done on a central server as an advantage depends on the use case and is not generally true. For example, it requires all information to be shared with the server, which is not possible for everyone that may be interested in the server. As such, one could imagine having a Docker with the webservice and method parameters in the R package to interact with servers with a different domain or IP address. This is, however, just a future feature request.\nSecond, regarding the data used in the functionality, I have three questions. How is the ordering of pathways in for KEGG, Reactome, and WikiPathways defined? For Reactome, I can imagine their own hierarchy is used. What about KEGG and WikiPathways? Is the Pathway Ontology used for the latter? A similar question applies when using custom GMT files? The ordering is then defined by the other in the file, I assume? Finally, in the Reproducibility section, but it is not clear to me why version information is not given for the data GO, KEGG, Reactome, etc. I would strongly suggest providing that information too. I would suggest clarifying the first two in the manuscript, but the last one may require additional functionality. Alternatively, the article should be clear that version information is only given for a subset of used resources.\nA third topic I like to ask about is the following: If g:Profile is an ELIXIR RIR, have you consider adding the option to use the Identifiers.org prefixes, e.g. \"wikipathways\" in addition to \"WP\"?\nFourth, the \"Use case\" uses packages that need to be installed. The keep symmetry with the description of gprofiler2 itself, please consider adding install instructions for these Bioconductor packages.\nLast, I collected a number of small textual suggestions and have annotated these with hypothes.is: https://hyp.is/rE4tRurJEeqMHW_RYqO0xA/f1000research.com/articles/9-709.\nThese suggestions include a number of title fixes in the bibliography where article titles are all converted to lower case, which looks weird for names of tools.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5946", "date": "17 Nov 2020", "name": "Hedi Peterson", "role": "Author Response", "response": "We thank the reviewer for thorough reading of the manuscript and for the suggestions to improve the gprofiler2 package and the g:Profiler tool itself. Reviewer's comments are in bold followed by our responses in regular font.The choice of the combination of having the code base under version control, archiving on Zenodo, and distribution via CRAN shows high-quality standards. A test suite, unfortunately, is missing, and adding it is recommended. We agree that testing is an important part of software development. However, since the main functionality of the gprofiler2 R package is to wrap POST requests to g:Profiler API, which has an extensive test suite, then we decided to not include additional tests to the package. Nevertheless, we use the R CMD checks for the package every time we submit a new version to CRAN. Furthermore, it could be interesting to explore curl as a replacement of RCurl, as I had trouble with SSL certificates with the RCurl package on Windows (but not essential).Thank you for the suggestion. The decision to use the RCurl package was made in 2012 when development started for the first version of the g:Profiler package. Replacing this dependency now can potentially cause issues for current users and external pipelines. Therefore, at the moment, we will keep this dependency as is, but we might consider the change in future updates. We are aware of the issues related to SSL certificates on Windows and due to that we provide access to g:Profiler both with and without SSL. I can build the package from source. When applying a regular CRAN check on the package I get a couple of warnings and notes, but none that are affecting this paper. I left two source code suggestions: https://github.com/egonw/r-gprofiler2/pull/1.Thank you for the suggestions, we included the suggested changes. I also like to note that I found out that the interactive plot does not seem to work in RStudio: I only get an empty window.It appears that this is a known issue with rendering interactive plotly images in the latest RStudio version (see https://github.com/ropensci/plotly/issues/1712 and https://github.com/rstudio/rstudio/issues/7507, for example). One of the publicly proposed solutions is to reinstall RStudio. We hope this solves the interactivity issue.Regarding the content of the article, I have the following questions. First, the argument that the computation is done on a central server as an advantage depends on the use case and is not generally true. For example, it requires all information to be shared with the server, which is not possible for everyone that may be interested in the server. As such, one could imagine having a Docker with the webservice and method parameters in the R package to interact with servers with a different domain or IP address. This is, however, just a future feature request.We agree that using a central server is problematic for users who cannot share their data. However, centralising computations enables us to maintain the g:Profiler ecosystem better and guarantees that the results from different access points (web tool, R package, Python package, Galaxy tools, custom API requests) remain consistent, which is beneficial for the users. To alleviate data sharing issue we do not store the input gene lists in g:Profiler unless the user explicitly requests their data to be stored for future access/reference via dedicated short links. This information is provided at the footer of the g:Profiler web page, and will be included within the next version of the R package (0.2.1). In addition, we added the following sentences (underlined) to the manuscript:“Relying on the central API also simplifies the maintenance of the g:Profiler interfaces and enables the R users to get access to the most up-to-date data without having to download the heavy annotation data files to their own devices. At the same time, g:Profiler respects users’ privacy and does not store input gene lists unless the user explicitly requests their data to be stored for future reference via dedicated short links (see section “Sending analysis from R to g:Profiler web interface”).”“In this case, the variable multi_gp_link is a character string that corresponds to a stable short-link to these enrichment results: https://biit.cs.ut.ee/gplink/l/0wgtcERnQT. We also note that the input gene lists will be stored in a database to provide short-link access.”Second, regarding the data used in the functionality, I have three questions. How is the ordering of pathways in for KEGG, Reactome, and WikiPathways defined? For Reactome, I can imagine their own hierarchy is used. What about KEGG and WikiPathways? Is the Pathway Ontology used for the latter? We use the order provided by the corresponding data source for all three of them. For example, pathways from KEGG are provided in the order of their identifiers, but pathways from Reactome are ordered by the name of the pathway term. We do not currently use the Pathway Ontology for WikiPathways. Thus, the only source for which we use hierarchical ontology order in the Manhattan plot is Gene Ontology, but the x-axis positions are fixed for all of the sources. This means that the terms in the Manhattan plot do not change the position along the x-axis when the input query changes. A similar question applies when using custom GMT files? The ordering is then defined by the other in the file, I assume? Yes, in case of custom GMT files, the order of the terms in the Manhattan plot is defined by their order in the GMT file. We included the following sentence (underlined) to the manuscript to clarify this:“The custom data source results can also be plotted using the Manhattan plots (Figure 3). In this case, the term position on the x-axis is defined by the order in the GMT file.” Finally, in the Reproducibility section, but it is not clear to me why version information is not given for the data GO, KEGG, Reactome, etc. I would strongly suggest providing that information too. I would suggest clarifying the first two in the manuscript, but the last one may require additional functionality. Alternatively, the article should be clear that version information is only given for a subset of used resources.Historically, g:Profiler versioning schema is based on Ensembl releases because we update our database according to their update schedule (with a time lag). The version fixes the exact extract of g:Profiler annotation database to give a point of reference for g:Profiler enrichment results and is not intended to cover all the numerous data source versions. However, we agree that the source specific version information might be useful for the users and it is available from the g:Profiler’s web page (under the “Data sources” section there is a link “Show data versions”). We included this information to the revised manuscript. Furthermore, based on this suggestion, we also added a new function get_version_info(organism) to the R package that will enable users to obtain the versions of all the data sources used for the set g:Profiler version and organism. The function is already in the source code repository https://gl.cs.ut.ee/biit/r-gprofiler2, but currently works only for the Beta version, i.e. if set_base_url(“http://biit.cs.ut.ee/gprofiler_beta”). This functionality will be included in the next CRAN release of the gprofiler2 R package (0.2.1). The amendments in the section “Reproducibility” to clarify the g:Profiler version info are underlined in the excerpt below. “The g:Profiler specific version number notes that the results were obtained using the state of the database that includes data from Ensembl release 99, Ensembl Genomes release 46 and WormBase ParaSite release 14, among other sources, and the g:Profiler codebase with the Git revision number f929183. The version number together with the details of applied parameters (available from multi_gp$meta$query_metadata) is enough to reproduce the enrichment results in g:Profiler. A more detailed information about the data source versions in a given g:Profiler version is available from the g:Profiler web page https://biit.cs.ut.ee/gprofiler under the link “Show data versions” in the “Data sources” section.“ A third topic I like to ask about is the following: If g:Profile is an ELIXIR RIR, have you consider adding the option to use the Identifiers.org prefixes, e.g. \"wikipathways\" in addition to \"WP\"?We have not considered the option to use the identifiers.org prefixes at the moment. Currently all the terms are linked inside the g:Profiler toolset and no external links are provided. However, we thank the reviewer for this suggestion and we will consider it for further g:Profiler releases.Fourth, the \"Use case\" uses packages that need to be installed. The keep symmetry with the description of gprofiler2 itself, please consider adding install instructions for these Bioconductor packages.We included the suggested installation instructions in the revised version of the manuscript. Last, I collected a number of small textual suggestions and have annotated these with hypothes.is: https://hyp.is/rE4tRurJEeqMHW_RYqO0xA/f1000research.com/articles/9-709. These suggestions include a number of title fixes in the bibliography where article titles are all converted to lower case, which looks weird for names of tools.Thank you very much for noticing the issues from bibliography parsing. We updated the manuscript according to the suggestions." } ] } ]	1	https://f1000research.com/articles/9-709
https://f1000research.com/articles/9-328/v1	04 May 20	{ "type": "Brief Report", "title": "COVID-19 testing capabilities at urgent care centers in states with greatest disease burden", "authors": [ "Walter Hsiang", "Howard Forman", "Siddharth Jain", "Akshay Khunte", "Grace Jin", "Laurie Yousman", "Michael Najem", "Alison Mosier-Mills", "Daniel Wiznia", "Howard Forman", "Siddharth Jain", "Akshay Khunte", "Grace Jin", "Laurie Yousman", "Michael Najem", "Alison Mosier-Mills", "Daniel Wiznia" ], "abstract": "While rapid and accessible diagnosis is paramount to monitoring and reducing the spread of disease, COVID-19 testing capabilities across the U.S. remain constrained. For many individuals, urgent care centers (UCCs) may offer the most accessible avenue to be tested. Through a phone survey, we describe the COVID-19 testing capabilities at UCCs and provide a snapshot highlighting the limited COVID-19 testing capabilities at UCCs in states with the greatest disease burden.", "keywords": [ "COVID-19", "urgent care center", "testing", "health services" ], "content": "Introduction\n\nWhile rapid and accessible COVID-19 diagnosis is paramount to monitoring and reducing the spread of disease, COVID-19 testing capabilities across the U.S. remain constrained. For many individuals, urgent care centers (UCCs) may offer the most accessible avenue to be tested. Using a phone survey, we describe the COVID-19 testing capabilities of UCCs in states with the greatest disease burden.\n\n\nMethods\n\nOur study received non-human research IRB exemption from the Yale School of Medicine and participant consent was not required. We identified ten states with the highest COVID-19 caseload as of March 19, 2020 according to the Centers for Disease Control (CDC)1. Using the Urgent Care Association “Find an Urgent Care” directory, we identified all UCCs within the state of interest and assigned each UCC a numeric identifier. A random number generator was used to select for a convenience sample of 25 UCCs per state. If the UCC was not able to be contacted, a new UCC was randomly selected and called. UCCs were classified into independent, hospital/health network, and academic categories.\n\nUsing a standardized survey script (Figure 1), trained investigators asked UCC receptionists about COVID-19 testing ability, testing criteria, time to test results, costs of tests and visits for insured/uninsured patients, and test referrals. All 250 calls were made on March 20, 2020 and were limited to 1 minute to minimize occupying clinic resources.\n\n\nResults\n\nOf 250 UCCs contacted, 57 (22.8%) offered COVID-19 testing. Hospital/health network-affiliated UCCs were more likely to offer COVID-19 tests compared to independent UCCs (odds ratio 3.69, 95% confidence interval 1.94–7.01, p<0.0001).\n\nOf UCCs that offered testing, 56 (98.2%) required the patient to be symptomatic (typically fever and respiratory symptoms) and 2 (0.4%) required a primary care physician referral. In total, 45 (86.5%) UCCs charged a fee to test uninsured patients, but no UCC could provide a definitive answer regarding test fees for insured patients given the shifting federal legislation. A total of 53 (94.6%) UCCs charged a visit fee in addition to the COVID-19 lab test fee. For the 49 centers that provided the wait time for test results, the median time was 120 hours (interquartile range 96 hours to 144 hours).\n\nOf UCCs that did not offer testing, 97 (51.3%) referred individuals to other clinics that could possibly test for COVID-19, and 37 (24.8%) directly referred individuals to a specific emergency department. Individual-level results for each UCC are available as Underlying data2.\n\n\nDiscussion\n\nIn the 10 states with the greatest COVID-19 caseload, only 23% of UCCs offered COVID-19 testing. Additionally, results would take approximately five days to be processed. Although time to test results at public/state labs are typically 24–48 hours (Table 1), time to test results at UCCs were longer as most samples are sent to external labs. However, it remains unclear whether UCC ability to obtain test samples may be unmatched by the ability to process tests. This finding underscores the importance of point-of-care testing that can rapidly detect COVID-19, particularly because severe disease peaks at approximately ten days from onset of initial symptoms3.\n\n*Time to results at state/public health labs obtained from the respective state’s Department of Public Health website as of March 20.\n\nFees and cost-sharing for COVID-19 tests remain unclear. The Families First Coronavirus Response Act, which passed on March 18, mandated all group and individual health plans cover COVID-19 testing and gave states the option to use Medicaid coverage for testing uninsured patients4. Although this study could not definitively define test fees, most UCCs stated they would charge test fees, contrary to recent federal regulations, in addition to fees for the urgent care visit itself as of March 20. Test and visit fees at UCCs may discourage patients from seeking COVID-19 testing.\n\nThis report has limitations. The small number of UCCs contacted per state may not accurately represent the state’s urgent care climate. Additionally, the rapidly changing nature of the COVID-19 pandemic may affect these findings. However, this study serves as an important snapshot that highlights the limited COVID-19 testing capabilities at UCCs in the most heavily burdened states.\n\n\nData availability\n\nHarvard Dataverse: COVID-19 Testing Capabilities at Urgent Care Centers in States with Greatest Disease Burden. https://doi.org/10.7910/DVN/SJSNZ64.\n\nThis project contains the individual-level responses of each urgent care center to each question from the call script (JMP and XLSX file formats.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nCoronavirus Disease 2019 Cases in the U.S. CDC. Published 2020. Accessed 2020. Reference Source\n\nHsiang W, Forman H, Jain S, et al.: \"COVID-19 Testing Capabilities at Urgent Care Centers in States with Greatest Disease Burden\". Harvard Dataverse, V2. 2020. http://www.doi.org/10.7910/DVN/SJSNZ6\n\nPan F, Ye T, Sun P, et al.: Time Course of Lung Changes On Chest CT During Recovery From 2019 Novel Coronavirus (COVID-19) Pneumonia. Radiology. 2020; 200370. PubMed Abstract \| Publisher Full Text\n\nH.R.6201 - Families First Coronavirus Response Act. In: Congress US, ed 2020. Reference Source" }	[ { "id": "67613", "date": "21 Jul 2020", "name": "Siddharth Sridhar", "expertise": [ "Reviewer Expertise Emerging infectious diseases", "clinical virology", "hepatitis E" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study provides a snapshot of the COVID-19 testing landscape in ambulatory clinics in the United States in March 2020. Overall, only 22.8% of UCCs offered COVID-19 testing. Turn around time was up to several days in several centres.\n\nTesting and tracing are vital means to control community COVID-19 epidemics. Testing is the first step in the test-isolate-quarantine paradigm that has successfully contained COVID-19 in many parts of the world. Deployment of testing to a wide section of symptomatic individuals in the communities is a challenge. UCCs are a potentially convenient location where such testing can be performed; widespread testing at these centres would also generate vital surveillance data. The fact that less than a quarter of all UCCs in affected states offered COVID-19 testing represents a lost opportunity and a lesson for future pandemic preparedness.\n\nSome suggestions for improvement are as follows:\n\nThis data is now 4 months old. While it would be too much to ask to repeat the survey, it would be good if the authors can provide any available updates on the number of UCCs offering COVID-19 testing or turn around time of state laboratories. Is the low availability of testing at UCCs improving in these states, especially in heavily affected areas like Florida?\n\nBriefly discuss the obstacles to UCCs offering COVID-19 tests.\n\nHow does this snapshot in March reflect the situation today in July? Do the authors observe an association between a higher proportion of testing UCCs in March with better COVID-19 control today?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "6100", "date": "24 Nov 2020", "name": "Walter Hsiang", "role": "Author Response", "response": "This data is now 4 months old. While it would be too much to ask to repeat the survey, it would be good if the authors can provide any available updates on the number of UCCs offering COVID-19 testing or turn around time of state laboratories. Is the low availability of testing at UCCs improving in these states, especially in heavily affected areas like Florida? While it is difficult to repeat this study under the previous condition, we attempted to provide additional clarity regarding this question by repeating the phone calls to the same 25 UCC locations in Florida in October. In Florida, the availability of testing improved from 28% to 64%, however, the mean wait time increased from 4 days to 7 days. Briefly discuss the obstacles to UCCs offering COVID-19 tests. We have added several statements on the obstacles to UCC testing in 3rd paragraph of the discussion section. How does this snapshot in March reflect the situation today in July? Do the authors observe an association between a higher proportion of testing UCCs in March with better COVID-19 control today? It is difficult to say whether higher availability of testing is associated with better COVID-19 control, since numerous factors affect the ability to control the spread of infection. However, as we continue into an additional wave of COVID-19 infections, sufficient availability of and accessibility to testing remains paramount." } ] }, { "id": "68237", "date": "30 Jul 2020", "name": "Buqing Liang", "expertise": [ "Reviewer Expertise Neurosurgery" ], "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 study, the authors conducted a phone interview to investigate the COVID-19 testing capabilities at urgent care centers in states with the greatest disease burden. This is an interesting and meaningful topic, especially during this pandemic crisis. However, there are some issues that need to be addressed.\nHow did the authors validate the data from the UCCs to ensure no wrong information was conveyed? Even at the same center, two receptionists may give different answers to the same question. Is it possible to double confirm with two different receptionists?\n\nAs the authors state that UCCs were randomly selected. How to differentiate big city centers vs rural area centers? High burden states still have rural areas that may have a very low incidence of COVID infection that does not supplement with UCCs with test capability. What is the percentage of these UCCs?\n\nBased on the above-mentioned reason, wouldn't investigating UCCs in cities with high COVID-19 incidence be a more reasonable design?\n\nLastly, in the discussion part, the authors merely analyzed the results but did not put forward how it can be utilized for improvement. Namely, what's the meaning of this study?\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6101", "date": "24 Nov 2020", "name": "Walter Hsiang", "role": "Author Response", "response": "How did the authors validate the data from the UCCs to ensure no wrong information was conveyed? Even at the same center, two receptionists may give different answers to the same question. Is it possible to double confirm with two different receptionists? We cannot guarantee that receptionists may or may not convey the correct information, but contacting offices in a randomized approach with a large sample has been a validated way to collect survey information. We cannot double confirm with receptionists given the changing landscape since March. However, this caller approach has been used and validated in many our similar survey methodologies: doi.org/10.1097/SLA.0000000000004373; doi.org/10.1177/0046958019838118; doi.org/10.1016/j.surg.2018.03.013; doi.org/10.1016/j.arth.2015.03.015 As the authors state that UCCs were randomly selected. How to differentiate big city centers vs rural area centers? High burden states still have rural areas that may have a very low incidence of COVID infection that does not supplement with UCCs with test capability. What is the percentage of these UCCs? We have added an urban vs rural variable in our analysis based off the UCC’s zip code in the 2010 U.S. Census. UCCs were all located in urban-designated areas. The urban-designation can be broken down into subcategories, and 4 centers were located in smaller towns with an urban cluster. However, these UCCs are still considered urban. It is well known that UCCs tend to locate in wealthier, urban areas. The results section and data sharing section have been updated accordingly. Based on the above-mentioned reason, wouldn't investigating UCCs in cities with high COVID-19 incidence be a more reasonable design? Given that the primary locations of UCCs at the time of the study were in urban metropolitan areas, we feel confident that our sample essentially represented cities with high COVID-19 incidence. Lastly, in the discussion part, the authors merely analyzed the results but did not put forward how it can be utilized for improvement. Namely, what's the meaning of this study? Thank you for this feedback. We have added two areas of improvement in the 3rd paragraph of the discussion." } ] } ]	1	https://f1000research.com/articles/9-328
https://f1000research.com/articles/9-344/v1	11 May 20	{ "type": "Software Tool Article", "title": "CusVarDB: A tool for building customized sample-specific variant protein database from next-generation sequencing datasets", "authors": [ "Sandeep Kasaragod", "Varshasnata Mohanty", "Ankur Tyagi", "Santosh Kumar Behera", "Arun H. Patil", "Sneha M. Pinto", "T. S. Keshava Prasad", "Prashant Kumar Modi", "Harsha Gowda", "Sandeep Kasaragod", "Varshasnata Mohanty", "Ankur Tyagi", "Santosh Kumar Behera", "Arun H. Patil", "Sneha M. Pinto", "T. S. Keshava Prasad" ], "abstract": "Cancer genome sequencing studies have revealed a number of variants in coding regions of several genes. Some of these coding variants play an important role in activating specific pathways that drive proliferation. Coding variants present on cancer cell surfaces by the major histocompatibility complex serve as neo-antigens and result in immune activation. The success of immune therapy in patients is attributed to neo-antigen load on cancer cell surfaces. However, which coding variants are expressed at the protein level can’t be predicted based on genomic data. Complementing genomic data with proteomic data can potentially reveal coding variants that are expressed at the protein level. However, identification of variant peptides using mass spectrometry data is still a challenging task due to the lack of an appropriate tool that integrates genomic and proteomic data analysis pipelines. To overcome this problem, and for the ease of the biologists, we have developed a graphical user interface (GUI)-based tool called CusVarDB. We integrated variant calling pipeline to generate sample-specific variant protein database from next-generation sequencing datasets. We validated the tool with triple negative breast cancer cell line datasets and identified 423, 408, 386 and 361 variant peptides from BT474, MDMAB157, MFM223 and HCC38 datasets, respectively.", "keywords": [ "Next-generation sequencing", "Variant protein database", "NGS-pipeline" ], "content": "Introduction\n\nCancer genome sequencing projects have revealed thousands of genomic variations in cancers (Forbes et al., 2010; Tomczak et al., 2015; Tate et al., 2019; Zhang et al., 2011). It is reasonable to presume that genomic variations in cancers might result in several new proteoforms. Genomic variations in coding regions may encode mutant proteins in cancer cells. These mutant proteins are known to drive proliferation in various cancers. For example, amino acid substitution of arginine to leucine at position 858 (L858R) in the epidermal growth factor receptor (EGFR) gene has been observed in 17% of pulmonary adenocarcinoma patients (Morgensztern et al., 2015). In addition, a mutation in gene BRAF V600E is known to result in increased possibility of metastatic melanoma (Chapman et al., 2011). Some of these mutant proteins are proteolytically processed in cancer cells, resulting in major histocompatibility complex (MHC) presentation of mutant peptides. These mutant peptides serve as neo-antigens that recruit T cells and result in immune activation (Kreiter et al., 2015). Therefore, it is important to identify mutant proteins expressed by cancer cells. However, there are no easy-to-use pipelines for biologists to identify such coding variants, which alter the protein sequences and may play an important role in tumorigenesis. Detection of cancer-specific proteoforms has been studied by several research teams using proteogenomics methods. This approach integrates proteomics data with genome sequences to identify novel protein-coding regions (Menschaert & Fenyö, 2017; Nesvizhskii, 2016; Ruggles & Fenyö, 2016), which can be easily adopted in the field of cancer biology for the detection of genomic aberrations and coding single nucleotide polymorphisms (SNPs). Cancer is a heterogeneous disease with a large number of genomic variations. However, detection of mutations and SNPs at the proteomic level will aid in further investigations on altered molecular networks and functional ramifications and may provide candidate molecules for novel therapeutic interventions (Subbannayya et al., 2016) for various types of cancers.\n\nSeveral qualitative and quantitative proteomics studies have been reported, which identify altered expression of proteins in cancers (Subbannayya et al., 2015). Often, general workflows were used in such investigations, wherein a reference protein database was used to search the experimentally derived tandem mass spectrometry data for the identification and quantification of the proteins (Kelkar et al., 2014). However, such a reference database is usually deprived of sample-specific amino acid variations brought about by genomic aberrations and coding SNPs reported for the various cancers. The publicly available databases such as dbSNP (Sherry et al., 2001), COSMIC (Forbes et al., 2015) and UniProt (Apweiler et al., 2004) can be used to identify the variant peptides (Alfaro et al., 2017) but millions of protein-variants from these databases might increase the probability of identifying false positives. Therefore, there is a need for an improved specific method to characterize germline and somatic variations at the proteomic level (Alfaro et al., 2017) specific to the cancer type. Hence, we developed CusVarDB with an in-built pipeline for genomics suite in deriving variants and creating custom variant protein databases.\n\n\nMethods\n\nCusVarDB is available at https://sourceforge.net/projects/cusvardb/ and http://bioinfo-tools.com/Downloads/CusVarDB/V1.0.0/. Our tool supports graphical user interface (GUI) for the easy execution of the next-generation sequencing (NGS) pipelines. The GUI was developed using Microsoft Visual Studio Community edition 2017. Linux commands were executed through the Python program (terminal.py) developed by Linwei available on GitHub. A portable version of Perl was used to execute the script for downloading the annotation database and executing the variant annotation. A python script was used to generate the custom variant database; the scripts were made portable using PyInstaller. The dry-run concept is implemented in the tool to customize the commands according to user need and run in batches.\n\nCusVarDB inherits different NGS pipelines for genomics, RNA-Seq and exome-seq datasets. The tool performs the following steps: i) alignment of genomic data to a reference genome; ii) variant calling; iii) variant annotation; and iv) generate variant protein database (Figure 1). Burrows-Wheeler Aligner (BWA) is executed for alignment of genome and exome (Li & Durbin, 2009) data, while HISAT2 is used for RNA-Seq data (Kim et al., 2015). Subsequent steps involving sample labelling, variant calling and annotation are performed using Picard, GATK (McKenna et al., 2010) and ANNOVAR (Wang et al., 2010), respectively. CusVarDB substitutes the amino acid variations from the previous steps to generate a custom variant protein database.\n\nSchematic representation of the workflow.\n\nCusVarDB runs on Windows 10 system. It requires Linux Bash Shell and ANNOVAR tool to be enabled and installed by the user. Minimum system requirements include Intel i5 or i7, having at least four cores with 8 GB of RAM and 1 TB hard drive. High performance processors such as Intel i9 or Xeon and large quantity of RAM can enable faster execution of tasks.\n\nThe tool requires FASTA, FASTQ and dbSNP database as input files. FASTA file will be loaded at configuration panel and indexed using bwa index or hisat2-build command. The quality control panel loads the FASTQ file and produces a quality check report using the FastQC tool. The third panel asks the user to load the FASTQ file(s), set the number of threads and RAM to perform the alignment and variant calling steps. This step produces the raw VCF file, which will be used for annotation in annotation tab. The annotation panel annotates the variants and incorporates those variants to the protein database to create a custom variant protein database. Detailed information of tool installation and execution of a test dataset are available in the manual (Manual.pdf) available to download here and on Zenodo (Kasaragod et al., 2020b).\n\nThe genomics datasets downloaded from the SRA repository were subjected to a quality check using the FastQC tool. Poor quality reads with Phred score less than twenty were trimmed using fastx_trimmer. Trimmed reads were mapped to human reference genome version 19 (hg19). AddOrReplaceReadGroup and Markduplicates operations from GATK were performed to add label information and to remove polymerase chain reaction (PCR) artefacts. Further, post alignment processing steps such as IndelRealigner and BaseRecalibrator were performed to correct the mapping errors made by alignment tool to increase variant calling accuracy. Variant calling was performed using HaplotypeCaller. Finally, raw variants were annotated using ANNOVAR and the annotated variants were incorporated to the protein database to create custom variant protein database.\n\nProteomic searches were carried out using Proteome Discoverer 2.3 (Thermo Scientific, Bremen, Germany). Searches could also be carried out using freely available open source alternatives such as MaxQuant, MsFragger, MS-GF+, MyriMatch or OMSSA. Mass spectrometry raw files were obtained from the PRIDE archive (PXD008222) and searched against the customized variant protein database using the SEQUEST-HT search algorithm. Trypsin and LysC were set as proteolytic enzymes with a maximum missed cleavage of one. Carbamidomethylation of cysteine was set as a fixed modification, and acetylation of protein N-terminus and oxidation of methionine were set as variable modifications with a minimum peptide length of seven amino acids. The precursor mass tolerance was fixed as 10 ppm, and 0.05 Da for fragment ions. The mass spectrometry data were searched against the decoy database with a 1% false discovery rate cut-off at the peptide level.\n\nWe have provided a dataset for users to test the tool. The test dataset was taken from the study SRR7418758 archived on the NCBI Sequence Read Repository (SRA) and aligned using human reference genome version 19 (hg19). Using samtools view command, reads mapped to chromosome 22 were extracted and converted to FASTQ files (paired-end). We have also provided chromosome 22 nucleotide sequences from hg19 and corresponding variant information from dbSNP database.\n\n\nUse cases\n\nTo determine the functionality of our tool, we have taken exome (Daemen et al., 2013) and proteome datasets (Lawrence et al., 2015). We incorporated 12,429; 13,923; 12,386 and 11,600 non-synonymous SNPs from BT474 (accession number SRR925752), MDMAB157 (accession number SRR925788), MFM223 (accession number SRR925796) and HCC38 (accession number SRR925778) cells, respectively, to the protein database (Figure 2A). These non-synonymous SNPs were incorporated to the reference protein database (Human RefSeq release 93) to create the customized variant protein database. The mass spectrometry-based raw files were searched against their respective custom variant protein database, which resulted in the identification of 423, 408, 386 and 361 variant peptides from BT474, MDMAB157, MFM223 and HCC38, cell line datasets (Figure 2B). The resultant variant peptide lists are available as Underlying data (Kasaragod et al., 2020a). The data generated from the tool demonstrates the usefulness and the ease of detection of variant peptides in an integrated omics analysis.\n\nA) Number of nonsynonymous variants incorporated to protein database. B) Mutant peptides identified across four cell lines (BT474, MDMAB157, MFM223 and HCC38) where the mass spectrometry raw files were searched against custom mutant protein database.\n\n\nConclusions\n\nCusVarDB generates customized variant protein database from NGS datasets. To our knowledge, this is the first tool which uses the Linux Bash Shell to execute the NGS tools on a Windows OS. The tool provides the additional feature of dry-run, making the commands highly customizable. The variant protein database generated by this tool is highly compatible for use as a reference protein database for analysis of variants at the proteomic level.\n\n\nData availability\n\nWhole exome data from Daemen et al. (2013) on BioProject, Accession number PRJNA210427\n\nMass spectrometry proteome data from Lawrence et al. (2015) on PRIDE, Accession number PXD008222: https://identifiers.org/pride.project:PXD008222\n\nTest dataset on SRA, Accession number SRR7418758: https://identifiers.org/insdc.sra:SRR7418758\n\nZenodo: CusVarDB: A tool for building customized sample-specific variant protein database from Next-generation sequencing datasets. http://doi.org/10.5281/zenodo.3747108 (Kasaragod et al., 2020a)\n\nThis project contains the following underlying data:\n\n- Gowda_CusVarDB_Supplementary_table1.xlsx (This table contains the resultant variant peptides along with the wild-type peptides from BT474, MDMAB157, MFM223, and HCC38 datasets. Along with mutant peptides, this section also provides additional information such as peptide-spectrum match [PSM], Protein accession, cross-correlation value from the search [Xcorr] and retention time [RT])\n\n- Gowda_CusVarDB_Supplementary_table2.xlsx (This table provides the complete details of the resultant peptides. Here the mutant and corresponding wild-type peptides are mentioned in different sheets. For a given mutant peptide its wild-type peptide and corresponding information can be mapped using the VLOOKUP function in Excel by keeping column A [Sl.No] as lookup parameter.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSoftware available from: https://sourceforge.net/projects/cusvardb/ and http://bioinfo-tools.com/Downloads/CusVarDB/V1.0.0/\n\nSource code available from: https://github.com/sandeepkasaragod/CusVarDB\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3780645 (Kasaragod et al., 2020b)\n\nLicense: Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank Mr. Hitesh Ugaram Kore, Ph.D. student, Cancer Precision Medicine Group, QIMR Berghofer Medical Research Institute, Australia for his assistance with testing the tool and fixing the bugs which has greatly improved the performance of the tool.\n\n\nReferences\n\nAlfaro JA, Ignatchenko A, Ignatchenko V, et al.: Detecting protein variants by mass spectrometry: a comprehensive study in cancer cell-lines. Genome Med. 2017; 9(1): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nApweiler R, Bairoch A, Wu CH, et al.: UniProt: the Universal Protein knowledgebase. Nucleic Acids Res. 2004; 32(Database issue): D115–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChapman PB, Hauschild A, Robert C, et al.: Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011; 364(26): 2507–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaemen A, Griffith OL, Heiser LM, et al.: Modeling precision treatment of breast cancer. Genome Biol. 2013; 14(10): R110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForbes SA, Tang G, Bindal N, et al.: COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer. Nucleic Acids Res. 2010; 38(Database issue): D652–D657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForbes SA, Beare D, Gunasekaran P, et al.: COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res. 2015; 43(Database issue): D805–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKasaragod S, Mohanty V, Tyagi A, et al.: CusVarDB: A tool for building customized sample-specific variant protein database from Next-generation sequencing datasets [Data set]. Zenodo. 2020a. http://www.doi.org/10.5281/zenodo.3747108\n\nKasaragod S, Mohanty V, Tyagi A, et al.: CusVarDB: A tool for building customized sample-specific variant protein database from Next-generation sequencing datasets: First release (Version 1.0.0). Zenodo. 2020b. http://www.doi.org/10.5281/zenodo.3780645\n\nKelkar DS, Provost E, Chaerkady R, et al.: Annotation of the zebrafish genome through an integrated transcriptomic and proteomic analysis. Mol Cell Proteomics. 2014; 13(11): 3184–98. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63336", "date": "23 Jul 2020", "name": "Suresh Mathivanan", "expertise": [ "Reviewer Expertise Cancer proteomics and bioinformatics" ], "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 manuscript that describes an important tool.\n\nThe authors may need to discuss other proteogenomic tools similar to this and provide the advantages. If such tools don't exist, perhaps make a strong case. Zhang lab tools can also be discussed with references.\n\nCan the authors also discuss and refer previous attempts on onco-proteogenomics? One of the first paper on onco-proteogenomics came in 2012: Mathivanan, S., Ji, H., Tauro, B.J., Chen, Y.S. and Simpson, R.J. (2012) Identifying mutated proteins secreted by colon cancer cell lines using mass spectrometry. Journal of Proteomics. 76, 141-91.\n\nDoes CurVardb provide only non-synonymous point mutations? What about frame shit mutations? Is that also provided or perhaps will be added in next version.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "63338", "date": "26 Aug 2020", "name": "Richard Kumaran Kandasamy", "expertise": [ "Reviewer Expertise My research expertise include proteomics", "proteogenomics", "bioinformatics and systems immunology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Kasaragod et al., describes a software application for developing sample-specific protein sequence database that takes into account the protein-coding variants, for interpretation of mass spectrometry data to see what portion of the variants are seen at the protein expression level. This is an important aspect in exemplifying the use of genomic data on proteomics datasets associated with cancer samples.\nThe software is made available for download and enough documentation has been provided for potential users.\nThe authors use this data set to identify protein-level evidence for expression of variants in four different cancel cell lines.\nThe manuscript can be accepted for indexing. I have one important point that I would like the authors to address:\nWhile the findings are potentially interesting, it would be nice if the authors pick a few important examples specific to these four cell lines.\nThis would give the users a better appreciation of the biological insights given by this application.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-344
https://f1000research.com/articles/7-1141/v1	26 Jul 18	{ "type": "Research Article", "title": "A systematic performance evaluation of clustering methods for single-cell RNA-seq data", "authors": [ "Angelo Duò", "Mark D. Robinson", "Charlotte Soneson", "Angelo Duò", "Mark D. Robinson" ], "abstract": "Subpopulation identification, usually via some form of unsupervised clustering, is a fundamental step in the analysis of many single-cell RNA-seq data sets. This has motivated the development and application of a broad range of clustering methods, based on various underlying algorithms. Here, we provide a systematic and extensible performance evaluation of 12 clustering algorithms, including both methods developed explicitly for scRNA-seq data and more general-purpose methods. The methods were evaluated using 9 publicly available scRNA-seq data sets as well as three simulations with varying degree of cluster separability. The same feature selection approaches were used for all methods, allowing us to focus on the investigation of the performance of the clustering algorithms themselves. We evaluated the ability of recovering known subpopulations, the stability and the run time of the methods. Additionally, we investigated whether the performance could be improved by generating consensus partitions from multiple individual clustering methods. We found substantial differences in the performance, run time and stability between the methods, with SC3 and Seurat showing the most favorable results. Additionally, we found that consensus clustering typically did not improve the performance compared to the best of the combined methods, but that several of the top-performing methods already perform some type of consensus clustering. The R scripts providing an extensible framework for the evaluation of new methods and data sets are available on GitHub (https://github.com/markrobinsonuzh/scRNAseq_clustering_comparison).", "keywords": [ "Clustering", "Single-Cell RNA-seq", "RNA-seq", "Benchmarking" ], "content": "Introduction\n\nRecent advances in single-cell RNA-seq (scRNA-seq) technologies have enabled the simultaneous measurement of expression levels of thousands of genes across hundreds to thousands of individual cells1–8. This opens up new possibilities for deconvolution of expression patterns seen in bulk samples, detection of previously unknown cell populations and deeper characterization of known ones. However, computational analyses are complicated by the high variability, low capture efficiency and high dropout rates of scRNA-seq assays9–11, as well as by strong batch effects that are often confounded by the experimental factor of interest12.\n\nGiven a collection of single cells, a common analysis task involves identification and characterization of subpopulations, e.g., cell types or cell states. With lower-dimensional single-cell assays such as flow cytometry, cell type detection is often done manually, by visual inspection of a series of two-dimensional scatter plots of marker pairs (“gating”) and subsequent identification of clusters of cells with specific abundance patterns. With large numbers of markers, such strategies quickly become unfeasible, and they are also likely to miss previously uncharacterized cell populations. Instead, subpopulation detection in higher-dimensional single-cell experiments such as mass cytometry (CyTOF) and scRNA-seq is often done automatically, via some form of clustering. As a consequence, a large number of clustering approaches specifically designed for or adapted to these types of assays are available in the literature.\n\nWhile extensive evaluations of clustering methods have been performed for flow and mass cytometry data13,14, there are to date fewer such studies available for scRNA-seq. The latter is complicated by the large number of different data generation protocols available for scRNA-seq, which in turn has a big effect on the data characteristics. Menon15 specifically evaluated three methods (Seurat16, WGCNA17 and BackSPIN18), illustrating their different behavior in low and high read depth data. A recent preprint19 compared 11 clustering tools on scRNA-seq from the 10x Genomics platform, showing that different methods generally produced clusterings with little overlap. An overview of several different types of clustering algorithms for scRNA-seq data is given by Andrews and Hemberg20.\n\nIn this paper, we extend these initial studies to a broader range of data sets with different characteristics, and additionally consider simulated data with different degrees of cluster separability. We evaluate 12 clustering algorithms, including both methods specifically developed for scRNA-seq data, methods developed for other types of single-cell data, and more general approaches, on a total of 12 different data sets. In order to focus on the performance of the clustering algorithms themselves, we use the same preprocessing approach (specifically cell and gene filtering) for all methods, and investigate the impact of the preprocessing separately. In addition to investigating how well the clustering methods are able to recover the true partition if the number of subpopulations is known, we evaluate whether they are able to correctly determine the number of clusters. Further, we study the stability and run time of the methods and investigate whether performance can be improved by generating a consensus partition based on results from multiple individual clustering methods, and the impact of the choice of methods to include in such an aggregation.\n\nWe observed large differences in the clustering results as well as in the run times of the different methods. SC3 and Seurat generally performed favorably, with Seurat being several orders of magnitude faster. In addition, Seurat typically achieved the best agreement with the true partition when the number of clusters were the same, while other methods, like FlowSOM, achieved a better agreement with the truth if the number of clusters was higher than the true number. Finally, we show that generally, combining two methods into an ensemble did not improve the performance compared to the best of the individual methods.\n\nGiven the high level of activity in methods research for preprocessing, clustering and visualization of scRNA-seq data, it is expected that many new algorithms (or new flavors of existing ones) will be proposed. In order to facilitate re-assessment as new innovations emerge and to provide extensibility to new methods and data sets, we provide the complete code to run all analyses in this study (https://github.com/markrobinsonuzh/scRNAseq_clustering_comparison). The current system uses a Makefile to run a set of R scripts for clustering, summarization and visualization of the results. In addition, all filtered (and unfiltered) data sets used in this study are readily available from the links provided in the GitHub repository.\n\n\nMethods\n\nThree real scRNA-seq data sets were downloaded from conquer21 and used for our evaluations: GSE60749-GPL13112 (here denoted Kumar22), SRP073808 (Koh23) and GSE52529-GPL16791 (Trapnell24). Table 1 and Supplementary Figure 1 give an overview of all data sets used in this study. For each of the data sets from conquer, the gene-level length-scaled TPM values (below referred to as “counts” since they are on the same scale as the raw read counts) and the phenotype were extracted from the MultiAssayExperiment25 object provided by conquer and used to create a SingleCellExperiment object. We also estimated transcript compatibility counts (TCCs) for each of these data set using kallisto26,27 v0.44, and used these as an alternative to the gene-level count matrix as input to the clustering algorithms.\n\nThe selected cell phenotype was used to define the \"true\" partition of cells when evaluating the clustering methods. For the Kumar data set, we grouped the cells by the genetic perturbation and the medium in which they were grown. For the Trapnell data set we used the time point (after the switch of growth medium) at which the cells were captured, and for the Koh data set we used the cell type annotated by the data collectors (obtained through FACS sorting). We note that the definition of the ground truth constitutes an intrinsic difficulty in the evaluation of clustering methods, since it is plausible that there are several different, but still biologically interpretable, ways of partitioning cells in a given data set, several of which can represent equally strong signals. By using ground truths that are defined independently of the scRNA-seq assay, we avoid artificial inflation of the signal that could result if the truth was derived from the scRNA-seq data itself.\n\nIn addition to the data sets from conquer, we obtained UMI counts from the Zheng data set5, generated by the 10x Genomics GemCode protocol, from https://support.10xgenomics.com/single-cell-gene-expression/datasets. We downloaded counts for eight pre-sorted cell types (B-cells, naive cytotoxic T-cells, CD14 monocytes, regulatory T-cells, CD56 NK cells, memory T-cells, CD4 T-helper cells and naive T-cells) and combined them into three data sets. For the data set denoted Zhengmix4eq, we combined randomly selected B-cells, CD14 monocytes, naive cytotoxic T-cells and regulatory T-cells in equal proportions (1,000 cells per subpopulation). For the Zhengmix4uneq data set, we combined the same four cell types, but in unequal proportions (1,000 B-cells, 500 naive cytotoxic T-cells, 2,000 CD14 monocytes and 3,000 regulatory T-cells). For the Zhengmix8eq data set, we combined cells from all eight populations, in approximately equal proportions (400–600 cells per population). For these data sets, we used the annotated cell type (obtained by pre-sorting of the cells) as the true cell label.\n\nUsing one subpopulation of the Kumar data set as input, we simulated scRNA-seq data with known group structure, using the splatter package28 v1.2.0. We generated three data sets, each consisting of 500 cells, with varying degree of cluster separability. For the SimKumar4easy data set, we generated 4 subpopulations with relative abundances 0.1, 0.15, 0.5 and 0.25, and probabilities of differential expression set to 0.05, 0.1, 0.2 and 0.4 for the four subpopulations, respectively. The SimKumar4hard data set consists of 4 subpopulations with relative abundances 0.2, 0.15, 0.4 and 0.25, and probabilities of differential expression 0.01, 0.05, 0.05 and 0.08. Finally, the SimKumar8hard data set consists of 8 subpopulations with relative abundances 0.13, 0.07, 0.1, 0.05, 0.4, 0.1, 0.1 and 0.05, and probabilites of differential expression equal to 0.03, 0.03, 0.03, 0.05, 0.05, 0.07, 0.08 and 0.1, respectively. The GitHub repository (https://github.com/markrobinsonuzh/scRNAseq_clustering_comparison) contains a link to a countsimQC report29, comparing the main characteristics of the simulated data sets to those of the underlying Kumar data set.\n\nThe scater package30 v1.6.3 was used to perform quality control of the data sets. Features with zero counts across all cells, as well as all cells with total count or total number of detected features more than 3 median absolute deviations (MADs) below the median across all cells (on the log scale), were excluded. Depending on the availability of manual annotation, we filtered out cells that were classified as doublets or debris. The scater package was also used to normalize the count values, based on normalization factors calculated by the deconvolution method from the scran package31 v1.6.2, and to perform dimension reduction using PCA32 and t-SNE33. Either the raw feature counts or the log-transformed normalized counts were used as input to the clustering algorithms.\n\nWe evaluated three methods for reducing the number of genes provided as input to the clustering methods. For each filtering method, we retained 10% of the original number of genes (with a non-zero count in at least one cell) in the respective data sets. First, we retained only the genes with the highest average expression (log-normalized count) value across all cells (denoted Expr below). Second, we used Seurat16 to estimate the variability of the features and retained only the most highly variable ones (HVG). Finally, we used M3Drop34 to model the dropout rate of the genes as a function of the mean expression level using the Michaelis-Menten equation (M3Drop). The gene-wise Michaelis-Menten constants are computed and log-transformed, and the genes are then ranked by their p-value from a Z-test comparing the gene-wise constants to a global constant obtained by combining all the genes. After filtering, we used scran to renormalize each data set, excluding cells with negative size factors. Supplementary Figure 2 shows the overlap between the retained genes with the different filtering methods, for each of the 12 data sets, and Supplementary Table 1 provides the number of cells retained after each type of filtering.\n\nTwelve clustering methods were evaluated in this study (see Table 2 for an overview). We included general-purpose clustering methods, such as hierarchical clustering and K-means, as well as methods developed specifically for scRNA-seq data, such as Seurat and SC3.\n\nAll methods except Seurat allow explicit specification of the desired number of clusters (k). Seurat instead requires a resolution parameter, which indirectly controls the number of clusters. For each data set, we ran each method with a range of k values (from 2 to either 10 or 15, depending on the true number of subpopulations in the data set). We ran Seurat with a range of resolution parameter values, approximately corresponding to the range of k values evaluated for the other methods. A subset of the methods provide an estimate of the true number of clusters; we record this estimate for comparison with the true number of subpopulations. For each choice of k (or resolution), we ran each method five times, allowing us to investigate the intrinsic stability of the obtained partitions. Note that the data is the same for all five instances, and thus only the stochasticity of the clustering method affects our stability evaluation. All parameter values except for the number of clusters were set to reasonable values following the authors’ recommendations or the respective manuals (Table 2). Gene and cell filtering within the clustering methods were disabled whenever possible, since these steps were performed in a uniform way during the preprocessing and gene selection steps.\n\nIn order to evaluate how well the inferred clusters recovered the true subpopulations, we used the Hubert-Arabie Adjusted Rand Index (ARI) for comparing two partitions46. The metric is adjusted for chance, such that independent clusterings have an expected index of zero and identical partitions have an ARI equal to 1, and was calculated using the implementation in the mclust R package v5.4. We also used the ARI to evaluate the stability of the clusters, by comparing the partitions from each pair of the five independent runs for each method with a given number of clusters.\n\nWe used a normalized Shannon entropy47 to evaluate whether the methods preferentially partitioned the cells into clusters of equal size, or whether they preferred one large and many small clusters. Given proportions p1, …, pN of cells assigned to each of N clusters, the normalized Shannon entropy is defined by\n\n\n\nSince the true degree of equality of the cluster sizes varies between data sets, we subtracted the normalized entropy calculated from the true partition to obtain the final performance index.\n\nTo evaluate the similarities between the partitions obtained by different methods, we first calculated a consensus partition from the five independent runs for each method, using the clue R package48 v0.3-55. Next, for each data set and each imposed number of clusters, we calculated the ARI between the partitions for each pair of methods, and used hierarchical clustering based on the median of these ARI values across all data sets to generate a dendrogram representing the similarity among the clusters obtained by different methods. To investigate how representative this dendrogram is, we also clustered the methods based on each data set separately, and calculated the fraction of such dendrograms in which each subcluster in the overall dendrogram appeared.\n\nFinally, we investigated whether clustering performance was improved by combining two methods into an ensemble. For each data set, and with the true number of clusters imposed, we calculated a consensus partition for each pair of methods using the clue R package, and used the ARI to evaluate the agreement with the true cell labels. We then compared the ensemble performance to the performances of the two individual methods used to construct the ensemble.\n\n\nResults\n\nThe 12 methods were tested on real data sets as well as simulations with a varying degree of complexity (Table 1) and across a range of the number of subpopulations. Focusing on the agreement between the true partitions and the clusterings obtained by imposing the true number of clusters showed a large difference between data sets as well as between methods (Figure 1; a summary across different numbers of clusters can be found in Supplementary Figure 3).\n\nEach row corresponds to a different data set, each panel to a different gene filtering method, and each column to a different clustering method. The methods and the data sets are ordered by their mean ARI across the filterings and data sets. Some methods failed to return a clustering with the correct number of clusters for certain data sets (indicated by white squares).\n\nAs expected, excellent performances were achieved for the well-separated data sets with a strong difference between the groups of cells (Kumar, KumarTCC and SimKumar4easy). When filtering by expression or variability, close to all methods achieved a correct partitioning of the cells in these data sets, while the M3Drop filtering led to poorer performance for the simulated data set. On the other hand, all methods failed to recover the partition of the cells by time point in the Trapnell data sets, where the ARIs were consistently below 0.5. This indicates that there are other, stronger, signals in this data set that dominate the clustering.\n\nWe note that the M3Drop filtering consistently led to worse performance for the simulated data sets, while the performance was more similar to the other filterings for the real data sets, which may indicate that the simulated dropout pattern is not consistent with the one being modeled by the M3Drop package. Due to negative size factor estimates, a larger number of cells had to be excluded in the Zhengmix data sets after the M3Drop filtering compared to the expression or HVG filtering (Supplementary Table 1). At most just over 20% of the cells in the expression and HVG filtering and up to approximately 40% of the cells for the M3Drop filtering were excluded, making a direct comparison between the filterings difficult. Furthermore, the genes retained in the M3Drop and expression filterings showed a low degree of overlap in many of the data sets (Supplementary Figure 2). Overall, only small differences were seen between the results for the data sets containing gene abundances and those containing transcript compatibility counts (TCCs).\n\nWhile none of the methods consistently outperformed the others over the full range of the imposed numbers of clusters in all data sets, SC3 and Seurat often showed the best performance. These methods were also the only ones that achieved a good separation of the cell types in the droplet-based Zhengmix data sets, which have a much higher degree of sparsity and a larger number of cells than the other data sets. This is consistent with a previous study15 showing that Seurat performed better than other types of algorithms on data with low read depth. Generally, the performance of Seurat was also not strongly affected by the gene filtering approach (except for the simulated data sets), while other methods, like SAFE, were more sensitive to the choice of input genes for some data sets. FlowSOM showed a poor performance for the true number of clusters (see Supplementary Figure 4 for an illustration, together with a selection of other data set/method combinations with poor ARI values). However, if the number of clusters was increased, the performance of FlowSOM improved considerably, and if the methods instead were compared at the number of clusters that gave the optimal performance for each method, FlowSOM showed a moderate performance (Supplementary Figure 5). RtsneKmeans, a general-purpose method, showed a higher average performance across the data sets and filterings than many of the clustering algorithms specifically developed for scRNA-seq data. Compared to SC3 and Seurat, RtsneKmeans showed poorer performance for the SimKumar8hard and Zhengmix4uneq data sets. The subpopulations in these data sets are nested in the t-SNE space, explaining the difficulty in clustering for the K-means algorithm (Supplementary Figure 1).\n\nWe also investigated whether the number of detected features per cell differed between the clusters, using a Kruskal-Wallis test49. No strong association was found for the simulated data sets (Supplementary Figure 6), indicating that there is low inherent bias in the clustering algorithms. For most of the real data sets we found highly significant differences in the number of detected features between cells in different clusters. However, it is unclear whether this represents a technical effect or a biological difference between the cell populations.\n\nWe measured the elapsed time for each run, using a single core and excluding the time to estimate the number of clusters if this was done via a separate function. Since the run times are strongly dependent on the number of features and cells in a data set, we represent them as normalized run times, by dividing with the time required for RtsneKmeans for the same data set (Figure 2A). Seurat was the fastest method, while pcaReduce, SAFE and SC3 were the slowest, sometimes by a large margin. Clustering only half of the cells with SC3 and predicting the class of the others with a Support Vector Machine (SC3svm) gave slightly shorter run times than applying the SC3 clustering to all cells. The method could potentially be accelerated by using a lower proportion of cells as a training subset. A detailed overview of the run time and the dependence on the number of clusters is given in Supplementary Figures 7 and 8. Apart from SC3 and SC3svm, the imposed number of clusters did not affect the run time.\n\n(A) Normalized run times, using RtsneKmeans as the reference method, across all data set instances and number of clusters. (B) Run time versus performance (ARI) for a subset of data sets and filterings, for the true number of clusters.\n\nPlotting the run time versus the Adjusted Rand Index for a subset of the data sets (excluding the ones with the strongest signal, where all methods found the correct clusters, and the TCC data sets) (Figure 2B) further illustrated the variability between the methods. Interestingly, Seurat was generally the fastest method, especially for the droplet-based data sets, but at the same time provided among the best partitionings of the data.\n\nFigure 1 illustrated the average performance of each method across the five runs on each data set, for the true number of clusters. By comparing the partitions obtained in the individual runs, we could also obtain a measure of the stability of each method (Figure 3A).\n\n(A) Median stability (ARI across different runs on the same data set) for the methods, with the annotated number of clusters imposed. Some methods failed to return a clustering with the correct number of clusters for certain data sets (indicated by white squares). (B) The difference between the normalized entropy of the obtained clusterings and that of the true partitions, across all data sets and for the annotated number of clusters. (C) The difference between the number of clusters giving the maximal ARI and the annotated number of clusters, across all data sets.\n\nCIDR, PCAHC, TSCAN, ascend and Seurat returned the same clusters in all five instances for all data sets, while the stability of the other methods depended on the data set. Again, the stability was lower for the simulated data sets after gene filtering by M3Drop (note that the same genes were used in all five runs), indicating that the selection of genes may be suboptimal.\n\nA summary of the variability both within and between the different filterings is shown in Supplementary Figure 9. It is worth noting that comparing the performances between the different filtering approaches is difficult for two reasons: first, the variability of the clustering runs for a given filtering might exceed the variation between the filterings, and second, filtering with M3Drop led to the exclusion of a large number of cells in the Zhengmix data sets, and these cells can not be used for the comparison. For the stable methods CIDR, TSCAN, ascend and PCAHC, the type of filtering had a relatively large impact on the clustering solutions, and often filtering on the mean gene expression and the gene variability gave more similar clusters than filtering with M3Drop. The stochastic methods showed both a high variability between the individual runs for a given filtering and between runs with different filterings.\n\nQualitative differences between cluster characteristics\n\nBy computing the Shannon entropy for the various partitions, we obtained a measure of the equality of the sizes of the clusters (Figure 3B). Since the true degree of cluster size uniformity as well as the number of clusters are different between data sets, we compared the normalized Shannon entropy of the clusterings to that of the true partitions. Thus, a positive value of this statistic indicates that a method tends to produce more equally sized clusters than the true ones, and a negative value instead indicates that the method tends to return more unequal cluster sizes, e.g., one large cluster and a few small ones. Most methods gave cluster sizes that were compatible with the true sizes for most data sets (a statistic close to 0), while especially FlowSOM was more variable, and often tended to group the cells into one large cluster and a few very small ones (see Supplementary Figure 4 for an example). One consequence of this was that FlowSOM often showed higher ARI values for a larger number of clusters, while the performance of many of the other methods decreased with increasing k (Supplementary Figure 3). These methods tended to have more equally sized clusters for larger numbers of clusters than the true number, leading to a higher disagreement between the true classification and the clusterings (the entropy across the range of k is shown in Supplementary Figure 10).\n\nAbove, we investigated the performance and stability of the methods when the true number of clusters (the number of different labels in the partitioning considered as the ground truth) was imposed. Whether this number of clusters actually provided the highest ARI value (i.e., the best agreement with the ground truth) mainly depended on the difficulty of the clustering task (Figure 3C), and the choice of method. No method achieved the best performance at the annotated number of clusters in all the data sets, although generally, the methods reached their maximum performance at or near the annotated number of clusters. The notable exception was FlowSOM, which required a relatively large number of clusters to reach its maximal performance.\n\nSC3, CIDR, ascend, SAFE and TSCAN all have built-in functionality for estimating the optimal number of clusters. In most cases, the estimated number was close to the true one; however, ascend and CIDR had a tendency to underestimate the number of clusters, while SC3 and TSCAN instead tended to overestimate the number (Supplementary Figure 11). The tendency of SC3 to overestimate the cluster number is consistent with a previous publication15. The agreement with the true partition at the estimated number of clusters is shown in Supplementary Figure 12. SC3 is still the best-performing method in this situation.\n\nThe similarity between each pair of methods was quantified by means of the ARIs for each pair of consensus clusterings (across the five runs of each method for each data set and number of clusters). Figure 4 shows a dendrogram of the methods obtained by hierarchical clustering based on the average ARI values across all data sets for the true number of clusters. The numbers shown at the internal nodes indicate the stability of the subclusters, that is, the fraction of the corresponding dendrograms from the individual data sets where a particular subcluster could be found. In general, the groupings of the methods shown in the dendrogram were unstable across data sets and number of clusters, indicated by the low stability fractions of all subclusters. This is consistent with previous studies showing generally poor concordance that varied across data sets19,42. Even SC3 and SC3svm had surprisingly different clusterings; in less than a third of the data sets, these two methods showed the most similar clusterings. In addition, no apparent association between the similarity of the clusterings and the type of input or the dimension reduction or underlying type of clustering algorithm was found.\n\nNumbers on internal nodes indicate the fraction of dendrograms from individual data sets where a particular subcluster was found.\n\nNext, we investigated whether we could improve the clustering performance by combining methods into an ensemble. For each pair of methods, we generated a consensus clustering and evaluated its agreement with the true partition using the ARI. In general, the performance of the ensemble was worse than the better of the two combined methods, and better than the worse of the two methods (Figure 5A), suggesting that we would obtain a better performance by choosing a single good clustering method rather than combining multiple different ones. This is largely consistent with a recent study evaluating the combination of four methods (SC3, CIDR, Seurat, tSNE+Kmeans), where the ensemble performance was generally on par with the best individual method42. It is still possible that an ensemble method could provide a general improvement over a given single method, since it is unlikely that the same method will be the best performing in all conceivable data sets. In fact, among the methods we evaluated, both SC3 and SAFE combine multiple individual methods to achieve the final clustering result. Studying individual combinations in more detail, we observed that combining SC3 or Seurat with almost any other method led to a worse performance than obtained by these methods alone (consistent with the observation that they were among the methods giving the best performance). On the other hand, methods like CIDR, FlowSOM and TSCAN could often be improved by combining them with another method (Figure 5B).\n\n(A) Difference between the ARI of each ensemble and the ARI of the best (left) and worst (right) of the two methods in the ensemble, across all data sets and for the true number of clusters. (B) Difference between the ARI of each ensemble and each of the components, across all data sets and for the true number of clusters. The histogram in row i, column j represents the differences between the ARIs of the ensemble of the methods in row i and column j and the ARI of the method in row i on its own.\n\n\nDiscussion and conclusions\n\nIn this study, we have evaluated 12 clustering methods on both real and simulated scRNA-seq data. There were large differences in the ability of the methods to recover the annotated clusters, and performance was also strongly dependent on the degree of separation between the true classes. SC3 and Seurat, two clustering methods developed specifically for single-cell RNA-seq data, delivered the overall best performance, and were the only ones to properly recover the cell types in the droplet-based data sets. There was, however, a large difference in the run time, with SC3 being several orders of magnitude slower than Seurat. Another difference between these two methods is that SC3 includes a method for estimating the number of clusters (which has a tendency towards overestimation), while Seurat will determine the number of clusters based on a resolution parameter set by the user.\n\nThe same preprocessing steps and fixed gene sets were used for all clustering methods. This enabled us to investigate the impact on the clustering algorithm itself, rather than entire pipelines or workflows. The selection of the filtering approach had an impact on the majority of the methods and resulted in different clustering solutions. Specifically for the more difficult data sets there was a higher dissimilarity. However, this did not necessarily affect the performances of the methods.\n\nThe stability of clustering algorithms can be evaluated by generating perturbed subsamples of the data set and redoing the clusterings. These subsamples can be created in several ways, e.g., by random subsampling with or without replacement, by adding noise to the original data50 or by simulating technical replicates51. Freytag19 showed that SC3, Seurat, CIDR and TSCAN were stable under cell-wise perturbations. In our study, we evaluated the methods with respect to their sensitivity to random starts. Overall, the methods showed a high degree of stability across all data sets, except for the simulated data sets in combination with the M3Drop filtering, where the stochastic methods showed a decrease in stability. This may be due to a disagreement between the mean-dropout relationship in the simulated data and the one assumed by M3Drop, leading to a suboptimal gene selection.\n\nThe evaluated methods are based on a broad spectrum of approaches for dimensionality reduction and clustering. We note that the majority of the methods use PCA or PCoA for dimension reduction or Euclidean distances as the distance metric (ascend allows for other alternatives). Thus, no clear advice on the type of algorithm that is best suited for clustering single-cell RNA-seq data can be made based on our results. In fact, the two best-performing methods, SC3 and Seurat, rely on very different underlying clustering algorithms.\n\nWe investigated the impact of changing the imposed number of clusters for the different methods, which revealed that a subset of the methods, in particular FlowSOM, consistently showed a better agreement with the true subpopulations if the number of clusters was increased beyond the true number. The reason for this appears to be that FlowSOM tends to split off a few very small clusters. In addition to the number of clusters, most methods rely on other hyperparameters. In this study, we have fixed these to reasonable values. However, additional investigations into the effect of these hyperparameters on the results would be an interesting direction for future research.\n\n\nData availability\n\nThe R scripts providing an extensible framework for the evaluation of new methods and data sets are available on GitHub. All filtered (and unfiltered) data sets used in this study are readily available from the links provided: https://github.com/markrobinsonuzh/scRNAseq_clustering_comparison.\n\nArchived R scripts as at time of publication are available from https://doi.org/10.5281/zenodo.131474352 under an MIT license.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nWe acknowledge funding support from the Swiss National Science Foundation (Grant Number 310030_175841 to MDR) and the Chan Zuckerberg Initiative (Grant Number 182828 to MDR).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank the members of the Robinson group at the UZH for valuable input.\n\n\nSupplementary material\n\nSupplementary File 1: PDF file containing Supplementary Figures 1–12 and Supplementary Table 1.\n\nClick here to access the data.\n\n\nReferences\n\nTang F, Barbacioru C, Wang Y, et al.: mRNA-Seq whole-transcriptome analysis of a single cell. 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Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1314743" }	[ { "id": "36544", "date": "27 Jul 2018", "name": "Jean Fan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview\nDuo et al compare multiple single-cell RNA-seq clustering approaches on real and simulated single-cell RNA-seq datasets.\n\nMajor comments\n- Quite a number of single-cell RNA-seq datasets are available for benchmarking but only a few were explored here. While an exhaustive interrogation of all single-cell RNA-seq datasets available is beyond the scope of this paper, it would be worthwhile for the readers if the authors could comment briefly on the appropriateness of the datasets used here in terms of their cell-type diversity or other factors that may impact benchmarking. As the authors note, a method's performance is inherently tied to the degree to which the tested subpopulations are truly (or artificially) transcriptionally distinct. In particular, I am concerned about the appropriateness of the Trapnell dataset, as it was originally intended for pseutotime/trajectory inference and may not even contain discrete transcriptional subpopulations. The poor performance as noted in Figure 1 for this dataset may simply arise from different methods cutting along this continuous trajectory in different ways. Similarly, for the Zheng 10x datasets, since each cell-type was sorted and sequenced separately, there is inevitably some degree of confounding of cell-type specific effects with batch effects that could make clustering much easier.\n\n- As datasets get bigger, the scalability of each method will be an important consideration. The authors provide a preliminary look into this via the different run time of each method in Figure 2, but how this run time depends on the number of cells is unclear. Readers will be interested in whether some methods scale better than others. It is worth having an additional figure of run time as a function of number of cells (via downsampling cells and then extrapolating to larger datasets) to fully capture the scalability of each method.\n\n- With regard to the stability between cluster runs, some methods may internally set various random seeds to ensure reproducibility. Please double check that the stability observed in Figure 3 is not simply the result of which methods uses random seeds. If a method does use an (or likely multiple) internal random seed, the seed must be changed to accurately assess stability.\n\nMinor comments\n- There are quite a number of single-cell RNA-seq clustering approaches and the list keeps growing (https://github.com/seandavi/awesome-single-cell). Only a fraction is represented in this comparison. While an exhaustive comparison of all methods is beyond the scope of this paper, the authors should comment briefly on how these particular 12 clustering algorithms were chosen.\n- While nearly all methods assessed use dimensionality reduction as a first step, it is unclear why some were allowed to reduce to 30 dimensions while others 50. It seems that particularly as datasets get larger with presumably more cell-types captured in each datasets, we will likely want to increase the number of PCs to fully capture the variation present in the data. While the authors have left the investigation into the effects of the number of PCs to future research, they should briefly note the reason for the choice of PCs used for each method.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\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?\nPartly\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? Partly", "responses": [ { "c_id": "3938", "date": "10 Sep 2018", "name": "Charlotte Soneson", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your constructive comments. Below are point-by-point responses to the individual comments. Quite a number of single-cell RNA-seq datasets are available for benchmarking but only a few were explored here. While an exhaustive interrogation of all single-cell RNA-seq datasets available is beyond the scope of this paper, it would be worthwhile for the readers if the authors could comment briefly on the appropriateness of the datasets used here in terms of their cell-type diversity or other factors that may impact benchmarking. As the authors note, a method's performance is inherently tied to the degree to which the tested subpopulations are truly (or artificially) transcriptionally distinct. In particular, I am concerned about the appropriateness of the Trapnell dataset, as it was originally intended for pseutotime/trajectory inference and may not even contain discrete transcriptional subpopulations. The poor performance as noted in Figure 1 for this dataset may simply arise from different methods cutting along this continuous trajectory in different ways. Similarly, for the Zheng 10x datasets, since each cell-type was sorted and sequenced separately, there is inevitably some degree of confounding of cell-type specific effects with batch effects that could make clustering much easier. There is indeed a large (and increasing) number of public scRNA-seq data sets available, generated with many different types of protocols. However, the main issue (especially with droplet-based data sets) is that no independent annotation of the cells is available, which implies that they are not suitable for unbiased benchmarking like we are doing here. Many public droplet-based data sets do contain “cell type labels”, but these are typically inferred by clustering the cells based on the scRNA-seq data itself, and thus any evaluation risks being biased in favor of methods similar to the one used to derive the labels in the first place. This is the main reason behind the selection of these data sets. We agree that the Trapnell data set was not generated with the purpose of finding cell types - however, we still find it useful to illustrate the performance of the methods in a data set where the “true clusters” (defined as the time point at which the cells where collected) do not represent the main/strongest signal in the data (see e.g. the t-SNE plots in Supplementary Figure 1). For the Zheng data set, it’s true that there could be confounding with batch effects, and ambiguous cells may be excluded, which would also make clusters more distinct. For our Zhengmix data sets, we therefore included both very different (e.g., B-cells and T-cells) and more similar (e.g., different types of T-cells) cell types (Supplementary Figure 1). We have expanded the discussion in the “Methods-Real data sets” section of the revised paper to clarify these issues. As datasets get bigger, the scalability of each method will be an important consideration. The authors provide a preliminary look into this via the different run time of each method in Figure 2, but how this run time depends on the number of cells is unclear. Readers will be interested in whether some methods scale better than others. It is worth having an additional figure of run time as a function of number of cells (via downsampling cells and then extrapolating to larger datasets) to fully capture the scalability of each method. Thanks for pointing this out. We have included a plot illustrating the scalability, investigated by downsampling of the largest data set, in Supplementary Figure 9. With regard to the stability between cluster runs, some methods may internally set various random seeds to ensure reproducibility. Please double check that the stability observed in Figure 3 is not simply the result of which methods uses random seeds. If a method does use an (or likely multiple) internal random seed, the seed must be changed to accurately assess stability. Two of the methods (TSCAN and monocle) set random seeds internally and do not allow these to be changed by the user. Other methods (SC3, Seurat and RaceID2) set a random seed but let the user specify it. For these methods, we explicitly set the random seed to different values in the five runs. We have clarified this in the “Results-High stability between clustering runs” section of the revised text. There are quite a number of single-cell RNA-seq clustering approaches and the list keeps growing (https://github.com/seandavi/awesome-single-cell). Only a fraction is represented in this comparison. While an exhaustive comparison of all methods is beyond the scope of this paper, the authors should comment briefly on how these particular 12 clustering algorithms were chosen. The methods were chosen to represent the most common types of algorithms used for clustering of scRNA-seq data. We have tried to include the most widely used methods, but also to include methods from tangential fields as well as more traditional clustering methods to serve as a baseline. We have clarified this in the text. While nearly all methods assessed use dimensionality reduction as a first step, it is unclear why some were allowed to reduce to 30 dimensions while others 50. It seems that particularly as datasets get larger with presumably more cell-types captured in each datasets, we will likely want to increase the number of PCs to fully capture the variation present in the data. While the authors have left the investigation into the effects of the number of PCs to future research, they should briefly note the reason for the choice of PCs used for each method. We extracted 50 principal components for the methods that performed an additional dimension reduction (by t-SNE), and 30 principal components for methods where the clustering was done in the principal component space. The only exception was FlowSOM; this was unintentional and has been harmonized in the revised version to use the same number of PCs as the rest of the methods." } ] }, { "id": "36545", "date": "03 Aug 2018", "name": "Saskia Freytag", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview\nThe authors present comprehensive benchmarking of clustering tools in R on real and simulated single-cell RNA-seq datasets. Their work includes performance, stability and run time analysis. Furthermore, they also investigate whether combining results from different methods increases performance.\n\nMajor comments\n\nThroughout the entire manuscript the authors should make it clear that only clustering tools available in R were investigated. This is important, as there are quite a number of popular python applications for clustering of single cell RNA-seq data available. Like Jean Fan, I am concerned about the appropriateness of the Trapnell et al. dataset and the Zheng et al. 10x datasets. Furthermore for the Zheng et al. dataset, I would like to know why the authors did not use all 10 pre-sorted cell populations available? Furthermore, how did the authors choose which cell populations to combine for their Zhengmix4 and Zhengmix8 datasets?\n\nMinor comments\nThe authors show nicely that Seurat is not very strongly affected by gene filtering. Could this be a result of its clustering approach being based on the 500 most variable genes? On page 7 in the paragraph “Run Times vary widely between methods” the authors use Adjusted Rand Index instead of its already introduced abbreviation Could the size of Figure 5 be increased? Why did some methods get raw and some methods log-transformed normalized counts? Consider changing Supplementary Figure 2 to a visual representation that represents size differences between sets, like UpSetR plots. On page 10 the authors say: ”In addition, no apparent association between the similarity of the clusterings and the type of input or dimension reduction or underlying type of clustering algorithm was found.” Could the authors explain in more detail how this analysis was performed. On page 6, the authors speculate that there are stronger signals that dominate clustering in the Trapnell et al dataset that are not time points. What could these be? Have the authors investigated cell cycle?\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? Partly\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": [ { "c_id": "3937", "date": "10 Sep 2018", "name": "Charlotte Soneson", "role": "Author Response", "response": "Thank you for reviewing our manuscript and for your constructive comments. Below are point-by-point responses to the individual comments. Throughout the entire manuscript the authors should make it clear that only clustering tools available in R were investigated. This is important, as there are quite a number of popular python applications for clustering of single cell RNA-seq data available. This has been clarified in the Abstract as well as in the Methods part of the text. Some of the most widely used clustering methods implemented in Python (e.g., scanpy) implement the same or similar clustering methods as those evaluated in this study, and could thus be considered to be implicitly investigated. Also, the evaluation system we provide (via the code in the GitHub repository and the associated data package) is not strictly limited to methods implemented in R; other methods can be included e.g. using system() calls. Like Jean Fan, I am concerned about the appropriateness of the Trapnell et al. dataset and the Zheng et al. 10x datasets. Furthermore for the Zheng et al. dataset, I would like to know why the authors did not use all 10 pre-sorted cell populations available? Furthermore, how did the authors choose which cell populations to combine for their Zhengmix4 and Zhengmix8 datasets? We agree that the Trapnell data set was not generated with the purpose of finding cell types - however, we still find it useful to illustrate the performance of the methods in a data set where the “true clusters” (defined as the time point at which the cells where collected) do not represent the main/strongest signal in the data (see e.g. the t-SNE plots in Supplementary Figure 1). We have clarified this in the “Methods-Real data sets” section of the revised paper. For the Zhengmix data sets, our aim was to generate data sets with a mix of well-separated (e.g., B-cells vs T-cells) and similar cell types (e.g., different types of T-cells). In addition, we wanted to investigate if the number of cell populations and/or the equality of the population sizes had an impact on the performance. The included cell type combinations were selected to allow us to address these questions; however, given the richness of this data set, there are certainly many more possible combinations to explore. We have expanded the description in the “Methods-Real data sets” section a bit to highlight these goals. The authors show nicely that Seurat is not very strongly affected by gene filtering. Could this be a result of its clustering approach being based on the 500 most variable genes? In all our investigations, we preselect the genes that are used as input for each clustering algorithm using three different variable selection methods, and internal variable selection or filtering steps are disabled. Specifically, for Seurat we perform the PCA using all the genes remaining after our filtering, and the clustering is then performed in the principal component space. Thus, the stability of Seurat should be affected in the same way as that of the other methods by the selection of variables. On page 7 in the paragraph “Run Times vary widely between methods” the authors use Adjusted Rand Index instead of its already introduced abbreviation Thanks for noticing this, we now use the abbreviation also here. Could the size of Figure 5 be increased? We have increased the size of Figure 5B. Why did some methods get raw and some methods log-transformed normalized counts? The methods are based on different distributional assumptions and underlying models, affecting the type of values that are most suitably used as input. We followed the recommendations of the authors of the respective methods, and the type of input used for each method is summarized in Figure 4. Consider changing Supplementary Figure 2 to a visual representation that represents size differences between sets, like UpSetR plots. We have replaced the Venn diagrams in Supplementary Figure 2 with UpSet plots. On page 10 the authors say: ”In addition, no apparent association between the similarity of the clusterings and the type of input or dimension reduction or underlying type of clustering algorithm was found.” Could the authors explain in more detail how this analysis was performed. This conclusion is drawn based on Figure 4, where no association between the clustering of methods by cluster similarity and any of the method characteristics can be seen. This has been clarified in the “Results-Inconsistent degree of similarity between methods” section of the revised paper. On page 6, the authors speculate that there are stronger signals that dominate clustering in the Trapnell et al dataset that are not time points. What could these be? Have the authors investigated cell cycle? We have not explicitly investigated the interpretation of the strongest signal in the Trapnell data set. However, Supplementary Figure 1 suggests that the annotation that we used to define the “true” clusters (the time at which the cells were collected) does not fully explain the grouping of the cells in the t-SNE visualization (in particular, the T12 and T24 groups are intermingled). As noted above, the main purpose of including this data set was to investigate the behaviour of the various methods in a data set where the clusters were less apparent." } ] } ]	1	https://f1000research.com/articles/7-1141
https://f1000research.com/articles/9-1336/v1	16 Nov 20	{ "type": "Opinion Article", "title": "From research to rapid response: mass COVID-19 testing by volunteers at the Centre for Genomic Regulation", "authors": [ "Ritobrata Ghose", "Álvaro Aranguren-Ibáñez", "Niccolò Arecco", "Diego Balboa", "Marc Bataller", "Sergi Beltran", "Hannah Benisty", "Angèle Bénard", "Edgar Bernardo", "Sílvia Carbonell Sala", "Eloi Casals", "Ludovica Ciampi", "Livia Condemi", "Alberto Corvó", "Marta Cosín-Tomás", "Mirabai Cuenca-Ardura", "Juan Manuel Duran Serrano", "María Isabel Espejo Díaz", "Marcos Fernandez Callejo", "Antoni Gañez-Zapater", "Raquel Garcia-Castellanos", "Romina Garrido", "Gil Henkin", "Toni Hermoso Pulido", "Xavier Hernandez-Alias", "Jorge Herrero Vicente", "Matthew Ingham", "Wei Ming Lim", "Sílvia Llonch", "Elena Marmesat Bertoli", "Irene Miguel-Escalada", "Ariadna Montero-Blay", "Cristina Navarrete Hernández", "Maria Victoria Neguembor", "Róisín-Ana Ní Chárthaigh", "Natalia Pardo-Lorente", "Laura Pascual-Reguant", "Sílvia Pérez-Lluch", "Reyes Perza", "Martina Pesaresi", "Daniel Picó Amador", "Paula Pifarré", "Davide Piscia", "Marcos Plana-Carmona", "Julia Ponomarenko", "Leandro Radusky", "Ezequiel Rivero", "Malgorzata Rogalska", "Guillem Torcal Garcia", "José Wojnacki", "Álvaro Aranguren-Ibáñez", "Niccolò Arecco", "Diego Balboa", "Marc Bataller", "Sergi Beltran", "Hannah Benisty", "Angèle Bénard", "Edgar Bernardo", "Sílvia Carbonell Sala", "Eloi Casals", "Ludovica Ciampi", "Livia Condemi", "Alberto Corvó", "Marta Cosín-Tomás", "Mirabai Cuenca-Ardura", "Juan Manuel Duran Serrano", "María Isabel Espejo Díaz", "Marcos Fernandez Callejo", "Antoni Gañez-Zapater", "Raquel Garcia-Castellanos", "Romina Garrido", "Gil Henkin", "Toni Hermoso Pulido", "Xavier Hernandez-Alias", "Jorge Herrero Vicente", "Matthew Ingham", "Wei Ming Lim", "Sílvia Llonch", "Elena Marmesat Bertoli", "Irene Miguel-Escalada", "Ariadna Montero-Blay", "Cristina Navarrete Hernández", "Maria Victoria Neguembor", "Róisín-Ana Ní Chárthaigh", "Natalia Pardo-Lorente", "Laura Pascual-Reguant", "Sílvia Pérez-Lluch", "Reyes Perza", "Martina Pesaresi", "Daniel Picó Amador", "Paula Pifarré", "Davide Piscia", "Marcos Plana-Carmona", "Julia Ponomarenko", "Leandro Radusky", "Ezequiel Rivero", "Malgorzata Rogalska", "Guillem Torcal Garcia", "José Wojnacki" ], "abstract": "The COVID-19 pandemic has posed and is continuously posing enormous societal and health challenges worldwide. The research community has mobilized to develop novel projects to find a cure or a vaccine, as well as to contribute to mass testing, which has been a critical measure to contain the infection in several countries. Through this article, we share our experiences and learnings as a group of volunteers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain. As members of the ORFEU project, an initiative by the Government of Catalonia to achieve mass testing of people at risk and contain the epidemic in Spain, we share our motivations, challenges and the key lessons learnt, which we feel will help better prepare the global society to address similar situations in the future.", "keywords": [ "COVID-19 testing", "volunteers", "health", "collaboration" ], "content": "Introduction: CRG in Orpheus’ shoes\n\nThe recent turn of events worldwide, brought on by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uprooted the world of all normality, presented a tremendous challenge. In tragic unison, COVID-19 became the viper to humanity’s Eurydice, and the world of researchers and medics, her Orpheus, in our attempt to revive and reset. With a name inspired by this mythological allegory, the Catalan government launched the ‘ORFEU’ project – a mass testing initiative to identify infected individuals – in an attempt to reduce infection rates and facilitate an easier transition into a post-confinement era. At the time that the government decided to launch the ORFEU project, the epidemic in Catalonia was at a critical high. In April 2020, the infection rates and mortality were peaking, and the health system was rapidly overwhelmed. As a result, no systematic testing initiatives were in place in various critical settings such as nursing homes, prisons or clinical institutions due to the vast demand for tests for symptomatic people in hospitals.\n\nWith the aim of performing 170,000 PCR-based COVID-19 tests, the ORFEU project united some of the major research institutes across Catalonia. There were two main sample-processing nodes with close to 200 volunteers collectively – one at the Barcelona Biomedical Research Park (PRBB) orchestrated by the Center for Genomic Regulation (CRG) and the other at Barcelona Science Park (PCB), jointly coordinated by IRB Barcelona, IBEC and the CNAG-CRG (Figure 1). Both nodes were supported by the CNAG-CRG informatics team and the whole process coordinated by the CRG director. The team at PRBB comprised a total of 106 volunteers including principal investigators, post-doctoral researchers, technicians, and administration personnel (Figure 2). Here, in this short article, we, as volunteers at the PRBB node, share our motivations, and briefly outline the processes and challenges that were important to adapt from a basic research centre to an analytical and diagnostic centre for COVID-19 so that other global institutes may benefit from our experience.\n\n(Top) Modifications to the workflow, which greatly influenced our ability to handle a larger number of samples. (Bottom) Cumulative number of samples processed from the start to the end of the ORFEU project.\n\nWe confirm that we have obtained written informed consent to use images of the individuals included in this presentation.\n\n\nThe motivation\n\n“Extraordinary times call for extraordinary measures. We saw a need that needed to be filled and we stepped in to help.” - Benet Wilson\n\nThe motivations of all the volunteers were diverse. While for some it was curiosity to better understand the on-ground scene, others hoped to get a much-desired alternative to in-house confinement. For others less fortunate, the motivation was fuelled by personal experiences with loved ones having suffered through the infection. But most of all, and unanimously, the underlying motivation was to help society fight against the pandemic by offering our specific skill sets and spreading the knowledge needed to battle the crisis. Together, obstacles such as reduced public transport, commuting distances and the inherent human fear factor, were overcome by our will to curb the pandemic through our contributions in the ORFEU project.\n\n\nOrganisation and pipeline\n\nAt the outset, once called into the ORFEU project, the CRG health and safety department achieved the mammoth task of adapting the research centre into an analytical and diagnostic centre. There were two major phases to this. First, a biosafety protocol outlining key information such as SARS-CoV-2 sample collection and handling, based on the procedures from the World Health Organisation1 and Centre for Disease Control and Prevention2 were established. In addition, a Biosafety Level 2 facility was modified by introducing level 3 measures, such as exclusive changing rooms and protective equipment disposal protocols, which was pivotal in our ability to deal with SARS-CoV-2 samples due to the need for adequate disinfection and correct storage. In the second phase, given our inexperience in handling such an emergency, adequate training of volunteers was critical. To start, a core team of volunteers of around 10 members were instructed on biosafety measures through an online training session given by an external expert in Biosafety Level 3 followed by a hands-on in situ training in smaller groups. All members were trained equally in sample collection and disinfection, sample registration, RNA extraction, PCR and validation, allowing fail-safes and contingencies in cases when members were not able to carry on for any reason. Eventually more volunteers were recruited based on sample load and divided into specialised groups led by a member from the core team. The entire workflow was monitored using a laboratory information management system (LIMS) developed ad hoc by the Bioinformatics Unit at CNAG-CRG and a user interface program for sample registration developed by the CRG bioinformatics unit. Together, the LIMS allowed for the smooth and rapid management of samples in an efficient and errorless process between various teams. Expert clinical microbiologists, from various collaborating hospitals, interpreted the PCR results online in a user-friendly module of the LIMS, and carried out the final task of diagnosis.\n\nThe ultimate aim was to automate the process for speedy sample processing as well as reproducibility using high-throughput TECAN robots (one of which was kindly loaned to the CRG by the company) and multiple RT-PCR machines (one kindly lent by the University Pompeu Fabra). Nevertheless, the importance of manual tasks and supervision was paramount, and required approximately three shifts of 4–5 hours every other day, with approximately 4–5 volunteers per shift. Having started at a few hundred samples processed per day, by the end of the project, the CRG was in a position to process 2500–3000 samples per day, with a turnaround time from sample reception to results in about 24–48 hours (Figure 1). The workflow can be followed in an infographic created by CRG3.\n\n\nKey challenges\n\nTo ensure the systematic running of the entire unit, it was important to establish a general workflow based on the process mentioned above. Through our implementation of the setup4, we identified key steps to assure process standardization and optimisation. Here we highlight some of the major points.\n\nA project of this scale requires navigating through a large amount of administrative and legal procedures, as well as coordinating many different organisations and teams. Resolving each of these issues caused an initial delay between the proposed and actual start of the project, resulting in a lower overall turnaround of test results than intended. Moreover, the sporadic receipt of samples from various parts of Catalonia presented a challenge in terms of organising shifts, adequate storage and handling, and quick turnaround time (ideally 24–48h). The CRG now has a go-to model ready for use in future situations to share with other organizations4.\n\nOne of the main challenges during the project was the simultaneous development of an online informatics platform, whilst the laboratory workflow was being designed. Our aim was to develop a platform that enabled tracking of all samples throughout the process internally and also provided an interactive and user-friendly analysis environment for third-party use by collaborating microbiologists to interpret the results. To achieve this, the CNAG-CRG, together with CRG bioinformaticians, developed a LIMS that included a diagnosis validation tool.\n\nThe PCR testing protocol had two major aspects – RNA extraction and real-time quantitative PCR. Identifying the optimal combination of reagents with sample processing equipment was arguably the most challenging part and is a must to be tackled at the earliest. The initial phase was subject to continual switching between different RNA extraction kits and equipment, which called for exhaustively long working hours and time-consuming daily training.\n\nCommunication and transfer of knowledge posed major challenges as well. Using dedicated social media channels and live cloud-based services helped in sharing status updates between shifts and quick responses to unexpected technical issues.\n\nA continuous logistical challenge throughout the course of the project was the limited availability of reagents and materials. In order to circumvent this issue, reduce the pressure on manufacturing units and optimise overall project costs, the CRG organised a protein production team that performed expression and purification of all the necessary proteins as a backup in the event that the supply chain broke completely. Protocols were initially tested on a small scale by the CRG’s Protein Technologies Unit and further expanded by volunteers. Moreover, extensive further validation and certification of these proteins in PCR mixes or as other reagents are limiting steps prior to them being available for analytical use.\n\nOften taken for granted, one of the main challenges during the coordination of a crisis initiative such as the ORFEU project was taking into account personal and professional lives. On one hand, at a personal level, the stress of the lockdown and having children at home or living with high-risk individuals made it difficult to volunteer. Accommodating for lockdown-induced anxiety and mental health needs was very important. On the other hand, from a professional point of view, the delays and late start of the project, which coincided with the reopening of many labs, made it difficult to balance between the two. In addition, and very importantly, as basic researchers, we needed to accept a new responsibility, that results from this project would directly impact people’s lives.\n\n\nSuccess stories\n\nThe outcomes we achieved in the ORFEU project would not have been possible without some key aspects that contributed to its success.\n\nAbove all, the motivation, commitment and effort of everyone involved, working towards a common objective, was a very strong driving force that kept us on track, despite the obstacles. The trust of the administration in the volunteers allowed us to take the lead on various aspects of the project, enabling productivity and creating an atmosphere of trust and motivation.\n\nTeamwork and cooperation were, as always, critical. Working together in a friendly, supportive and cooperative environment made it easier to cope with stress and difficult situations without unnecessary conflict. The level of mutual respect was very high and communication was transparent, preventing any tension within the team despite varying opinions, changing protocols and long working hours.\n\nWhile the entire team’s effort was very important, it is necessary to highlight that without the dedication and perseverance of the initial organising team, and the various principal investigators, the project would not have been possible. Their tireless commitment for the optimisation of protocols and round-the-clock availability were indispensable stepping-stones for the rest of the team to be able to deliver on the project targets.\n\nA major turning point in our ability to handle large volumes of samples came through the tireless efforts of highly skilled personnel at the CRG core facilities who worked to overcome technical hurdles and managed, for instance, to convert a robot that was processing 96 samples every 90 min to processing 384 samples in the same time.\n\nDelegation of tasks and specialised teams became a critical factor in speeding up not only the sample-processing rate but also the training and induction of different groups of volunteers. Given the clear communication and coordination between specialised teams, typically overseen by rotating team leaders, the whole process adopted a very smooth and high-throughput functioning capacity.\n\n\nLessons learned\n\nConverting a research centre into an analytical platform is not easy: Despite the resources and technical expertise, setting-up an analytical platform from scratch and under a state of emergency was a complex process. Added to the challenges intrinsic to the novel social responsibilities, and the professional responsibility of delivering accurate results, the preparation of this platform required various obstacles to be negotiated.\n\nCreate a collaborative work environment: A strong team built through collaboration between researchers, the government and health professionals, with a varied skill set and good morale proved to be essential. Stratified decision-making, accountability, and distribution of tasks can promote the efficient functioning of the unit. Moreover, cross-institute collaborations, which arise from such projects, are long-term connections that can present opportunities for collaboration on various projects across disciplines and sectors, for instance between basic researchers, clinicians, health authorities and policymakers.\n\nTechnical assurance is critical: The transition from the creation of LIMS to its usability was an important process requiring optimization and often limiting sample processing rates. During the user interface design, it was critical to avoid making assumptions. The entire development process benefited from monitoring usage of the tool after an initial training session, as well as through the course of the project. Documenting workflows and database schemas was fundamental. Additionally, prioritization and differentiation between critical and non-critical features allowed the optimal use of limited software development personnel.\n\nImportance of good communication: Communication is one of the cornerstones of an efficient system. Establishing communication channels quickly through social media, live cloud-based services or internal institute-based communication systems available to everyone involved was very important to guarantee an effective workflow, and to ensure transparency, robustness and stability.\n\nProtocol standardization: The initial stages of the ORFEU project witnessed a lot of changing protocols and volunteers were required to adapt very quickly. Multiple tests were needed to optimize both processes, and as mentioned above technical developments were needed to increase the capacity of the RNA extraction robots. Although not discussed here, we found numerous artefacts in the PCR reactions. For instance, something as banal as minor traces of ethanol in the reaction could induce artifactual amplification, or the contamination of neighbouring wells in plates where a patient had a very large viral load. See 4 for our final protocol.\n\nOpen access and availability of organizational workflow and protocols: All protocols, as well as optimization trials (including negative results), from situations such as the COVID-19 pandemic, would gain from being shared openly by enabling other research centres and institutions to make better use of their resources5. Moreover, these are important opportunities to initiate dialogue and collaboration between local and global research institutions, alongside local governments and health authorities. This would allow them to put in place emergency plans and workflows that can prevent loss of precious time and scarce resources, as was exemplified in the initial phases of the ORFEU project.\n\nResearchers and research are important to society: Beyond knowledge creation, researchers can mobilize quickly to produce tangible outcomes for the benefit of society. Proven across Catalonia, and globally, researchers can respond and adapt rapidly and effectively to emergencies, collaborating with local healthcare systems. As a result, a greater consideration for scientific research from governments, health authorities and society would better leverage resources during a health crisis.\n\n\nOur top 10 recommendations to be prepared for the next pandemic\n\n1. A general but adaptable workflow should initially be established by a core team of administrators, researchers and informaticians. Additional teams should closely follow to prevent exhaustion of the leading team and sustain productivity. To ensure readiness, an emergency response team should revisit the entire workflow annually and adapt it to address any potential issues based on current infrastructure, availability of personnel, and other possible complications.\n\n2. Define clear tasks and responsibilities as soon as possible. The combination of specialized teams and a small group of people with broader knowledge ensures efficiency and robustness.\n\n3. An initial database schema should be agreed upon ahead of time followed by suitable data transfer formats and protocols between systems (e.g. LIMS and Electronic Health Records). To ensure the systematic transfer of samples and eventually results, set up a common platform with the health authorities to assure transparent communication of all necessary logistic information.\n\n4. Set up dedicated communication channels that allow fast information transfer and updates.\n\n5. Volunteers should be recurrently tested, and not exclusively at the start of the programme. This will ensure mental peace and health safety of all directly or indirectly involved, as a result, ensuring sustainability of the project.\n\n6. Set up a pipeline to produce your own reagents (RT-PCR master mix and RNA extraction kits), so as not to solely rely on external sources to acquire them.\n\n7. Open access protocols should be shared across research centres and health institutions as soon as they are standardised. Problems arising during their development should also be communicated to avoid needless repetition.\n\n8. Equipment and pipelines should not be dismantled until normality has been reinstated globally to prepare for future outbreaks and the possible need for testing protocols.\n\n9. In emergencies, requirements are dynamic and processes can change very quickly. Hence, the volunteer recruitment process should continue as a backup for as long as possible.\n\n10. Despite being volunteer work, all participants worked at their own projects’ expense. Understanding from supervisors and recognition from funding agencies and government bodies, for instance by extending fellowships, would attract more support.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nWe thank all volunteers from both nodes – PRBB and PCB for their participation and hard work in the ORFEU project. A list of all participants can be found below. In addition, we specifically extend our thanks to Jochen Hecht (CRG Genomics Unit Head) and Carlo Carolis (CRG Biomolecular Screening & Protein Technologies Unit Head) for their tireless work throughout the ORFEU project. We also thank the collaborating microbiologists who were instrumental in the final diagnosis process.\n\nThe testing phase would not have been possible without the efforts of the biosafety team at CRG to adapt the facilities, to establish protocols and to maintain the safety conditions.\n\nOur thanks to Luis Serrano Pubul (Director, CRG), Juan Valcárcel (Group Leader, CRG), Guillaume Filion (previously Group Leader at CRG) and Mònica Morales Ballús (Core Facilities Programme Coordinator, CRG) for coordinating and organizing the ORFEU project. Finally, we thank Michela Bertero (Head of International & Scientific Affairs, CRG), who was a major driving force towards the creation of this manuscript.\n\nList of volunteers from CRG: Marta Agostinho, Álvaro Aranguren Ibáñez, Niccolò Arecco, Carme Arnan, Leonor Avila, Borja Balbastre, Diego Balboa, Magalí Bartomeus, Marc Bataller, Sergi Beltran, Hannah Benisty, Marta Benito, Aina Bernal Martínez, Edgar Bernardo, Elvan Boke, Elisabetta Broglio, Sílvia Carbonell Sala, Carlo Carolis, Eloi Casals, Ludovica Ciampi, Olga Coll, Livia Condemi, Marta Cosín-Tomás, Pia Cosma, Mirabai Cuenca-Ardura, Natalia Dave, Montse Diaz, Juan Manuel Duran Serrano, María Isabel Espejo Díaz, Imma Falero, Rute Fernandes, Marcos Fernandez Callejo, Ana Belen Fernandez Llorente, Guillaume Filion, Antoni Gañez-Zapater, Ximena Garate, Raquel Garcia-Castellanos, Romina Garrido, Fátima Gebauer, Ritobrata Ghose, Juan Carlos Gomez Escobar, Julia Grawenhoff, Thomas Guegan, Jochen Hecht, Gil Henkin, Toni Hermoso Pulido, Xavier Hernandez-Alias, Jorge Herrero Vicente, Matthew Ingham, Jonas Juan Mateu, Isabel Jurado, Damjana Kastelic, Jonas Krebs, Alejandra Laguillo Diego, Claire Lastrucci, Wei Ming Lim, Sílvia Llonch, Andrew Macrae, Elena Marmesat Bertoli, Elena Martín Rodríguez, Rocco Mazzolini, Neus Mestre Farràs, Christel Michel, Irene Miguel-Escalada, Belén Miñana, Ivano Mocavini, Magda Monfort, Ariadna Montero-Blay, Cristina Navarrete Hernández, Maria Victoria Neguembor, Róisín-Ana Ní Chárthaigh, Natalia Pardo-Lorente, Laura Pascual-Reguant, Sílvia Pérez-Lluch, Reyes Perza, Martina Pesaresi, Daniel Pico Amador, Paula Pifarre, Davide Piscia, Marcos Plana Carmona, Julia Ponomarenko, Matja Popovic, Anna Puig, Leandro Radusky, Anna Ribó Rubio, Ezequiel Rivero, Natalia Rodrigo, Malgorzata Rogalska, Jacopo Scrofani, Sandrine Schwartz, Anna Sillero, Silvia Speroni, Nicholas Stroustrup, Chelsea Szu Tu, Guillem Torcal Garcia, Eduard Valera Zorita, José Wojnacki, Ivan Zadra, Roser Zaurin Quer\n\nList of volunteers at the PCB node: CNAG – Paola Pisacane, Laetitia Casano, Lidia Sevilla Fortes, Patricia Lorden Rodriguez, Ana Gonzalez, Nuria Aventin, Domenica Marchese, Maite Rico, Sara Ruiz Gil, Maria Gallo, Angèle Bénard, Marta López Parra, Ginevra Caratu, Javier Gutiérrez Cuesta, Julie Blanc, Marta Gut, Katia Kahlem, Matthew Ingham, Eloi Casals, Elena Marmesat, Davide Piscia, Daniel Pico Amador, Alberto Corvó, Marcos Fernández Callejo, Sergi Beltran (*participation at the PRBB node as well); IRB – Victor Gonzalez, Anna Pijuan, Núria Villegas, Uxue Urdiroz, Victor Alcalde, Valentina Ramponi, Saska Ivanova, Vanessa Lopez, Berta Duran, Clara Morral, Marta Lovera, Clara Borras, Isabel Calvo, Laura Novellasdemunt, Ester Saus, Eduardo Pauls, Paloma Solá, Adrian Gabriel Torres, Cian Lynch, Joana Fort i Baixeras, Pasquale Pellegrini, Thomas Mortimer, Noelia Alcazar, Adria Nicolas, Blazej Baginski, Juan Felipe Slebe, Marc Furriols, Monica Torras, Nicolas Martin, Panagiotis Giannios, Lorena Gonzalez, Laura Gonzalez, Maria Sanchiz, Raquel Bernad, Eduard Puig, Alba Millanes, Erika Lopez, Isabelle Heath, Mariona Nadal, Israel Ramos, Claudia Arnedo, Patrick Aloy, Jesús Sánchez; IBEC – Ignasi Casanellas, Mireia Seuma, Albert Manzano, Anabel-Lise Le Roux, Sefora Conti, Adrian Lopez Canosa, Marina Pavlova, Isabela Santos Fortunato, Andrea García Lizarribar, Juanma Fernandez, Julia Rodriguez Comas, Swapnil Sanmukh, Francesco de Chiara, Nuria Camarero Palao, Gerard Rubi, Ferran Velasco, Ignasi Granero, Alba Rubio, Andrés Marco Giménez, Rafael Mestre, Marta Badia, Marc Molina, Elisabet Urrea.\n\n\nReferences\n\nWorld Health Organization: Laboratory biosafety guidance related to coronavirus disease (COVID-19). Reference Source\n\nCenters for Disease Control and Prevention: Guidance for General Laboratory Safety Practices during the COVID-19 Pandemic. Reference Source\n\nCentre for Genomic Regulation: ORFEU PROGRAMME. Reference Source\n\nCentre for Genomic Regulation: ORFEU PROGRAMME, SARS-CoV-2, Testing Protocol. Reference Source\n\nCentre for Genomic Regulation: Guidelines for Open Science on COVID-19 Research. Reference Source" }	[ { "id": "74857", "date": "16 Dec 2020", "name": "Ricardo Leite", "expertise": [ "Reviewer Expertise Genomics", "sequencing", "single cell", "bioinformatics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion article is truly an example on how scientists have assisted society during COVID-19 pandemic, by adapting and perform wide-scale testing in their communities. Scientists have a set of skills and knowledge of protocols, techniques and methodology, and these resources are sometimes ignored and left out by health authorities. That was not the case of Catalan government decided to launch ORFEU program, to avail the existing expertise and test capability and scale it up with the help of many volunteers that mobilize to perform a massive effort of COVID-19 testing. This article documents the insight vision, point of view, motivation and challenges of the volunteers from PRBB during this process.\nBesides the success stories, the learned lessons and perspectives from this transition and this period are very important and they can be explored in less troubled times.\nIt is important to pinpoint some of these lessons such as: balancing mental health and anxiety with new responsibilities; creating a collaborative work environment based on trust and cross institutes collaborations; good communication and mechanisms for sharing open data (protocols, workflows) and the effort on standardization and automatization of protocols. Although not fully discussed, the protocols and guidelines are referenced along the text and are available for consultation.\nThe ten recommendations are clear and solid and undoubtedly resulting from an intensive planification effort and posterior discussion and it can be directly transferred to other institutes, to be consider in new upcoming events and future outbreaks.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "96371", "date": "02 Nov 2021", "name": "Umair Ahmed Bargir", "expertise": [ "Reviewer Expertise Immunology", "Genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGhose et al., in this opinion article, highlight the success behind the mammoth responsibility which was imposed upon the Catalan government and the role played by volunteers and researchers of the ORFEU project.\nThe authors have highlighted the key challenges faced and the lessons learned while successfully implementing this mammoth task. To overcome similar challenges that can arise in the future, authors have given a ten-point recommendation to be prepared for the subsequent pandemics.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1336
https://f1000research.com/articles/9-996/v1	18 Aug 20	{ "type": "Research Article", "title": "Stage 2 Registered Report: How subtle linguistic cues prevent unethical behaviors", "authors": [ "Wen Guo", "Huanxu Liu", "Jingwen Yang", "Yuqi Mo", "Can Zhong", "Yuki Yamada", "Wen Guo", "Huanxu Liu", "Jingwen Yang", "Yuqi Mo", "Can Zhong" ], "abstract": "Background: Differences in descriptions can influence people’s evaluations and behaviors. A previous study by Bryan and colleagues suggested that subtle linguistic differences in ethical reminders can differentially prevent readers’ unethical behavior. The present study tried to replicate the previous finding in the Japanese context (Experiment 1); additionally, we explored the influence of unfamiliar Japanese instruction words that captured participants’ attention (Experiment 2). Methods: In two online experiments, participants were asked to make 10 coin-tosses and report the number of “heads” results, which would indicate the amount of money that they could earn. In Experiment 1, we analyzed the difference in the number of “heads” results as reported by 768 participants under three conditions with different instructions (“Don’t cheat” vs. “Don’t be a cheater” vs. baseline as a control). In Experiment 2, we conducted an extended experiment with an additional task in which more attention was directed toward the text. Results: In Experiment 1, we successfully replicated the results of the original experiment. The results of Experiment 2 showed no evidence that the results in Experiment 1 were influenced by attentional factors. Conclusions: In conclusion, the results of the present study supported the hypothesis that self-identity-related words of moral reminder curb unethical behaviors more effectively. Stage 1 report: https://doi.org/10.12688/f1000research.20183.4", "keywords": [ "cheating", "persuasion", "attention", "moral", "self construal", "labeling" ], "content": "Introduction\n\nWhen people behave dishonestly, they usually downplay the seriousness of the dishonest act (e.g., Monin & Jordan, 2009; Steele, 1988), weakening the link between the dishonesty and one’s self-identity (e.g., Bandura, 1999) to avoid the correspondent inference (Jones & Nisbett, 1972; Ross, 1977) that one is the kind of person who behaves dishonestly. According to self-concept maintenance theory, individuals in general strive to create and maintain an image of themselves as good and ethical people (Markus & Wurf, 1987; Mazar et al., 2008).\n\nIn general, we believe that highlighting a self-identity word will prevent unethical behaviors to some degree. According to Blasi (1984), a moral person is one for whom moral categories and moral notions are central, essential, and important to self-understanding. Morals cut deeply to the core of what and who such people are as individuals. However, one study revealed that highly constructed self-identities are associated with more unethical behaviors (Cojuharenco et al., 2012).\n\nRegarding ethical behavior, a moral-character model has been proposed, where moral character consists of motivation, ability, and identity elements (Cohen & Morse, 2014). Moral identity here refers to being disposed toward valuing morality and wanting to view oneself as a moral person. This disposition should be considered when attempting to understand why people who behave unethically tend to apply a variety of strategies to weaken the behavior–identity link (Bandura, 1999). The use of “euphemistic labeling” to describe one’s attributes and weaken the link regarding language should also be included in this disposition.\n\nDifferent ways of description can easily influence people’s evaluation and judgment about something, even if they have a wealth of previously established knowledge (Fausey & Boroditsky, 2010). For instance, using a transitive verb (agentive description, e.g., “Timberlake ripped the costume”) to describe an accident makes participants significantly more likely to blame the actor compared to the same description with the words changed to an intransitive verb (nonagentive description, e.g., “The costume ripped”). Another study found that, for children aged 5–7 years old, when a noun label was employed to describe a character (e.g., “She is a carrot-eater”) rather than a verbal predicate (e.g., “She eats carrots whenever she can”), their judgment about those characteristics would be more stable over time (Gelman & Heyman, 1999). The same phenomenon has been demonstrated regarding self-perception (Walton & Banaji, 2004). It is possible that language has some effect in this category (Gelman et al., 2000) because when nouns are used to refer to something, one may have a deeper understanding of it, which is noted to “enable inductive inferences” (Gelman & O’Reilly, 1988).\n\nOnce the subtle description is used to refer to oneself, a noun label may have a stronger effect. Bryan et al. (2011) found that more people would choose to vote if they heard the words “be a voter” rather than “to vote” on the day before election day. Additionally, research showed that, compared to “helping,” “being a helper” encouraged more children to conduct kind behaviors toward others (Bryan et al., 2014). However, subsequent research found that although “being a helper” can lead to more kind behaviors initially, once there is a setback, the backlash may also be stronger accordingly (Foster-Hanson et al., 2020). The reason underlying this phenomenon is as follows: as category labels, nouns bear a strong link to identity and may lead to self-doubt once one fails.\n\nAccording to Bryan et al. (2011), the effect of noun expression comes from a motivation-driven process. When a noun is involved with a positive identity such as “voter” and “helper,” people simply see themselves as voters or helpers and they produce more correlated behaviors; When the noun is involved with undesirable (negative) identities, however, these kind of words should cause people to avoid correlated behaviors.\n\nIn social psychology, experiments of priming of unethical behaviors and its subsequent prevention typically involve money or time (Gino & Mogilner, 2014; Gino & Pierce, 2009; Mogilner & Asker, 2009; Vohs et al., 2006). A mere exposure to money is associated with unethical outcomes (Kouchaki et al., 2013). In Gino et al.’s experiment (2014), participants were asked to complete a scrambled-sentences task using some money-related words or time-related words; results showed that priming time (rather than money) makes people behave more ethically.\n\nIn contrast, another experiment by Bryan et al. (2013) allowed experimenters to prevent unethical behaviors through semantic priming. They manipulated the task’s instructions by changing the use of verbs (“Don’t cheat”) to noun labels (“Don’t be a cheater”) to inhibit participants from engaging in unethical behaviors. The self-identity related group (“don’t be a cheater”) had significantly lower proportion of unethical cheating behaviors.\n\nIn the present study, we aim to replicate Experiment 3 of Bryan et al. (2013), for the following reasons:\n\nFirst, the participants in Experiment 1 in Bryan et al. (2013) were asked to think of a number from 1 to 10. If the number was even, they were paid $5; if it was odd, there was no reward. Bryan et al. (2013) paid for even numbers because it has been reported that participants typically show a strong bias toward odd numbers in a random number generation task (Kubovy & Psotka, 1976), but this oddness bias had not been confirmed for betting behaviors. Furthermore, an even or odd number participants think of is just imaginary, occurring in one’s inside world, not an external real event; hence, it is difficult to use it as an index of falsification. An index used for cheating should emphasize that participants’ reports can differ from the fact. Thus, we abandoned the method of Bryan et al. (2013) Experiment 1. In their Experiments 2 and 3, they used a coin-tossing task: participants were asked to toss a coin and receive a reward corresponding to the result of their coin flips. We choose this method for our experiment because tossing a coin induces a real external event, which is more objective and operable, and hence it is better than thinking of a number to measure cheating behavior. In addition, compared with Experiment 2 in the original study, which just used two conditions, “cheater” and “cheating”, a baseline group was included in Experiment 3, which made Experiment 3 more complete in its design—an approach we followed also.\n\nMoreover, we found that the effect size in Experiment 3 was small (f = 0.302 in GPower (significance level α = 0.05, power level 1-β = 0.95), meaning that Experiment 3 required at least 174 participants; in fact, only 99 people joined the original research. From this, we suppose that the effect size in Experiment 3 was overvalued.\n\nAccording to the above review, high levels of self-identity and the willingness of individuals to maintain a positive self-view should prevent unethical behaviors. We predict that the self-relevant noun “cheater” will curb cheating behaviors more significantly than the verb “cheating” and the baseline condition (in which there is no reminder in the instruction).\n\n\nMethods\n\nOur experiment was conducted online in a private and impersonal way, which means that participants did not meet or be expected to meet the experimenters. We aimed to replicate Experiment 3 of Bryan et al. (2013), in which there were three conditions: “cheater,” “cheating,” and “baseline”; in the baseline condition, a reminder about cheating was not mentioned.\n\nParticipants. Participants were users of the Yahoo! Crowdsourcing Service in Japan. Participants were required to meet the a priori criterion that they were native Japanese speakers. We conducted a pilot test to determine the shortest time in which one could reasonably participate in the experiment in good faith. This pilot test is detailed in a later section (Outlier extraction). Participants were excluded if they completed the experiment faster than the pilot test time. Repeat participation was prevented.\n\nProcedure. At the beginning of the experiment, demographic information of participants’ age and gender was collected. Online instructions indicated that a recent controversial article has claimed to report the first scientific evidence for paranormal phenomena (Bem, 2011); this was the same cover story used in the original study.\n\nWe asked the participants to find a coin at home and, while trying to influence the outcome of each toss with their minds, flip the coin 10 times, making it land on “heads” as often as possible. They were asked to ensure that they were “properly motivated.” For every toss landing on “heads,” they received 10 T-points (≈ 10 yen) as a reward. The instructions indicated that participants would receive a reward corresponding to the result of their coin flips. Noting that the average Yahoo! Crowdsourcing user receives two T-points for one task, the reward we set was five times that amount. Even those participants who reported a chance level of five “heads” received 25 times more points than for a normal task. This reward should be large enough for some participants to engage in cheating behavior.\n\nParticipants were randomly assigned the three conditions. All three conditions were posted on the crowdsourcing website, and a person who participates in one of the conditions cannot participate in the others. This resulted in passive randomization. In the “cheater” and “cheating” conditions, the manipulation was embedded in the instructions. We referred to the instructions in the original experiment (Bryan et al., 2013) and translated it into Japanese, as follows:\n\nNOTE: Please don’t [cheat/be a cheater] and report that one or more of your coin flips landed heads when it really landed tails! Even a small [amount of cheating/number of cheaters] would undermine the study, making it appear that psychokinesis is real.\n\n[嘘をつかない/嘘つきにならない]でください。コイントスの結果，実際にはコインの裏が出たのに表が出たと報告しないでください。\n\nたとえわずかでも[嘘があると/嘘つきがいると]，この研究を台無しにし，念力が実在するように見せてしまいます。\n\nThe manipulation was also embedded in the instructions on the next page, where participants log the outcomes of their 10 coin-flips. At the top of the page, a message was displayed in large red words: “嘘をつかないでください/嘘つきにならないでください” This means “PLEASE DON’T [CHEAT/BE A CHEATER]” as in the original experiment (Bryan et al., 2013).\n\nIn the baseline condition, the instructions were the same as above, except that the cheating message was not mentioned.\n\nPower analysis and sample size. Because Experiment 3 of Bryan et al. (2013) did not report the effect size, η 2, first, we calculated the effect size of the analysis of variance (ANOVA) result from the F and df values. Bryan et al. (2013) reported the statistics of their one-way ANOVA as F(2, 96) = 4.38, p = .015. Hence, we calculated η 2 based on Cohen’s (1973) method, as η ２＝.0836. Then, we calculated the effect size, f, as follows: f = √(η 2/(1 – η 2) = 0.302. The small sample size may overestimate the effect size so, as a replication convention (e.g., Nitta et al., 2018), we halved the effect size of the original experiment, and used GPower 3.1.9.3 (Faul et al., 2009) to conduct a power analysis (i.e., to 0.151). In GPower, we set the significance level α = 0.05, power level 1-β = 0.95, and effect size f = 0.151. According to the conditions of the original experiment, we divided the participants into three groups. The required total sample size was 681, with 227 participants in each group; therefore, we tried to recruit at least 681 participants, and data collection did not exceed 810 participants. This stopping rule was set because it was difficult for us to limit the number of participants to exactly 681, due to the characteristics of the simultaneous participatory online recruitment system; therefore, we allowed for up to 120% of the required sample size (i.e., 810). If more than 810 people participated in the experiment, we selected the data of the first 810 participants based on the time stamp and used this for the analysis. Also, we set the number of participants (max. 365 males and 445 females) to match the gender distribution of the original study (male: female = .45:.55).\n\nData analyses. In this study, the dependent variable was the mean number of “heads” reported. In the original experiment, a one-way ANOVA and t-test were performed. Specifically, the ANOVA was performed for analyzing the main effect of the three groups. A problem in the original study was that the authors did not report adjustments for any significance level in subsequent multiple comparisons. Therefore, in the present study, we used a one-way ANOVA and Tukey’s method for the multiple comparisons. Additionally, in order to check the cheating in each group, the original study performed one-sample t-tests between the mean number of “heads” reported and the chance level (i.e., 50%; 5 times out of 10 flips). These analyses were performed using jamovi (version 1.0.5). The original results are summarized in Table 1.\n\nMoreover, as the dependent variable was based on the counts of “heads” reported and that the 10 coin tosses were nested within each participant, a quasi-Poisson or Poisson regression was used for exploratory analyses. In the (quasi-)Poisson model, the variance was assumed to be the mean multiplied by a dispersion parameter (Ma et al., 2014). Dispersion parameters with a value greater than one indicate that overdispersion exists; in this case, quasi-Poisson regression will be performed. Thus, which analysis to used depends on the result of variance and the mean of “heads” counts. We first tested the original hypothesis. Then, information of gender and age were added as predictors to establish a regression model.\n\nOutlier extraction. For our online experiment, we established a minimum completion time (MCT) for inclusion in the final sample by asking five colleagues who were unfamiliar with this experiment to complete the experiments as fast as possible, then calculating the mean completion time. Specifically, each colleague performed a coin toss ten times; after each toss they recorded the result on the experiment website. This pilot test did not include the attempt to motivate psychokinesis and measured only the required time of the coin toss and recording. Bryan et al. (2013) also used the MCT as an extraction criterion. We excluded those participants who completed it faster than the MCT, because they might have rushed through the experiment and failed to complete it in good faith.\n\nThis experiment was employed as an extended, conceptual replication of Experiment 3 in the original study (Bryan et al., 2013). Our Experiment 2 was only performed when the results of Experiment 1 successfully replicated those of the original experiment. In the original experiment, the numbers of heads claimed in the “cheater” condition was significantly lower than that in the “cheating” and baseline conditions, but no difference was found between the “cheating” and baseline conditions. Here we cannot easily interpret the non-significant results based on self-identity alone. We aimed to test whether lower levels of attention to the instruction in the “cheating” condition reduced the effectiveness of preventing dishonest behaviors in our Experiment 1. Thus, we conducted Experiment 2, adding a “cheating” with task condition in which we used tasks concerning an instruction to ensure that participants’ attention was captured (e.g., Folk et al., 1992; Folk et al., 2002). When we translated the instruction into Japanese, we felt the unfamiliarity of a “cheater” condition in a Japanese language situation. Participants in our experiment might find that the reminder “don’t be a cheater” commands extra attention because of this sense of deviation. Therefore, even if the result of the original experiment was completely reproduced in our Experiment 1, it would not fully support the finding of the original experiment, as the reason for the possible different dishonest behavior rates between the “cheating” and “cheater” conditions in our Experiment 1 might be that the participants in the “cheating” group paid relatively less attention to the instruction; for this reason, “cheating” might have worked weakly as a moral reminder in this condition. Because the experiments are conducted online, it was difficult to ensure that the participants have actually seen and understood the instruction; in addition, it was also possible that the participants ignored the instructions of Experiment 1 due to satisficing, (e.g., Chandler et al., 2014; Oppenheimer et al., 2009; Sasaki & Yamada, 2019), further diminishing the effect of the unattended reminder (i.e., “cheating”). In this Experiment 2 we addressed these attention-related effects.\n\nNoticeably, the main difference between our Experiment 1 and the original Experiment 3 lay in the different language used in the instruction. Thus, if our Experiment 1 was a successful replication, we would then choose to focus on the expression used in the Japanese instruction, rather than the English instruction of the original Experiment 3.\n\nTo support this approach, we conducted a preliminary experiment, asking participants to evaluate their familiarity with certain expressions in Japanese. The expressions “Don’t cheat” and “Don’t be a cheater” were translated into Japanese, and native speakers evaluated their familiarity with them (1: not familiar to 5: very familiar) via an Internet survey on Yahoo! Crowdsourcing. The protocol of this experiment was registered on the Open Science Framework (Guo et al., 2020). The results showed that the familiarity rating score in the “cheater” condition was significantly lower than that in the “cheating” condition, t(64) = 6.73, p < .001, Cohen’s d = 0.834. Hence, we conjectured that the anticipated difference in the results between the “cheating” and “cheater” conditions in Experiment 1 may partly occur due to differences in attention paid to the instruction, instead of the preservation of a positive self-image proposed by the original study (Bryan et al., 2013). This meant that part of the effect of the “cheater” condition was due to the unfamiliar expression, which attracts people’s attention then played a role in preventing them from conducting unethical behavior. See Extended data for details about this experiment.\n\nIn our Experiment 2, we manipulated the way in which participants saw the instructions to explore the differences between the “cheating” and baseline conditions. Experiment 2 comprised three conditions: “cheating,” “cheating” with task, and baseline. We predicted that the “cheating” with task condition would be more effective in curbing unethical behaviors than the “cheating” and baseline conditions, because the task would arouse more attention. While the instruction in the “cheating” condition was in large red capital letters, this should entail no significant difference compared with baseline.\n\nProcedure. The procedure for Experiment 2 was identical to that of Experiment 1, except for important differences in two aspects. In Experiment 2, we focused on whether the participants read the instructions as diligently as we expect. First, we deleted the original “cheater” condition and added another “cheating” condition (i.e., “cheating” with task condition). Second, in the “cheating” with task condition, we added a task page in which participants were asked to choose the exact expression (i.e., “Don’t cheat”) that appeared on the screen from three sample sentences. We reminded participants of this task in advance to ensure they read the instructions carefully.\n\nPower analysis and participants. Because the power analysis of Experiment 2 was the same as in Experiment 1, we intended to recruit participants in the same way as Experiment 1. The minimum completion time was also established for participants to be included in the final sample. This exclusion standard was similar to that in Experiment 1.\n\nData analyses. In Experiment 2, the dependent variable was the mean number of “heads” reported. We still used a one-way ANOVA and Tukey’s method for the multiple comparisons. To check the cheating rate in each group, a one-sample t-test between the mean number of “heads” reported and the chance level (50%) was analyzed. The data of participants who failed to provide the right answer to the attention task were not used for further analysis. Another analysis by a (quasi-)Poisson regression model was also performed to explore the contribution factors of cheating counts.\n\n\nStudy timeline\n\nAfter Stage 1 acceptance, our colleagues were asked to complete the pilot test to calculate the MCT. Then, we posted our experiments on the Yahoo! Crowdsourcing Service to recruit participants. The experiments and subsequent analysis were conducted from March 29, 2020 to July 10, 2020.\n\nThe present study received approval from the psychological research ethics committee of the Faculty of Human-Environment Studies at Kyushu University (approval number: 2019-004). Completion of experiments by participants was regarded as consent to participate; they also had the right to withdraw from the experiment at any time without providing a reason. In addition, we protected participants’ personal information. Because this study was conducted online, even if participants engaged in cheating behaviors, we could not identify them or meet the participants face-to-face.\n\n\nResults\n\nWe recruited 1298 users of the Yahoo! Crowdsourcing service who clicked on a survey called “Coin Game.” Of these participants, 803 met our minimum completion time (MCT) criterion for good faith during the course of the experiment. Moreover, we deleted the data of participants exceeding the limit we set to match the gender distribution of the original study; finally, we used data from 768 participants (males = 315, females = 445, no response = 8, Mage = 45.68) for analyses.\n\nIn this study, the dependent variable was the mean number of “heads” reported. We performed a one-way ANOVA and Tukey’s method for the post hoc comparisons. The results of ANOVA showed a significant main effect of condition (F (2,765) = 5.86, p = .003, f = 0.123). Multiple comparisons showed that participants in the “cheater” condition reported having obtained significantly lesser “heads” than the participants in the “cheating” condition (t (765) = 2.554, p = .029, Cohen’s d = 0.22) and the baseline condition (t (765) = 3.24, p = .004, Cohen’s d = 0.29). The “heads” reported in the baseline condition had no significant difference with the “cheating” condition (t (765) = 0.754, p = .731, Cohen’s d = 0.07). The results of Experiment 1 suggested that participants instructed with “cheater” had a significantly lesser cheating rate than those instructed with “cheating” and those that were uninstructed (Figure 1A). Hence, Experiment 1 successfully replicated the results in the original study (Bryan et al., 2013).\n\nThe mean number of “heads” in each instruction condition in Experiment 1 (Panel A) and Experiment 2 (Panel B). Error bars indicate 95% confidence intervals of the mean.\n\nMoreover, we compared each condition to a chance level (i.e., five times) to check bias in each group. The “cheating” (M = 5.22) and baseline (M = 5.33) groups had a significantly higher rate than five (t (265) = 2.20, p = .029, Cohen’s d = 0.135 and t (241) = 3.31, p = .001, Cohen’s d = 0.212, respectively). The number of “heads” in the “cheater” (M = 4.86) group did not significantly differ from five (t (259) = 1.36, p = 0.176, Cohen’s d = 0.084). In this respect too, we succeeded in replicating the results of the original experiment.\n\nThe dependent variable was based on counts of reported “heads” and 10 trials were nested within each participant. A Poisson regression analysis on the number of reported “heads” was performed. We first added “condition” as a factor into the model. The results indicated that condition (χ2 (2) = 6.23, p = 0.044) significantly contributed to explaining variance related to the number of “heads.” In contrast, when participants’ gender and age were used as predictors for the regression model, the results indicated that gender (χ2 (2) = 1.11, p = .575) and age (χ2 (58) = 26.40, p = 1.00) did not significantly predict the number of “heads.”\n\nAlthough we successfully replicated the results in the original study, it was still premature to conclude that the subtle difference in the instruction affected the cheating behavior. In particular, as there is a significantly different degree of familiarity between the words “cheater” and “cheating” in the Japanese instruction (see our preliminary experiment), there was still a possibility that attentional factors might have influenced the cheating. Therefore, we conducted Experiment 2.\n\nWe recruited 1255 users of the Yahoo! Crowdsourcing service. Among these, 800 (males = 365, females = 425, Mage = 43.35) met the MCT criterion for good-faith participation and were used for analyses. The number of participants by gender did not exceed the maximum sample size we had set.\n\nIn Experiment 2, three conditions, the “Don’t cheat,” “Don’t cheat” with attention task, and baseline groups, respectively, were employed. The analysis was identical to Experiment 1. The result of the one-way ANOVA did not show a significant main effect of condition [F (2, 797) = 0.667, p = .514, f = 0.045 (Figure 1B)]. The cheating behavior in the attention task condition did not significantly decrease more than the baseline and “cheating” conditions (t (797) = 0.957, p = .604, Cohen’s d = 0.01 and t (797) = -1.034, p = .555, Cohen’s d = 0.09).\n\nMoreover, we also performed a t-test between each condition and a chance level of five. The “cheating with attention task” (M = 5.25) and baseline (M = 5.23) conditions had a significantly larger number than five (t (242) = 2.380, p = .018, Cohen’s d = 0.153 and t (286) = 2.389, p = .018, Cohen’s d = 0.141, respectively). The number of “heads” in the “cheating” condition had no significant difference with five (t (269) = 0.937, p = .350, Cohen’s d = 0.057).\n\nA Poisson regression analysis on the number of reported “heads” was also performed. Condition [χ2 (2) = 0.628, p = .731] did not significantly contribute to explain variance related to the number of “heads.” Additionally, gender [χ2 (2) = 0.728, p = .695] and age [χ2 (57) = 27.139, p = 1.00] did not significantly predict the number of “heads.”\n\nThese findings do not suggest that the settings made for participants to pay full attention to the reminder instruction significantly reduced the incidences of cheating. Therefore, it is reasonable to assume that the significant effect of “cheater” in Experiment 1 was not due to excessive attention to the instruction.\n\n\nDiscussion\n\nIn this study, we directly replicated Experiment 3 of Bryan et al. (2013) (Experiment 1) and addressed potential attentional effects related to translation (Experiment 2) using a Japanese sample. These registered experiments were perfectly performed without any deviation from the protocol. As a result, in Experiment 1, we successfully replicated the original results of Bryan et al. (2013). We found a significant main effect of condition similar to the original study, and the “Don’t be a cheater” had a minimal number of reported “heads” (M=4.86), which meant participants in this group had the lowest rate of dishonest behavior. However, we could not conclude that the participants in the “cheater” group have lesser cheating behaviors than those in the “cheating” group. As we predicted in the Stage 1 protocol and confirmed in a preliminary experiment, the unfamiliar expression of “Don’t be a cheater” in the Japanese context would have made participants pay more attention to this sentence; hence, it would have made for a more effective reminder. In other words, “Don’t cheat” in the Japanese context might be ignored by some participants and lose its role as a reminder. Therefore, there were more cheating behaviors in the “cheating” condition.\n\nTo examine the attentional factor on cheating behaviors, we added an additional “Don’t cheat” condition with a recognition task for the reminder. We aimed to test whether the effect of the reminder was enhanced by participants’ excessive attention to the unfamiliar expression in the Japanese context. One of the mechanisms of the theory of self-concept maintenance relies on the attention to standards of one’s conduct (Mazar et al., 2008). As Mazar et al. (2008) found, when reminded of their standards, people had to face their actions, and therefore, could be more honest. However, in Experiment 2, no significant main effect of conditions was shown, which clarified that the proper attention to the reminder “Don’t cheat” was still ineffectual in curbing the cheating behavior. We did not obtain results consistent with Mazar et al. (2008). Thus, we excluded the role of the attentional factor in verb-based and noun-based reminders in the Japanese context. The results supported the hypothesis proposed by Bryan et al. (2013) that, in such cases, undesired self-relevant nouns can cause people to avoid a particular behavior and such nouns influence behavior by emphasizing its implications for identity (Bryan et al., 2011).\n\nAlthough we successfully replicated the original study, some differences were present in terms of results. Note that the effect size of the present study was considerably smaller than the original study (f = 0.302 for the original study and f = 0.123 for the present study). Similarly, in the multiple comparisons, as per the trend (“cheating” – “cheater” = 0.71 and “cheater” – baseline = 0.66 for the original study vs. “cheating” – “cheater” = 0.22 and “cheater” – baseline = 0.29 for present study), it is likely that there was some inflation of effect size due to the small sample size in previous studies (99 participants in the original study vs. 768 participants in the present study). Another replication of the original study by Savir & Gamliel (2019) also obtained results with a lower effect size. They added positive-valence appeals to check the effectiveness of self-appeals in reducing unethical behavior. In their meta-analysis, “cheater” was actually more effective than “cheating”, whereas they reported a lower effect size (overall estimate of 0.17) as compared with Bryan et al. (2013) (overall estimate of 0.80). In the present study, the effect size was comparable to the previous replication study (Savir & Gamliel, 2019).\n\nBesides the comparison between conditions, to check bias in each group, we compared the “heads” times in each condition in both experiments to a chance level. Inconsistent with the original experiment and Experiment 1, in Experiment 2, there was no significant difference between the “cheating” condition and the chance level. This discrepancy is not important because it is not relevant to the claimed hypothesis. However, whether this \"cheating\" reminder does or does not affect behavior is inconsistent across studies, and hence it will be interesting to see what factors define the difference. Note that the present study showed that the effect size of the reminder itself is very small, so it is clear that no promising hypothesis exists at this point as a crud factor (Orben & Lakens, 2020).\n\nThe present study contributes to research across cultures and to the generalization of the original effect. Previously published studies employing a similar method have studied this effect in US and Israel (Bryan et al., 2013; Savir & Gamliel, 2019), but to the best of our knowledge, the subtle linguistic effects of moral reminders on cheating have not been investigated in Asia. The results of this study also provide a cross-cultural validity of the inhibitory effects of different lexical-based warnings on unethical behaviors.\n\nIn the present study, we selected a sample of Japanese people in Asia (instead of Americans in the original experiments) and set up an authentic online survey environment as in the original study (instead of a laboratory experiment) to ensure that participants behave naturally. Given that in the two language contexts (Japanese and American English) self-identity words curb unethical behaviors more effectively, we expect that our results with users of a crowdsourcing platform can be generalized to other online contexts. However, given that the priming of unethical behavior in the present study (the same as Bryan et al., 2013) was based on the coin flip paradigm, the pattern of results, which might hold only for the instant coin flip paradigm, do not generalize to curbing all kinds of unethical behaviors. It should also be noted that linguistic expressions conveying the same meaning may still vary greatly in two different language families, depending on the cultural habits and the language structure itself (as in our preliminary experiment, “Don’t be a cheater” was not a familiar expression in Japanese, as it is not usually used in that way); therefore, it is still necessary to consider the generality of this effect in countries with a similar linguistic system in future research. Considering the results of the Poisson regression analysis, we have no reason to believe that the results depend on other characteristics of the participants. It is unclear at this time whether other materials and contextual factors could be constraints on generality (Simons et al., 2017).\n\nA limitation that we want to discuss is the considerably high rate of dropout: 495 people of 1298 (38.1%) and 455 people of 1255 (36.2%) in the two experiments, respectively. In future online surveys, we may need to recruit participants over 150% of the required sample size to ensure an adequate number of participants. There were advantages and disadvantages to conducting the cheating experiment online; because of anonymity, participants were free from social expectations and could cheat more easily (Grym & Veronica, 2016). However, we did not have a true grasp of whether participants completed the experiment in good faith (e.g., Sasaki & Yamada, 2019).\n\n\nConclusion\n\nIn conclusion, this study successfully replicated the results of Experiment 3 of Bryan et al. (2013) as a registered report without any deviation from the protocol, and excluded the possibility of the attentional factor when the reminder was translated to Japanese. We closely followed their experimental design and analytic methods. The results of our study supported the hypothesis that self-identity-related nouns of moral reminder curbed unethical behaviors more effectively than when the same meaning was expressed using verbs; furthermore, they supported the cross-linguistic validity of these findings. Moreover, we found that the attentional factor showed no effect in decreasing unethical behaviors in the Japanese context. Future research should also consider personal traits when focusing on the inhibiting effects of language on individual unethical behaviors.\n\n\nData availability\n\nOpen Science Framework: How subtle linguistic cues prevent unethical behaviors, https://doi.org/10.17605/OSF.IO/AXDHV (Guo et al., 2020).\n\nThis project contains the following underlying data:\n\nData collected for Experiment 1 (XLSX format, uploaded 15/07/2020)\n\nExplanation for dataset of Experiment 1 (RTF format, uploaded 13/07/2020)\n\nData collected for Experiment 2 (XLSX format, uploaded 13/07/2020)\n\nExplanation for dataset of Experiment 2 (RTF format, uploaded 13/07/2020)\n\nOpen Science Framework: How subtle linguistic cues prevent unethical behaviors, https://doi.org/10.17605/OSF.IO/AXDHV (Guo et al., 2020).\n\nThis project contains the following extended data:\n\nProtocol for the pilot study conducted for Experiment 2 (PDF format, uploaded 31/07/2019)\n\nData collected for the pilot study conducted for Experiment 2 (XLSX format, uploaded 23/07/2019)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBandura A: Moral disengagement in the perpetration of inhumanities. 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Publisher Full Text\n\nVohs KD, Mead NL, Goode MR: The psychological consequences of money. Science. 2006; 314(5802): 1154–1156. PubMed Abstract \| Publisher Full Text\n\nWalton GM, Banaji MR: Being what you say: The effect of essentialist linguistic labels on preferences. Soc Cogn. 2004; 22(2): 193–213. Publisher Full Text" }	[ { "id": "69708", "date": "22 Sep 2020", "name": "Sergio Cervera-Torres", "expertise": [ "Reviewer Expertise Cognitive & e-Health Psychology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 nicely replicates, in a Japanese sample, the findings by Bryan et al. (2013) about how subtle linguistic cues prevent unethical behaviour based on a coin-tossing task.\n\nAs a reviewer of Stage 1 of this study, I must say that all my major concerns have been successfully amended. I only have minor suggestions:\n- Introduction: I still think that the storyline could be improved a bit. I find unclear whether the statement \"In general, we believe that highlighting a self-identity word...\" at the beginning of the second paragraph is an especial belief of the authors or an argument. Maybe it could suit better at the end of the paragraph \"According to Bryan et al. (2011)...\".\n- Experiment 1. I would use Inclusion (or exclusion) criteria as a heading instead of \"outlier extraction\" and move the section before the \"data analyses\" section.\n\n- Experiment 2. You use three different expressions to name the new condition: \"cheating\" with task condition, \"cheating with attention task\" condition, and \"don't cheat\" condition with a recognition task. I suggest being consistent through the paper and use, \"cheating with attention task\". In the procedure, I would use different wording for \"..we deleted the original \"cheater condition and added...\" such as \"...we omitted the original cheater condition and included instead...\"\n- Discussion. In the second paragraph, you state \"we aimed to test whether the effect of the reminder was enhanced by participants' excessive attention to the unfamiliar expression [cheater] in the Japanese context\". This is not exactly what you did. As you state in the introduction of Experiment 2 \"We aimed to test whether lower levels of attention to the instruction in the “cheating” condition reduced the effectiveness of preventing dishonest behaviours in our Experiment 1\". The difference is subtle but important.\n\nAre the data able to test the authors’ proposed hypotheses by satisfying the approved outcome-neutral conditions? Yes\n\nAre the introduction, rationale and stated hypotheses the same as the approved Stage 1 submission? Yes\n\nDid the authors adhere precisely to the registered experimental procedures? If not, has an explanation been provided regarding any change? Yes\n\nAre any unregistered post hoc analyses added by the authors justified, methodologically sound and informative? Not applicable\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": [ { "c_id": "6080", "date": "03 Nov 2020", "name": "Yuki Yamada", "role": "Author Response", "response": "Thank you for reviewing our registered report again! We have responded to each of your comments as follows. 1.- Introduction: I still think that the storyline could be improved a bit. I find unclear whether the statement \"In general, we believe that highlighting a self-identity word...\" at the beginning of the second paragraph is an especial belief of the authors or an argument. Maybe it could suit better at the end of the paragraph \"According to Bryan et al. (2011)...\". Reply: In the second paragraph, we wanted to state how the self-identity influences ethical behaviors through the moral core of a person. However, this topic was also discussed in Bryan et al. (2013) and it makes sense to put it at the end of the sixth paragraph of the introduction as your suggestion. As this is a revision of a part of the manuscript of stage 1, please let us add a footnote before the “Data Availability” part to explain this modification does not change the logic or the hypothesis that we registered in our stage 1 manuscript. 2.- Experiment 1. I would use Inclusion (or exclusion) criteria as a heading instead of \"outlier extraction\" and move the section before the \"data analyses\" section. Reply: Thank you for your suggestion. We have replaced the “Outlier extraction” with “Exclusion criteria” and set the “Exclusion criteria” section before the “Data analyses” section. 3.- Experiment 2. You use three different expressions to name the new condition: \"cheating\" with task condition, \"cheating with attention task\" condition, and \"don't cheat\" condition with a recognition task. I suggest being consistent through the paper and use, \"cheating with attention task\". In the procedure, I would use different wording for \"..we deleted the original \"cheater condition and added...\" such as \"...we omitted the original cheater condition and included instead...\" Reply: According to your suggestion, the expression of the new condition has been unified as “cheating” with attention task. Besides, we also modified the corresponding part in the manuscript. 4.- Discussion. In the second paragraph, you state \"we aimed to test whether the effect of the reminder was enhanced by participants' excessive attention to the unfamiliar expression [cheater] in the Japanese context\". This is not exactly what you did. As you state in the introduction of Experiment 2 \"We aimed to test whether lower levels of attention to the instruction in the “cheating” condition reduced the effectiveness of preventing dishonest behaviors in our Experiment 1\". The difference is subtle but important. Reply: The purpose of Experiment 2 was to explore the role of the attention factor on unethical behavior in different samples from Experiment 1. As you said, we should revise the expression to describe our work more clearly, so we have rephrased the description of the discussion as follows: We aimed to test whether the lower levels of attention caused by a familiar instruction (“Don’t cheat”) in the “cheating” condition reduced the effectiveness of preventing unethical behavior in our Experiment 1." } ] }, { "id": "69709", "date": "26 Oct 2020", "name": "Michiko Asano", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study nicely replicates Experiment 3 of Bryan et al. (2013), which showed that a self-relevant noun of moral reminder (i.e., “Please don’t be a cheater”) curbed unethical behaviours more effectively than a verb (i.e., “Please don’t cheat”) did, in an online survey environment with Japanese speakers. The results of Experiment 2 of the current study, which addressed potential attentional effects related to translation, strengthened their claim.\nI only have minor suggestions, as described below, and approve this manuscript for publication.\nMinor suggestions:\nThe last sentence of the second paragraph of Introduction “However, one study revealed that highly constructed self-identities are associated with more unethical behaviors (Cojuharenco et al., 2012).”: It is not very clear to me what point the authors are trying to make by citing this study here.\n\nThe fourth paragraph of Discussion: “Inconsistent with the original experiment and Experiment 1, in Experiment 2, there was no significant difference between the “cheating” condition and the chance level. This discrepancy is not important because it is not relevant to the claimed hypothesis... Note that the present study showed that the effect size of the reminder itself is very small, so it is clear that no promising hypothesis exists at this point as a crud factor (Orben & Lakens, 2020).”: I understand that the lack of a significant difference between the “cheating” condition and the chance level is not relevant to the hypothesis of Experiment 2, and the effect size of the reminder itself is small. However, I still feel the authors’ claim here (“This discrepancy is not important”) is too strong and perhaps needs to be toned down because it is the prerequisite for this line of study that cheating behaviours occur in the “cheating” condition.\n\nAre the data able to test the authors’ proposed hypotheses by satisfying the approved outcome-neutral conditions? Yes\n\nAre the introduction, rationale and stated hypotheses the same as the approved Stage 1 submission? Yes\n\nDid the authors adhere precisely to the registered experimental procedures? If not, has an explanation been provided regarding any change? Yes\n\nAre any unregistered post hoc analyses added by the authors justified, methodologically sound and informative? Not applicable\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": [ { "c_id": "6081", "date": "03 Nov 2020", "name": "Yuki Yamada", "role": "Author Response", "response": "Thank you for reviewing our registered report again! We have responded to each of your comments as follows. Minor suggestions: 1. The last sentence of the second paragraph of Introduction “However, one study revealed that highly constructed self-identities are associated with more unethical behaviors (Cojuharenco et al., 2012).”: It is not very clear to me what point the authors are trying to make by citing this study here. Reply: In Cojuharenco et al. (2012), high levels of self-identities construction elicited more unethical behaviors. This finding was inconsistent with the conclusion of the experiment in Bryan et al. (2013) that we intended to replicate, thus here we cited this research with different conclusions in order to place more emphasis on the importance of replicating the results of Bryan's experiment. However, to avoid excessive and unnecessary changes of the introduction (the stage 1 manuscript), we decided only to explain it here, but do not add extra explanations to the manuscript. 2. The fourth paragraph of Discussion: “Inconsistent with the original experiment and Experiment 1, in Experiment 2, there was no significant difference between the “cheating” condition and the chance level. This discrepancy is not important because it is not relevant to the claimed hypothesis... Note that the present study showed that the effect size of the reminder itself is very small, so it is clear that no promising hypothesis exists at this point as a crud factor (Orben & Lakens, 2020).”: I understand that the lack of a significant difference between the “cheating” condition and the chance level is not relevant to the hypothesis of Experiment 2, and the effect size of the reminder itself is small. However, I still feel the authors’ claim here (“This discrepancy is not important”) is too strong and perhaps needs to be toned down because it is the prerequisite for this line of study that cheating behaviours occur in the “cheating” condition. Reply: Thank you for your constructive suggestion. As you pointed out, we have to admit that we discussed the “cheating” condition in Experiment 2 with a too strong tone. It is meaningful to explore the effectiveness of “don’t cheat” reminder in different circumstances, thus here we cannot assert that this discrepancy is not important. We toned down the discussion and modified the corresponding part of the manuscript as follows: This inconsistency in the results between the present experiments does not affect the interpretation of the main hypothesis we tried to test here. Yet, whether this “cheating” reminder does or does not affect behavior is inconsistent across studies as well, and hence it will be interesting to further investigate what factors define the difference." } ] } ]	1	https://f1000research.com/articles/9-996
https://f1000research.com/articles/9-659/v1	30 Jun 20	{ "type": "Case Report", "title": "Case Report: Renal potassium wasting in SARS-CoV-2 infection", "authors": [ "Holly Mabillard", "Hilary Tedd", "Ally Speight", "Christopher Duncan", "David A. Price", "John A. Sayer", "Holly Mabillard", "Hilary Tedd", "Ally Speight", "Christopher Duncan", "David A. Price" ], "abstract": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with many potentially fatal complications. Renal involvement in various forms is common in addition to serum electrolyte disturbances. Early reports suggest that hypokalaemia may frequent those with SARS-CoV-2 infection and various aetiological factors may cause this electrolyte disturbance. A Chinese retrospective study has demonstrated renal potassium wasting in patients infected with SARS-CoV-2, however, it is not known if these patients were receiving diuretic therapy which may be a contributing factor. This case report illustrates an example of renal potassium wasting in SARS-CoV-2 infection in the absence of diuretics and extra-renal mechanisms with important lessons learned.", "keywords": [ "COVID-19", "hypokalaemia", "potassium", "tubulopathy" ], "content": "Introduction\n\nSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with many potentially fatal complications1. Renal involvement in various forms is common in addition to serum electrolyte disturbances2. Renal pathologies identified so far include acute tubular injury, proteinuria, rhabdomyolysis, secondary focal segmental glomerulosclerosis and possible renin-angiotensin-aldosterone system (RAS) activation2.\n\nEarly reports suggest that hypokalaemia may frequent those with SARS-CoV-2 infection3 and various aetiological factors may cause this electrolyte disturbance such as gastrointestinal potassium loss, diuretic-induced potassium wasting, renal tubulopathy and RAS activation. An early Chinese retrospective study has demonstrated renal potassium wasting in patients infected with SARS-CoV-2, however it is not known if these patients were receiving diuretic therapy which may have been a contributing factor3. This case report illustrates an example of renal potassium wasting in SARS-CoV-2 infection in the absence of diuretics and extra-renal mechanisms in addition to lessons learned from an important and potentially dangerous complication of the disease.\n\n\nCase report\n\nOur patient, an 82 year old Caucasian female, was admitted to the medical admissions unit with confusion and drowsiness in April 2020. She was noted to be pyrexial (38.6 degrees Celsius) and hypoxic (oxygen saturations 86% on room air) on admission and had a Glasgow coma score of 14/15. She had a chronic cough with intermittent clear sputum. Bilateral inspiratory crackles were reported on chest auscultation and she looked hypovolaemic. Blood pressure was 90/60 mmHg and heart rate was 100 bpm. Her comorbidities include chronic obstructive pulmonary disease, previous dynamic hip screw following a right fractured neck of femur, osteoarthritis, hypertension, thoracoabdominal aortic aneurysm and osteoporosis. Usual medications included oral colecalciferol, diazepam, lercanidipine, omeprazole, sertraline, tolterodine and a salbutamol inhaler. Her chest x-ray did not show evidence of infection. Her diazepam and antihypertensives were suspended and she was commenced on oxygen therapy aiming for saturations between 88 and 92%. Her coronavirus disease 2019 (COVID-19) RT-PCR test from a deep throat swab was positive. The patient developed systemic symptoms the next day and a rise in C-reactive protein (CRP) was seen (Table 1) so she was commenced on doxycycline 100mg once daily for five days to cover a lower respiratory tract infection. Both urine and blood cultures were negative. 3 Days after admission she developed hypokalaemia which was treated with 72 mmol oral SandoK (potassium bicarbonate and potassium chloride therapy) daily (in divided doses) and she was given 1L intravenous Hartman’s solution (containing 5 mmol/L potassium) daily for three days (Figure 1). Six days after admission the patient’s oxygen requirements had returned to baseline but hypokalaemia (serum potassium 3.4 mmol/L) persisted despite treatment. A trans-tubular potassium gradient was found to be significantly elevated at 10.7. Her serum potassium level normalised to 3.9 mmol/L eight days later. Clinical improvement was noted at this point and the patient received physiotherapy until her mobility was safe enough to be discharged after a total of 20 days in hospital. Given her COVID-19 positive status she received four weeks of rivaroxaban therapy post discharge to reduce the chance of venous thromboembolism. At no point did our patient have diarrhoea or receive any medications that would interfere with her serum or urine potassium such as RAS inhibitors or loop or thiazide diuretics. Her renal function remained at baseline throughout her admission (serum Creatinine 60–77 µmol/L and eGFR 70–82 mL/min/1.73m2).\n\n\nDiscussion\n\nThe renal complications that are known from this devastating disease so far include proteinuria, acute tubular injury, rhabdomyolysis, secondary focal segmental glomerulosclerosis and possible RAS activation (both directly and indirectly)2. This case demonstrates evidence of renal potassium wasting as an example of a further renal complication of SARS-CoV-2 infection which has, so far, only been reported in China yet without the knowledge of contribution from diuretics. Although, the patient’s chronic obstructive pulmonary disease would naturally lead us to speculate this as a cause for her metabolic alkalosis; her serum bicarbonate has always been normal, her COPD was mild, she had an elevated blood pH despite high pCO2 levels and renal potassium wasting is not a known sequela of COPD.\n\nA retrospective pre-print Chinese study highlighted hypokalaemia as a complication early on in the pandemic which contributed to the momentum in speculation of RAS involvement3. This was due to chronic kidney disease, hypertension, cardiovascular disease and diabetes mellitus being more commonly observed in those with severe SARS-CoV-2 infection along with the knowledge that SARS-CoV-2 enters host cells through the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of pulmonary type 2 alveolar cells. The Chinese data (n = 175) reported the presence of hypokalaemia in 62% of patients with SARS-CoV-2 infection and 22% of all patients had severe hypokalaemia (serum potassium <3.0 mmol/L). Many (28%) had concomitant metabolic alkalosis and of the 20 patients who had a spot urine potassium/creatinine ratio, their degree of urine potassium wasting was significantly higher than those with normokalaemia and that this correlated with the degree of infection severity. It was also noted in two patients that their renal potassium wasting persisted until complete clinical recovery from the virus which was the case with our patient. Hypokalaemia was also documented in 41.3% of a cohort of SARS-CoV infection during the 2003 epidemic but it should be noted that the largest SARS-CoV-2 case series to date (n = 1099) did not find a major difference in serum potassium between those with mild and severe disease.\n\nResearch into the renin-angiotensin-aldosterone system has shown that genetic polymorphisms in the ACE2 gene are associated with a difference in blood pressure response to sodium and potassium intake4. One study reported that ACE2 SNPs are associated with a blood pressure reduction in response to potassium intake during a period of high salt intake where blood pressure would typically be expected to rise and that ‘potassium sensitivity’ of blood pressure may exist5. Further research using the 100 000 genomes project has suggested that ACE2 polymorphisms could explain the higher risk of severe disease and death amongst males and those of African and East Asian origin in SARS-CoV-2 infection. The Chinese SARS-CoV-2 study could reflect the literature which suggests subjects with ‘severe disease’ may represent a sub-group with ACE2 polymorphisms6. It is already known that rs182366225 and rs2097723 polymorphisms are more frequent in the East Asian population and increase the expression of ACE2. It may therefore be that certain ACE2 polymorphisms are a risk factor for hypokalaemia. It should also be noted that, if this is the case, then there may be a greater prevalence of hypokalaemia amongst certain ethnic populations and that the study by Chen, D. et al. demonstrates a higher prevalence of hypokalaemia than in other studies because of this.\n\nComplications from hypokalaemia can be life-threatening7. Hypokalaemia can result in muscle weakness and cramps, thirst and paraesthesia but when serum potassium drops below 3.0 mmol/L arrhythmias such as QTc prolongation, torsades de pointes, ventricular fibrillation and sudden cardiac death can occur8. The incidence of ventricular fibrillation is five-fold higher in those with hypokalaemia in comparison to hyperkalaemia9. Inadequate management of hypokalaemia in hospitalised patients has been reported in as much as 24% in one study demonstrating an at-risk group of patients and a need for therapeutic vigilance here10.\n\nThe SARS-CoV-2 pandemic gives us many reasons to be vigilant with the detection and management of hypokalaemia. First, many of the SARS-CoV-2 clinical trial drugs prolong the QTc interval which adds to the arrhythmogenic effect of hypokalaemia. Some of the trial drugs can induce hypokalaemia themselves risking severe electrolyte disturbances. Second, arrhythmia induction has more than one risk factor in this patient population, for example, hypoxia-mediated, cytokine-storm-syndrome and direct viral cardiac myocyte damage which all make these patients vulnerable to cardiac complications11. Third, the use of diuretics to improve oxygenation in those with acute respiratory distress syndrome also risks hypokalaemic complications. Finally, the data for return of spontaneous circulation (ROSC) during cardiac arrest in a retrospective study (n = 136) is unfortunately very poor (13.2% achieved ROSC and less than 3% were alive at 30 days post-cardiac arrest) which reiterates the importance of vigilance for electrolyte disturbance in this patient population12.\n\nThe popular mechanism for hypokalaemia remains RAS system activation and it should be noted that not all patients with RAS activation are hypokalaemic, due to the renal potassium switch mechanism13,14. Early reports demonstrate good histopathological evidence of tubular injury which seem to have a multifactorial aetiology in SARS-CoV-2 patients15,16. These include acute tubular necrosis from cytokine-storm syndrome, direct virion invasion of tubular cells, peritubular congestion, drug toxicity and pigment-cast nephropathy from rhabdomyolysis. Based on these mechanisms it seems likely that tubular injury is difficult to avoid in these patients.\n\nCurrent evidence for mechanisms of renal injury in SARS-CoV-2 infection is early and much of which is based on speculation, case reports and case series. It is clear that we need more studies that measure all components of the RAS pathway in SARS-CoV-2 patients to truly determine RAS involvement and more data on serum and urine electrolytes in infected patients including the trans-tubular potassium gradient. It is easy to measure the TTKG and measurement only requires serum and urine osmolality and potassium (urine potassium/serum potassium)/(urine osmolality/ serum osmolality). Estimation is accurate providing urine is not dilute and that urine sodium concentration is >25mmol/l so that distal sodium delivery is not a limiting factor17.\n\n\nConclusion\n\nHere we summarise a case of severe SARS-CoV-2 infection with evidence of significant renal potassium wasting resulting in hypokalaemia. Complications from hypokalaemia can be life-threatening, especially during critical illness, thus we advocate checking the trans-tubular potassium gradient in patients with moderate-severe infection. This should help identify those at risk of hypokalaemia so appropriate monitoring and electrolyte replacement can be promptly instituted to prevent this potentially fatal complication.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nJAS is supported by Kidney Research UK and Northern Counties Kidney Research Fund.\n\n\nReferences\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBatlle D, Soler MJ, Sparks MA, et al.: Acute kidney injury in COVID-19: Emerging evidence of a distinct pathophysiology. J Am Soc Nephrol. 2020; 31(6). PubMed Abstract \| Publisher Full Text\n\nChen D Jr, Li X, Song Q Sr, et al.: Hypokalaemia and Clinical Implications in Patients with Coronavirus Disease 2019 (COVID-19). medRxiv. 2020. Publisher Full Text\n\nPatel SK, Velkoska E, Freeman M, et al.: From gene to protein—experimental and clinical studies of ACE2 in blood pressure control and arterial hypertension. Front Physiol. 2014; 5: 227. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao Q, Gu D, Kelly TN, et al.: Association of genetic variants in the apelin-APJ system and ACE2 with blood pressure responses to potassium supplementation: the GenSalt study. Am J Hypertens. 2010; 23(6): 606–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhayat AS, de Assumpcao PP, Khayat BMC, et al.: ACE2 polymorphisms as potential players in COVID-19 outcome. MedRxiv. 2020. Publisher Full Text\n\nCrop MJ, Hoorn EJ, Lindemas J, et al.: Hypokalemia and subsequent hyperkalemia in hospitalized patients. Nephrol Dial Transplant. 2007; 22(12): 3471–3477. PubMed Abstract \| Publisher Full Text\n\nWidimsky P: Hypokalaemia and the heart. E-journal of Cardiology Practice. 2008; 7(9). Reference Source\n\nClausen TG, Brocks K, Ibsen H: Hypokalemia and ventricular arrhythmias in acute myocardial infarction. Acta Med Scand. 1988; 224(6): 531–537. PubMed Abstract \| Publisher Full Text\n\nKjeldsen K: Hypokalemia and sudden cardiac death. Exp Clin Cardiol. 2010; 15(4): e96–9. PubMed Abstract \| Free Full Text\n\nZheng YY, Ma YT, Zhang JY, et al.: COVID-19 and the cardiovascular system. Nat Rev Cardiol. 2020; 17(5): 259–260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShao F, Xu S, Ma X, et al.: In-hospital cardiac arrest outcomes among patients with COVID-19 pneumonia in Wuhan, China. Resuscitation. 2020; 151: 18–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamel KS, Schreiber M, Halperin ML: Renal potassium physiology: integration of the renal response to dietary potassium depletion. Kidney Int. 2018; 93(1): 41–53. PubMed Abstract \| Publisher Full Text\n\nMabillard H, Sayer JA: The Molecular Genetics of Gordon Syndrome. Genes (Basel). 2019; 10(12): 986. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSu H, Yang M, Wan C, et al.: Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China. Kidney Int. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiao B, Wang C, Wang R, et al.: Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection. MedRxiv. Publisher Full Text\n\nElisaf M, Rizos E, Siamopoulos K: Potassium excretion indices in the diagnostic approach to hypokalaemia. QJM. 2000; 93(5): 318–9. PubMed Abstract \| Publisher Full Text" }	[ { "id": "68472", "date": "17 Aug 2020", "name": "Shabbir Moochhala", "expertise": [ "Reviewer Expertise Clinical renal disease", "renal tubular disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well-observed, useful and much needed case and comment on an important renal complication of SARS-CoV-2 infection in a co-morbid patient.\nMy main concern is that the possibility of this being a primary metabolic alkalosis (with secondary hypokalaemia) has not been adequately refuted. The blood gas data show a metabolic alkalosis with respiratory compensation, and simultaneously a relatively modest hypokalaemia for this degree of alkalosis. Contraction alkalosis (due to pyrexia causing volume depletion/hypotension) or hypercapnoeic metabolic alkalosis (hypoxic on admission with COPD) might be explanations for this1. Results for chloride and CK, if available, may be helpful.\n\nI agree that the results do not support aldosterone-mediated hypokalaemia.\n\nIn fig 1, Hartmann's and sando K on day 3 should be mentioned together for clarity.\n\nI am not clear about when TTKG was measured. Presumably this was before potassium replacement on day 3 to avoid confounding, but this should be mentioned.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "71137", "date": "07 Oct 2020", "name": "Ming-Hsien Tsai", "expertise": [ "Reviewer Expertise nephrology" ], "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 reported a case with hypokalemia may relate to SARS-Cov-2 infection. This case is well written but still, some concerns need to be clarified.\n\nMajor:\nThe timeline in this case is important for a comprehensive understanding. I suggest that the authors can modify Table 1 to show the baseline data and consequent laboratory change.\n\nDid the authors have the result of urine nitrogen, creatinine, sodium, potassium, calcium, Mg, phosphate, and osmolarity levels? If yes, please provide.\nMinor:\nThe dosed of medications that she used in OPD should be provided in the part of the case report.\n\nThe author should focus on this case and clearly tell us that what is the most possible etiology of renal wasting in this case.\n\nThe urine K/Cr ratio can offer a more quick and reliable interpretation of the hypokalemia. I suggest the author can discuss this.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-659
https://f1000research.com/articles/9-732/v1	20 Jul 20	{ "type": "Research Article", "title": "Investigating a novel multiplex proteomics technology for detection of changes in serum protein concentrations that may correlate to tumor burden", "authors": [ "Annie He Ren", "Ioannis Prassas", "Antoninus Soosaipillai", "Stephanie Jarvi", "Steven Gallinger", "Vathany Kulasingam", "Eleftherios P. Diamandis", "Annie He Ren", "Ioannis Prassas", "Antoninus Soosaipillai", "Stephanie Jarvi", "Steven Gallinger", "Vathany Kulasingam" ], "abstract": "Background: To account for cancer heterogeneity, we previously introduced the concept of “personalized” tumor markers, which are biomarkers that are informative in subsets of patients or even a single patient. Recent developments in various multiplex protein technologies create excitement for the discovery of markers of tumor burden in individual patients, but the reliability of the technologies remains to be tested for this purpose. Here, we sought to explore the potential of a novel proteomics platform, which utilizes a multiplexed antibody microarray, to detect changes in serum protein concentration that may correlate to tumor burden in pancreatic cancer. Methods: We applied the Quantibody® Human Kiloplex Array to simultaneously measure 1,000 proteins in sera obtained pre- and post-surgically from five pancreatic cancer patients. We expected that proteins which decreased post-surgery may correlate to tumor burden. Sera from two healthy individuals, split into two aliquots each, were used as controls. To validate the multiplexed results, we used single-target ELISA assays to measure the proteins with the largest serum concentration changes after surgery in sera collected pre- and post-surgically from the previous five patients and 10 additional patients. Results: The multiplexed array revealed nine proteins with more than two-fold post-surgical decrease in at least two of five patients. However, validation using single ELISAs showed that only two proteins tested displayed more than two-fold post-surgical decrease in one of the five original patients. In the independent cohort, six of the proteins tested showed at least a two-fold decrease post-surgery in at least one patient. Conclusions: Our study found that the Quantibody® Human Kiloplex Array results could not be reliably replicated with individual ELISA assays and most hits would likely represent false positives if applied to biomarker discovery. These findings suggest that data from novel, high-throughput proteomic platforms need stringent validation to avoid false discoveries.", "keywords": [ "proteomics", "ELISA", "multiplex", "immunoassay", "pancreatic cancer", "protein technologies" ], "content": "Introduction\n\nCancer manifests through alterations in the expression and interaction of proteins, which taken together gives rise to hallmarks such as unchecked cellular proliferation and metastasis1. Many studies have thus tried to dissect the cancer proteome, with the long-standing goal of finding biomarkers that predict disease status. However, there has been limited success in bringing new cancer protein biomarkers to the clinics2,3, partly due to limitations of the popular and robust technique used for proteomics - mass spectrometry (MS). Despite vast progress in MS-based methods over the past decade, the dynamic range of assays, especially in complex mixtures such as serum, may still lack the sensitivity to detect rare tumor-related proteins in biological fluids4,5. When it comes to sensitivity, there is perhaps no better tool than the enzyme-linked immunosorbent assay (ELISA). The technique however is restricted in multiplexing power due to limited availability of antibodies and cross reactivity. Recently, innovative ELISA-based microarrays overcame this problem by using nanofluidics to bridge multiplexing with unforeseen sensitivity, and as a result can reasonably measure thousands of proteins simultaneously6–9. We can now leverage such high throughput and resolution to study the cancer proteome, even in single patients.\n\nProtein biomarker research has long focused on a one-size-fits-all approach, where the utility of biomarkers is largely based on how well they perform in the majority of patients of a cancer type. However, the existing mindset surrounding biomarker discovery has recently been challenged by an increasing knowledge on a vastly heterogeneous cancer landscape. With more and more genomics studies showing that tumor heterogeneity exists between patients, within metastatic and primary sites and even within the primary tumor10–13, it has become evident that past research may have overlooked the array of distinct tumor markers that may exist in each patient. Searches for new cancer biomarkers that work for most patients may thus be in vain, and a shift to profiling tumor-related proteins in individuals is warranted. In the light of this, our study seeks to test a novel concept, which we had previously introduced and coined as “rare” or “personalized” tumor markers14. These biomarkers may not be very informative for the majority of patients of a cancer type, but may nevertheless be highly precise and sensitive at predicting tumor response to therapy and relapse in a small subset (10–15%) of patients, or even in a single patient14. In the future, a comprehensive panel of such personalized tumor markers would allow for rapid and high throughput screening of blood-based samples using clinically trusted immunoassays, in order to identify the most informative biomarkers tailored to monitor therapeutic response and relapse in each patient14,15. This notion is analogous to the routinely used molecular approach, for which whole genome sequencing is used to identify actionable mutations for individual patients for therapy selection. In an age of precision medicine, our vision holds remarkable potential for advancing individualized cancer treatment by personalizing tumor surveillance in each patient to determine the optimal trigger points for switching therapy or initiating second-line treatment – especially in the numerous cancer types where traditional biomarkers fall short.\n\nHere, we aim to take the first step toward examining the promise of new and exciting multiplexed protein technologies for identifying changes in serum proteins that could correspond to tumor burden in individual patients, using pancreatic cancer as a model. Pancreatic cancer is the fourth-leading cause of cancer deaths, with pancreatic ductal adenocarcinoma (PDAC) being the most lethal and common form (making up 90% of all cases)16,17. CA 19-9 is currently used for monitoring treatment efficacy, but it cannot detect PDAC early and is not expressed in patients who lack the Lewis antigen (about 10% of the Caucasian population)18,19. CA 19-9 can also be elevated in many disorders such as other gastrointestinal cancers and pancreatitis18,19. With an improved selection of new chemotherapeutics and immunotherapies on the horizon17,20, it is more critical than ever to assess which patient populations would benefit from initiating a new therapy, and when personalized tumor markers which closely monitor therapeutic efficacy and relapse could aid in guiding future treatment strategies and improve survival outcome on a patient-to-patient basis.\n\nIn this study, we used the high-throughput, multiplexed immunoassay, Quantibody® Human Kiloplex Array, to concurrently quantify 1,000 inflammation-related proteins (including CA 19-9) in the serum of five PDAC patients obtained pre- and post-tumor resection (n=10). As crosstalk between inflammation-related molecules is a key component of tumorigenesis21–23, we theorized that proteins which drastically drop in level following optimal tumor debulking surgery may be correlated to the change in tumor burden in the patients. These proteins were then quantified in a larger cohort of serum collected from 15 PDAC patients pre- and post-surgery (n=30) using commercially available ELISAs, in order to validate the reliability, accuracy, and reproducibility of the multiplexed array results. Our findings provide preliminary insights into the utility and challenges of applying emergent multiplexed proteomics instruments in identifying changes in serum protein levels in cancer.\n\n\nMethods\n\nSerum samples from PDAC patients were retrospectively obtained from the University Health Network BioBank with approval by the Research Ethics Board of University Health Network, Toronto, Canada (Approval number 10-0591). Informed consent forms outlining the serum collection process and research study were signed by patients prior to sample collection. Samples were stored at -80°C prior to analysis. Sera from five PDAC patients collected within one week before surgery and at four weeks after surgery were analyzed (n=10). One serum sample from a normal male and one from a normal female, each split into two identical aliquots (n=4), were used as controls.\n\nSerum samples from another 10 PDAC patients collected within one week pre-surgery and at four weeks post-surgery, along with the previous cohort, were tested for validation (n=30). Sera from a normal male and female (n=2) were used as controls.\n\nThis multi-analyte immunoassay offered by RayBiotech uses nanotechnology to combine 25 nonoverlapping arrays, essentially performing 1,000 sandwich ELISAs simultaneously. The microfluidics system boasts absolute quantification with good precision (intra-assay CV = 7–10%; inter-assay CV = 10–15%) and sensitivity (pg/mL). The biological relevance of the 1,000 molecules tested (available online at https://www.raybiotech.com/human-kiloplex/) is mostly inflammation-related, including cytokines, tumor markers, and transcription factors among others.\n\nIn each array, standards with predetermined concentrations were assayed alongside the samples to provide a standard curve. The samples were evaluated in technical quadruplicates, and the disease and control status were undisclosed to the technical personnel. Each sample was diluted two-fold to a final volume of 3 mL for use in all 25 arrays. The workflow, described in the manufacturer’s manual, is akin to that of a sandwich ELISA. In brief, capture antibody was bound to the glass surface. After blocking and sample incubation, nonspecific binding was washed away, and a biotin-labeled detection antibody was then added. Next, streptavidin-conjugated Cy3 equivalent dye was added, and the resulting fluorescent signals were read via a microarray laser scanner (GenePix 4200A, Molecular Dynamics, Sunnyvale, CA, USA). Q-Analyzer software (Raybiotech) was used to compute an absolute quantification by calculating the average of the quadruplicate values, and accounting for intra- and inter-slide normalization.\n\nFor all ELISAs, PDAC sera were tested in singleton due to limited sample volume, while standards were tested in duplicate. DuoSet IC ELISA kits (R & D Systems, Minneapolis, MN, USA) were used to measure serum levels of CEACAM-1 [Catalogue number (Cat #) DY2244] and IL-17RA (DY177), according to the manufacturer’s protocol. In brief, the assay was performed at room temperature (RT) with three washes between steps. First, 96-well microtiter plates were coated with capture antibody (4 µg/mL) and incubated overnight. Plates were blocked for 1 h before samples (1:6 dilution for CEACAM-1; 1:3 for IL-17RA) and standards were loaded and incubated for 2 h. Biotinylated detection antibody (100 ng/mL) was added and incubated for 2 h. Streptavidin-HRP was then loaded and incubated for 20 min in the dark. Enhanced K-Blue® TMB substrate (Neogen, Lexington, KY, USA) was next added, followed by 20 min incubation in the dark. Finally, the fluorescent signal was visualized by adding 2N H2SO4 and then measuring with the Wallac EnVision 2103 Multilabel Reader (Perkin Elmer, Waltham, MA, USA).\n\nThe DuoSet IC ELISA kit was also used to quantify PD-L2 (Cat # DY1224) serum levels according to manufacturer’s instructions with the following changes. Plates were not blocked. Samples (1:6 dilution) and standards were prepared in 6% BSA at 50 μl/well. Then, a 1:1 mixture of Assay Buffer A (60 g/L BSA, 37 g KCl, 25 ml/L normal mouse serum, 100 ml/L normal goat serum and 10 g/L bovine IgG in 50 mM Tris, 0.005% (v/v) Tween-20, pH 7.8) and 6% BSA was added at 50 μl/well before a 2 h incubation. Detection antibody was prepared in Assay Buffer B, where buffer content was the same as Assay Buffer A but minus KCl.\n\nSerum levels of DSCAM (Cat # ELH-DSCAM-1), GATA-4 (ELH-GATA4-1), C1QTNF9 (ELH-C1QTNF9-1), CREG1 (ELH-CREG-1), and CRISP2 (ELH-CRISP2-1) were measured using RayBio® ELISA kits (RayBiotech) following the manufacturer’s protocol. In brief, the assay was performed at RT with four washes between the following steps. Pre-coated plates were blocked prior to loading samples (1:2 dilution) and standards, followed by a 2.5 h incubation. Biotinylated detection antibody was then loaded and incubated for 1 h. Next, streptavidin-HRP was added and incubated for 45 min. TMB substrate, followed by 30 min incubation in the dark, and 2N H2SO4 were added, and finally the fluorescence signal was read using the microplate reader.\n\nWe mined the Human Protein Atlas for the nine candidate proteins to study the mRNA and/or protein expression in pancreatic cancer. For each protein, we focused on examining the Cell Atlas for RNA expression in different cell types, as well as the Pathology Atlas which provides data on RNA and protein expression in different cancer types.\n\nStatistical analyses were performed using the GraphPad Prism software (version 4.02). Pearson correlation coefficients were computed to evaluate intra-assay and inter-individual variation. Shapiro-Wilks test was used to assess data normality. A two-tailed paired t-test was performed for proteins where data was normally distributed, while Wilcoxon test was used otherwise. P-values of less than 0.05 were considered to be statistically significant.\n\n\nResults\n\nWe first sought to assess the reliability of the Quantibody® Human Kiloplex Array using scatterplot analysis (Figure 1). Concentrations of the 1,000 proteins analyzed were log10 transformed to best visualize the large range of values. Intra-assay variability was assessed by testing two identical aliquots of one male and one female control sample (Figure 1A, B). The Pearson correlation coefficients (r) for intra-assay variability between the two aliquots of the male (Figure 1A) and female control sample (Figure 1B) were 0.862 (P<0.001) and 0.853 (P<0.001) respectively, signifying overall good reproducibility of this assay. The inter-individual variability in the healthy controls was also assessed. For each male and female control, we computed an average value for the two aliquots, and visualized the correlation between the average values through a scatterplot (Figure 1C). The Pearson correlation coefficient in this case was 0.821 (P<0.001), denoting minimal inter-individual variability.\n\nSince the Quantibody® array includes the classical PDAC biomarker, CA 19-9, we also compared the post-surgical fold change in serum CA 19-9 level reported by the clinic to those obtained from the Quantibody® array in two of the five patients, where clinical data was available (Figure 2A, B). Following tumor resection, patient 3 (P3) had a 5.7-fold decrease, while patient 4 (P4) saw a 4.7-fold decrease in CA 19-9 according to clinical assessment (Figure 2C). On the other hand, the Quantibody® array data displayed a post-surgical 0.8- and 4.7-fold decrease in CA 19-9, in P3 and P4, respectively (Figure 2C).\n\n(A) Strong intra-assay correlation between the two identical male control samples (aliquot 1 vs. aliquot 2) that were assayed in a blinded fashion (P<0.001). Similarly, (B) shows strong correlation between the two identical female control samples (P<0.001). (C) Strong correlation between the average value of the two male control samples and that of the two female control samples (P<0.001). Pearson correlation coefficient (r) was used for analysis.\n\n(A) Clinical value of CA 19-9 before and after surgery in two PDAC patients. (B) CA 19-9 values obtained from the Quantibody® multiplexed array (Raybiotech) in the same patients. (C) The fold decrease seen in the two PDAC patients according to clinical and multiplexed data.\n\nRaw data for each figure, in addition to Quantibody® output data, are available as Underlying data24.\n\nFor the 1,000 proteins tested by the Quantibody® Kiloplex Array, we calculated the fold change in serum concentration from the pre-surgical to post-surgical value for each patient. Post-surgical values that were below the limit of detection were normalized to equal the value of limit of detection. We found nine proteins that were decreased by at least two-fold in at least two of the five patients (Figure 3). Three proteins (GATA-4, CREG, and PD-L2) showed a drastic drop to undetectable levels after surgery in patients who initially expressed them before surgery (Figure 3A–C). Serum levels of CEACAM-1, PON1, and C1qTNF9 decreased by at least five-fold in patients who highly expressed the protein before surgery (Figure 3D–F). Finally, DSCAM, IL-17RA and CRISP-2 levels dropped by at least two-fold in patients who showed high levels before surgery (Figure 3G–I).\n\nSubsequently, we performed a group analysis by comparing the collective patient samples obtained pre-surgically (n=5) to samples obtained post-surgically (n=5) for the nine proteins (Figure 4). Out of the nine proteins selected from the multiplex assay results, only IL-17RA levels dropped significantly post-surgery when comparing the patient cohort as a whole (P=0.01, paired t-test) (Figure 4I).\n\nBar graphs depict normalized serum level of proteins in five PDAC patients (P1-P5), before and after surgery, and in two controls (C1 and C2). Red dashed line depicts limit of detection. Error bars represent 95% confidence intervals based on the technical quadruplicate values. * Post-surgical decrease of at least two-fold. ** Post-surgical decrease of at least five-fold. (A–C) Levels of CREG, GATA-4 and PD-L2 dropped to undetectable levels in patients who expressed the protein prior to surgery. (D–F) PON1, C1qTNF9 and CEACAM-1 levels decreased by at least five-fold in patients who highly expressed the protein pre-surgery. (G–I) DSCAM, IL-17RA and CRISP-2 levels decreased by at least two-fold in at least two of five patients.\n\nWilcoxon or paired T-test was used to collectively compare paired patient samples obtained pre-surgery (n=5) and post-surgery (n=5). The healthy group consists of all aliquots from the controls (n=4). Only IL-17RA levels were significantly reduced post-surgery (P<0.01, paired T-test).\n\nWe aimed to perform single-target ELISA assays to quantitatively measure the expression levels of the nine proteins that was selected from the multiplex assay results in a larger cohort of PDAC patients. Sera collected pre- and post-surgically from an independent cohort of 10 PDAC patients were used (n=20). In order to compare data between the ELISA and Quantibody® array, we included sera from the original five PDAC patients (n=10, pre- and post-surgery). One of the proteins, PON1, was not tested as there was no reliable ELISA assay for quantifying in serum. Amongst the eight proteins assessed in the original patients (P1-P5), only CEACAM-1 and CRISP-2 showed a post-surgical fold decrease that aligned with the Quantibody® array data (Figure 5). Specifically, P5 displayed a two-fold decrease in CEACAM-1, while P1 showed a drop in CRISP-2 to undetectable levels following tumor resection (Figure 5D, F). In the independent cohort (P6-15), at least one patient showed a greater than two-fold decrease post-surgery in six proteins (CREG, PD-L2, CEACAM-1, C1qTNF9, DSCAM, and IL-17RA) (Figure 5). However, this observed fold change requires further validation.\n\nBar graphs depict serum level of the proteins in fifteen PDAC patients (P1-P15), before and after surgery, and in two controls (C1 and C2). Red dashed line depicts limit of detection. * Post-surgical decrease of at least two-fold. ** Protein level dropped to undetectable level following surgery.\n\n\nDiscussion\n\nCancer is not a homogeneous disease, but rather displays genetic and phenotypic variations between not only patients but even within a single tumor25,26. Considering the inter- and intra-heterogeneity of tumors, we previously introduced the concept of “rare” or “personalized” tumor markers, which are biomarkers that predict tumor load in small subsets of patients or even in a single patient14. With the advent of multiplexed antibody-based microarrays, we can potentially search for such rare tumor markers in individual patients at never-before-seen depth, in a small volume (less than 1 mL) of blood-based sample. Here, we wanted to explore the feasibility of using a multiplexed proteomics platform to quantify serological proteins in pancreatic cancer that may be present at low concentrations (in the pg/mL range) and may change in serum level in correlation to changes in tumor burden after surgery. Pancreatic cancer, particularly the subtype PDAC, currently lacks appropriate biomarkers for detecting tumor presence early and sensitively16,17,19. Patients may benefit from personalized biomarkers that sensitively monitor therapeutic response and recurrence in order to optimize treatment plans and improve patient outcome. With the emergence of various multiplex proteomics technologies, there is eagerness to identify personalized tumor markers in the serum of cancer patients. However, the first step to this long journey would be to test the feasibility and reliability of the new technology for accurately detecting changes in serum level of proteins in cancer patients in relation to a distinct change in tumor burden, such as the removal of the tumor through surgery. In this study, we applied the Quantibody® Human Kiloplex Array to determine the expression profiles of 1,000 proteins in serum of PDAC patients collected pre- and post-surgically. We postulate that proteins which decrease significantly in level after tumor resection may correspond to tumor burden in the patient, depending on the reliability and accuracy of the new multiplex technology at hand.\n\nWe first evaluated the reliability of the Quantibody® Kiloplex Array by examining the intra-assay variability. Samples from one healthy male and female, each split into two aliquots (n=4), were included as technical and biological controls. Although the overall correlation between the aliquots of male and female control samples was strong, many proteins showed zero value for one aliquot and high value for the other (Figure 1A, B). This suggests that some proteins in this assay exhibited high technical variation even after normalization for intra- and inter-slide differences. In fact, the technical quadruplicate values obtained prior to normalization unveiled high inconsistency with apparent outliers for many proteins (data not shown, available as Underlying data)24. A study that used a similar Quantibody® array to concurrently measure 174 cytokines in serum of ovarian cancer patients had seen very low variability, with the Pearson correlation coefficients for intra-slide and inter-slide reproducibility being 0.923 (P<0.001) and 0.899 (P<0.001) respectively27. As the first to report the intra-assay variability of the Quantibody® Kiloplex array, our findings suggest that this technology may lose reproducibility in some targets when scaled to 1,000 proteins. Additionally, we analyzed the inter-individual variability between the male and female control samples. Once again, the overall correlation was strong, but some proteins showed large variation in concentration between the two individuals (Figure 1C). Since we are interested in finding tumor markers in single patients, any inter-individual variation is unlikely to affect the analysis. Finally, we compared the CA 19-9 values reported by the clinic with those obtained from the Quantibody® Kiloplex array for two patients. Although both methods showed distinct post-surgical decreases in CA 19-9 in P4 (Figure 2B, C), P3 did not show a decrease in CA 19-9 post-surgery compared to before surgery according to the Quantibody® data (Figure 2A), which was not in concordance with the clinical data (Figure 2C). This discrepancy may be due to either technical variation, day-to-day variation as the sera were taken at different time points, or a combination of both factors.\n\nDelving into the selection of proteins with the largest change in serum level after surgery, we mainly took into account the fold change seen in protein level post-surgery compared to pre-surgery in each patient. This approach generated nine proteins whose serum concentration decreased by at least two-fold in at least two of the five patients (Figure 3). Of the nine proteins that showed decline in serum level after surgery, GATA-4, PON1, CEACAM-1 and DSCAM have already been studied in pancreatic cancer and were found to be associated with immune suppression, aggressive phenotype, cancer progression and/or metastasis28–31. Remarkably, polymorphisms in the PON1 gene have been associated with increased PON1 expression in a subset of pancreatic cancer patients30. Our other protein that showed decrease in serum level after surgery, CREG (gastric cancer), IL-17RA (gastric/colorectal cancer), and PD-L2 (breast/liver cancer) have been associated with other cancers32–35. Furthermore, overexpression of C1qTNF9 has been proposed to activate aberrant AKT and MAPK signaling pathways in cancer cells33. On the other hand, CRISP2 is mainly expressed in the testes and has not been studied in cancer36. Additional mining of the Human Protein Atlas for the nine proteins further confirmed overexpression at the mRNA and/or protein level in a proportion of pancreatic cancer patients.\n\nTo validate the concentration changes we observed in the proteins using the multiplex assay, we used commercially available and verified single target ELISAs to accurately measure each protein in the sera of 15 PDAC patients taken before and after surgery. For comparison with the Quantibody® Kiloplex Array data, we included sera from the same five patients who were previously screened using the multiplexed assay. Of the eight proteins tested, a post-surgical fold decrease of at least two-fold, which would align with the multiplexed array results, was only observed in CEACAM-1 and CRISP2 in one patient each (Figure 5D, F). This unfavorable outcome suggests that the multiplexed platform may suffer from high false discovery rate, where the observed fold change may reflect technical inconsistency as discussed in our evaluation of the intra-assay reproducibility. To date, seven studies have used similar Quantibody® arrays at smaller scales to measure a range of six to 320 proteins simultaneously in the serum of various diseases37–40. Notably, Green et al. used a Quantibody® array to concurrently examine the level of 10 cytokines in the serum of head and neck squamous cell carcinoma patients (n=101) taken before and after tumor treatment37. The study found six cytokines that were significantly reduced post-treatment, however the results were neither validated via a different approach nor in an independent cohort37. Only one other study published to date has used the Quantibody® Kiloplex array, where Platonov et al. leveraged it to delineate the protein pathways resulting from KISS1 gene expression in human breast cancer cell lines41. Although they identified numerous secreted proteins that were disregulated in conditioned media upon KISS1 activation, the results were not validated using a secondary analytical method41. All in all, we are the first to employ the Quantibody® Kiloplex array to simultaneously measure 1,000 proteins in sera of cancer patients. Moreover, we further attempted to validate the array results using a trusted independent approach. Our efforts suggest that the use of new multiplexed proteomics platforms, such as the Quantibody® Kiloplex array, in biomarker discovery may still be nascent and pose significant questions in terms of precision and reproducibility. Nevertheless, a handful of revolutionary tools remains to be explored. Dionne et al. have aimed to compare the Quantibody® array with the magnetic bead-based MILLIPLEX® multiplex assay to simultaneously detect the levels of 40 cytokines in extracted tear samples, remarking that each method was superior at detecting a specific subset of cytokines42. Furthermore, two studies have separately employed multi-analyte technologies (Proseek® proximity extension assay versus the ELISA-based Simple Plex™ assay) to assemble a panel of biomarkers for the early detection of ovarian cancer43,44. Data from the Proseek® assay for CA125 values (known biomarker for ovarian cancer) had displayed a strong correlation with those obtained from clinical assays43. The study showed promise for multiplexed tools to replicate results obtained from established clinical assays, and to discover new candidate biomarkers in cancer43.\n\n\nConclusion\n\nLimitations in MS-based techniques (which has been traditionally been used for proteomics) has instigated an explosion of novel, multiplexed proteomics technologies, each promising high precision and resolution while only using miniscule amounts of blood-based samples (<1 mL). However, the jury is still out for whether novel multiplexed technologies are accurate and sensitive enough for biomarker discovery in complex biological fluids. We observed a high rate of potential false positives using the Quantibody® Kiloplex array for identifying changes in protein concentration in the serum of pancreatic cancer patients. Our findings suggest that while the influx of pioneering proteomics tools may bring excitement around a new era of personalized biomarker discovery, it is imperative to corroborate results and findings through independent trusted techniques in order to minimize false discoveries. Studies that fail to validate the candidate biomarkers resulting from multiplex proteomics platforms using independent approaches may be unknowingly presenting a high rate of false positives. Moving forward, our proposed emergent concept of developing a comprehensive panel of “rare” or “personalized tumor markers” seeks to challenge the existing mentality surrounding cancer biomarkers. Our study aimed to provide novel information for where proteomics and cancer biomarker research is going, and encourage future research on the feasibility of using pioneering proteomics platforms, outside of mass spectrometry, for personalized cancer biomarker discovery. With further exploration of novel proteomics platforms and their promise in biomarker research, we envision that unveiling a truly precise and sensitive technology can make personalized proteomics a reality – where thousands of proteins can be precisely quantified using a drop of serum in order to identify the most informative personalized tumor marker for an individual patient.\n\n\nData availability\n\nHarvard Dataverse: Investigating a novel multiplex proteomics technology for detection of changes in serum protein concentrations that may correlate to tumor burden. https://doi.org/10.7910/DVN/N9K3OM24.\n\nThis project contains the following underlying data:\n\nF1000R_Raw data_5.19.20 (XLSX). (Raw data for the present study, arranged by Figure.)\n\nMount Sanai Hospital Service Report (XLSX; 8 files). (Output files from Raybiotech ELISA assays; each file contains data for approximately 100 proteins.)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe thank Dr. Davor Brinc for discussions on determining the analytical parameters of the ELISA assays for PD-L2, CEACAM-1, and IL-17RA.\n\n\nReferences\n\nPaltridge JL, Belle L, Khew-Goodall Y: The secretome in cancer progression. Biochim Biophys Acta. 2013; 1834(11): 2233–2241. PubMed Abstract \| Publisher Full Text\n\nBelczacka I, Latosinska A, Metzger J, et al.: Proteomics biomarkers for solid tumors: Current status and future prospects. Mass Spectrom Rev. 2019; 38(1): 49–78. PubMed Abstract \| Publisher Full Text\n\nDiamandis EP: The failure of protein cancer biomarkers to reach the clinic: why, and what can be done to address the problem? BMC Med. 2012; 10: 87. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70939", "date": "10 Sep 2020", "name": "Georgios Pampalakis", "expertise": [ "Reviewer Expertise Clinical diagnosis", "biochemistry", "activity-based probes", "organophosphates", "proteases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well-established that cancer exhibits a high degree of heterogeneity. The authors recently introduced the concept of personalized tumor markers to study tumor burden in individual patients. Here they attempted to explore this idea using a new commercially available antibody microarray. This microarray is based on a nanotechnology-based microfluidic multiplex sandwich ELISA for 1000 protein analytes. This system was developed to overcome certain limitations of mass spectrometry applied in clinical diagnosis. The specific aim is to identify changes of proteins in the serum of individuals before and after surgery that can be used to monitor therapeutic response or relapse. These proteins may differ between different patients and could be specific for even a single patient. The microarray used was a commercially available platform and importantly the authors validated the findings with classical ELISA assays. The authors analyzed serum samples obtained pre- and post-surgery in 5 patients. They found that the commercially available assay exhibits good reproducibility but the results could not be fully replicated with classical ELISA indicating that future improvements in the microarray platform are necessary in order to use it in clinical diagnosis. The work is clearly presented, and the methods are sufficiently detailed. All conclusions are fully supported by the data. Some drawbacks of the study are presented by the authors. Overall the study is clear and important since it compares a commercial high-throughput assay for reproducibility and accuracy.\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": [ { "c_id": "5965", "date": "24 Sep 2020", "name": "Eleftherios P Diamandis", "role": "Author Response F1000Research Advisory Board Member", "response": "We would like to thank Dr Pampalakis for the constructive feedback." } ] }, { "id": "70938", "date": "22 Oct 2020", "name": "Anna E Lokshin", "expertise": [ "Reviewer Expertise Cancer biomarkers", "multiplexed immunoassay", "pancreatic cancer" ], "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 study, the authors explored a highly multiplexed antibody microarray platform, Quantibody Human Kiloplex array to detect changes in serum protein concentrations in pancreatic cancer patients before and after surgery to identify biomarkers that correlate that may correlate with tumor burden. The study is important in that it found that the Quantibody® Human Kiloplex Array results could not be reliably replicated with individual ELISA assays and most hits would likely represent false positives if applied to biomarker discovery. However, to directly compare two immunoassays, the same antibodies should be used. Additionally, the conclusions are based on too few cases.\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? Partly\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?\nPartly\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? Partly", "responses": [ { "c_id": "6104", "date": "24 Nov 2020", "name": "Eleftherios P Diamandis", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Dr Lokshin, Thank you for your constructive feedback. We found your insights helpful for improving the clarity of the manuscript. We have included in the revised manuscript information on the antibodies. The antibodies used in 5 single target ELISAs (DSCAM, GATA-4, C1QTNF9, CREG1, and CRISP2) were obtained from RayBiotech and are the same antibodies used in the Quantibody Kiloplex Array. The antibodies used for CEACAM-1, IL-17RA, and PD-L2 in the Kiloplex Array are not reported by Raybiotech, so we do not know whether the antibodies in the 3 DuoSet IC ELISA kits from R & D Systems were the same. However, our findings show that the Kiloplex Array data did not show concordance with results obtained from the verified, single-target ELISAs, even in the 5 ELISAs that used the same antibodies. We also provided more information on the validation of the single-target ELISAs in human serum. We agree that the previous version of the manuscript only partly supported the conclusions drawn. Therefore, in addition to adding information on the ELISAs, we edited the conclusion to stress that our findings are in a small sample size and do not conclusively determine the technical reliability of the multiplex technology. We cannot decisively state whether it generates false hits for biomarker discovery. Thus, we only state that we did not find concordance in the Kiloplex Array results compared to single-target ELISAs, which could mean that studies that use multiplex proteomics technologies may benefit from validating their candidates using independent methods. We hope our response address your concerns. Thank you for taking the time to review our work." } ] } ]	1	https://f1000research.com/articles/9-732
https://f1000research.com/articles/9-1143/v1	16 Sep 20	{ "type": "Case Report", "title": "Case Report: Spontaneous perforation of a bicornuate uterus with concomitant sarcoma", "authors": [ "Soobin Yim", "Inji Yeo", "Myunghwa Lee", "Kyu-Sang Kyeong", "Hye-yon Cho", "Jung Bae Kang", "Min Sun Kyung", "Soobin Yim", "Inji Yeo", "Myunghwa Lee", "Kyu-Sang Kyeong", "Hye-yon Cho", "Jung Bae Kang" ], "abstract": "A 47-year-old nulliparous, virginal woman presented to the emergency department with acute abdominal pain. Emergency pelvic ultrasonography and abdominal CT were taken, which showed a significant amount of hemoperitoneum and a bicornuate uterus with about 18cm x 10cm mass on the left uterus. Since the mass had increased vascularity and irregular margins, we thought that the mass could be a uterine sarcoma. Pelvic MRI and PET/CT were taken additionally for oncologic evaluation before surgery. Intraoperative findings showed a ruptured bicornuate uterus with a large mass within the left uterine horn. Total abdominal hysterectomy with bilateral salpingo-oophorectomy was performed. Pathologic analysis confirmed an undifferentiated uterine sarcoma. Therefore, we report a case of spontaneous rupture of bicornuate uterus with concomitant sarcoma occurred in a 47-year-old woman.", "keywords": [ "Bicornuate uterus", "Uterine rupture", "Uterine sarcoma" ], "content": "Introduction\n\nSpontaneous uterine rupture occurs most commonly with labor and delivery1. When it does occur, the most common cause of rupture is dehiscence of a previous transmyometrial surgical incision, such as that from a cesarean section scar2. Spontaneous rupture of a uterus without a previous surgical scar is very uncommon and significantly less is known2.\n\nBicornuate uterus is a common type of congenital uterine malformation; it takes the form of a double uterus with a single cervix and vagina3. When a zygote is implanted in a horn of a bicornuate uterus, it is unable to expand as a normal uterus does to accommodate a growing fetus4. The walls of the anomalous uterus tend to become abnormally thin as pregnancies advance. Implantation of the zygote in a rudimentary horn is considered an independent risk factor for uterine rupture5.\n\nUterine sarcoma is a rare and aggressive soft tissue neoplasm in women of all ages. It usually presents with abdominal or pelvic pain, vaginal bleeding; sarcoma does not typically cause uterine rupture and hemoperitoneum. To our knowledge, there have been only five cases reported in the literature that describe a uterine sarcoma presenting with rupture and induced hemoperitoneum6.\n\nHerein, we report a case from diagnosis to surgical treatment of a 47-year-old woman with bicornuate uterus who had no previous history of spontaneous uterine rupture or uterine sarcoma. Preoperative pelvic ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET)/CT were performed for diagnosis. We hypothesize that as her uterine sarcoma advanced, our patient experienced uterine rupture as a result of her congenitally malformed, bicornuate uterus.\n\n\nCase report\n\nA 47-year-old nulliparous, virginal woman presented to the emergency department with a 2-day fever and acute abdominal pain. She also complained of a 2-month history of foul-smelling vaginal bleeding. She had pyrexia, with a temperature of 38°C (100.4°F), and marked tenderness of the whole abdomen. She has a past medical history significant for breast cancer, which was treated with six cycles of CAF (cyclophosphamide, [doxorubicin] Adriamycin, fluorouracil), adjuvant radiotherapy, and tamoxifen for 4 years; the doses are uncertain since she had taken those therapies in the other institutes. She has not had a gynecological evaluation for over 8 years since the end of her breast cancer treatment, and she had discontinued her tamoxifen.\n\nLaboratory investigation demonstrated a hemoglobin level of 12.3 g/dL (normal range, 12–16 g/dL), a white blood cell count of 12,600/mm3 (normal range, 4,000–10,000/mm3) and a C-reactive protein of 157.9 mg/L (normal range, 0.1–5.0 mg/L). Mild elevation of her liver enzymes was observed, with a total bilirubin of 2.2 mg/dL (normal range, 0.2–1.4 mg/dL) and a direct bilirubin of 0.7 mg/dL (normal range, 0.0–0.5 mg/dL).\n\nA dynamic CT scan of the liver was performed to evaluate for biliary infection as this patient had been known to have a history of gallbladder stones for years along with complaints of upper abdominal pain. The CT scan revealed a 16-cm uterine mass and increased free fluid in the pelvic cavity, including both paracolic gutters, the perihepatic space, and the perisplenic space. Consequently, she was referred to our obstetrics and gynecology department.\n\nTransrectal ultrasonography showed a bicornuate uterus and an 18.6- × 9.1-cm mass with heterogenous echogenicity within the left uterus. The mass appeared to extend through the cervix into the vaginal cavity. Its irregular-shaped margins and increased blood flow suggested the possibility of malignancy (Figure 1). Because her vital signs were stable (aside from fever), the patient received an oncological evaluation and antibiotic treatment before surgery. Since F-18 FDG were ordered and needed time to arrive, PET/CT were planned two days later, and the surgery was planned for the next day. Antibiotics (piperacillin 4g, tazobactam 0.5g, metronidazole 0.5g) were injected intravenous every 8 hours, and administered for about 48 hours until just before surgery.\n\nUltrasonography shows a 1.5-cm-thick endometrial layer in the right uterus (short arrow) and up to 2.69 cm in the left uterus (long arrow). The mass was connected to the left uterus and appeared to be extending into the vaginal cavity. Internal blood flow was increased.\n\nPelvic MRI was performed on the day after the patient’s initial presentation, revealed underlying uterus didelphys with an approximately 15- × 9- × 17-cm mass with mixed signal intensity in the lower abdominal area (Figure 2) and an approximately 6- × 2.7- × 3-cm mass of the left cervix and lower uterine body on T2-weighted imaging. These MRI findings suggested the possibility of hemoperitoneum or cancer peritonei due to rupture of (1) endometrial cancer, (2) uterine sarcoma, or (3) large myoma with degeneration.\n\nThis scan shows an approximately 15- × 9- × 17-cm mass with mixed high and low signal intensity and irregular margins in the lower abdomen on coronal view.\n\nA whole-body PET/CT scan was ultimately performed (two days after presentation) and showed an intensely hypermetabolic subserosal mass with accompanying hemorrhage and possible rupture; further, hypermetabolic peritoneal nodules and peritoneal infiltration with ascites were observed (Figure 3).\n\nAn approximately 13-cm intensely hypermetabolic hemorrhagic mass is noted in the pelvic cavity, suggestive of a subserosal uterine mass possibly connected with a cervical lesion. There are regions of hypermetabolic peritoneal thickening and nodules with a small amount ascites.\n\nThe patient underwent diagnostic laparotomy three days after presentation. Surgical exploration revealed a ruptured bicornuate uterus with a large mass within the left uterine horn (Figure 4). The mesentery of the small bowel and appendix were partially adhered to the uterine mass. Total abdominal hysterectomy with bilateral salpingo-oophorectomy was performed, and invasive cancer implants within the bowel were removed. Estimated surgical blood loss was approximately 1500 mL.\n\nA significant amount of hemoperitoneum was observed.\n\nPathological analysis confirmed an undifferentiated uterine sarcoma with tumor size up to 23 × 13 × 6 cm. Because a tumor in her left fallopian tube and one of her bowel mass implants tested positive for cancer, she was diagnosed with FIGO Stage IIIA (from the International Federation of Gynecology and Obstetrics cancer staging system)7.\n\nPostoperatively, the patient was treated with a combination of etoposide (150 mg/m2 for 3 days), ifospamide (1.5 g/m2 for 3 days), and cisplatin (70mg/m2 for 1 day) once every 3 weeks. Chest and abdomen CT were performed for follow up 2 weeks after. There were no sign of cancer recurrence in the chest and abdominal CT.\n\n\nDiscussion\n\nSpontaneous uterine rupture presents most commonly with labor and delivery1. The most common cause of uterine rupture is dehiscence of a previous transmyometrial surgical incision, typically the result of a cesarean section2. However, spontaneous rupture of a uterus without a surgical scar is very uncommon and significantly less well known2. Other significant risk factors for uterine rupture include oxytocin-induced labor, antepartum fetal death, and first trimester miscarriages8. There have been several reported cases of uterine rupture in Müllerian anomalies where the zygote implants within a rudimentary horn. Higher rates of uterine rupture have been reported in patients with Müllerian duct abnormalities who elect to undergo a trial of labor after cesarean delivery when compared with patients without Müllerian duct abnormalities, suggesting that these anomalies may be an independent risk factor for uterine rupture5.\n\nThe uterus is formed by the fusion of two paramesonephric ducts (Müllerian ducts) during embryogenesis. The separate ducts fuse into a single uterine body between the sixth and eighth weeks of gestation9. Failure of complete fusion of the Müllerian ducts leads to various types of malformations of the female genital tract10. The incidence of uterine anomalies is 0.06% to 38% in the general population11. Bicornuate uterus is a common type of uterine malformation, taking the form of a double uterus with a single cervix and vagina. Each uterus has a single horn linked to an ipsilateral fallopian tube that faces its ovary3. The bicornuate uterus often has unusually thick and strong round ligaments along with a thick vesicorectal fold running between them12. This fibrous band in the form of a rectovesical ligament has a restrictive effect on the expansion of the pregnant horn of a bicornuate uterus, thereby weakening the medial aspect of the horn13. Rupture of a bicornuate uterus during a growing pregnancy occurs due to inability of the malformed uterus to expand as a normal uterus would4. The walls of the anomalous uteri tend to become abnormally thin as the pregnancy advances. Rudimentary horn rupture is likely to occur in the late first trimester or even in the second trimester3.\n\nSome cases are associated with leiomyoma of the uterus. Potential etiologies for spontaneous rupture of leiomyomas are rupture of the superficial overlying vessels, degeneration or sarcomatous changes of uterine leiomyomas. Uterine sarcoma is a rare neoplasm; therefore, uterine rupture as a result of sarcoma is even more unusual. Most uterine sarcomas are diagnosed after surgery by postoperative biopsy14.\n\nUterine rupture with resulting hemoperitoneum has been described particularly for leiomyomas, and approximately 100 cases have been reported. Hemoperitoneum is the result of spontaneous rupture of the superficial veins overlying leiomyomas15 or the result of avulsion of a pedunculated myoma by trauma16,17. The most likely mechanism behind spontaneous rupture of a leiomyosarcoma is tumor necrosis. A patient with uterine rupture typically presents in hypovolemic shock with severe abdominal pain that requires emergency care. Because a patient's condition can deteriorate rapidly after uterine rupture, most patients will need immediate blood and fluid replacement therapy and an exploratory laparotomy to be done without a clear preoperative diagnosis before surgery14. There are very few cases described in the literature of uterine sarcoma resulting in a life-threatening clinical scenario such as uterine rupture or hypovolemic shock6.\n\nWe formulated two hypotheses regarding the etiology of uterine rupture in our patient: (1) the sarcoma itself had necrotic changes that led to uterine rupture and (2) the patient had an underlying bicornuate uterus, which exerted a restrictive effect on the ability of the myometrium to expand in the setting of a growing sarcoma.\n\nPelvic ultrasonography or CT imaging is commonly used for preoperative diagnosis of uterine sarcoma. Ultrasonography can also be the initial examination measure performed urgently in the setting of acute pelvic symptoms. However, ultrasonography remains insufficient to differentiate between a remodeled myoma and a sarcoma based on morphologic criteria alone. As a result, color Doppler ultrasonography can be used to measure resistance indexes which reveal a significant difference between leiomyomas (resistance index 0.01–0.59) and sarcomas (resistance index 0.06–0.41)18\n\nCT imaging has good diagnostic value, because extravasation of contrast enhancement can be used to evaluate for active bleeding. Further, a diagnosis of hemoperitoneum can be made by assessing the Hounsfield unit (HU), a measure of radiodensity. The attenuation values of unclotted extravascular blood usually measure between 30 and 45 HU, whereas clotted blood is between 45 and 70 HU because of its high protein content19. However, a CT imaging is not sensitive enough to distinguish between a leiomyosarcoma and a necrotic leiomyoma20.\n\nPelvic MRI is a useful diagnostic tool for the detection and characterization of uterine sarcoma, as well as for an assessment of disease staging. MRI images of uterine sarcomas generally have irregular margins and are isointense or hypointense on T1-weighted imaging (compared to the signal from the myometrium) with possible hemorrhagic zones. In T2-weighted imaging, uterine sarcomas have an intermediate-to-high signal. In contrast, leiomyomas have regular margins and low-to-intermediate signals on both T1-weighted and T2-weighted imaging. Although the radiological findings of these lesions can overlap, characteristic features can help narrow the differential diagnosis and guide adequate treatment selection and follow-up. In addition to morphologic features, diffusion-weighted imaging seems to be a potentially useful tool in the characterization of large uterine lesions21.\n\nIn our patient case, it was uncertain whether the uterine rupture was due to endometrial cancer, uterine sarcoma, or degenerated myoma. The mass appeared to be extending through the cervix into the vaginal cavity, but vaginal examination and endometrial biopsy could not be performed because the patient is a virgin. The patient had stable vital signs other than pyrexia, and her pain was not severe. CT imaging revealed that she had a bicornuate uterus, therefore, we decided to perform an pelvic MRI and PET/CT scan.\n\nThe treatment of uterine sarcomas is surgical intervention. It includes a total hysterectomy with bilateral adnexectomy in the event of a tumor limited to the uterine body. The indication for adjuvant treatment remains a topic of debate22. The prognosis for uterine sarcomas remains poor. The probability of survival at 5 years is estimated at 30%, all stages combined23.\n\nThough uncommon, uterine rupture in the setting of a bicornuate uterus with concomitant sarcoma should be highlighted as a possible cause of hemoperitoneum when a woman presents with severe abdominal pain, particularly in the setting of a pelvic mass and uterine anomaly with intact ovaries detected on imaging. CT imaging is useful when ultrasonography shows no definite evidence of uterine rupture. We recommend the use of MRI imaging to help differentiate between leiomyoma and sarcoma before surgical intervention takes place.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWe received written informed consent from the patient for the use and publication of the patient’s data.", "appendix": "References\n\nGibbins KJ, Weber T, Holmgren CM, et al.: Maternal and fetal morbidity associated with uterine rupture of the unscarred uterus. Am J Obstet Gynecol. 2015; 213(3): 382 e1–382 e6. PubMed Abstract \| Publisher Full Text\n\nGhanshyam Verma P, Padmawar A, Gore S, et al.: Spontaneous rupture of an unscarred uterus in nongravid horn of bicornuate uterus. Indian Journal of Obstetrics and Gynecology Research. 2020; 7(1): 126–8. Publisher Full Text\n\nJayaprakash S, Muralidhar L, Sampathkumar G, et al.: Rupture of bicornuate uterus. BMJ Case Rep. 2011; 2011: bcr0820114633. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKore S, Pandole A, Akolekar R, et al.: Rupture of left horn of bicornuate uterus at twenty weeks of gestation. J Postgrad Med. 2000; 46(1): 39–40. PubMed Abstract\n\nRavasia DJ, Brain PH, Pollard JK: Incidence of uterine rupture among women with müllerian duct anomalies who attempt vaginal birth after cesarean delivery. Am J Obstet Gynecol. 1999; 181(4): 877–81. PubMed Abstract \| Publisher Full Text\n\nÖzcan J, Dülger Ö, Küpelioğlu L, et al.: Uterine sarcoma in a 14 year-old girl presenting with uterine rupture. Gynecol Oncol Rep. 2014; 10: 44–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMbatani N, Olawaiye AB, Prat J: Uterine sarcomas. Int J Gynaecol Obstet. 2018; 143 Suppl 2: 51–8. PubMed Abstract \| Publisher Full Text\n\nAl-Zirqi I, Daltveit AK, Forsen L, et al.: Risk factors for complete uterine rupture. Am J Obstet Gynecol. 2017; 216(2): 165 e1–e8. PubMed Abstract \| Publisher Full Text\n\nNg'ang'a N, Ratzersdorfer J, Abdelhak Y: Vaginal birth after two previous caesarean deliveries in a patient with uterus didelphys and an interuterine septal defect. BMJ Case Rep. 2017; 2017: bcr2016219149. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgu PU, Okaro JM, Mbagwu UK, et al.: Spontaneous rupture of gravid horn of bicornuate uterus at mid trimester--a case report. Niger J Med. 2012; 21(1): 106–7. PubMed Abstract\n\nChan YY, Jayaprakasan K, Zamora J, et al.: The prevalence of congenital uterine anomalies in unselected and high-risk populations: a systematic review. Hum Reprod Update. 2011; 17(6): 761–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhattachary TK, Sengupta P: Rudimentary Horn Pregnancy. Med J Armed Forces India. 2005; 61(4): 377–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNitzsche B, Dwiggins M, Catt S: Uterine rupture in a primigravid patient with an unscarred bicornuate uterus at term. Case Rep Womens Health. 2017; 15: 1–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim WH, Cohen SC, Lamaro VP: Intra-abdominal haemorrhage from uterine fibroids: a systematic review of the literature. BMC Surg. 2020; 20(1): 70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButtery BW: Spontaneous haemoperitoneum complicating uterine fibromyoma. Aust N Z J Obstet Gynaecol. 1972; 12(3): 210–3. PubMed Abstract \| Publisher Full Text\n\nSule AZ: Traumatic rupture of uterine fibroid: an uncommon cause of post traumatic haemoperitoneum. West Afr J Med. 2000; 19(2): 158–9. PubMed Abstract\n\nDrutman J, Fruechte DM: Hemoperitoneum due to traumatic avulsion of a pedunculated uterine leiomyoma. AJR Am J Roentgenol. 1992; 158(6): 1410. PubMed Abstract \| Publisher Full Text\n\nAviram R, Ochshorn Y, Markovitch O, et al.: Uterine sarcomas versus leiomyomas: gray-scale and Doppler sonographic findings. J Clin Ultrasound. 2005; 33(1): 10–13. PubMed Abstract \| Publisher Full Text\n\nLubner M, Menias C, Rucker C, et al.: Blood in the belly: CT findings of hemoperitoneum. Radiographics. 2007; 27(1): 109–25. PubMed Abstract \| Publisher Full Text\n\nMegibow AJ, Balthazar EJ, Hulnick DH, et al.: CT Evaluation of Gastrointestinal Leiomyomas and Leiomyosarcomas. AJR Am J Roentgenol. 1985; 144(4): 727–31. PubMed Abstract \| Publisher Full Text\n\nSantos P, Cunha TM: Uterine sarcomas: clinical presentation and MRI features. Diagn Interv Radiol. 2015; 21(1): 4–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanjeekal S, Chambers A, Fung MFK, et al.: Systemic therapy for advanced uterine sarcoma: a systematic review of the literature. Gynecol Oncol. 2005; 97(2): 624–37. PubMed Abstract \| Publisher Full Text\n\nGonzález Bosquet E, Martínez Palones JM, González Bosquet J, et al.: Uterine sarcoma: a clinicopathological study of 93 cases. Eur J Gynaecol Oncol. 1997; 18(3): 192–5. PubMed Abstract" }	[ { "id": "73429", "date": "22 Oct 2020", "name": "Jin-Sung Yuk", "expertise": [ "Reviewer Expertise Endocrinologic Gynecologist" ], "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 second paragraph on page 1: it would be helpful to first explain the relationship between the bicornuate uterus and the uterine rupture in the third sentence.\nFigure 4 on page 4: The anatomical structure of the photograph cannot be distinguished. I think it will be helpful to put text on the picture to distinguish it.\nThe first paragraph of the discussion: It is very similar to the first paragraph of the introduction. Variations such as changing the form of the sentence or describing the content in more detail are needed.\nThe 3rd paragraph o discussion: \"rupture of the superficial overlying vessels\" is the result, not the cause of rupture of leiomyoma.\nThe 4th paragraph on discussion: These sentences (\"Uterine rupture with ~ by trauma.\") fit the previous paragraph rather than this one. It would be nice to integrate with the previous paragraph and reduce the content.\nThe 4th paragraph on page 5: In the conclusion of the last paragraph, it was argued that CT is helpful when sono cannot discriminate. Write something to support this. For example, \"CT is superior than sono in circumstances that ~~~\".\nThe 7th paragraph on page 5: This sentence (\"In our patient~~or degenerated myoma.) appears to be the result of the MRI reading. From the point of view, it would be better to move it backwards than the CT at the end of this paragraph.\nThe 7th paragraph on page 5: In the last sentence (\"CT imaging revealed~ PET/CT scan)\", it is unclear why the MRI and PET/CT scans were performed. (bicornuate uterus? The distinction between leiomyoma and sarcoma? The distinction between rupture?)\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6106", "date": "24 Nov 2020", "name": "Inji Yeo", "role": "Author Response", "response": "Thank you for your very informative and appropriate comments. Reviewer's comment 1: The second paragraph on page 1 -> we changed the order of sentences to explain the relationship between the bicornuate uterus and the uterine rupture Reviewer's comment 2: Figure 4 on page 4 -> we put text on picture, and added description Reviewer's comment 3: The first paragraph of the discussion -> We added normal structure of uterus, and changed the form as commented Reviewer's comment 4: The 3rd paragraph of discussion -> we removed that sentence Reviewer's comment 5: The 4th paragraph on discussion -> We changed the order of sentence, and integrated the 4th paragraph with 5th paragraph Reviewer's comment 6: The 4th paragraph on page 5 -> we removed the sentence that is in argue, and added pros of CT over sono. Reviewer's comment 7: The 7th paragraph on page 5: -> we moved above sentence backward to the end of paragraph about MRI reading Reviewer's comment 8: The 7th paragraph on page 5 -> We performed the PET/CT to make distinction between leiomyoma and sarcoma before surgery." } ] }, { "id": "72608", "date": "23 Oct 2020", "name": "Pallav Sengupta", "expertise": [ "Reviewer Expertise Reproductive Endocrinology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report on 'Spontaneous perforation of a bicornuate uterus with concomitant sarcoma' by Yim et al is describing an interesting finding. The authors have described the physical and laboratory investigations and operative and post-operative procedures well. The report can be accepted in F1000Research. However, the presentation of the literature in various sections can be improved further to improve the readability of the article.\nAbstract: This section is well-presented. However, it is preferable to discuss some information on operative as well well as post-operative procedures with the lab investigations. Please also try to rewrite the last sentence which reads repetitive.\n\nIntroduction: Nicely written. The last paragraph should discuss the objective specifically, rather than mentioning the methods performed.\n\nCase Report: Well described.\n\nDiscussion: Please start with the discussion of normal uterine structures and development (as done in the second paragraph) and continue with the structural abnormalities and so on. The rest has been discussed well.\n\nThe authors are requested to provide the ethical approval to publish the study along with the patient consent details (already provided).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6107", "date": "24 Nov 2020", "name": "Inji Yeo", "role": "Author Response", "response": "Thank you for your very informative and appropriate comments. Reviewer's comment 1 : Abstract -> We added her post-operative progress, and changed the form of the last sentence not to reads repetitive Reviewer's comment 2: Introduction -> We added more contents about objective of this article Reviewer's comment 3: Discussion -> We added contents about normal uterine structures as commented" } ] } ]	1	https://f1000research.com/articles/9-1143
https://f1000research.com/articles/9-1318/v1	12 Nov 20	{ "type": "Data Note", "title": "The complete genome sequences of 22 parrot species (Psittaciformes, Aves)", "authors": [ "Taylor Hains", "Kathleen O'Neill", "Jafet Velez", "Nancy Speed", "Susan Clubb", "Taras Oleksyk", "Stacy Pirro", "Taylor Hains", "Kathleen O'Neill", "Jafet Velez", "Nancy Speed", "Susan Clubb", "Taras Oleksyk" ], "abstract": "The parrots (Psittaciformes, Aves) are a group of colorful, intelligent, long-lived birds with a wide range of body sizes and plumage colors and patterns. One third of the parrot species is threatened with extinction due to habitat loss and the pet trade, a larger percentage than any other comparable bird order. We present the complete genome sequences of 22 species of parrots from 14 genera and 3 families: Anodorhynchus hyacinthinus, Ara ararauna, Ara chloropterus, Ara glaucogularis, Ara militaris, Aratinga solstitialis, Aratinga weddellii, Cacatua leadbeateri, Eclectus roratus, Eupsittula pertinax, Guaruba guarouba, Lorius garrulus, Myiopsitta monachus, Nymphicus hollandicus, Pionus senilis, Psittacus erithacus, Psittacus timneh, Psitteuteles goldiei, Pyrrhura frontalis, Pyrrhura griseipectus, Pyrrhura molinae, Pyrrhura perlata. Genomic data can be used to better understand species identity, hybridization, genetic diversity, and identification of animal products possibly derived from endangered species.", "keywords": [ "Parrots", "birds", "Psittaciformes", "cockatoos", "genomes" ], "content": "Introduction\n\nThe parrots (Psittaciformes, Aves) are an order of 370 extant species (Clements et al., 2019) characterized by high intelligence (Pepperberg, 2017), diverse plumage colors and patterns (Berg & Bennett, 2010), and clawed zygodactyl feet (Botelho et al., 2014). A third of all parrot species are threatened by extinction due to habitat loss and the practice of capturing young birds for the pet trade (Butchart et al., 2004).\n\nComplete genome sequences for parrot species will benefit such diverse fields as biodiversity, evolutionary studies, species hybridization, and research into the development of brain function. Genomic data will also aid in the development of molecular markers to identify products, which may be derived from endangered species (Yang et al., 2018).\n\n\nMethods\n\nSamples from 22 parrot species were obtained from molted feathers from pet birds, and blood samples from birds from captive breeding programs (Table 1). Where blood samples were used, they were obtained by a licensed veterinarian coinciding with a standard health check. Animals were handled in a manner consistent with accepted practices (NRC, 2011). In the case of molted feathers, pulp was excised from the shaft of a large tail or primary wing feather prior to extraction.\n\nDNA extraction was performed on blood and feather pulp using the Qiagen DNAeasy genomic extraction kit, using the manufacturer’s process. A paired-end sequencing library was constructed using the Illumina TruSeq kit, according to the manufacturer’s instructions. The library was commercially sequenced at Genewiz (New Jersey, USA) on an Illumina Hi-Seq platform in paired-end, 2 × 150bp format.\n\nThe resulting fastq files were trimmed of adapter/primer sequence and low-quality regions with Trimmomatic (v0.33) (Bolger et al., 2014). The trimmed sequence was assembled by SPAdes (v2.5) (Bankevich et al., 2012). This was followed by a finishing step using RagTag (v1.0.0) (Alonge, 2020) to make additional contig joins based on conserved regions in other parrot species: Melopsittacus undulatus (GCA_012275295), Amazona guildingii (GCA_013399615), Agapornis roseicollis (GCA_002631895), and other species in this study.\n\n\nResults\n\nThe genome sizes and NCBI Assembly accessions for each species are shown in Table 1.\n\nGenome sizes ranged from 1.07 – 1.21 G (scaffolded), with no clear delineation between the three represented families (Psittacidae, Psittaculidae, Cacatuidae).", "appendix": "Data availability\n\nAccession numbers for all genome sequences in GenBank can be found in Table 1.\n\n\nReferences\n\nAlonge M: Ragtag: Reference-guided genome assembly correction and scaffolding. GitHub Archive. 2020. Publisher Full Text\n\nBankevich A, Nurk S, Antipov D, et al.: SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012; 19(5): 455–477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerg ML, Bennett ATD: The evolution of plumage colouration in parrots: a review. Emu - Austral Ornithology. 2010; 110(1): 10–20. Publisher Full Text\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\nBotelho JF, Smith-Paredes D, Nuñez-Leon D, et al.: The developmental origin of zygodactyl feet and its possible loss in the evolution of Passeriformes. Proc Biol Sci. 2014; 281(1788): 20140765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButchart SH, Stattersfield AJ, Bennun LA, et al.: Measuring global trends in the status of biodiversity: red list indices for birds. PLoS Biol. 2004; 2(12): e383. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClements JF, Schulenberg TS, Iliff MJ, et al.: The eBird/Clements Checklist of Birds of the World: v2019. 2019. Reference Source\n\nNational Research Council: Guide for the Care and Use of Laboratory Animals. Eighth edition, 2011. Reference Source\n\nPepperberg IM: \"Birdbrains\" should not be ignored in studying the evolution of g. Behav Brain Sci. 2017; 40: e216. PubMed Abstract \| Publisher Full Text\n\nYang F, Ding F, Chen H, et al.: DNA barcoding for the identification and authentication of animal species in traditional medicine. Evid Based Complement Alternat Med. 2018; 2018: 5160254. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77387", "date": "19 Feb 2021", "name": "Paweł Mackiewicz", "expertise": [ "Reviewer Expertise bioinformatics", "genomics", "phylogenetics", "molecular evolution" ], "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 was submitted to the section Data Note. It presents very briefly the description of nuclear genomes from 22 parrot species. Although the importance of these genomes is crucial for various analyses, the presentation should be improved. The title indicates that the genomes are complete, whereas the obtained sequences are not assembled and are still organized in scaffolds. Therefore, the title should be changed and the authors should describe in more detail the data set including information about incompleteness and gaps in the sequences. The limitations of the datasets should necessary be presented. The authors should state which type of analyses can be performed on such incomplete data set and which further steps should be performed to assembly the sequences. Could you answer for the following questions: Which problems prevented the obtaining the complete genomes? Are the authors going to assembly and study these genomes in the future? Moreover, the authors should present in detail sources for each studied sample, e.g. zoo, natural environment with detailed localization, individual breeders, reservation etc. It is essential. Describe in detail the captive breeding programs in the context of the samples, which you mentioned. Ethics policies should be also included. Were the samples obtained and transferred according to the species protection law? It should be clearly stated. Although my review may seem harsh, actually I think that the obtained genomic sequences are valuable for various studies and the data set is interesting for scientific community. I am very willing to accept the manuscript after the inclusion of information and explanations that I mentioned in my review.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] }, { "id": "82331", "date": "08 Apr 2021", "name": "Devon DeRaad", "expertise": [ "Reviewer Expertise Phylogenetics", "genomics", "birds" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThese are potentially very valuable data for avian genomics, as they increase the number of parrot genomes many times. These data will provide valuable resources for evolution and conservation genomics study.\nThis study has a few limitations, detailed below:\nThe biggest limitation is the inability to link the genomic information with certainty to a taxonomic name because of the lack of a voucher specimen and uncertainty about the possible hybrid status of pet parrots. The samples come from either pet birds or birds in captive breeding programs. While birds in captive breeding programs are more likely to be genetically pure, pet parrots could come from anywhere (even the illegal pet trade). Parrot species are known to hybridize in introduced habitats, in the wild, and in captivity—and can even produce viable offspring between genera (Hernandez-Brito et al. 2021 in Ibis1). More details on provenance might alleviate some concerns, but it will be hard to get away from some uncertainty about whether these genomes are accurate representations of their taxonomic identifiers.\n\nThe genomes are not complete, since the assemblies come from Illumina short reads and likely do not cover certain, more difficult regions of the genome. Information on depth of sequencing and contiguity (e.g., N50) is available on NCBI, but should also appear in the table to allow readers to assess genome quality more directly.\n\nThe introduction would be enriched by a little more information on the rationale for the study. Specifically, what is currently missing from parrots studies that these genomes could provide? Describe existing genomic resources and then narrow to a knowledge gap that the authors hope to fill with these genomes. Was there a rationale for the specific species chosen?\n\nLikewise, the Discussion would benefit from more elaboration on the broad utility of these datasets, e.g., mapping whole genome re-sequence data or reduced representation sequence data for population genetics studies, etc. Specifically, which uses might not be diminished by some uncertainty surrounding taxonomic assignment?\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "96540", "date": "26 Oct 2021", "name": "Roberto Ambrosini", "expertise": [ "Reviewer Expertise Bird ecology (RA) and genomics (SS)." ], "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 by Hains and co-authors provides NCBI accession numbers for 22 scaffold-level parrot genome assemblies. These data have the potential to add a consistent number of parrot genomes to the current literature and are therefore very valuable. However, there are a series of points that we think can be improved.\nDespite this work being published as a data note and no detailed results are therefore expected, the data description is too brief, in our opinion.\nMore details on the quality and contiguity of the assemblies should be provided, at least the N50 or the NG50 statistics, the number of scaffolds, and some QV statistics, like those produced by Merqury (Rhie et al. 2020)1.\nThe title also claims that the genomes are complete, but the paper does not provide any metrics about it. Usually, short Illumina reads can not resolve the repetitive regions of the genome. We understand that the authors probably could not reach the chromosome-level due to technical limitations (e.g. Hi-C data to join the scaffolds seem not available), still, we suggest providing some measure of the completeness of their assemblies both at structural (k-mer completeness form Merqury) and functional (from Busco analysis (Manni et al. 2021)2) level. For similar reasons, we think that the title should be changed by replacing the term “complete” with “scaffold-level” as the latter describes the quality of the described genomes more accurately.\nWe also suggest adding more information about how samples were acquired (permissions and ethic statements). I am also concerned by the fact that the parrots can hybridize in captivity, so detailed information about the (potential) hybrid status of each individual should be provided.\nWe hope our suggestions can help the authors to further improve their paper.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1318
https://f1000research.com/articles/9-1243/v1	15 Oct 20	{ "type": "Case Report", "title": "Case Report: Complete heart block as a manifestation of cardiac metastasis of oral cancer", "authors": [ "Andrianto Andrianto", "Eka Prasetya Budi Mulia", "Denny Suwanto", "Dita Aulia Rachmi", "Mohammad Yogiarto", "Eka Prasetya Budi Mulia", "Denny Suwanto", "Dita Aulia Rachmi", "Mohammad Yogiarto" ], "abstract": "Metastatic tumors of the heart presenting with complete heart block (CHB) is an extremely uncommon case. There are no available guidelines in managing CHB in terminal cancer. Permanent pacemaker implantation in such cases is a challenge in terms of clinical utility and palliative care. We report a case of a 24-year-old man suffering from tongue cancer presenting with CHB. A intracardiac mass and moderate pericardial effusion were present, presumed as the metastatic tumor of tongue cancer. We implanted a temporary pacemaker for his symptomatic heart block and cardiogenic shock, and pericardiocentesis for his massive pericardial effusion. We decided that a permanent pacemaker would not be implanted based on the low survival rate and significant comorbidities. Multiple studies report a variable number of cardiac metastasis incidence ranging from 2.3% to 18.3%. It is rare for such malignancies to present with CHB. The decision to implant a permanent pacemaker is highly specific based on the risks and benefits of each patient. It needs to be tailored to the patient’s functional status, comorbid diseases, prognosis, and response to conservative management.", "keywords": [ "tongue cancer", "cardiac metastasis", "complete heart block", "case report" ], "content": "Introduction\n\nCardiac metastasis is the least common presentation in malignant cancer. Primary cardiac tumors are also rare (on postmortem analysis commonly between 0.01% to 0.1%). However, the frequency of secondary metastatic tumors to the pericardium, myocardium, great vessels, and coronary arteries are between 0.7% to 3.5% in the general population and up to 9.1% in patients with a history of malignancies1.\n\nComplete heart block (CHB) as the primary clinical presentation of heart metastasis is very unusual2. There are currently no guidelines for the management of CHB in terminal stage of cancer.\n\nWe report a case of CHB caused by cardiac metastasis and review the literature to further help the management of our patient.\n\n\nCase presentation\n\nA 24-year old Asian man was admitted the cardiology department with CHB and hypotension. The patient was a chef and had a history of tongue cancer for six months and had undergone 30 cycles of radiotherapy. A week before, the patient came to the emergency department (ED) because of oral bleeding and general weakness. The patient denied any history of cardiovascular disease.\n\nThe patient was presented with chest discomfort and general weakness. He was hypotensive and bradycardic with blood pressure of 80/40 mmHg, regular heart rate of 44 beats per minute, respiratory rate of 18 breaths per minute, and oxygen saturation of 97% on room air. Chest auscultation was clear, and no murmurs heard. Electrocardiogram showed CHB with a junctional escape rhythm at 44 bpm (Figure 1). Echocardiography showed normal left ventricle kinetic, normal left ventricular ejection fraction (62%), and normal right ventricle systolic function. There were moderate pericardial effusion and intracardiac masses (2.1 × 0.9 cm and 1.8 × 0.8 cm) in the right atrial and septal leaflet of tricuspid. Hyperechoic areas in the annulus of tricuspid, lateral wall of right atrium and right ventricle, and interventricular septum were also found in an echocardiogram (Figure 2 and Figure 3). Laboratory finding revealed anemia (hemoglobin 8.5 g/dL; normal range 13.3-16.6 g/dL), leukocytosis (white blood count 18,470/mL; normal range 3,370-10,000/mL), hypoalbuminemia (albumin 2.6 g/dL; normal range 3.4-5.0 g/dL), hypokalemia (potassium 3.4 mmol/L; normal range 3.5-5.1 mmol/L), and hypercalcemia (calcium 16.2 mg/dL, corrected calcium 16.9 mg/dL; normal range 8.6-10.3 mg/dL).\n\nHyperechoic areas were found in the annulus of tricuspid, lateral wall of right atrium and right ventricle, and interventricular septum (red arrows).\n\nPericardial effusion was found in (A) anterior, posterior, (B) inferior, (C) base, and (D) left-lateral of the heart.\n\nPrevious magnetic resonance imaging (MRI) of head and neck, six months before this admission, revealed malignant tongue mass (staging AJCC 2010 of lip and oral cavity mass: T4N1Mx) and bilateral nasal cavity thickening. Multi-slice computed tomography (CT) scan of the head revealed an enhancing mass 1×1.3×1.5 cm at the base of the tongue and multiple lymph node enlargements subcentimeter in the upper and lower paratracheal. Histopathology examination of tongue biopsy confirmed poorly differentiated squamous cell carcinoma.\n\nA temporary pacemaker was immediately implanted as the patient showed symptomatic heart block and cardiogenic shock. CHB persisted despite corrected electrolyte imbalance. On the 14th day of admission, the patient developed pleural effusion, and worsening pericardial effusion with echocardiogram showed massive pericardial effusion and sign of tamponade. Further chest X-ray evaluation on the 14th day of treatment (which includes electrolyte imbalance correction, supportive treatment of general weakness condition, anemia, hypoalbuminemia, and infection) showed left parahilar ground glass appearance with suspicions of lung metastasis and pleural effusion (Figure 4). Pericardiocentesis was then performed with pericardial fluid showing hemorrhagic typical for malignant disease.\n\nPericardial fluid pigtail was already inserted for drainage of pericardial effusion.\n\nBecause the patient showed general improvement after supportive treatment for seven days and no symptoms of CHB, it was decided that the temporary pacemaker would be extracted. Considering the poor prognosis of this cancer, risk of permanent pacemaker (PPM) implantation, and severe comorbidities, we decided not to implant a PPM after acquiring the patient’s and his family’s consent. The patient died due to respiratory failure and septic shock 20 days after showing the first symptoms of cardiac metastasis.\n\n\nDiscussion\n\nFrom the literature, we acquired a total of 14 articles regarding heart block as a manifestation of cardiac metastasis, as summarized in Table 12–15. Oral cavity, uterus, and thyroid are the most common primary cancers that metastasize to the heart. Squamous cell carcinoma was the primary histologic finding. Heart metastasize may be present with clinically silent symptoms to an alarming presentation of hemoptysis and syncope. Locations of metastasis were mostly in the right ventricle, supporting the hypothesis of the hematologic spread of cancer cells. PPM implantation was performed in 10 cases, yet only one case reported a significant lifespan after PPM implantation.\n\nThe prevalence of cardiac metastasis, in general, is arguably low. However, multiple studies reported a variable number of cardiac metastasis incidence ranging from 2.3% to 18.3%. The prevalence of malignancy originating from the oral cavity was 5.3%. The involvement of pericardium made up two-thirds of all cardiac metastasis. Myocardium and endocardium involvement each made up one-third of all cardiac metastasis. Only 5% involved the endocardium. The most common site of metastasis for squamous cell carcinomas is epicardium (41.4%)16.\n\nMyocardial infiltration by cancer cells may present with arrhythmias, such as atrial flutter or fibrillation, premature beats, or ventricular arrhythmias. Conduction system involvement may induce various degree of atrioventricular blocks16.\n\nThe presence of right atrial mass in our patient supports the possibility of hematologic spreading of metastatic cancer cells into the endocardium. Pericardial effusion may represent metastasize or inflammatory reaction toward the malignancy. The presence of CHB suggests the infiltration of the heart conduction system.\n\nValves are an uncommon site for metastasis because of the absence of vessels in the physiological valvular stroma and the constant cusp motion. Bussani et al. reported, out of over a thousand of postmortem examination, there was only one case of valve involvement16. The mass in the septal leaflet of tricuspid valves appeared in echocardiography examination in this patient showed valve involvement of cardiac metastasis.\n\nAsymptomatic in the early stages, cardiac metastasis could lead to a wide range of signs and symptoms, such as cardiac failure, conduction disturbances, angina, and pain as it progresses. Disruption of the cardiac conduction system by cardiac metastases can lead to lethal arrhythmias, including atrial fibrillation with a rapid ventricular response, CHB, or ventricular fibrillation1. From the literature, we acquired 14 cases reporting CHB as a manifestation of cardiac metastasis originating from various malignancies, three of which are from the oral cavity.\n\nCardiac masses in our case presented with features favoring tumor, such as echo density similar to myocardium, normal wall motion, valvular lesion, no history suggestive of coronary artery disease, and a clinical history of oral cancer as primary site suspected to metastasize to the heart.\n\nCardiac MRI is the best imaging modality and, along with positron emission tomography scanning, are mostly used in investigating the extent of infiltration by malignant cells2,17. Patients with specific cardiac devices, such as pacemaker and defibrillators, would be disqualified from undergoing MRI, an important consideration given the frequency at which arrhythmia complicates cardiac metastasis17. The temporary pacemaker implanted during the early stages of CHB made MRI impractical for our patient.\n\nIn our patient, CHB was initially thought to be the result of electrolyte imbalance. However, as the electrolyte was restored to its normal level without any improvement of CHB, it suggested that CHB was caused by infiltration of the metastatic cell to the conduction system of the heart. The presence of hemorrhagic pericardial effusion supports the suggestion of pericardial metastasis. Regardless of the lack of histological confirmation, we suggest that this case was cardiac metastasis diagnosed antemortem.\n\nPredominantly, after a reversible or transient cause of bradycardia is excluded, cardiac pacing indication is decided by bradycardia severity, instead of its etiology. The European Society of Cardiology (ESC) guidelines state that some types of persistent bradycardia require permanent pacing. In acquired AV block, pacing is indicated in patients with second-degree type 2 or third-degree AV block regardless of symptoms (class I)18. Similar to the ESC guidelines, the American College of Cardiology/American Heart Association/Heart Rhythm Society guidelines also state that patients with transient or reversible causes of AV block should receive medical and supportive treatment if necessary, including temporary transvenous pacing, prior to confirmation of the need for permanent pacing (class of Recommendation/COR I). In addition, for patients with acquired CHB not associated with physiologic or reversible etiology, permanent pacing is recommended regardless of symptoms (COR I)19.\n\nIn our case, we assume that the CHB was persistent after trying to correct possible external causes, such as hypercalcemia and hypokalemia, and after a sufficient waiting period. Moreover, it is still also unknown whether CHB would resolve after cancer treatment2,20. Our literature review showed that there are only two cases of metastatic CHB that are reversible after undergoing chemotherapy for cardiac metastasis8,12, while several other cases showed that CHB is irreversible2–7,9–11,13–15. The short life expectancy in patients with metastatic CHB also makes it difficult to follow up on CHB reversibility. Our literature review showed only one metastatic CHB case reported a significant lifespan more than two years after PPM implantation8, while other cases reported a lifespan of no longer than one year2–7,9–15.\n\nOur patient was expected to continue the radiotherapy cycle. Radiotherapy (RT) itself can induce pacemaker malfunction. Software impairments are the primary manifestation of malfunction during RT. This will perhaps lead to pacemaker reset, leaving only device basic function. Radiation dosage appears to contribute less to inducing pacemaker malfunctions than the beam energy of the RT21.\n\nFor patients with indications for permanent pacing, but accompanied with significant comorbidities or who are anticipated having a shortened lifespan because of terminal progressive illness, the implantation of a PPM should not be performed if it is unlikely to deliver significant clinical benefits or if it hinders the main therapy for the patient’s goal of care. Even though pacemaker implantation risks are rather low, the risk-benefit ratio is not favorable if the possible benefit is also quite low19. Thus, after discussing with our patient and his family, we decided not to implant a PPM after acquiring the patient’s and his family’s consent.\n\n\nConclusion\n\nCHB in a patient with oral cancer should increase the physician's suspicion of cardiac metastasis. It is rare for such malignancies to present with CHB. The decision to implant a permanent pacemaker is highly specific based on the risks and benefits of each patient. It needs to be tailored to the patient’s functional status, comorbid diseases, prognosis, and response to conservative management. Even though the pacemaker implantation risks are rather low, the risk-benefit ratio is not favorable if the possible benefit is also quite low. A permanent pacemaker was not implanted in our patient because of the poor prognosis, severe comorbidities, and low expected lifespan.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient’s parent.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nGoldberg AD, Blankstein R, Padera RF, et al.: Tumors Metastatic to the Heart. Circulation. 2014; 128(16): 1790–1794. PubMed Abstract \| Publisher Full Text\n\nKumar D, Mankame P, Sabnis G, et al.: A case report: metastatic complete heart block. Eur Heart J Case Rep. 2018; 2(4): yty131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuckberg GD, Fowler NO: Complete Atrioventricular Block due to Cardiac Metastasis of Bronchogenic Carcinoma. Circulation. 1961; 24: 657–661.\n\nPark YM, Shin JO, Kim M, et al.: Cardiac metastasis of leiomyosarcoma complicated with complete atrio-ventricular block and ventricular tachycardia. Korean Circ J. 2016; 46(2): 260–263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshihiro T, Tsuchihashi K, Kusaba H, et al.: Cardiac metastasis of squamous cell carcinoma of the thyroid gland with severe disseminated intravascular coagulation: A case report. Mol Clin Oncol. 2017; 6(1): 91–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKasai T, Kishi K, Kawabata M, et al.: Cardiac metastasis from lung adenocarcinoma causing atrioventricular block and left ventricular outflow tract obstruction. Chest. 2007; 131(5): 1569–1572. PubMed Abstract \| Publisher Full Text\n\nCho JY, Kim KH, Park H, et al.: Complete atrioventricular block as an initial manifestation of recurred oral cavity cancer : a case report. BMC Cardiovasc Disord. 2018; 18(1): 142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClifford SM, Guerra SM, Mangion JR: Massive metastatic intracardiac lymphoma presenting with complete heart block with resolution following chemotherapy. Echocardiography. 2003; 20(2): 201–202. PubMed Abstract \| Publisher Full Text\n\nMocini D, Longo R, Colivicchi F, et al.: A complete atrioventricular block secondary to myocardial metastases of lung cancer . A case report. Ital Hear J. 2005; 6(11): 931–932. PubMed Abstract\n\nFerraz JGG, Martins ALM, De Souza JF, et al.: Metastatic Tumor of Squamous Cell Carcinoma From Uterine Cervix to Heart: Ante-Mortem Diagnosis. Arq Bras Cardiol. 2006; 87(4): e104–7. PubMed Abstract \| Publisher Full Text\n\nOzyuncu N, Sahin M, Altin T, et al.: Cardiac metastasis of malignant melanoma : a rare cause of complete atrioventricular block. Europace. 2006; 8(7): 545–548. Publisher Full Text\n\nKnowles JW, Elliott AB, Brody J: A case of complete heart block reverting to normal sinus rhythm after treatment for cardiac invasive Burkitt’s lymphoma. Ann Hematol. 2007; 86(9): 687–690. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRathi VK, Williams RB, Yamrozik J, et al.: Cardiovascular magnetic resonance of the charcoal heart. J Cardiovasc Magn Reson. 2008; 10(1): 37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin CK, Cochet A, Lewi JE: Complete Heart Block in a Patient With Metastatic Papillary Thyroid Carcinoma. Fed Pract. 2015; 32(1): 34–35. PubMed Abstract \| Free Full Text\n\nYoneda T, Kase K, Amino Y, et al.: A case of gingival cancer with pulmonary metastases that developed complete atrioventricular block and ventricular fibrillation as a result of myocardial metastases. Clin Case Rep. 2016; 4(12): 1075–1081. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBussani R, Abbate A, Silvestri F: Cardiac metastases. J Clin Pathol. 2007; 60(1): 27–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLichtenberger JP, Reynolds DA, Keung J, et al.: Metastasis to the Heart: A Radiologic Approach to Diagnosis With Pathologic Correlation. AJR Am J Roentgenol. 2016; 207(4): 764–772. PubMed Abstract \| Publisher Full Text\n\nBrignole M, Auricchio A, Baron-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\nKusumoto FM, Schoenfeld MH, Barrett C, et al.: 2018 ACC/AHA/HRS Guideline on the Evaluation and Management of Patients With Bradycardia and Cardiac Conduction Delay: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Rhythm Society. Circulation. 2019; 140(8): e382–e482. PubMed Abstract \| Publisher Full Text\n\nFotouhi Ghiam A, Dawson LA, Abuzeid W, et al.: Role of palliative radiotherapy in the management of mural cardiac metastases: who when and how to treat? A case series of 10 patients. Cancer Med. 2016; 5(6): 989–996. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaremba T, Jakobsen AR, Søgaard M, et al.: Radiotherapy in patients with pacemakers and implantable cardioverter defibrillators: A literature review. Europace. 2016; 18(4): 479–491. PubMed Abstract \| Publisher Full Text" }	[ { "id": "73101", "date": "27 Oct 2020", "name": "Radityo Prakoso", "expertise": [ "Reviewer Expertise Pediatric Cardiology and Congenital Heart Disease" ], "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\nSome typos and grammars should be evaluated. In the presentation, what does author mean about \"improvement\"? Why did TPM extracted by improvement but the complete heart block was actually not improving? I mean its 7th days were they sure that the worsening condition not due to the low cardiac output because of too low of heart rate? Maybe the author could add some suggestions of using medication to increase heart rate if there were significant reasons to \"must take\" the TPM?\nConclusion So what does the author advise if one day there were similar patients came with complete heart block? Should the author suggest to implant the TPM or maybe using medication to increase the heart rate, such as sympathomimetic amines.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6079", "date": "11 Nov 2020", "name": "Andrianto Andrianto", "role": "Author Response", "response": "Some typos and grammars should be evaluated. In the presentation, what does author mean about \"improvement\"? Why did TPM extracted by improvement but the complete heart block was actually not improving? I mean its 7th days were they sure that the worsening condition not due to the low cardiac output because of too low of heart rate? Maybe the author could add some suggestions of using medication to increase heart rate if there were significant reasons to \"must take\" the TPM? Answer: Thank you for your comment. We have revised come typos and grammar. We also added a clearer definition in general condition, supportive treatment including dopamine use, and reason the TPM was extracted in section case presentation paragraf 4-6. Conclusion So what does the author advise if one day there were similar patients came with complete heart block? Should the author suggest to implant the TPM or maybe using medication to increase the heart rate, such as sympathomimetic amines. Answer: Thank you for your comment. We have added our suggestion of TPM use in such cases in section conclusion paragraf 1." } ] } ]	1	https://f1000research.com/articles/9-1243
https://f1000research.com/articles/9-1311/v1	10 Nov 20	{ "type": "Research Article", "title": "Use of 16s rRNA to identify non-lactose-fermenting bacilli and molecular detection of ESBL resistance genes associated with hospital-acquired infection in Soba University Hospital, Sudan", "authors": [ "Wissam Ahmed Al Hag", "Hana Elbadawi", "Muzamil Mahdi Abdel Hamid", "Hana Elbadawi", "Muzamil Mahdi Abdel Hamid" ], "abstract": "Background: Non-lactose-fermenting gram-negative bacilli (NLFGNB) have become significant nosocomial pathogens and often exhibit intrinsic multidrug resistance. Sequencing of 16s rRNA genes could be utilized for robust identification of NLFGNB. This study aimed to identify resistant NLFGNB associated with hospital-acquired infections using 16s rRNA sequencing and to detect the extended-spectrum β-lactamase (ESBL) genes of isolates in Soba Hospital, Khartoum State, Sudan. Methods: A prospective, cross-sectional, laboratory-based study was conducted from October 2017 to March 2018 at the Microbiology Department of Soba University Hospital. A total of 100 randomly selected NLFGNB samples were isolated from blood and urine during the time of the study. All the isolates were identified using standard biochemical tests and antimicrobial sensitivity testing, 16s rRNA gene sequencing, and bioinformatics techniques. Results: The biochemical tests revealed that, out of the 100 NLFGNB isolates, the Pseudomonas species was predominant (57 isolates), followed by gram-negative bacilli (33 isolates), Coccobacilli (9 isolates) and Coliform (1 isolate) species. Sequencing of 16s rRNA genes identified all the resistant isolates at the species level: Pseudomonas aeruginosa (26%), Acinetobacter baumannii (22%), Burkholderia cepacia (13%), Stenotrophomonas maltophilia (10%), Enterococcus species (E. faecalis, E. faecium) (10%), and other GNB (Acinetobacter variabilis, Klebsiella pneumoniae, Morganella morganii, Escherichia fergusonii, Enterobacter hormaechei and Pseudomonas stutzeri) (19%). The antimicrobial susceptibility tests indicated that 31 isolates were resistant to at least three classes of antibiotics and contain the highest level of ESBL resistance genes. Conclusions: Pseudomonas aeruginosa and Acinetobacter baumannii were the most widely recognized NLFGNB identified from hospital-acquired infections in Soba hospital. Among the NLFGNB, antimicrobial resistance and ESBL resistance genes were observed at a high frequency.", "keywords": [ "Gram-negative bacilli", "non-lactose fermenting", "16S rRNA", "ESBL", "hospital-acquired infection", "Pseudomonas aeruginosa", "Acinetobacter baumannii", "nosocomial infection." ], "content": "Introduction\n\nHospital-acquired infections, also known as nosocomial infections (NIs), by gram-negative bacteria are a major threat to health worldwide (Karaiskos & Giamarellou, 2014). These bacteria are consistently evolving and developing novel mechanisms of resistance to commonly used antibiotics, especially when these antibiotics are overused, which generates a selection pressure (Peleg & Hooper, 2010). Increasingly, non-lactose-fermenting gram-negative bacilli (NLFGNB) have become significant pathogens in healthcare facilities (Akbar et al., 2014; Hiltunen et al., 2017). The public health risk of NLFGNB is growing rapidly worldwide; consequently, warnings of the disturbing spread of anti-infection-resistant microorganisms causing NIs, which have cost lives and resources, have also risen (Djordjevic et al., 2017; Leski et al., 2016; Ranjbar et al., 2018).\n\nThe over-prescription and use of antimicrobial agents may be playing a major role in the escalating number of NIs (Mataseje et al., 2016; Prasert et al., 2020; Rabirad et al., 2014). Bacteria are identified phenotypically and characterized by biochemical tests; however, these tests are not as accurate as required (Bush & Jacoby, 2010). Advanced techniques in molecular biology and sequencing approaches are needed for the discovery, identification, and characterization of emerging and re-emerging pathogens (Wilson et al., 2014). 16s rRNA gene sequencing offers a practical and reliable molecular identification method (Brown-Elliott et al., 2006; Clarridge, 2004). 16s rRNA gene sequence analysis can better identify poorly described, recognize inadequately depicted, infrequently isolated, or phenotypically deviant strains, can be routinely utilized for identification of bacteria, and can prompt the acknowledgment of novel pathogens and non-cultured bacteria (Case et al., 2007; Crosby & Criddle, 2003; Dahllöf et al., 2000; Reller et al., 2007). A significant function for 16s rRNA gene sequencing is to identify precisely assembled living organisms for additional study (Goldenberger et al., 1997; Jalava et al., 1995). Gram-negative organisms are involved in both network- and emergency clinic-procured contaminations (Mataseje et al., 2016; Prasert et al., 2020). NLFGNB infections cause a significant public health problem in hospitals, especially when they develop multi-drug resistance (Kaye & Pogue, 2015; Lochan et al., 2017). These microorganisms can develop several mechanisms of resistance, including β-lactamase creation, overexpression of multi-drug efflux pumps, target site mutations, and reducing the permeability of the external membrane (Djordjevic et al., 2017).\n\nThe correct identification of these bacteria by conventional microbiology methods is often limited by phenotypic misidentification (AbdulWahab et al., 2015; Alby et al., 2013; Plongla et al., 2016). The spread of extended-spectrum β-lactamase (ESBL)-making strains restricted the range of antimicrobial agents that could be used to treat patients effectively and raised concerns for control of infections caused by NFGNB and limitation of treatment to prohibitively expensive antibiotics (Manyahi et al., 2017; Ranjbar et al., 2018; Zhang et al., 2009). The mutation of TEM-1, TEM-2, and SHV-1 β-lactamases produced the ESBLs, which were discovered over 1980–1990 and first detected in Western Europe (Bubpamala et al., 2018; Bush et al., 1995). DNA sequencing technologies offered a sensitive diagnostic tool that improved the detection, identification, and characterization of drug-resistant bacteria (Livermore, 1995; Ranjbar et al., 2018).\n\nNLFGNB is commonly associated with hospital-acquired infections; therefore, identification of the species and the study of antimicrobial susceptibility patterns are imperative. ESBL genes are the most common resistance genes associated with gram-negative bacilli. Thus, it is essential to determine both the prevalence of ESBL among NLFGNB and the recognizable proof of microscopic organisms at the species level, which is essential to determine the most effective treatment and best guide case management (Bush et al., 1995; Livermore, 1995).\n\nIn Sudan, recognizable proof of NLFGNB at the species level is difficult to achieve by conventional methods. Alternative strategies, (e.g., API or VITEK) are not affordable; therefore, the current study was undertaken to provide more sensitive and accessible methods that require 16s rRNA to detect and confirm the bacterial pathogens separated from patient samples in Soba University Hospital in Khartoum, Sudan.\n\n\nMethods\n\nA prospective, cross-sectional, laboratory-based study was conducted from September 2017 to February 2018. The study conducted in the Microbiology Department of Soba University Hospital and the Institute of Endemic Diseases, University of Khartoum, Sudan. A total of 100 clinical isolates from specimens of urine and blood were collected from hospitalized patients (Pourhoseingholi et al., 2013). All isolates that are NLFGNB were included and isolates that are gram positive and/or lactose fermenter excluded in this study. Blood isolates totaled 64 (64%) and urine totaled 36 (36%). All 100 strains of NLFGNB sub-cultured on blood, chocolate, and MacConkey agar and incubated at 37°C overnight for re-identification, at which point they were assessed using standard microbiological strategies (such as morphology, microscopy, and biochemical tests set out by Nagy et al., (2018)). Quality control strains were utilized in biochemical tests and antimicrobial susceptibility testing of E. coli (ATCC #25922) and P. aeruginosa (ATCC #27853). NFLGB colonies and gram-negative bacteria were selected. Antibiotic susceptibility testing was accomplished by way of Kirby-Bauer disk-diffusion; all isolates were swabbed on Muller-Hinton agar, placed on antibiotic disks, and incubated at 37°C for 18–24 hours. All isolates were tested against the following antibiotics: ceftriaxone (CRO; 30 μg), ceftazidime (CAZ; 30 μg), cefuroxime (CXM; 30 μg), cefotaxime (CXM; 30 μg), cotrimoxazole (COT; 25 μg), amikacin (AK; 10 μg), ciprofloxacin (CIP; 5 μg), Cefepime combined with amoxycillin/clavulanate (AMC; 30 μg), and imipenem (IPM; 10 μg). Results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (Eigner et al., 2005; Moubareck et al., 2009).\n\nDNA was extracted from cultured specimens using guanidine chloride and underwent PCR using a thermal cycler (Analytik Jena Biometra TADVANCED , Germany), by using specific primers, such as the universal primer 16s rRNA, and specific primers for ESBL genes, such as CTX-M, SHV, and TEM (Table 1). PCR reaction was completed in a total reaction volume of 25 μL (5 μL Master mix of Maxime RT PreMix kit [iNtRON Biotechnology, Seongnam, Korea], 0.6 μL forward primer, 0.6 μL reverse primer, 2 μL DNA, and 16.8 μL deionized sterile water). The following cycle conditions were used: initial denaturation step at 94°C for 5 min, after which there were 30 cycles of denaturation at 94°C for 45 seconds, primer toughening temperature (as indicated in Table 1) for 45 seconds, and extension at 72°C for 60 seconds, with a final elongation step at 72°C for 5 min. Products were electrophoresed in 2% agarose gel in 1X TBE containing 2.5 μL of ethidium bromide (20 mg/mL) at 100 V for 40 min. The amplified product was distinguished by contrasting with a 100-base-pair standard DNA ladder (iNtRON BIOTECHNOLOGY, Seongnam, Korea) and the bands were visualized under UV (analytikjena® Biometra BDAcompact, Germany). Purified the PCR results of 16SrRNA and Sanger sequencing was performed by Macrogen Company (Seoul, Korea). Then nucleotides sequences of the genes 16SrRNA accomplished were scanned for similarity sequence using nucleotide BLAST for species identification (Petti, 2007).\n\nNucleotide sequences of the 16s rRNA gene were assessed for sequence similarity and species identification using NCBI Nucleotide BLAST. MEGA X software was used for conducting statistical analysis of molecular evolution and for constructing phylogenetic trees of bacteria identified in the study.\n\nData were analyzed using SPSS version 20.0 and Microsoft Excel. Cross tabulation was used to present the relation data, quantitative data were performed to discover the contrasts between bacterial isolates with resistance to at least one class of antibiotics by samples (blood and urine).\n\nThe study protocol was approved by the ethics committee of the Institute of Endemic Diseases, University of Khartoum, and the permission was obtained from the managers of Soba University Hospital, under reference number IEND_REC 12/2017. Participation in this study was entirely voluntary and all participants read and signed informed consent forms; where children were assessed, their parents provided written informed consent.\n\n\nResults\n\nThe largest proportion of specimens were collected from the NICU ward, comprising 42 blood and 4 urine specimens (91% and 8.7%, respectively). In total, ten blood and nine urine specimens were then collected from the pediatric ward (52.6% and 47%, respectively), followed by specimens from the renal unit (six blood and five urine samples), the medicine ward (one blood and nine urine samples), the ICU unit (five blood and three urine samples), and the surgery ward, which held the lowest number of specimens (six urine and no blood samples). The number of samples collected from inpatients from different wards are shown in Table 2. Demographic details, alongside identification of bacterial isolates and resistance genes, are available as Underlying data Al Hag, 2020.\n\nA total of 60 out of 100 isolates (60%) were recognized by traditional and biochemical strategies; the remaining 40% could not be identified. The isolate Pseudomonas spp. (54%) was most common, followed by other GNB (40%) then Coccobacilli (6%). Molecular identification using 16s rRNA sequencing showed that the most common Acinetobacter spp. that was isolated was Acinetobacter baumannii (25.3%), while the most common oxidase positive isolate was Pseudomonas aeruginosa (34.8%). Pseudomonas spp. (12.6%), Burkholderia cepacia (6.8%), Stenotrophomonas maltophilia (4.8%), and other gram-negative bacilli, including Morganella morganii and Enterobacter hormaechei, account for 10%. The isolates of Enterococcus spp. account for (3.3% of E. faecium and E. faecalis), Escherichia fergusonii and Klebsiella pneumoniae (2.4%) were excluded from the results (Figure 1).\n\nOther gram-negative bacilli include Morganella morganii and Enterobacter hormaechei.\n\nNLFGNB isolates with antimicrobial resistance pattern are shown in Figure 2. Isolates tested using the disk diffusion method showed the highest percentage of resistance to be 98% and 94.5% against ampicillin and cefotaxime, respectively, followed by cephalexin (90.7%), amoxicillin/clavulanic acid (90.1%), ceftriaxone (88.4%), and ceftazidime (84%). Co-trimoxazole and nitrofurantoin resistance were recorded to be present in 74.3% and 75.1% of isolates, respectively. Resistance rates to ciprofloxacin was 50.1%, gentamicin was 52.5%, and amikacin was 22.3%. Imipenem and meropenem were the most efficient antibiotics tried, with resistance rates of 17.2% and 20.2%, respectively. The examination of the antimicrobial susceptibility pattern of these isolates demonstrated that NLFGNB showed high rates of multidrug resistance, being resistant to most third-generation cephalosporins, as observed in P. aeruginosa and A. baumannii (99% and 98%, respectively) (Testing, 2017).\n\nNames of antimicrobial agent were abbreviated as follows: imipenem (IPM), meropenem (MER), ceftazidime (CAZ), ceftriaxone (CTR), cefotaxime (CXM), cephalexin (CN), ciprofloxacin (CIP), gentamicin (GEN), cotrimoxazole (COT), nitrofurantoin (NIT), amoxicillin/clavulanic acid (AMC), ampicillin (AMP), and amikacin (AK).\n\nPCR amplifications were performed to recognize three virulence genes using their appropriate primers. The most frequent gene found in species is SHV (~51% of total isolates), followed by CTX-M (~43%) and TEM (~20%). All bacterial species that were isolated had one or two ESBL genes; therefore, they show a high level of antibiotic resistance (Table 3) (Rhoads et al., 2012).\n\nThe alignment of DNA sequences for NLFGNB isolates identiﬁed them at the species level and contrasted with public databases resulted in the retrieval of two sequences of various species which displayed identical similarity scores; thus, the isolate was not assigned to a single taxon but was reported as belonging to either of the two species. The sequence of an isolate showed 99.99% identity to sequences of Pseudomonas stutzeri and Pseudomonas spp., whereas B. cepacia represented 99.86%, S. maltophilia represented 99.88%, E. fergusonii represented 99.72%, and other gram-negative bacteria represented 95.88% identity.\n\n\nDiscussion\n\nNLFGNB were previously considered to be contaminants but have now emerged as significant healthcare-associated pathogens (Malini et al., 2009). Pseudomonas and Acinetobacter species are known to be the regular nosocomial pathogens (Gales et al., 2001; Tortoli et al., 2001). In this study, the most common NLFGNB isolated was P. aeruginosa, followed by A. baumannii, which is similar to the results obtained by Sarkar et al. (2018) and Malini et al. (2009), who reported P. aeruginosa as the most common isolate, followed by A. baumannii (Malini et al., 2009; Wauters & Vaneechoutte, 2015). Other NLFGNB, such as B. cepacian, S. maltophilia, and Stenotrophomonas pavanii vary from study to study, both in terms of their genera and prevalence. However, their role as opportunistic pathogens in immunocompromised and debilitated individuals has been invariably established (Chawla et al., 2013). Strains isolated by biochemical methods were recorded a Pseudomonas spp., Coccobacilli, and other GNB. The sequencing test identified the isolates to many strains, such as P. aeruginosa, A. baumannii, B. cepacian, S. maltophilia, and other GNB. The prevalence of ESBLs in this study showed SHV to be the highest level of the total sample, followed by CTX-M, with TEM being the lowest of the resistance isolates. ESBLs are enzymes that demonstrated resistance to the third generation of cephalosporin. As these enzymes are plasmid-encoded, the spread of bacterial resistance disseminates rapidly (Jena et al., 2017). ESBLs are responsible for resistance against β-lactam antibiotics, such as penicillin, cephalosporin, monobactams, and sometimes also carbapenems (Cantón et al., 2008). Resistance of NLFGNB and multidrug resistance have risen widely according to numerous studies. B. cepacia is another NLFGNB that colonizes and infects patients with chronic bacteremia and UTI. It is known to cause infections in hospitalized patients and, once infected, is hard to eliminate (Akbar et al., 2014; Chawla et al., 2013; Plongla et al., 2016).\n\nIn this study, the isolate of B. cepacian recorded 6.8% of the non-fermenters isolated from blood and urine culture. B. cepacian is known for its inherent resistance to numerous beta-lactam drugs, including aminoglycosides, colistin and polymyxin B, first-line therapeutics against serious pseudomonas infections (Rahbar et al., 2010). This isolate showed maximum (100%) susceptibility to imipenem, in accordance with Sidhu et al. who also reported 100% susceptibility to imipenem. Similarly, in a study done by Grewal et al., (2017) and Gautam et al., (2009). B. cepacia isolates showed excellent susceptibility to imipenem and Meropenem.\n\nThree strains of S. maltophilia were isolated during the study; it is now considered a common non-fermenter that causes infection in hospital settings (Verweij et al., 1998). Our study has shown the isolation of this bacterium in 6.4% of isolates. Earlier A’Court and Garrard reported S. maltophilia to account for 5% of nosocomial pneumonias (A’Court & Garrard, 1992). It showed 100% susceptibility to some of the antibiotics, notably ciprofloxacin. Similar results were obtained by Malini et al. (2009).\n\nThe majority of our isolates were resistant to ampicillin (98%), followed by cefotaxime (94.5%) then amoxicillin/clavulanate acid (90.1%). Resistance rates also rise in ciprofloxacin (50.1%), gentamicin (52.5%), and amikacin (22.3%). Imipenem and meropenem were the most effective antibiotics tested (Malini et al., 2009; Rahbar et al., 2010), with 100% sensitivity to ampicillin, cefotaxime, and ciprofloxacin.\n\nA phylogenetic tree for the most common NLFGNB shows the relationships among various species, with their phylogeny based on similarities and differences in their genetic characteristics.\n\nThis study covered this gap by using advanced molecular techniques to identify, characterize, and determine the spread of hospital-associated bacterial infections to permit infection control personnel to recognize potential sources of pathogens more effectively, and encourage physicians, healthcare workers, and stakeholders to develop treatment and control strategies against these rapidly evolving organisms.\n\n\nConclusion\n\nIn conclusion,NLFGNB in communities and hospitals can help inform antibiotic treatment. This study showed a significantly higher prevalence of P. aeruginosa and A. baumannii among NLFGNB associated with hospital infections. The isolation of MDR (P. aeruginosa) and MDR (A. baumannii) in the present study raises the concern of rapidly emerging antibiotic resistance in this group of bacteria in Sudan. Proper screening of non-fermenters in nosocomial settings and regular assessment of their antibiotic susceptibility profiles are suggested for effective management of infections and limitation of the emergence of multidrug resistance. Assessment of 16s rRNA gene sequences permits bacterial identiﬁcation that is stronger, more reproducible and more exact than that obtained by phenotypic testing. Thus, when ordinary methods do not yield an excellent (or very good) species identiﬁcation, non-fermenter GNB should be exposed to 16s rRNA gene sequencing if adequate species assignment is of concern. Since the spread of MDR and ESBL-producing NLFGNB isolates reduces treatment options and increases hospital cost, it is important to keep up to date on prevailing resistant patterns of any locality, which will help determine a suitable antimicrobial therapy.\n\n\nData availability\n\nThe 16S rRNA sequences generated in this study are available from GenBank under sequential accession numbers MW034199– MW034227.\n\nFigshare: Data infromation 1_original.xlsx. https://doi.org/10.6084/m9.figshare.13077398.v1 Al Hag, 2020.\n\nThis project contains the following underlying data:\n\nData infromation 1_original.xlsx. (Demographic information with details on bacterial strain and resistance profile.)\n\nData infromation 2_original.xlsx. (Results of PCR genotyping for each sample.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe would like to thank the technical staff of Medical Microbiology Department in Soba University Hospital, University of Khartoum for their assistance in isolates and data collection.\n\n\nReferences\n\nA’Court C, Garrard CS: Nosocomial pneumonia in the intensive care unit: mechanisms and significance. Thorax. 1992; 47(6): 465–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdulWahab A, Taj-Aldeen SJ, Ibrahim EB, et al.: Discrepancy in MALDI-TOF MS identification of uncommon Gram-negative bacteria from lower respiratory secretions in patients with cystic fibrosis. Infect Drug Resist. 2015; 8: 83–8. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Marrs CF, Simon C, et al.: Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Sci Total Environ. 2009; 407(12): 3702–6. PubMed Abstract \| Publisher Full Text" }	[ { "id": "74558", "date": "25 Feb 2021", "name": "Asim Elnour", "expertise": [ "Reviewer Expertise Professor of Clinical medicine and expert clinical pharmacist. Specialized in all clinical research and peer review of scientific content and merits of publications." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current study is a cross-sectional, laboratory-based study. Approved methods such as biochemical tests and antimicrobial sensitivity testing, 16s rRNA gene sequencing, and bioinformatics techniques were all used in the study. The current study has used variable methods and analysis tools like; conventional methods, antimicrobial sensitivity testing, molecular technique, and Bioinformatics software. The statistical analysis and its interpretation were appropriate. However, the small sample size of 100 isolates may hinder some limitations on the current study. Possible sensitivity analysis on data for molecular identification, and bioinformatics analysis may have improved the uptake of the current study findings. The use of outcome measure as quantitative data was performed to discover the contrasts between bacterial isolates with resistance to at least one class of antibiotics by samples (blood and urine). The source data underlying the results were available to ensure full reproducibility of the study. For instance, the 16S rRNA sequences generated in this study were available from GenBank under sequential accession numbers MW034199– MW034227.\nThe conclusions of this current original research were consistent with data reported, and the paper has cited current literature, for instance relevant papers published in 2020. The current research has identified resistant Non-Lactose-Fermenting Gram-Negative Bacilli (NLFGNB) (100 sample) associated with hospital-acquired infections using 16s rRNA sequencing (57% Pseudomonas species). The current work has detected the extended-spectrum βlactamase (ESBL) genes of isolates. Resistant was evident in 31 isolates (Pseudomonas aeruginosa and Acinetobacter Baumannii). This study showed a significantly higher prevalence of Pseudomonas aeruginosa and Acinobacter baumannii among NLFGNB associated with hospital infections. The isolation of MDR Pseudomonas aeruginosa and MDR Acinobacter baumannii in the present study raises the concern of rapidly emerging antibiotic resistance in this group of bacteria particularly in Sudan. The resistance of NLFGNB represents a major challenge for hospital acquired bacterial infection with possibility of multi-drug resistant. Therefore, attempts to identify the genetic pattern and expression of the associated genes in the above-mentioned resistance can have implications on the detection of the relevant genes of extended-spectrum β-lactamase (ESBL).\nThe above-mentioned findings may assist in improving our understanding of the resistant pattern of NLFGNB which is associated with many hospital-acquired infections. The current study showed important clinical relevant information that is relevant to teaching and could be of value to critical thinking learning. The study findings may contribute to future clinical research specially in academic centres with distinct investigations on bacterial resistant.\nFinally, the uptake of the current study needs further research work to generalize the research findings that could be emulated by similar clinical trials.\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/9-1311
https://f1000research.com/articles/9-1309/v1	10 Nov 20	{ "type": "Research Article", "title": "On the transformation of MinHash-based uncorrected distances into proper evolutionary distances for phylogenetic inference", "authors": [ "Alexis Criscuolo" ], "abstract": "Recently developed MinHash-based techniques were proven successful in quickly estimating the level of similarity between large nucleotide sequences. This article discusses their usage and limitations in practice to approximating uncorrected distances between genomes, and transforming these pairwise dissimilarities into proper evolutionary distances. It is notably shown that complex distance measures can be easily approximated using simple transformation formulae based on few parameters. MinHash-based techniques can therefore be very useful for implementing fast yet accurate alignment-free phylogenetic reconstruction procedures from large sets of genomes. This last point of view is assessed with a simulation study using a dedicated bioinformatics tool.", "keywords": [ "MinHash", "p-distance", "evolutionary distance", "substitution model", "phylogenetics", "genome", "simulation" ], "content": "Introduction\n\nTo estimate the level of proximity between two non-aligned genome sequences x and y, recent methods (e.g. 1–7) have focused on decomposing the two genomes into their respective sets Kx and Ky of non-duplicated nucleotide k-mers (i.e. oligonucleotides of size k). A pairwise similarity may then be easily estimated based on the Jaccard index j = \|Kx ∩ Ky\| / \|Kx ∪ Ky\|8. The Jaccard index between two sets of k-mers is a useful measure for two main reasons. First, it can be quickly approximated using MinHash-based techniques (MH9), as implemented in e.g. Mash2, sourmash3, Dashing4, Kmer-db6, FastANI5, or BinDash7. Such techniques select a small subset (of size σ) of hashed and sorted k-mers (called sketch) from each Kx and Ky, and approximate j by comparing these two subsets (for more details, see 2,9–12). Second, the proportion p of observed differences between the two aligned genomes (often called uncorrected distance or p-distance) can be approximated from j (therefore without alignment) with the following formula (e.g. 13,14):\n\n\n\nprovided that both sizes σ and k are large enough, and j is not too low (see below).\n\nAs a consequence, a pairwise evolutionary distance d can be derived from the Jaccard index j using transformation formulae of the following form:\n\n\n\nwhere p is obtained using Equation (1). Parameters b1 and b2 can be defined according to explicit models to estimate the number d of nucleotide substitutions per character that have occurred during the evolution of the sequences x and y, e.g. 15–24. When b1 = b2 = 1, Equation (2) corresponds to the Poisson correction (PC; e.g. 21) distance. Although it is based on a simplistic model of nucleotide substitution1,16,25,26, PC is the p-distance transformation implemented in many MH tools (e.g. Mash, Dashing, FastANI, Kmer-db, BinDash). However, more accurate distance estimates may be obtained by using substitution models based on more parameters. Among these models, equal-input (EI, sometimes called F8118,19,24,27–29) takes into account the equilibrium frequency πr of each residue r in Σ = {A, C, G, T}. An EI distance can be estimated using Equation (2) with b1=1−∑r∈Σπr2 and b2=1−∑r∈Σπrxπry, where πrx and πry are the frequencies of r in the two sequences x and y, respectively20. Further assuming that the heterogeneous replacement rates among nucleotide pairs and sites can be modelled with a Γ distribution, an EI distance d can be derived from p using the following formula:\n\nwhere a > 0 is the (unknown) shape parameter of the Γ distribution, e.g. 22,24,30–33. It is worth noticing that when a is high, Equation (2) and Equation (3) yield very similar distance estimates (for any fixed b1 and b2).\n\nThe aim of this study is to assess the accuracy of Equation (2) and Equation (3) in transforming a MH p-distance p^, where p^ is derived from the MH Jaccard index j^ using Equation (1). In the following, analyses of large sets of simulated nucleotide sequences show three complementary results. First, current MH implementations enable p-distances to be conveniently estimated under several conditions. Second, PC and EI transformations (2) and (3) of MH p-distance estimates can suitably approximate evolutionary distances derived from general time reversible (GTR; e.g. 34) models of nucleotide substitution. Third, PC and EI distances derived from MH estimates enable accurate phylogenetic tree reconstruction from unaligned nucleotide sequences.\n\n\nResults and discussion\n\nVarying d from 0.05 to 1.00 (step = 0.05), a total of 200 nucleotide sequence pairs with d substitution events per character were simulated under the models GTR and GTR+Γ. Each model was adjusted with three different equilibrium frequencies: equal frequencies (f1; πA = πC = πG = πT = 25%), GC-rich (f2; πA = 10%, πC = 30%, πG = 40%, πT = 20%), and AT-rich (f3; πA = πT = 40%, πC = πG = 10%). The GTR substitution rates and the Γ shape parameters were obtained based on a maximum likelihood (ML) analysis of 142 real-case phylogenomics datasets. Overall, ML estimates of Γ shape parameters were quite low (i.e. varying from 0.162 to 0.422, with an average of 0.314), confirming that the heterogeneity of the substitution rates across sites is a non-negligible factor when studying evolutionary processes. Every simulation was completed with indel events, resulting in sequences > 3 Mbs with relative lengths (i.e. longer/shorter) varying from 1.0196 (d = 0.05) to 1.1117 (d = 1.00), on average.\n\nFor each of the 2 (GTR, GTR+Γ) × 3 (f1, f2, f3) × 20 (d = 0.05, 0.10, ..., 1.00) × 200 = 24,000 simulated sequence pairs x and y, the corresponding p-distance was estimated using three MH tools: Mash, BinDash and Dashing. Of note, the accuracy of a MH estimate j^ of the Jaccard index between Kx and Ky is mainly dependent on two parameters: the k-mer size k and the sketch size σ. The size k should be large enough to minimize the probability q of observing a random k-mer shared by x and y by chance alone. Such a value can be obtained from q by k = ⌈log\|Σ\| (g(1−q)/q) − 0.5⌉, where g is the length of the largest sequence2,29,35. The size σ should be large enough to minimize the error bounds of j^2, but also to avoid the inconvenient estimate j^ = 0. Following 29, σ was set by the proportion s of the average sequence length.\n\nTo investigate the impact of both parameters σ and k on the accuracy of the MH estimates, each MH tool was used with s = 0.2, 0.4, 0.6, 0.8 and q = 10−3, 10−6, 10−9, 10−12. As in simulated sequences, g ranges from 4.99 Mbs (d = 0.05) to 3.38 Mbs (d = 1.00) on average, s translates into moderately to very large sketch sizes σ, and q into k-mer sizes k = 16, 21, 26, 31.\n\nTwo statistics were calculated to assess the linear relationship between the MH estimate p^ (derived from j^≠ 0) and the ’true’ p-distance p: the coefficient of determination R2 and the slope β of the linear least-square regression p^ = βp. Let Ψ(pmax) be the subset of pairs (p, p^) such that p ≤ pmax. Varying pmax from 0.10 to 0.55, R2 and β were estimated from Ψ(pmax) (Figure 1). The cumulative proportions fj^=0 of MH Jaccard index j^ = 0 within [0, pmax] were also measured (Figure 1). Finally, every value pr>0.99 was estimated, where pr>0.99 is defined as the highest p-distance such that the subset Ψ(pr>0.99) provides a coefficient of correlation r > 0.99 (as assessed by a Fisher transformation z-test with p-value < 1%; Figure 1). The highest values pr>0.99 were obtained with parameters k = 26 (q ≤ 10−9) and s = 0.8 (illustrated in Figure 2).\n\nFor each sketch size (columns; set by s = 0.2, 0.4, 0.6, 0.8) and each k-mer size (rows; k = 16, 21, 26, 31), three line charts represent different statistics determined with Mash (green), BinDash (red), and Dashing (blue). For pmax ranging from 0.10 to 0.55 (x-axes), represented statistics are (i) the coefficient of determination R2 (up; y-axis ranging from 0.85 to 1.00) and (ii) the slope of the linear least-square regression through the origin (middle; y-axis ranging from 0.8 to 1.2) computed from estimated p^ and corresponding ’true’ p-distances p ≤ pmax, as well as (iii) the cumulative proportion fj^=0 of estimated Jaccard index j^ = 0 within [0, pmax] (bottom; y-axis ranging from 0.0 to 0.3). Circles in R2 line charts (up) indicate the largest value pr>0.99 such that the subset of pairs (p,p^) defined by p ≤ pr>0.99 provides a coefficient of correlation r > 0.99.\n\nThe p-distances p^ estimated by Mash (up left), BinDash (up right) and Dashing (bottom left) with k = 26 (q = 10−9) and s = 0.8 are plotted against the ’true’ p-distances p between 24,000 pairs of nucleotide sequences simulated under six scenaria of evolution: GTR with equilibrium frequencies f1 = (0.25, 0.25, 0.25, 0.25) (green points), f2 = (0.10, 0.30, 0.40, 0.20) (red) and f3 = (0.40, 0.10, 0.10, 0.40) (blue), and GTR+Γ with f1 (cyan), f2 (orange) and f3 (magenta). Points corresponding to j^ = 0 are not represented. Each scatter plot is completed with the least-square regression line through the origin (dashed black line) estimated from the subset of points (p,p^) such that p ≤ pr>0.99, where pr>0.99 = 0.345 (Mash), 0.335 (BinDash) and 0.330 (Dashing).\n\nOne important result (Figure 1) is that current MH implementations return suitable estimates of p as long as p ≤ 0.25, provided that k is sufficiently large. Indeed, when k ≥ 21 (and any s ≥ 0.2), the statistics pr>0.99 are higher than 0.25 (Figure 1), therefore showing that p and p^ are highly linearly correlated when p ≤ 0.25 (see e.g. Figure 2). Interestingly, when p ≤ 0.25, the worthless estimate j^ = 0 was almost never observed with the different selected parameters s and q (Figure 1).\n\nFurthermore, when p > 0.25, large k-mers are required to obtain satisfactory estimates, i.e. k > 21 or q < 10−6 (Figure 1). However, dealing with k > 21 involves using large sketch sizes to minimize the cases j^ = 0 (see fj^=0 in Figure 1). Simulation results suggest that k = 26 (i.e. q = 10−9) and s > 0.4 yield suitable estimates of p, obtained from sequences of lengths > 4 Mbs with pairwise p < 0.35 (see Figure 1 and Figure 2). Indeed, when p ranges between 0.25 and 0.35, small sizes k (e.g. k ≤ 21 or q ≥ 10−6) always provide underestimated p^ (with any s). Large size k (e.g. k = 31 or q = 10−12) results in the same trend, but also in high numbers of useless estimates j^ = 0 (even with large σ; see fj^=0 in Figure 1).\n\nWhen p ≥ 0.35, MH tools always underestimate the p-distances between the sequences simulated for this study (Figure 1 and Figure 2). One could suggest that more accurate MH estimates p^ will be expected with larger sketch sizes σ. Nevertheless, results represented in Figure 2 (i.e. q = 10−9 and s = 0.8, providing the highest pr>0.99) are based on average σ varying from ∼2.7 × 106 (d = 1.00) to ∼3.6 × 106 (d = 0.35), which are larger than some real genomes.\n\nWhen a pairwise p-distance p can be estimated from unaligned nucleotide sequences, it may be transformed into an evolutionary distance d, based on Equation (2) or Equation (3). The relationship between p and d was represented in Figure 3 for different distance estimators: PC transformation (2) (b1 = b2 = 1), and EI transformations (2) and (3) with equilibrium frequencies f1 (b1 = b2 = 0.75 under homogeneous substitution pattern20), f2 (b1 = b2 = 0.70) and f3 (b1 = b2 = 0.66). Parameter a in EI Equation (3) was estimated by least-square regression from the pairs (p, d) derived from the sequences simulated under the models GTR and GTR+Γ (see above).\n\nThe six charts represent the evolutionary distance d(y-axis ranging from 0.00 to 1.00) against the p-distance p (x-axis ranging from 0 to 0.55). Gray points (p, d) are derived from the simulation of sets of 4,000 sequence pairs, each under six different scenaria of evolution: GTR (top) and GTR+Γ (bottom) with equilibrium frequencies f1 = (0.25, 0.25, 0.25, 0.25) (left), f2 = (0.10, 0.30, 0.40, 0.20) (middle) and f3 = (0.40, 0.10, 0.10, 0.40) (right). PC and EI versions of Equation (2) are represented with red and green curves, respectively. EI version of Equation (3) is represented with blue curves for a = 1, and with black curves for the values a = 4.590 and a = 0.291 determined by least-square regression from the gray points derived from the models GTR (top) and GTR+Γ (bottom), respectively.\n\nPC and EI p-distance transformations (2) result in improper underestimates as the expected distance d increases. Indeed, when compared with realistic GTR-based distances d, PC and EI transformations (2) give distance estimates that are always lower than d, especially under GTR+Γ and when d is large (e.g. d > 0.1; Figure 3). This downward bias is somewhat expected, knowing that PC and EI transformations (2) are based on less parameters than both models GTR and GTR+Γ. However, the additional parameter a in Equation (3) may help dealing with heterogeneous substitution rates among residue pairs (e.g. 36). Hence, the relationship between GTR distances d and the corresponding p-distances p can be approximated by the EI transformation (3) with a = 4.590 (Figure 3). Moreover, as d returned by Equation (3) is inversely proportional to a (for any fixed p), the relationship between d and p under the model GTR+Γ (with Γ shape parameter of 0.314, on average) can also be approximated by the EI transformation (3) with a = 0.291 (Figure 3).\n\nThese results show that complex distance measures can be approximated by simple analytical formulae based on few parameters. In practice, nucleotide frequencies (four parameters) can be trivially computed and p-distances (a fifth parameter) can be estimated using MH tools (see above). Therefore, the evolutionary distance d between two sequences that have evolved under the parameter-rich model GTR+Γ can be approximated from these only five parameters using (3) with a ≤ 4.590 (Figure 3).\n\nAt this point, it should be stressed that MH p^ tends to be overestimated. Indeed, MH estimates are of the form p^ ≈ βp with slope β varying from 1.08 (BinDash, k = 31, s = 0.2) to 1.15 (Dashing, k = 26, s = 0.2) when p ≤ pr>0.99 (Figure 1). This has a direct impact on the derived distances: using PC and EI transformations (2) on p^ = βp with β = 1.15 and p ≤ 0.35 provide distance estimates that are quite comparable to the ones returned by Equation (3) with a ranging from 1.000 to 4.590 (see Figure 4 for the equilibrium frequencies f2; similar results were observed with f1 and f3 – not shown). The PC transformation (2) on the upward biased MH p^ returns distances that are then comparable to some complex distance measures (e.g. derived from a GTR model), therefore justifying its use by many MH tools. Nevertheless, the EI transformation (3) remains necessary when dealing with distantly related sequences (e.g. p > 0.2) and strong heterogeneity of the substitution rate across sites (e.g. often observed Γ shape parameter < 1.000). In such cases, the value of the parameter a should always be slightly increased to compensate the MH upward bias. For instance, EI transformation (3) on p with a = 0.291 (i.e. GTR+Γ distance least-square fitting in Figure 3) can be approximated by the same Equation on p^ = βp with β = 1.15 and a = 0.431.\n\nThe relationship between the p-distance p (x-axis ranging from 0.00 to 0.35) and the corresponding evolutionary distance d (y-axis ranging from 0.00 to 0.70) is represented when using PC (red dots) and EI (with equilibrium frequencies f2; green dots) transformations (2) on p^ = β p with β = 1.15. For ease of comparison with Figure 3, EI (f2) transformation (3) on p are represented with a = 1.000 (blue curve) and a = 4.590 (black curve).\n\nTo assess whether MH p-distance transformations may translate into reliable phylogenetic trees, additional simulations were performed. A total of 142 sets of sequences was simulated under the model GTR+Γ along reference phylogenetic trees. Representative GTR+Γ model parameters (same as above) and reference phylogenetic trees were obtained based on a ML analysis of real-case phylogenomics datasets. Sizes of the reference trees ranged from 10 to 154 taxa (31 on average), with diameters (i.e. maximum distance between any two leaves of a tree) varying from 0.204 to 2.883 (0.975 on average). Sequence lengths and indel events were simulated in the same way as the previous sequence pair simulations.\n\nThe script JolyTree v2.0 was used to reconstruct phylogenetic trees from the simulated sequences. For each pair of unaligned sequences, this script estimates the MH p-distance using Mash, and transforms it into an evolutionary distance. Using these MH-based distances, JolyTree next reconstructs a minimum evolution phylogenetic tree with confidence supports at branches, based on a ratchet-based hill-climbing procedure (for more details, see 29). To obtain accurate MH p-distance estimates, JolyTree was run with parameters q = 10−9 and s = 0.5 (see above). Evolutionary distances were estimated using the PC and EI transformations (2), as well as the EI transformation (3). To observe the impact of the parameter a, the EI transformation (3) was computed with a varying from 0.05 to 10.0. The accuracy of each p-distance transformation for phylogenetic inference was assessed by the percentage of recovered reference trees, i.e. identical topologies (Figure 5).\n\nThe percentage of recovered reference trees (y-axis ranging from 50% to 100%) is represented (light blue dots) in function of the parameter a (x-axis ranging from 0.0 to 10.0) in EI formula (3). The overall trend of these dots is illustrated using a moving average (dark blue curve). Dashed lines represent the percentages of recovered reference trees obtained with the PC (red) and EI (green) transformations (2).\n\nUsing JolyTree with EI transformations improves the percentage of recovered reference trees (Figure 5). In spite of their limitations, PC distances result in the recovery of 75.3% of the 142 reference trees, but EI transformation (2) increases this percentage to 76.7% (Figure 5). Furthermore, the EI transformation (3) generally provides better results in a large range of a, i.e. up to 83.1% of recovered reference trees (Figure 5). Low a-values (e.g. a ≤ 0.3) translate into many incorrect tree topologies, whereas high ones (e.g. a > 6) tend to provide the same reference tree recovering percentage as the EI transformation (2) (Figure 5). Most suitable values of a (corresponding to the highest reference tree recovering percentages, e.g. 80%) seem to range in the interval [1.0, 2.0] (Figure 5).\n\nThese simulation results are consistent with two views which are somehow contradictory. On the one hand, accurate (parameter-rich) distance estimates are required, because biased ones (i.e. corresponding to a concave or convex function of the actual evolutionary distances) may result in incorrect phylogenetic trees23,37. On the other hand, simple (underparameterized) distance estimates should often be preferred, because they frequently result in more accurate tree topologies21,38–42. Here, the simple PC and EI transformations (2) (one and five parameters, respectively) enable many reference trees to be recovered (Figure 5). However, the EI transformation (3) is able to approximate realistic distance measures (e.g. GTR+Γ) by using only one supplementary parameter a (Figure 3). It therefore enables more reference trees to be recovered (Figure 5).\n\nIn line with 43, most suitable values of a (e.g. between 1.0 and 2.0) are all higher than the Γ shape parameter values used for simulating the sequence datasets (i.e. varying from 0.162 to 0.422, with an average of 0.314). This can be explained by the MH upward bias (see above), but also by the large variance of the estimate (3) when a becomes low. Reminding that the Γ shape parameters (and the reference trees) used in these simulations were inferred from real-case datasets, these results suggest that using EI transformation (3) with a ≈ 1.5 may be suitable to infer genus phylogenetic trees. In light of this, it should be stressed that the article29 describing the first distributed version of JolyTree (v1.1) incorrectly stated that the Mash output is the MH estimate p^ (instead of its PC transformation). As JolyTree v1.1 uses the EI transformation (2), this misinterpretation translates into the odd transformation formula δ = −b1 loge (1 + loge(1 − p^) / b2). However, as δ can be approximated on p^ ≤ 0.35 by the EI transformation (3) with a = 1.208, this explains the overall accuracy of JolyTree v1.1 despite its use of δ29.\n\n\nConclusions\n\nAlignment-free phylogenetic inference from pairwise MH-based distance estimates is a promising approach. It enables phylogenetic trees to be quickly reconstructed from a large number of genomes without the burden of multiple sequence alignments (see e.g. 2,29,44–52). This report confirms this view by showing that proper evolutionary distances can be easily derived from MH p-distance estimates, therefore enabling accurate phylogenetic inferences.\n\nFirst, although implemented to approximate nearest neighbors in sequence sets, current MH tools (e.g. Mash, BinDash, Dashing) were shown to be able to conveniently estimate pairwise p-distances p up to p ≈ 0.35. In practice, as p is very similar to the one-complement of the Average Nucleotide Identity (ANI; e.g. 29,53,54), MH estimates of p can then be obtained between genomes gathered from many bacteria, archaea or eukaryota genera, i.e. with pairwise ANI > 65%.\n\nSecond, the EI p-distance transformation (3) was proven efficient to approximate complex distance measures, e.g. derived from GTR model with heterogeneous substitution rates across sites. Because of an upward bias observed in MH p-distance estimates, simpler transformations (based on few parameters, as the commonly used PC) still provide distance measures that are comparable to GTR ones, but with (unrealistic) homogeneous substitution rates across sites. However, thanks to its supplementary parameter a, EI transformation (3) remains necessary to approximate distance measures between distantly related sequences that have arisen from more realistic substitution events.\n\nThird, as proper evolutionary distances can be derived from MH p-distance estimates, their efficiency in phylogenetic inference was established using the dedicated tool JolyTree29. In particular, the EI transformation (3) with a ≈ 1.5 enables accurate phylogenetic trees to be inferred.\n\n\nMethods\n\nTo simulate the evolution of nucleotide sequences according to realistic substitution processes, the 187 genus datasets compiled in 29 (available at https://doi.org/10.3897/rio.5.e36178.suppl2) were first considered to infer a representative range of GTR parameter values. For each of the 187 genera, the associated genome assemblies were processed using Gklust v0.1 to obtain one representative genome assembly for each putative species. This analysis provided 142 sets of representative genome assemblies after discarding genera containing < 10 putative species. For each of these 142 sets, coding sequences were clustered using Roary v3.1255. Each cluster with at least four coding sequences was used to build a multiple amino acid sequence alignment using MAFFT v7.40756. Multiple sequence alignments were back-translated at the codon level and concatenated, leading to 142 supermatrices of nucleotide characters. A phylogenetic tree was inferred from each supermatrix of characters using IQ-TREE v1.6.7.257 with evolutionary model GTR+Γ. All data related to these analyses are publicly available as Extended data at https://doi.org/10.5281/zenodo.403424458.\n\nTo assess the accuracy of different pairwise distance estimates, a simulation of sequence pairs was performed under both models GTR and GTR+Γ with three different sets (πA, πC, πG, πT) of equilibrium frequencies: f1 = (0.25, 0.25, 0.25, 0.25), f2 = (0.10, 0.30, 0.40, 0.20), and f3 = (0.40, 0.10, 0.10, 0.40). For each of these six scenaria and for each d varying from 0.05 to 1.00 (step = 0.05), the program INDELible v1.0359 was used to simulate the evolution of 200 sequence pairs with d substitution events per character. Initial sequence length was 5 Mbs, and an indel rate of 0.01 was set with indel length drawn from [1, 50000] according to a Zipf distribution with parameter 1.5. For each simulated sequence pair, model parameters (i.e. GTR: six relative rates of nucleotide substitution; GTR+Γ: six rates and one Γ shape parameter) were randomly drawn from the 142 sets of estimated ones (see above). All simulated sequences are publicly available as Extended data at https://doi.org/10.5281/zenodo.403446160.\n\nTo compare the efficiency of p-distance transformations for phylogenetic reconstruction, the program INDELible v1.03 was also used to simulate the evolution of a sequence along each of the 142 phylogenetic trees previously inferred from different genera (see above). For each of the 142 genera, sequence evolution was simulated under the model GTR+Γ with the corresponding parameters (i.e. four nucleotide frequencies, six relative rates, and one Γ shape parameter). Sequence length and indel events were simulated as described above. The 142 simulated sequence sets are publicly available as Extended data at https://doi.org/10.5281/zenodo.403464361.\n\nMH p-distances were estimated with Mash v2.2, BinDash v1.0, and Dashing v0.3.4-11-gb44a. BinDash and Dashing were used with the MH b-bit ﬂavor with b = 18. Of note, as Mash and Bindash directly return the PC distance d, the corresponding p-distance was computed by p = 1 − e−d .\n\nPhylogenetic tree reconstructions from simulated sequences were performed with the script JolyTree v2.0. This version implements the PC and EI transformations (2) and (3) of the pairwise p-distances estimated by Mash. If any, missing evolutionary distances duv = ∅ (i.e. corresponding to j^ = 0 or p ≥ b2) between sequences u and v are approximated by JolyTree from the other non-missing evolutionary distances by duv=min⁡x≠u,v; dxu,dxv≠∅(dxu+dxv). This fast approximation is derived from the triangle inequality property duv ≤ dxu + dxv expected from the triplet of evolutionary distances induced by any sequence triplet u, v, x (see e.g. 62).\n\n\nData availability\n\nA list of the 14,244 genome assemblies used to build the 187 genus datasets (Supplementary material of 29). https://doi.org/10.3897/rio.5.e36178.suppl2.\n\nZenodo: Phylogenomic analyses of 142 prokaryotic genera. https://doi.org/10.5281/zenodo.403424458.\n\nZenodo: Simulated pairs of nucleotide sequences for testing (alignment-free) genome distance estimate methods. https://doi.org/10.5281/zenodo.403446160.\n\nZenodo: Model trees and associated simulated nucleotide sequences for testing phylogenetic inference methods. https://doi.org/10.5281/zenodo.403464361.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe author thanks Pascal Campagne for his meaningful comments on the manuscript. The author is also obliged to Sylvain Brisse and to the Hub de Bioinformatique et Biostatistique, Institut Pasteur, Paris (France), for their support. This work used the computational and storage services (TARS cluster) provided by the IT department at Institut Pasteur, Paris.\n\n\nReferences\n\nFan H, Ives AR, Surget-Groba Y, et al.: An assembly and alignment-free method of phylogeny reconstruction from next-generation sequencing data. BMC Genomics. 2015; 16(1): 522. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOndov BD, Treangen TJ, Melsted P, et al.: Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 2016; 17(1): 132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTitus Brown C, Irber L: sourmash: a library for MinHash sketching of DNA. Journal of Open Source Software. 2016; 1(5): 27. Reference Source\n\nBaker D, Langmead B: Dashing: Fast and accurate genomic distances with HyperLogLog. bioRxiv. 2019. 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Reference Source" }	[ { "id": "74633", "date": "26 Nov 2020", "name": "Brian Ondov", "expertise": [ "Reviewer Expertise High-Performance Computing", "Locality Sensitive Hashing", "Sequence Alignment" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes a series of simulations assessing various parametric transformations of MinHash-based genomic distance estimates into more robust evolutionary distances. It concludes that (1) MinHash methods accurately estimate nucleotide distances, (2) that accurate estimates of corrected evolutionary distances can be derived from these, with the more parameterized of two models performing better, and (3) that accurate phylogenies can be inferred from those estimates.\nThe manuscript is well written and generally clear, although some of the methods are described so technically as to be hard to follow (e.g. pmax and pr>0.99 in \"MinHash-based p-distance approximation\").\nMy main concern, which is not large, is the choice of the sketch size, s (or σ). The MinHash family of algorithms assumes s << G (genome size). The smallest sketch size tested here, however, is 1/5 of the average genome size. This size, described as \"moderately large,\" is actually two or three orders of magnitude larger than Mash's default, for example. The justification for this is to lower the error bounds. As a consequence, however, what is being tested here is, in a sense, the viability of pure k-mer counting a la Fan et al., rather than the MinHash approaches, which trade some accuracy of the Jaccard estimation for speed. This is still useful, but could be somewhat misleading for those that need the speed benefits of running MinHash with more typical parameters. Additionally, since the sweep of s is linear, but MinHash error bounds relate to an exponential of s, the sweep ends up being less informative than it could be, which is likely why the columns of Figure 1 are nearly identical.\nAs a simple remedy, I would suggest sweeping the parameter s exponentially rather than linearly, starting closer to the default sketch size for the tools. It could still end closer to the genome size to get a sense of the behavior of MinHash as it degenerates to the actual k-mer Jaccard score. Along these lines, it would also make sense for the later phylogenetic experiments to either use a smaller s or do another sweep of values.\nAs a minor point when listing relevant software in the introduction: Dashing is based on HyperLogLog sketching, which is similar in concept to MinHash but algorithmically distinct.\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": [] }, { "id": "74629", "date": "01 Dec 2020", "name": "Burkhard Morgenstern", "expertise": [ "Reviewer Expertise Software development for sequence comparison and phylogeny. Alignment-free sequence comparison." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author investigates how phylogenetic distances between genomic sequences, calculated by MinHash methods, can be transformed into more meaningful distances, based on complex models of nucleotide substitutions.\nIn the last few years, a number of alignment-free methods have been developed to calculate distances between genomic sequences. While earlier alignment-free methods used rough measures of sequence (dis)similarity, some methods have been proposed recently that accurately estimate distances - the number of substitutions per position since two sequences evolved from their last common ancestor - based on stochastic models of evolution (see below). These methods are restricted, however, to the simplest possible model, the Jukes-Cantor model.\nIn recent papers, MinHash techniques were proposed as an attractive way of estimating the p-distance between DNA sequences, i.e. the number of mismatches per position in an (unknown) alignment of the compared sequences.\n\nThe present paper shows how distances based on complex models of evolution (GTR, GTR + \\Gamma) can be approximated using MinHash-based p-distances. The approach is carefully evaluated based on simulated sequences.\nThe proposed method is a welcome and useful addition to existing alignment-free methods, extending them to more realistic models of evolution. The approach is novel, the paper is very well written and clear, and suitable references are given to the literature for more details. Therefore, I support indexing of the manuscript.\n\nA certain limitation is that the manuscript is restricted to MinHash distances. According to the author, these methods are accurate for p-distances roughly < 0.25 (for reasonable k-mer length), but are less accurate for larger distances. However, a number of other alignment-free methods have been proposed that accurately estimate distances for much larger distances, e.g. Kr (Haubold et al., 20091), FSWM (Leimeister et al., 20172), Phylonium (Klötzl and Haubold, 20193), Slope-SpaM (Röhling et al., 20204). All these methods estimate distances based on the simple Jukes-Cantor model; the program Co-phylog estimates non-corrected p-distances (Yi and Jin, 20135). It should be straight-forward to transform these distances to more complex models, as done in the present paper, and to compare them to the MinHash methods evaluated in the paper.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "74631", "date": "03 Dec 2020", "name": "Guy Perrière", "expertise": [ "Reviewer Expertise Phylogenetics", "comparative genomics and bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have no special criticism on the methodology and my comments on that part are really minor. First, what is the justification to the choice of a value equal to 1.5 for the shape of the Zipf distribution used to simulate the indels. As a user of INDELible myself I know very well how this value can be of importance so I would like to know how this value was chosen (arbitrary choice?). Second, I am not sure that the right reference for the computation of missing distances from the triangle inequality property is Guénoche and Grancolas (1999). The first paper I have seen on that topic is Lapointe and Kirsch (19951). Also, I have no concerns on the results presented and everything seems ok for me.\nThe conclusion of the manuscript presents the approach of phylogenetic inference from pairwise MH-based distance estimates as a possible alternative to classical methods of phylogenetic reconstruction (especially in bacteria). Indeed, a classical phylogenomic approach involves the chaining of a complex set of procedures: the identification of orthologous genes in multiple species; the alignment of these genes; and finally, the building of a tree from the concatenation of those alignments. I think that the inclusion in the paper of a comparison of two phylogenies obtained with: i) the MH-based distance and ii) a concatenation would strengthen this claim. A lot of concatenation datasets extracted from complete bacterial genomes are available and it would be not too difficult to do the experiment. I think this addition is of importance because I have no idea on the influence horizontal gene transfers can have on MH-based reconstructions when classical approaches try to minimize this influence.\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/9-1309
https://f1000research.com/articles/9-699/v1	13 Jul 20	{ "type": "Research Article", "title": "Workplace management initiatives and talent engagement in the Nigerian pharmaceutical industry", "authors": [ "Hezekiah O. Falola", "Opeyemi O. Ogueyungbo", "Oluwatunmise O. Ojebola", "Opeyemi O. Ogueyungbo", "Oluwatunmise O. Ojebola" ], "abstract": "Background: Talent engagement is increasingly gaining the attention of pharmaceutical industry, particularly in developing nations like Nigeria. The existing literature shows that the subject of workplace management initiatives and talent engagement in the Nigerian pharmaceutical industry has not been sufficiently researched. This study investigates the influence of workplace management initiatives on talent engagement in some selected pharmaceutical companies in Nigeria. Methods: In total, 600 respondents were surveyed across various departments and units of ten selected pharmaceutical companies in Nigeria using multiple sampling techniques. Only 429 copies of the questionnaire, representing a 71.5% response rate, were returned and analyzed using Smart PLS 3.0. Results: The outcomes of the statistical analysis show that recognition, employees’ wellbeing, learning and development as well as diversity and inclusion had significant influence on talent, emotional, cognitive and behavioural engagements. Conclusions: In line with the statistical results, the study concludes that workplace management initiatives influenced talent engagement. The study emphasized the need for the review of many workplace management initiatives in order to determine its suitability within the context of pharmaceutical industry in Nigeria.", "keywords": [ "Recognition", "wellbeing", "learning and development", "diversity and inclusion", "talent engagement" ], "content": "Introduction\n\nMost organizations in the 21st century experience stiff competitions that necessitates continuous changes currently observed in the global business environment. Organizations, particularly the pharmaceutical companies are now more concerned with fostering sustainable talent engagement initiatives (Ikon & Chika, 2017). Talent engagement is believed to be sustained through strategic management tactics that motivate and stimulate high and productive talent engagement (Falola et al., 2018a). In this regard, workplace management initiatives could play a more prominent role in driving talent engagement in the pharmaceutical industry.\n\nIt has been established in the literature that management initiatives drive employee engagement and high performance (Falola et al., 2018a; Ikon & Chika, 2017). Previous studies in this context identified various forms of management initiatives that drive work engagement. For instance, Oludayo et al. (2018) studied work-life balance and flexible work arrangements as management initiatives that can be adopted in the banking sector. Falola et al. (2020a) examined various forms of initiatives provided by the management of institutions of higher learning in driving job effectiveness such as research grants, conference support initiative, and pedagogy support among others. Falola et al. (2018a) also studied the influence of work engagement initiatives as predictors of academic staff behavioral outcomes in Nigeria Universities. Other researchers also studied diversity management initiatives, workplace stress management initiatives, emotional intelligence initiatives, and organizational energy initiatives among others (Aguwa et al., 2014; Ibidunni et al., 2019; Onyokoko et al., 2019; Oseni, 2017). Most of these studies were carried out mostly in institutions of higher learning, the banking industry, the fast moving consumer goods (FMCGs) manufacturing sector and the public health sector in Nigeria. This suggests that there is knowledge gap in the area of workplace management initiatives and talent engagement in Nigeria pharmaceutical industry. This current study investigates the influence of workplace management initiatives (recognition, wellbeing, learning and development as well as diversity and inclusion) on talent engagement (talent emotional engagement, talent cognitive engagement and talent behavioural engagement) in the pharmaceutical industry in Nigeria.\n\nMoreover, most existing empirical studies used correlation and regression for the analysis of the collected and coded data, however, the present study adopts Smart PLS 3.0 as the statistical tool for the analysis. This gap in the literature is given attention in this current study by looking at the influence of workplace management initiatives on talent engagement in the pharmaceutical industry in Nigeria with the following specific objectives. The objectives are to: investigate the influence of employee outstanding performance recognition on talent engagement; examine the effect of employees wellbeing initiative on talent engagement; determine the effect of learning and development initiatives on talent engagement and to investigate the influence of diversity and inclusion on talent engagement with the use of more advance analytical statistical tool. The insight offers by this study can potentially help the management of the pharmaceutical industry in Nigeria to improve on the workplace management initiatives that will enhance talent engagement in the industry. The findings of this study can be leveraged by management and other stakeholders in the industry for policy formulation and implementation that will enhance talent engagement.\n\n\nLiterature review\n\nWellbeing according to the World Health Organization (2020) is defined as the state of good physical health, mental, emotional, social and financial wellness. Wellbeing is the state of employee’s state of happiness, self-actualization, life satisfaction and positive mood in the workplace. Studies have shown that if employees are healthy, satisfied and emotionally stable, they will be more productively engaged (Bakker et al., 2014; Falola et al., 2018a; Smith & Smith, 2019). Thus, for organizations to earn employees engagement, their wellbeing must be prioritized. This can be done by putting in place initiatives in the areas of their financial, health, educational and financial wellness (Keeman et al., 2017). The financial wellness initiatives assist employees to manage their daily finances and achieve long-term financial goals. Organizations do offer financial counseling in the areas of budgeting, managing assets, building credit, reducing debt and saving for retirement to ensure employee financial wellness (Keeman et al., 2017).\n\nSome organizations provide employees with access to earned wages based on demand to help them solve cash flow problems, especially for low-wage workers (Bennett et al., 2017). All these are some of the initiatives that motivate employees to be more engaged in the world of work. Educational wellness on the other hand is a management initiative to encourage employees to further their education through full or partial scholarships (Bennett et al., 2017). Organizations also help to improve employee’s health status by conducting regular health screenings and tests to deal with preventable and chronic conditions (World Health Organization, 2020). Studies have shown that employees with high level of health, financial and educational wellbeing put in positive thought and effort into their job responsibilities in the work place (Keeman et al., 2017; Saks, 2016). This implies that organizational wellbeing initiatives could enhance employee engagement in their job roles and functions.\n\nRecognition is the informal, timely or formal acknowledgement of an individual or team's effort. If efforts of employees are adequately recognised with appropriate performance incentives, employees’ engagement will be enhanced (Falola et al., 2018b). Nazir & Islam (2017) classified employee recognition into four major types, these include personal recognition, result recognition, work practice recognition and job dedication recognition. Generally, everyone wants to be recognised. Masri & Elsuliman (2019) argued that the most important form of employee recognition is work practice recognition. Findings reveal that lack of work practice recognition is very risky because it contributes to workplace distress (Masri & Elsuliman, 2019; Nazir & Islam, 2017).\n\nRecognizing employee’s achievement sends a positive signal of being valued and cared for by the organizational management which engenders their dedication and engagement to organizational goals (Kahn, 1990). This is reflected in their acquisition of more knowledge and experiences (talent cognitive engagement), display of positive feelings and emotions towards their jobs, supervisors and colleagues (talent emotional engagement) and in physical actions put into jobs (talent behavioural engagement). Inegbenojie (2018) reiterated that employee recognition is a management tool used to achieve talent engagement in organizations. Workplace management initiatives such as recognition and employee wellbeing foster employees’ engagement in the world of work.\n\nTechnological advancement, e-commercialization and globalization have promoted diversity and inclusion of a diverse workforce. Diversity brings people of different ages, skills, language, education, values, competences, beliefs, sexual orientation, race, disability, religion, tribe, culture, gender, colour, ethnicity, nationality, lifestyle and economic status together (Akinnusi et al., 2017). Diversity in the organization is not tantamount to chaos and reduced output as a creative and engaged workforce with diverse ideas and talents can foster the accomplishment of organizational goals. For instance, an organization that has significant mix of male and female gender is considered a gender diverse organization (Onwuchekwa et al., 2019).\n\nManagement initiatives of managing diversity/inclusion is an organizational strategy used to foster an encouraging workplace environment. Diversity management initiatives must recognise, respect, tolerate and accept differences among employees (Akinnusi et al., 2017). Talent engagement according to Macey et al. (2019) is determined by many interactions in the workplace, one of which is diversity. Scholars like Macey et al. (2019); Onwuchekwa et al. (2019) asserted that employee engagement is dependent upon various factors in the workplace. Nevertheless, Inegbenojie (2018) opine that individual differences and effective inclusive management approach play a vital role in determining an employee's level of engagement.\n\nLearning and development is organizational consistent effort in enhancing employee acquisition of knowledge, job-related skills and improved employee behaviour. Employees are able to acquire, transfer, interpret and share information better and more engaged with their core job responsibilities (Chiekezie et al., 2016). Learning and development is an essential administrative function of human resource management (HRM) that helps the employees to be productively engaged. According to Adelere (2017) employees are the most important assets of any organization, superseding money, materials and machines. According to Siddiqui (2019) employee learning and development is considered as the most essential function of any proficient organization, including those in the pharmaceutical industry. As a result, the pharmaceutical industry has invested heavily in employee learning and development to operate the machines used for drug production processes, which in turn better facilitates employee engagement which is a major driver of competitive advantage (Ogueyungbo et al., 2019).\n\n\nMethods\n\nThe study used a survey for data collection which was performed between November 2018 and January 2020.\n\nRespondents profile: Gender: Male 249 (58%), Female 180 (42%); Age: Most of the respondents (284; 66%) were between 31–50 years; Marital Status: Single 111 (25.9%), Married 286 (66.7%) others 32 (7.5%); Work experience: Most of the respondents (330; 77%) had between 6–20 years work experience; Level: Middle management 236 (55%), Senior management 169 (38.9%), Executive management 26 (6.1%).\n\nThere are 115 registered pharmaceutical companies in Nigeria out of which over 50% are located in Lagos State (Pharmapproach, 2018). The ten best rated pharmaceutical companies were selected in Lagos State Nigeria. Lagos State was chosen because of the high concentration of pharmaceutical companies in the state. In addition, all the ten selected companies manufacture their products locally. The selection of the ten pharmaceutical companies was based the year of establishment, the number of brands, growth and expansion, the total number of products manufactured, and the corporate image (Pharmapproach, 2018; PMG-Man, 2019). These culminated into the level of performance, scientific innovation and quality service delivery of the selected companies. The justification for the criteria used are presented as follows:\n\ni. The selected companies are credible indigenous pharmaceutical organizations that have distinguished themselves in the pharmaceutical industry and have been in existence for a minimum of 40 years with uninterrupted production.\n\nii. They produce a minimum of 100 branded pharmaceutical products to their names, ranging from tablets, caplets, oral liquid syrups and other consumables.\n\niii. They also produce a minimum of 1 billion tablets, 250 million capsules, 20 million powder sachets annually. They have a presence in all the geo-political zones in Nigeria where they either produce, market or distribute pharmaceutical products that meet international standards to government at all levels and private health care providers.\n\niv. The criteria for determination of the corporate image include: (1) recognizable brand, (2) familiarization of people with the companies, (3) the level of partnership, and (4) perceived opinion of customers and employees of the selected companies.\n\nThe ten selected pharmaceutical companies have a total 3787 employees excluding casual, contract and lower level staff which represents the population of the study. Sample size was determined using Bartlett et al. (2001) table chart and the sample size accounted for 570 and was approximated to 600 to allow for unreturned copies of administered questionnaire at the margin of error of 0.05. Proportional affixation criterion (PAC) was used for the determination of the copies of questionnaire administered to each organization. Pharmaceutical companies sample in each stratum is proportional to the relative weight of the study population as depicted in Table 1.\n\nMultiple samplings (purposive, stratified and simple random) techniques were used in this study. Purposive sampling was used because only the permanent employees at the middle, senior and executive management level cadres with a minimum of 2 years in their current organizations participated in the survey in order to get more accurate and reliable information. Also, stratified sampling was adopted because the population comprises different strata and all employees in each stratum were giving equal chance of been selected with the use of a simple random sampling method. The study used a structured questionnaire adapted from the existing literature to collect data from the respondents with the use of 5-point Likert scale. Copies of questionnaire were administered by the research staff with the help of three research assistants during working hours.\n\nThe pharmaceutical organizations must be producing locally.\n\nRespondents must be employees of the selected pharmaceutical organizations.\n\nOnly middle management level employees and above could participate.\n\nRespondents must have spent a minimum of two years in their current pharmaceutical organizations.\n\nRespondents must be permanent staff of the selected pharmaceutical organizations.\n\nRespondents must be literate enough to read and write English.\n\nCasual, contract staff as well as lower level management were excluded from the survey.\n\nTo measure workplace management initiatives, the authors created a workplace management initiatives (WMI) scale by adapting the elements of Cannon (2015); Inegbenojie (2018) and Keeman et al. (2017) instruments. The researchers developed the measurement instruments into 12 items representing the four domains of (1) recognition, (2) employee wellbeing, (3) learning and development, and (4) diversity and inclusion. Each of the domain was measured with three items each on a 5 Likert scale of strongly agree (5), agree (4), indifference (3), disagree (2), and strongly disagree (1). Based on the response options categorization, the higher total scores on the scale indicate respondents' perception on the subject of workplace management initiatives The following are the examples of the items: “My organization takes time to publicly acknowledge my successes”, “My organization provides incentives, bonuses and other rewards for outstanding performance”, “I receive praise from my supervisor when I successfully reach performance goals or other targets”, “My organization gives adequate attention to employees’ wellbeing”, “The welfare packages for employees are motivating”, “My organization gives priority to safety of lives and property of the employees”, “My organization provides conducive environment that encourages learning and development”, “I have access to the learning and development I need to do my job well”, “This is great company for me to make a contribution to my development”, “Employees who are different from most others are treated fairly in my organization”, “My organization is committed to diversity and inclusion”, “In my organization, everyone has access to equal employment opportunities regardless of their differences”.\n\nTo measure talent engagement, the authors adapted the elements of Nazir & Islam (2017) instruments in developing the items. The measurement instruments were developed into nine items representing the three domains of (1) cognitive engagement, (2) behavioural engagement, and (3) emotional engagement. Each of the domain was measured with three items each on a 5 Likert scale of strongly agree (5), agree (4), indifference (3), disagree (2), and strongly disagree (1). Based on the response options categorization, the higher total scores on the scale indicate respondents’ perception on the subject of talent engagement. The examples the items are: “My job allows me to be creative in performing my responsibilities”, “My organization encourages me to use my skills and capabilities in the work process”, “I often think of ways and suggestions to improve my work”, “I feel a sense of belonging and identify with my organization”, I am proud to work in this organization”, “I feel like staying in this organization”, “I exert a lot of energy doing work”, “I feel energized and empowered at work”, “I stay at work till the job is finished”. Face and content validity of the items were checked while internal consistency reliability testing was carried out through composite reliability, average variance extracted (AVE) estimate as well as Cronbach's Alpha. A copy of the questionnaire is provided as extended data (Falola et al., 2020b).\n\nThe principal investigator submitted an application for ethical approval of research proposals to the Business Management Research Ethics Committee on 9th October 2018 for approval (Approval No: BMREC/18/21/102). Research team was given introduction letter that was presented to the selected organizations stating the purpose of the research. All the participants were adequately informed about the objective of this study. Only willing employees participated in the survey. All respondents were assured they would stay anonymous and assured that their responses will be treated with extreme confidentiality. It is equally imperative to note that verbal consent was gotten from the respondents of this research. The representatives of the selected pharmaceutical companies were consulted for research permission guidelines. Based on the information provided in principle, an application letter was written requesting permission to research their organizations with the objective of the study clearly stated. Also, the research ethics approval form was attached to the application letter. This type of research is categorised as exempt research that involves a survey with no or minimal risk i.e. level 1 research as presented in the Research Ethical Application Form. In social and management sciences, exempt research does not require signed consent from the participants rather implied consent is usually sufficient particularly in the spirit of anonymity and confidentiality. Through verbal consent, the researchers ensured that the respondents were well informed about the context and intent of this research and were kept abreast with the process and regime of participation.\n\nThe research instrument validity was determined through face and content validity by two distinguished professors in the field. Meanwhile factor loadings, compose reliability, average variance extracted (AVE) estimate as well as Cronbach Alpha were carried out to ascertain the reliability of the research instrument. A pilot study was conducted to ascertain the validity and reliability of the research instrument. As recommended by Baker (1994) the sample size for pilot study should be at least 10% of the study population. The sample size of the main study was expected to be 600 using Bartlett et al. (2001) table chart. In total, 60 copies of questionnaire were administered to a pharmaceutical company in Ota. The pilot result shows that data was normally distributed and the scale reliabilities (factor loadings, compose reliability, average variance extracted (AVE) estimate as well as Cronbach’s Alpha) were well above the benchmarks as recommended by Byrne (2001) and Tabachnick & Fidell (2007). The statistical outcomes are depicted in Table 2.\n\nStatistical Package for Social Sciences (SPSS) software version 22 was used for the coding of the retrieved data while Smart PLS 3.0 was used for the analysis of the data that shows the degree of influence of workplace management initiatives on talent engagement in the pharmaceutical industry. The smart PLS displays the algorithm and bootstrapping models. Algorithm model is a structure of regressions in terms of weight vectors that helps to determine the path co-efficient, the r-square values and the significance values. The bootstrapping helps in determining the significance testing of coefficient and t-values. The default bootstrapping in Smart PLS is 500 subsamples which helps to facilitate significant results. This was enhanced by increasing bootstrapping value to 5000 as suggested by (Ramayah et al. (2018). Meanwhile, linear regression analysis can as well be used as alternative statistical tool for the analysis. Also, the processes and procedures required for the assumptions of credible research analysis were systematically and carefully checked to be sure that the data presented are sufficiently adequate and accurate as recommended by Kline (2005). The assumptions include, appropriate research design, reliabilities, data normality and linearity. In total, 429 copies of questionnaire representing (71.5%) response rate were returned and used as final sample for this study.\n\n\nResults\n\nWorkplace management initiatives were measured with four constructs: recognition, wellbeing, learning and development, and diversity and inclusion while affective engagement, behavioural engagement, cognitive were used to measure talent engagement. For the purpose of the interpretation of the analysis, R-Square i.e the coefficient of determination, structural path co-efficient (B value), T-statistic value, and P-values are critical indicators of Smart PLS used for the determination of the results as depicted in Figure 1–Figure 3 (Falola et al., 2020b).\n\nPLS: partial least squares, PLS Algorithm is used for formative scales. R: recognition, EW: employee wellbeing, LD: learning & development, DI: diversity & inclusion.\n\nPLS: partial least squares, β: beta value, T values: calculated differences represented in units of standard error, R: recognition, EW: employee wellbeing, LD: learning & development, DI: diversity & inclusion.\n\nPLS: partial least squares, β: beta value, P values: calculated probability, R: recognition, EW: employee wellbeing, LD: learning & development, DI: diversity & inclusion.\n\nFigure 1 depicts PLS algorithm model of workplace management initiatives that drives talent engagement. The path depicts the degree of relationship between the observed variables. The R-square which determines the level of variance of talent engagement and workplace management initiatives. R-square could be substantial (>0.75), moderate (>0.50) or weak (>0.25.). Figure 1 shows that the R2 = 0.909 for workplace management initiatives. This implies that all the factor loading items of talent engagement weakly but significantly explain 90.9% of the variance in workplace management initiatives. In a related development, Figure 1 also indicates that talent engagement collectively explained 60.1% of the variability of talent engagement. This implies that the items of workplace management initiatives explain 60.1% of the variance in talent engagement.\n\nBootstrapping is recommended by Ramayah et al. (2018) was used to evaluate T-values. We also reran the analysis with the bootstrapping setting to 5000 as suggested by Henseler et al. (2012) and Garson (2016) for confirmatory purposes. Bootstrapping helps in calculating path coefficients, outer loading, outer weights, indirect effect and total effect as depicted in Figure 2. All the T-values presented in Figure 2 are above 1.96 while the p-values presented in Figure 3 are significant at 0.05. This suggests that workplace management initiatives have significant influence on talent engagement.\n\nThe β value which indicates the expected variance in talent engagement for a unit variation in the workplace management initiatives was used to test the significance of the formulated hypotheses. The greater the β value the more the substantial effect on workplace management initiatives. The significant effect of workplace management initiatives on talent engagement was verified through the T-statistical test. The path co-efficient of the observed variables are presented in Table 3 (Falola et al., 2020b).\n\n\nDiscussion\n\nFurther to the PLS statistical and empirical results presented in Table 3, it was observed that the structural path co-efficient of the measures of workplace management initiatives i.e recognition, employee wellbeing, learning and development and diversity and inclusion indicate significant relationship at 0.05. Similarly, the path co-efficient also revealed that employee outstanding performance recognition initiatives (R1) indirectly and significantly influenced talent engagement (β=0.463, T-value=4.427, P-value =0.000 <0.05). This implies that, if an employee outstanding performance recognition initiative is given priority by the management of pharmaceutical industry it will significantly enrich talent engagement. This finding corroborates Masri & El-suliman (2019) submission that recognition of employee outstanding performance through various performance incentives plays a significant role in the level of talent engagement. This suggests that for the pharmaceutical industry in Nigeria to enhance talent engagement, recognition of optimal and excellent performance should be given priority. This also supports the findings of (Inegbenojie, 2018) who noted that employee recognition of performance as a result of training provided enhances job engagement.\n\nThe path co-efficient value shows that employee wellbeing has an indirect significant influence on talent engagement (β=0.158, T-value=4.427, P-value =0.000 <0.05). This implies that employee wellbeing, if strengthened by the management of the pharmaceutical companies, will enhance talent engagement. This finding validates the submission of Keeman et al. (2017) and the World Health Organization (2020). The finding also corroborates the findings of Bennett et al. (2017) that if management are concerned about the wellbeing of their workforce, it will naturally motivate employees to be emotionally and cognitively attached to the organization with a sense of job satisfaction. Saks (2016) noted that if employees are satisfied with their job, the level of their engagement will be enhanced.\n\nSimilarly, learning and development also have indirect significant influence on talent engagement (β=0.464, T-value=9.978, P-value =0.000 <0.05). This finding validates the submissions of (Adelere, 2017; Chiekezie et al., 2016; Siddiqui, 2019). They found out that learning and development will increase the level of talent engagement considerably. The implication of this is that if management of the pharmaceutical companies invest in the learning and development of employees, their level of exposure to the best practices in their various specialisation will be enhanced. As noted by Siddiqui (2019) well exposed employees are likely to be more productively engaged.\n\nIn a related development, it was also revealed from the path co-efficient that diversity and inclusion has an indirect significant influence on talent engagement (β=0.270, T-value=6.135, P-value =0.000 <0.05). This implies that diversity and inclusion initiatives of the management in the pharmaceutical industry will promote and enhance talent engagement. This corroborates with the findings of Onyokoko et al. (2019) and Emeh et al. (2017). Akinnusi et al. (2017) also validates this finding and posited that diversity and inclusion give employees a sense of belonging. As noted by Schneider et al. (2017), if employees perceived that they have a sense of belonging, they tend to be more engaged. Table 3 also shows that learning and development has the highest beta value among other measures of workplace management initiatives that best predict talent engagement. This suggests that management of the pharmaceutical companies should continue to invest in the learning and development initiative that drives talent engagement. Largely, the path co-efficient shows that the level of influence of workplace management initiatives on talent engagement is statistically significant with a beta value of 0.775 with T-Statistical value of 19.815. This implies that workplace management initiatives have significant influence on talent engagement. The beta value of 0.775 suggests 77.5% influence on principal variable i.e. if one unit of workplace management initiatives increases, then 77.5% talent engagement will increase.\n\n\nConclusion\n\nWorkplace management initiatives in driving talent engagement in Nigeria pharmaceutical industry cannot be overemphasised. Therefore, investing in workplace initiatives that drive talent engagement can be regarded as a strategic approach to promoting engagement. Recognition of excellent performance through performance incentives, employee wellbeing packages, relevant learning and development initiatives as well as diversity and inclusion are some of the workplace management initiatives that can be leveraged by the management of the pharmaceutical industry in Nigeria to enhance talent emotional, cognitive and behavioural engagement.\n\n\nLimitations and suggestions for further studies\n\nThis study covers only ten outstanding pharmaceutical companies in Lagos, Nigeria. This implies that the scope of the selected organization is not wide-ranged. Therefore, future studies may broaden the scope of the study to include other pharmaceutical companies in other major cities and zones in Nigeria. This study investigated the influence of workplace management initiatives on talent engagement. Only four constructs such as recognition, wellbeing, learning and development as well as diversity and inclusion were used for the measurement of workplace management initiatives, future study can add to the number of constructs an also introduce moderating variable that may help in strengthening relationships between workplace management initiatives and talent engagement. Future study can also look at the possibility of using mixed method as against the quantitative method used in this current study for a more elaborate finding.\n\n\nData availability\n\nFigshare: SPSS DATA-WMI.sav. https://doi.org/10.6084/m9.figshare.12589571.v1 (Falola et al., 2020b)\n\nThis project contains the following underlying data:\n\n- SPSS DATA-WMI & TE.sav (SPSS data on workplace management initiatives and talent engagement)\n\nFigshare: SPSS DATA-WMI.sav. https://doi.org/10.6084/m9.figshare.12589571.v1 (Falola et al., 2020b)\n\nThis project contains the following extended data:\n\n- QUESTIONNAIRE-WMI & TE.docx (Study questionnaire)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAdelere M: Effect of staff training and development on organizational performance: evidence from Nigerian bottling company. 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Publisher Full Text\n\nSchneider B, Yost AB, Kropp A, et al.: Workforce engagement: What it is, what drives it, and why it matters for organizational performance. J Organ Behav. 2017; 39(4): 462–480. Publisher Full Text\n\nSiddiqui D: The impact of training & development and communication on employee engagement – A study of banking sector. Business Management and Strategy. 2019; 10(1): 2157–6068. Publisher Full Text\n\nSmith AP, Smith HN: Wellbeing at work and the lie of scale. Journal of Health and Medical Sciences. 2019; 2(1): 40–51. Publisher Full Text\n\nTabachnick BG, Fidell LS: Using multivariate statistics. (5th ed.). San Francisco: Allyn & Bacon. 2007. Reference Source\n\nWorld Health Organization: Mental health and psychosocial considerations during the COVID-19 outbreak. Geneva, Nations for Mental Health. 2020. Reference Source" }	[ { "id": "72011", "date": "12 Oct 2020", "name": "Yejide Olukemi Oseni", "expertise": [ "Reviewer Expertise Pharmacy administration", "public health", "health and pharmacy policy and regulation", "pharmacy practice", "health management" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle could read: \"Influence of workplace management initiatives on talent engagement in Nigerian Pharmaceutical Industry.\"\nIntroduction:\nThe specific objectives if need be stated should follow the main objective earlier stated.\n\nI could not see the reason for the specific objectives.\n\nThe use of different adjectives to capture the specific objectives has no meaning in the work done i.e. Investigate the influence of …, examine the effect…., determine the effect…\n\nIs using different analytical tools a gap? Are you saying the tool has never been used for a similar study in the past?\n\nLiterature review:\nFor flow in your discussion and alignment with your stated objectives, rearrange the discussion sub-head accordingly.\n\nIs job engagement the same as work engagement and employee engagement?\n\nUnder “Talent wellbeing and job engagement” subhead, corrected to “Employee wellbeing and job engagement”.\n\n“Employee recognition and talent engagement” subhead could be better captioned as “Employee performance and talent engagement”.\n\nThe last statement under “Talent wellbeing and job engagement” and “Employee performance and talent engagement” subheads look like your conclusion. I think should be expunged.\n\nThe second paragraph under “Diversity/ inclusion and talent management” subhead is a mix-up. 1st sentence looks like another conclusion. 3rd sentence could have been stated at the beginning of the subhead if necessary. 4th sentence is not necessary at all because that was what you were discussing.\n\nMethods:\nYou need to streamline your methods to show every important aspect of it: study design, study area, study period, sample size, research instrument, data collection method, data analysis, and ethical approval.\n\nYour study design is not stated.\n\nRespondent Profile as stated should be presented in the Result. Hence the Section A of the Questionnaire which is sociodemographic characteristics of the respondents should be discussed in the research instrument.\n\nThe last two statements in the sample size and sampling procedure should be part of your research instrument. Also the Pharmacists Council of Nigeria Register is the only official document that will give you an accurate number of manufacturing premises registered for that year.\n\nWhy did you use the ten best-rated pharmaceutical companies only?\n\nBullet iii under justification for criteria have two points merged.\n\nInclusion and exclusion criteria is a tautology. You have discussed it under sample size and sampling procedure.\n\nMeasures and variables are Section B of your research instrument. Present it with Section A.\n\nThe way this statement “The organisation encourages me to use my skills and capabilities in the work process” is drafted makes it more of a workplace management initiative measure rather than talent engagement measurement. Also this statement “This is great company for me to make a contribution to my development” is more of a talent engagement measure.\n\nResults:\nResult of the sociodemographic characteristics as presented in the Respondent Profile should come here.\n\nLine 3; “affective” used for the first time here. Be consistent. I think you mean emotional.\n\nLimitations:\nSeparate limitations and suggestions for further studies\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? Partly\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": [ { "c_id": "6044", "date": "21 Oct 2020", "name": "Hezekiah Falola", "role": "Author Response", "response": "Yejide Olukemi Oseni is deeply appreciated for taking her time to review our article. Her comments and observations are valued. Our responses to some of the issues raised are presented below: IntroductionThe authors decided to operationalize the independent and dependents variables to allow the readers to know the coverage of the constructs used for the measurement of both the independent and dependent variables. Therefore, the specific objectives that stemmed from the broad objective were carefully presented in the study. Literature ReviewJob engagement, work engagement, employee engagement, and talent engagement are similar concepts that can be used interchangeably, particularly in HRM parlance. Also, talent in HRM means employees with distinctive competencies. Therefore, talent wellbeing is more appropriate within the context of the study. In HRM employee recognition is not the same as employee performance. The influence of employee compensation in the prediction of talent engagement was the focus here. Therefore, the authors would like to retain the original subhead, i.e. Employee recognition and talent engagement. Methodology The robust methodology is one of the areas that F1000Research emphasized for article publication consideration. Streamlining the methodology aspect of the study might not be necessary. Besides, the authors explained all the essential elements of the research methods using appropriate justifications. Also, since the sociodemographic characteristics of the respondents were not part of the objective of the study, the authors decided to present the summary of the respondents' profile which was believed to be sufficient for the study. Results The dimensions of engagement as established in the literature are affective, behavioural and cognitive, among others. Using affective is not out of place, particularly in this type of study. LimitationsSince this is an article and not a thesis or dissertation, separating limitations and suggestions for future studies might not be necessary." } ] }, { "id": "73158", "date": "28 Oct 2020", "name": "Cecile Schultz", "expertise": [ "Reviewer Expertise Organisational behaviour and human resource management" ], "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\nAn interesting and technically neat study was presented. Recent and relevant sources were used to discuss the variables of the study.\n\nThe gap in the literature study was clear.\n\nAppropriate statistics were used to investigate the research objective.\n\nDiscussions were elaborative and supported by previous studies.\n\nThe authors must elaborate on the research philosophy(positivism perhaps?), research design (survey research design) and the research approach (quantitative).\n\nThe practical implications of the study must also be addressed.\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": [ { "c_id": "6075", "date": "10 Nov 2020", "name": "Hezekiah Falola", "role": "Author Response", "response": "Cecile Schultz is deeply appreciated for reviewing our article. The methodology has been revised as suggested and the implications of the study has been included in version 2 of the article as recommended. Thank you" } ] }, { "id": "73152", "date": "28 Oct 2020", "name": "Deepika Pandita", "expertise": [ "Reviewer Expertise human resources", "talent management", "employee engagement" ], "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\nIntroduction: 1. Though the main objective is stated explicitly specific objectives need to be highlighted. 2. Objectives need to be written carefully with suitable words like examine, investigate will make your research more important.\n\nLiterature review: The literature review needs to be arranged well. Is your definition of job engagement the same as work engagement and employee engagement? The entire sequencing of the constructs in the literature review needs to be re-examined.\n\nMethods: Create subheadings and emphasize study design, study area, study period, sample size, research instrument, data collection method, and analysis of the data. You have not mentioned the study design in your paper. The profile of the respondent is also not stated. Sampling frame and sample size should be a part of the research design. Why were the best rated pharma companies chosen? Any rationale behind that?\n\nResults: Results needs to be presented carefully. There is no mention of practical implications in your study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6074", "date": "10 Nov 2020", "name": "Hezekiah Falola", "role": "Author Response", "response": "We want to sincerely appreciate Deepika Pandita for the insightful comments and suggestions. All the issues raised have been effected in version 2 of the article. Thank you" } ] } ]	1	https://f1000research.com/articles/9-699
https://f1000research.com/articles/9-617/v1	16 Jun 20	{ "type": "Research Article", "title": "Frequency and clinical significance of prostatic involvement in men with febrile urinary tract infection: a prospective observational study", "authors": [ "Thayyil Shahilal Arjunlal", "Surendran Deepanjali", "Ramanitharan Manikandan", "Rajappa Medha", "Thayyil Shahilal Arjunlal", "Ramanitharan Manikandan", "Rajappa Medha" ], "abstract": "Background: Frequent asymptomatic involvement of the prostate has been demonstrated in men with febrile urinary tract infection (fUTI). In view of this, men with fUTI are often given a longer duration of antibiotic treatment; however, evidence to support this is limited. Methods: We prospectively studied adult men with fUTI admitted under the Department of Medicine in a tertiary care hospital in southern India. fUTI was defined as fever of ≥38°C with at least one symptom/sign of UTI and pyuria, requiring hospitalization. We estimated serum total prostate-specific antigen (PSA) levels at enrollment, one month and three months after treatment completion. We assessed prostatic volume by transrectal ultrasonography (TRUS) and estimated the serum high sensitivity C-reactive protein (hs-CRP) levels at baseline and after three months. Results: We enrolled 64 men (median [IQR] age 53 [45-60] years); 50 patients completed follow-up. At baseline, the median (IQR) serum PSA level was 2.15 (1.18-3.02) ng/mL and median (IQR) serum hs-CRP level was 2.43 (2.28-2.58) mg/L. At three months, serum PSA levels decreased by ≥25% in 47 (94%) of 50 patients. The median (IQR) of prostatic volume was 25.4 (18.9-34) mL at baseline, and ≥10% decrease in prostatic volume was observed in 24 (48%) of 50 patients at three months. The change in the serum PSA levels did not correlate with clinical findings like prostatic tenderness or with prostatic volume changes. Further, serum PSA levels did not correlate with hs-CRP levels. On follow-up, seven patients had lower urinary tract symptoms; only one of them had recurrent fUTI.\n\nConclusions: Asymptomatic prostatic involvement, although common in men with fUTI, does not seem to influence the treatment outcomes.", "keywords": [ "urinary tract infections", "prostate-specific antigen", "men", "prostatitis", "antibiotic treatment duration" ], "content": "Introduction\n\nUrinary tract infections (UTIs) in men are generally considered to be complicated UTIs because of increased prevalence of underlying structural and functional abnormalities1. One such abnormality is the possibility of involvement of the prostate gland during an episode of UTI. Symptomatic involvement of the prostate gland by acute bacterial infection, known as acute bacterial prostatitis (ABP), classically presents with fever and systemic symptoms along with pelvic pain and infravesical obstruction2. However, even in the absence of prominent voiding and storage symptoms, subclinical involvement of the gland is possible in men with febrile UTI (fUTI). A study of Swedish men with fUTI provided the major evidence for this, in the form of increased serum prostate-specific antigen (PSA) levels and prostatic volume during an episode of UTI3. Another study using 111indium-labelled leukocyte scintigraphy found that, although often clinically unrecognized, the prostate was involved in most cases of fUTI and acute pyelonephritis in men4.\n\nDifferentiating fUTI with subclinical prostatic involvement from classical ABP is important. First, some experts recommend that antibiotic treatment in men with fUTI should not only sterilize the urine but also achieve sufficient concentrations in the prostate. Fluoroquinolones and co-trimoxazole were recommended as optimal choices to achieve this aim5,6. If the increased levels of PSA are truly indicative of ABP, implementing this guidance would be pragmatically challenging in settings where antimicrobial resistance, especially to fluoroquinolones, is very common among uropathogens and other appropriate oral options are not available7. Second, while it is generally agreed that ABP requires antibiotic treatment for at least two to four weeks to prevent chronic prostatitis8, it is unclear whether subclinical prostatic involvement would also necessitate a longer treatment. We, therefore, aimed to study the longitudinal changes in serum PSA levels and prostatic volume in men with fUTI. We also aimed to study the association of these changes with clinical findings and short-term recurrence of UTI.\n\n\nMethods\n\nThe study protocol was reviewed and approved by the Institute Ethics Committee (Human Studies) of Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry (JIP/IEC/2016/29/970). Written informed consent was obtained from all study participants.\n\nWe conducted a prospective observational study in the medical wards of Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), a tertiary care hospital in Southern India, during July 2016 and May 2018. During this study period, we screened all consecutive male patients aged 18 years or over admitted under the Department of Medicine as suspected cases of fUTI for eligibility to participate in the study. We defined fUTI as documented fever of at least 38°C with at least one symptom or sign referable to the urinary tract such as dysuria, frequency, urgency, flank pain or renal angle tenderness. All patients had evidence of microscopic pyuria (>5 pus cells/hpf) or urine dipstick positive for leukocyte esterase. We excluded patients with catheter-associated UTI, urological procedures or surgery in the past four weeks, diagnosed with prostate carcinoma, or significant upper urinary tract obstruction evidenced by gross hydroureteronephrosis (HUN).\n\nAfter obtaining written informed consent, the investigator (AL) performed a standardized clinical evaluation. A digital rectal examination (DRE) was done to look for prostatic enlargement, tenderness or bogginess. Blood samples were drawn within 48 hours of admission for serum PSA and high sensitivity C-reactive protein (hs-CRP) estimation, and serum was stored at -80°C for batched analysis. Since DRE may lead to an increase in serum PSA levels, blood samples were drawn before performing DRE. Serum PSA levels were estimated in duplicate using a two-site immune-enzymatic sandwich assay that uses a mouse monoclonal anti-PSA antibody (Cat. No. 37200, Hybritech Prostate Specific Antigen, Beckman Coulter, Fullerton, CA) as described for the National Health and Nutrition Examination Survey 2001-20029. Serum hs-CRP was estimated in duplicate using a solid phase direct sandwich assay which uses a monoclonal antibody (Cat. No. CR120C, Calbiotech. Inc., El Cajon, CA) as per the manufacturer’s instructions. All patients underwent ultrasonographic examination of kidney, ureter and bladder. As soon as the patients became clinically stable, a transrectal ultrasound (TRUS) examination of the prostate and seminal vesicles was performed using Famio 8 SSA-530A (TOSHIBA), PVQ 641V 6 MHz probe by a urology senior resident. We collected data on the antibiotic regimen started, duration of therapy and clinical course during hospital stay.\n\nAt the first follow-up assessment after one month of treatment completion, serum PSA estimation was done in all patients. In those with persistent fever or urinary tract symptoms, urine dipstick leukocyte esterase test and microscopic examination were done. In those with evidence of significant pyuria, urine culture was repeated.\n\nAt the second follow-up visit after three months of treatment completion, clinical evaluation and repeat measurements of serum levels of PSA and hs-CRP were done. TRUS was also repeated to re-assess the size of prostate and to assess resolution or persistence of inflammation. As defined by Ulleryd et al.3, a fall of ≥25% in serum PSA levels at three months and a decrease in prostatic volume of ≥10% at three months were considered significant.\n\nAssuming that 80% of patients would have evidence of prostatic involvement3, 64 patients were required to estimate this proportion with 10% absolute precision.\n\nWe summarized categorical variables as n (%) and continuous variables as mean±SD or median (IQR) as appropriate. We applied the Wilcoxon signed rank test to assess the change in serum PSA and serum hs-CRP levels at three months compared to baseline. We performed the Friedman test to analyze the trend of serum PSA at admission, one month and three months. We applied the Wilcoxon rank sum test to compare baseline serum PSA and change in serum PSA levels at three months between patients with and without clinical features suggestive of prostatic involvement. We tested the correlation between baseline serum PSA levels and fall in its levels by three months and that between baseline serum PSA levels and the change in serum hs-CRP levels by three months using Spearman’s rank correlation. All analyses were performed using the statistical software package Stata/IC 12.1 for Windows, StataCorp LP, College Station, Texas, USA. All tests were two-sided, and P <0.05 was considered statistically significant.\n\n\nResults\n\nBetween July 2016 and May 2018, we screened 91 men with a diagnosis of fUTI and included 64 patients; 50 patients completed follow-up assessments at one month and three months. Figure 1 depicts the flow of subjects through the study. Clinical and laboratory characteristics of the study subjects at baseline are summarized in Table 1. Notably, 16 (25%) patients had presented with obstructive urinary symptoms. In addition, 25 (39%) patients had prostatic tenderness on DRE.\n\nTRUS, transrectal ultrasound; PSA, prostate-specific antigen; hs-CRP, high sensitivity C-reactive protein.\n\na = co-morbid conditions include coronary artery disease, malignancy, chronic liver disease, cerebrovascular accident.\n\nUTI, urinary tract infection; IQR, interquartile range; SD, standard deviation; hpf, high-power field.\n\nMean duration of fever was 6.7±4.4 days; 15 (23%) patients had received antibiotics prior to study enrollment. Using a strict diagnostic criteria of fever, dysuria, urinary retention and prostatic tenderness on DRE, eight (12%) of 64 patients could be classified as cases of ABP. Urine culture was sent prior to the first dose of antibiotic after admission in 45 (70%) patients. Eschericia coli was the major uropathogen, isolated in 25 (86%) patients.\n\nEmpirical antibiotic regimens used in the study population included ceftriaxone in 28 (44%), amikacin in 21 (33%), a combination of ceftriaxone and amikacin in seven (11%), cefaperazone/sulbactum in three (5%), a combination of piperacillin/tazobactum and amikacin in three (5%) patients and meropenem in one (2%) patient. A patient with Candida tropicalis fungemia and prostatic abscess was treated with fluconazole for 28 days. Modification of the empirical regimen based on susceptibility was done in six (9%) patients. Mean duration of antibiotic therapy was 8.6±3.6 days.\n\nOf the 64 patients included, 50 patients came for first follow-up visit after one month of treatment completion. Among them, persistent lower urinary tract symptoms (LUTS) were present in seven (14%) patients. One of them (patient 1), who had fungal prostatic abscess at initial admission, gave a history of recurrent fever. He had significant microscopic pyuria and urine culture showed significant growth of E. coli, which was susceptible to amikacin. He was re-admitted and was treated with amikacin for seven days. Of the remainder, two patients had significant microscopic pyuria, while four did not. Urine cultures in the two patients with pyuria showed Klebsiella spp in one patient (patient 2; treated with nitrofurantoin on ambulatory basis). The third patient’s (patient 3) urine culture was contaminated, and a repeat culture was sterile. Since his LUTS improved significantly with increased fluid intake, he was not treated with antibiotics.\n\nThe same set of 50 patients attended the three months follow-up. Of note, the three patients (patient1, 2 and 3) who had LUTS and microscopic pyuria during first follow-up visit continued to be symptomatic at this visit too, although none was febrile. They continued to have significant pyuria at this visit. While the repeat urine culture of patient 1 was sterile, patients 2 and 3 had significant bacteriuria, the organisms were different from previous cultures (Enterobacter spp and Enterococcus spp, respectively). No antibiotic therapy was prescribed in these three patients at this juncture. They were advised to maintain good hydration. All three patients had significant resolution of symptoms subsequently. Details of these patients are presented in Table 2.\n\nUTI, urinary tract infection; TRUS, transrectal ultrasound; PSA, prostate-specific antigen; hs-CRP, high sensitivity C-reactive protein.\n\nAt admission, 14 (22%) of 64 patients had serum PSA values ≥4 ng/mL. Among the 50 patients who followed up, PSA levels were ≥4 ng/mL in four (8%) and one (2%) at one month and three months, respectively. There was a significant decrease in serum PSA levels at one and three months compared to baseline (Table 3). The fall in serum PSA levels at three months was strongly correlated to the baseline serum PSA value (Spearman’s rho 0.93; P <0.001; Figure 2.) Of the 50 patients, 47 (94%) had ≥25% decline in serum PSA values at three months.\n\nPSA, prostate-specific antigen; hs-CRP, high sensitivity C-reactive protein; TRUS, transrectal ultrasound; IQR, interquartile range\n\nPanel A: Dotplot of serum prostate-specific antigen (PSA) levels at baseline, one month and three months of follow-up. Panel B: Correlation between baseline serum PSA level and the change in PSA levels at three months.\n\nSerum hs-CRP levels also decreased significantly at three months compared to baseline (Table 3, Figure 3). There was no correlation between serum PSA levels and hs-CRP levels either at baseline or at three months. However, the change in serum PSA levels had a weak correlation with the change in serum hs-CRP levels (Spearman’s rho 0.27; P = 0.054).\n\nTRUS was done in 64 patients at baseline, within two (2–4) days of hospitalization. There were no procedure-related complications. The most significant finding was the presence of prostatic abscess in one patient. Other findings on TRUS are presented in Table 3. Follow-up TRUS examination was done in 50 patients, 95±7 days after hospital discharge. Of the six patients who had focal hypoechogenicity at admission, three continued to have the finding at three months, while new hypoechogenic lesions were noticed in two other patients. However, none of the patients who had these lesions were symptomatic at three months.\n\nA decrease of in prostatic volume ≥10% was observed in 24 (48%) patients. The change in serum PSA levels at three months did not correlate with change in prostatic volume.\n\nSerum PSA levels at baseline did not differ significantly between patients with/without clinical features suggestive of prostatic involvement such as urinary retention, lower abdominal pain, prostatic tenderness on DRE or a possible diagnosis of ABP. Similarly, none of these clinical features were associated with the change in serum PSA levels compared to the baseline (Table 4).\n\nAll data presented as median (IQR), aDefined as presence of fever, dysuria, urinary retention and prostatic tenderness on DRE\n\nPSA, prostate-specific antigen; ABP, acute bacterial prostatitis; IQR, interquartile range; DRE, digital rectal examination\n\n\nDiscussion\n\nWe found that most men with fUTI requiring hospitalization had elevated serum PSA levels and nearly half of them had a decrease in prostatic volume on follow up. However, only a handful of them had clinical findings suggestive of prostatic involvement, and recurrence following treatment was uncommon.\n\nOur findings are in agreement with the seminal study by Ulleryd et al.3. Although there are a few more studies on PSA levels in men with fUTI, these studies had enrolled patients with ABP10,11. The median (IQR) serum PSA levels at admission in our patients was 2.15 (1.18–3.02) ng/mL, which is lower compared to the study by Ulleryd et al., which was 14 (range 0.54–140) ng/mL. We used a chemiluminescent immunoassay method, while Ulleryd et al. used a monoclonal fluoroimmunoassay. While mild assay-related variations are possible, the main reason for lower PSA levels in our study could be because of ethnic variations in PSA levels. Studies from India show that the mean serum PSA values in Indian men are lower compared to the Western population12–14.\n\nConventionally, elevated PSA levels and a change in prostatic volume have been proposed as definitive evidence of prostatic involvement in men with fUTI3,6. Based on this premise, often it is contended that fUTI in men should be treated with antibiotics for a duration of at least two weeks. However, interpreting changes in PSA levels and prostatic volume as reliable evidence of ‘prostatitis’ is questionable. ABP is a clinically defined entity classically characterized by fever, systemic symptoms, pelvic pain and urinary tract symptoms such as dysuria, urinary frequency, and urinary retention15. Although it is known that PSA levels become elevated in men with ABP, the converse may not be true. Our findings indicate that it might be fallacious to equate pauci-symptomatic elevation of serum PSA levels as definitive evidence of prostatitis due to following reasons.\n\nFirst, only a minority of patients with such changes actually had clinical findings to suggest prostatic involvement, and the PSA levels and prostatic volume changes were not related to prostatic symptoms. Second, we did not find a correlation between hs-CRP levels and the elevation in PSA levels and the change in prostatic volume. Notably, Ulleryd et al. also did not find a correlation between elevated serum PSA and markers of systemic inflammation. Third, despite the fact that 80% of patients were treated with antibiotics for seven days duration, recurrence of UTI was uncommon. Thus, while we confirm the findings that transient elevations in PSA levels and prostatic volume are very common in men with fUTI, we disagree with the interpretation of the clinical significance of these subclinical changes. It is quite possible that the elevated PSA levels indicate a physiological response to bacterial infection rather than indicating a pathological disease process. Townes et al. found that epithelial expression and release of PSA was increased by E. coli challenge16. A few other studies also show that elevated PSA levels represent an enhanced prostate innate host defence17,18. Serum PSA levels also rise during episodes of sexually transmitted infections and may remain elevated for several months after effective antibiotic therapy19. Other non-genitourinary infections like infectious mononucleosis and chikungunya also cause elevated serum PSA levels20,21. It is possible that elevated PSA level is a non-specific response to systemic inflammation caused by prostate cell damage and increased vascular permeability.\n\nWe found the presence of focal hypoechoic areas in prostate in a small proportion of patients, the significance of which is unclear — these patients also had good response to treatment and none had LUTS on follow up. Horcadaja et al. observed hypoechoic lesions in about 25% of patients with ABP22; these lesions persisted in about one-third of patients after antibiotic therapy for one month.\n\nThe practical application of clinically distinguishing fUTI from ABP is mainly to decide on the choice and duration of antibiotics. It would be fallacious to justify the need for prolonging the antibiotic treatment based solely on biochemical and TRUS changes which might possibly suggest prostatic involvement. While clinicians generally agree on the need to treat patients with ABP for at least two weeks, it needs to be pointed out that this duration is not based on good quality evidence23. In addition, considerable heterogeneity exists in the diagnosis and management of ABP among various clinical departments24. Even though a shorter duration of antibiotics was associated with an increased risk of recurrent prostatitis in observational studies, it could be because those patients had clinically manifest ABP and not just biochemical and/or ultrasonographic changes25. Indeed, even in ABP, some experts believe that the role of shorter treatment duration needs to be explored26. This is a very important aspect since longer treatment durations have been paradoxically associated with increased late recurrences of UTI in the outpatient setting27. In addition, longer antibiotic treatment durations do not augur well with the principles of antibiotic stewardship28.\n\nVery few clinical trials have addressed the issue of optimal treatment duration for fUTI in men without features of ABP. A recent trial from the Netherlands found that shorter duration resulted in lower clinical cure rates at short term in men29. Prostatic involvement was attributed as a possible reason for this. However, clinical cure at 70-84 days did not differ between genders, and shorter treatment did not result in more recurrence in men on long term. Another smaller trial from India comparing non-fluoroquinolone antibiotic therapy for seven or 14 days found no difference in re-treatment rates between males and females30. Average antibiotic treatment duration in the present study was less than 10 days. Yet, we did not find significant short-term recurrence of fUTI in them.\n\nPossible limitations of our study are: i) it would have been more informative if we had longer follow-up and assessment for chronic prostatitis in the study population; ii) measurement of prostatic volumes potentially could have been affected by inter-observer variability; and iii) we did not collect data on glandular vascularity, which could indicate the presence of inflammation31.\n\n\nConclusions\n\nIn conclusion, increase in serum PSA levels and certain ultrasonographic findings, which might possibly indicate subclinical prostatic involvement, were very common among men with fUTI. However, the clinical significance of these changes is uncertain.\n\n\nData availability\n\nFigshare: Prostatic involvement in male UTI. https://doi.org/10.6084/m9.figshare.12286865.v232\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors thank all the participating patients and the staff at JIPMER for their kind co-operation. The authors thank Dr. Tamilarasu Kadhiravan, Additional Professor, Department of Medicine for critical appraisal of the study findings and help with the figures in the manuscript.\n\n\nReferences\n\nRubin RH, Shapiro ED, Andriole VT, et al.: Evaluation of new anti-infective drugs for the treatment of urinary tract infection. Infectious Diseases Society of America and the Food and Drug Administration. Clin Infect Dis. 1992; 15 Suppl 1: S216–227. 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Reference Source\n\nEtienne M, Chavanet P, Sibert L, et al.: Acute bacterial prostatitis: heterogeneity in diagnostic criteria and management. Retrospective multicentric analysis of 371 patients diagnosed with acute prostatitis. BMC Infect Dis. 2008; 8: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoon BI, Han DS, Ha US, et al.: Clinical courses following acute bacterial prostatitis. Prostate Int. 2013; 1(2): 89–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaber KG: Management of bacterial prostatitis: what's new? BJU Int. 2008; 101(Suppl 3): 7–10. PubMed Abstract \| Publisher Full Text\n\nDrekonja DM, Rector TS, Cutting A, et al.: Urinary tract infection in male veterans: treatment patterns and outcomes. JAMA Intern Med. 2013; 173(1): 62–68. PubMed Abstract \| Publisher Full Text\n\nSpellberg B: The New Antibiotic Mantra -“Shorter Is Better”. JAMA Intern Med. 2016; 176(9): 1254–1255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Nieuwkoop C, van der Starre WE, Stalenhoef JE, et al.: Treatment duration of febrile urinary tract infection: a pragmatic randomized, double-blind, placebo-controlled non-inferiority trial in men and women. BMC Med. 2017; 15(1): 70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRudrabhatla P, Deepanjali S, Mandal J, et al.: Stopping the effective non-fluoroquinolone antibiotics at day 7 vs continuing until day 14 in adults with acute pyelonephritis requiring hospitalization: A randomized non-inferiority trial. PLoS One. 2018; 13(5): e0197302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKravchick S, Cytron S, Agulansky L, et al.: Acute prostatitis in middle-aged men: a prospective study. BJU Int. 2004; 93(1): 93–96. PubMed Abstract \| Publisher Full Text\n\nDeepanjali S, Thayyil A, Rajappa M, et al.: Prostatic involvement in male UTI. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12286865.v2" }	[ { "id": "68741", "date": "10 Aug 2020", "name": "Veeravan Lekskulchai", "expertise": [ "Reviewer Expertise Clinical Pathology", "Clinical Chemistry", "Clinical Toxicology" ], "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 entitled \"Frequency and clinical significance of prostatic involvement in men with febrile urinary tract infection: a prospective observational study\" and was written by Arjunlal et al. The authors studied chances of having acute bacterial prostatitis in male admitted with urinary tract infection. The manuscript was written appropriately and relevantly. However, I have a few major concerns for this work. First in the Results, though, 64 patients were enrolled and their baseline results were available, 14 of them were not followed up and were excluded after that. Consequently, the baseline results of these 14 patients should be excluded from this work (Table 3, Figure 3). The baseline results should come from 50 patients similar to the results in the next one and 3 months. Another concern is why they used hs-CRP in cases whose inflammation were obviously indicated. They should use CRP level. This point needs an explanation.My minor concern is the use of old references. If possible, they should be replaced by newer ones.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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": [ { "c_id": "5996", "date": "27 Oct 2020", "name": "Surendran Deepanjali", "role": "Author Response", "response": "Response to Dr. Veeravan Lekskulchai We thank Dr Lekskulchai for the very helpful and constructive comments. We have tried to address the concerns raised by Dr Lekskulchai and have made modifications in our manuscript accordingly. Please find below a point-by-point response to the comments. Comment: This work is entitled \"Frequency and clinical significance of prostatic involvement in men with febrile urinary tract infection: a prospective observational study\" and was written by Arjunlal et al. The authors studied chances of having acute bacterial prostatitis in male admitted with urinary tract infection. The manuscript was written appropriately and relevantly. However, I have a few major concerns for this work. Response: We are grateful for the kind compliments. We hope to address the concerns raised by Dr Lekskulchai. Comment: First in the Results, though, 64 patients were enrolled and their baseline results were available, 14 of them were not followed up and were excluded after that. Consequently, the baseline results of these 14 patients should be excluded from this work (Table 3, Figure 3). The baseline results should come from 50 patients similar to the results in the next one and 3 months. Response: We than Dr Lekskulchai for pointing this out. We have now presented the important baseline variables pertaining to the 50 patients separate from the total 64 recruited patients in Table 3. Also, we have modified Figure 3 accordingly to include data pertaining to these 50 patients only. Comment: Another concern is why they used hs-CRP in cases whose inflammation were obviously indicated. They should use CRP level. This point needs an explanation. Response: Dr Lekskulchai is correct in pointing out that serum CRP estimation would be better than hs-CRP in patients with obvious inflammation such as febrile UTI. However, a pre-specified follow-up at 3 months was planned in our study, and it was expected that UTI-associated inflammation would have substantially decreased in many patients on follow-up. Since a more sensitive assay would be required to demonstrate this low-grade inflammation with CRP concentrations often below 1.0 mg/dL, we used an hs-CRP assay rather than a CRP assay. Previous studies on subclinical prostatic inflammation have used hsCRP as a biomarker (Milbrandt M, Winter AC, Nevin RL, et al. Insight into infection-mediated prostate damage: Contrasting patterns of C-reactive protein and prostate-specific antigen levels during infection. Prostate. 2017;77:1325-1334.). We are also duty-bound to inform Dr Lekskulchai that the query on the utility of hs-CRP made us look closely at the hs-CRP primary data. We then identified an inadvertent data entry error. We realized that the optical density values of the ELISA read-outs were mistakenly entered as the actual hs-CRP values. We sincerely regret this inadvertent error. We have now uploaded a corrected version of the underlying data set containing the corrected hs-CRP values which can be found at Deepanjali, Surendran; Thayyil, Arjunlal; Rajappa, Medha; Ramanitharan, Manikandan (2020): Prostatic involvement in male UTI. figshare. Dataset. https://doi.org/10.6084/m9.figshare.12286865.v3. Accordingly, we also have now made necessary changes in descriptive data on serum hs-CRP as well as Figure 4. While this error does not change our findings and conclusions, we found that the weak correlation between change in serum PSA levels with change in serum hs-CRP levels was no longer significant when the analysis was repeated using corrected hs-CRP values. We have now provided this information in Version 2. Comment: My minor concern is the use of old references. If possible, they should be replaced by newer ones. Response: We ran an updated PubMed search using terms “serum PSA AND urinary tract infections”, “serum PSA and acute bacterial prostatitis” and “hs-CRP AND urinary tract infection”. However, we could not find any newer reference related to our manuscript. If Dr Lekskulchai could kindly point out any particular reference/references which needs to be checked for new information, we would be happy to do so. We sincerely hope Dr Lekskulchai finds our responses satisfactory. We would certainly try to address any further concerns if present. Thank you." } ] }, { "id": "68790", "date": "17 Aug 2020", "name": "Aneesh Basheer", "expertise": [ "Reviewer Expertise Infectious diseases", "tropical medicine", "hematology", "evidence based medicine", "medical education" ], "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\nRationale and research question The authors submit evidence from literature that supports involvement of the prostate in a high proportion of men with febrile UTI. These studies primarily used surrogate markers to identify prostate involvement such as elevated prostate specific antigen (PSA) or increased prostate volume by ultrasonography. When acute bacterial prostatitis (ABP) is diagnosed, treatment with appropriate antibiotics for 2 to 4 weeks would be needed. ABP typically presents with pelvic pain and voiding symptoms. If the prostatic involvement in febrile UTI is a subclinical ABP, this would need similar treatment. The authors tried to determine whether this subclinical involvement (evidenced by elevated PSA and prostate volume) had any association with short term UTI recurrence and clinical findings. The research question is not specified anywhere in the paper. It is suggested that a clear research question be added at the end of the introduction.\n\nMethods: The authors have used a prospective cohort design to address the issue. This is generally the ideal method to study and correlate variables longitudinally. The methods section is well written and describes the recruitment and follow up in great detail. The absence of an a-priori comparison group is a weakness, albeit minor. Instead the authors chose to analyze sub-groups from the single cohort of men with febrile UTI – those with features suggestive of clinical prostatitis and those without. A much better and internally valid design would have been the recruitment and follow up of 2 different groups in a similar manner.\n\nResults: The introductory paragraph on results has data pertaining to calculations based on 64 original participants, 50 finally available cases and a subgroup of cases where cultures were taken prior to antibiotics. The percentages provided therefore could confuse readers and hence it is suggested that authors clearly reframe the sentences to indicate the population from which these numbers and percentages were obtained. There were 25 patients with prostate tenderness; however, authors mention that only 8 could be classified as Acute bacterial prostatitis. It is not clear then under what banner, the remaining 17 patients fall?\n\nDiscussion: The authors state that most men with febrile UTI had elevated PSA. From the results it is not clear what cut-off was used to determine elevated PSA. Since PSA cut offs are generally age based, it would also be interesting to note the effect of age of the cases on the PSA levels. A regression analysis could probably provide useful information in this regard. Moreover, the results section states that only 14 (22%) of the participants had PSA levels more than 4 ng/mL. The median PSA of the patients was 2.15 which is also well within normal ranges for the lowest age groups used for PSA cut offs. This disparity between results and the discussion section needs to be clarified. Authors noted a statistically significant drop in the PSA levels at baseline and at 3 months. Further, 94% of patients had more than 25% drop which was the predefined significant change. The predefined significant change occurred. However, the baseline PSA itself was not high. The authors need to discuss this in their limitations. It is possible that the 25% reduction considered by authors may not be the “minimal clinically important difference”. The other possibility is that these effects have been affected by the low baseline prostate involvement that was presupposed as 80% for sample size calculation.\n\nOverall comments: Well written except for minor modifications suggested.\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? Partly\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?\nPartly\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": [ { "c_id": "5997", "date": "27 Oct 2020", "name": "Surendran Deepanjali", "role": "Author Response", "response": "Response to Dr Aneesh BasheerWe thank Dr Basheer for the very constructive comments, and we have modified the manuscript incorporating his suggestions. Please find below a point-by-point response to Dr Basheer’s comments and queries.Comment: Rationale and research question. The authors submit evidence from literature that supports involvement of the prostate in a high proportion of men with febrile UTI. These studies primarily used surrogate markers to identify prostate involvement such as elevated prostate specific antigen (PSA) or increased prostate volume by ultrasonography. When acute bacterial prostatitis (ABP) is diagnosed, treatment with appropriate antibiotics for 2 to 4 weeks would be needed. ABP typically presents with pelvic pain and voiding symptoms. If the prostatic involvement in febrile UTI is a subclinical ABP, this would need similar treatment. The authors tried to determine whether this subclinical involvement (evidenced by elevated PSA and prostate volume) had any association with short term UTI recurrence and clinical findings. The research question is not specified anywhere in the paper. It is suggested that a clear research question be added at the end of the introduction. The research question is not specified anywhere in the paper. It is suggested that a clear research question be added at the end of the introduction.Response: We thank Dr Basheer for summing up our study’s rationale very thoroughly. As per your suggestion, we have now explicitly spelt out the aim of our study at the end of Introduction. We write “ We, therefore, conducted the present study to answer the following research questions — i) What is the frequency of prostatic involvement in men with fUTI, and ii) Is prostatic involvement associated with recurrence of UTI?” Comment: Methods: The authors have used a prospective cohort design to address the issue. This is generally the ideal method to study and correlate variables longitudinally. The methods section is well written and describes the recruitment and follow up in great detail. Response: We thank Dr Basheer for the encouraging comments.Comment: The absence of an a-priori comparison group is a weakness, albeit minor. Instead the authors chose to analyze sub-groups from the single cohort of men with febrile UTI – those with features suggestive of clinical prostatitis and those without. A much better and internally valid design would have been the recruitment and follow up of 2 different groups in a similar manner.Response: We would like to clarify that the definition of prostatic involvement was specified a priori in our study protocol. We had given in the Methods section of version 1 that “As defined by Ulleryd et al [3], a fall of ≥25% in serum PSA levels at 3 months, and a decrease in prostatic volume of ≥10% at 3 months were considered significant.” However, for the sake of better understating, we have re-worded this as “We defined prostatic involvement as per the criteria suggested by Ulleryd et al [3]. A reduction of serum PSA by >25% irrespective of the initial PSA level, and/or a decrease in prostatic volume by >10% after 3 months was taken as evidence of prostatic involvement.” By nature of this definition, it is not possible to classify patients upfront at inception into those with and without prostatic involvement, as suggested by Dr Basheer. However, we agree with Dr Basheer that these patients could be followed up beyond 3 months to ascertain long term outcomes, which was not done in our study. Comment: Results:The introductory paragraph on results has data pertaining to calculations based on 64 original participants, 50 finally available cases and a subgroup of cases where cultures were taken prior to antibiotics. The percentages provided therefore could confuse readers and hence it is suggested that authors clearly reframe the sentences to indicate the population from which these numbers and percentages were obtained.Response: We thank Dr. Basheer for pointing this out. We have now modified the introductory paragraph taking care that the reference population for the percentages is clearly specified. Also, data presented in Table 3 now clearly demarcate information from all 64 recruited patients at baseline from those 50 in whom complete follow-up is available. Similarly Figures 2 & 3 have been modified to represent data from 50 patients only. Comment: There were 25 patients with prostate tenderness; however, authors mention that only 8 could be classified as Acute bacterial prostatitis. It is not clear then under what banner, the remaining 17 patients fall?Response: As we had pointed out in the Discussion section, it is known that there is much heterogeneity among clinicians from different disciplines in arriving at a diagnosis of ABP (Reference 24, version 1). While some clinicians might feel that prostatic tenderness alone is sufficient to diagnose ABP in men with fUTI, many physicians might not agree with this. Therefore, to ensure reproducibility we have adopted a classical syndromic definition for ABP in which only those patients with tender prostate as well as urinary retention (which is a voiding symptom) were classified as ABP. We chose this stricter definition to make the clinical diagnosis unambiguous, since we also intended to evaluate whether the diagnosis had any bearing on baseline serum PSA levels and its longitudinal changes (Table 4, version1). While it is quite possible that the remaining 17 patients had a forme fruste of ABP, the present study was not designed to answer this question. Moreover, even when the analysis was based on individual symptoms, we did not find any association with serum PSA levels (Table 4). Comment: Discussion: The authors state that most men with febrile UTI had elevated PSA. From the results it is not clear what cut-off was used to determine elevated PSA. Since PSA cut offs are generally age based, itwould also be interesting to note the effect of age of the cases on the PSA levels. A regressionanalysis could probably provide useful information in this regard. Moreover, the results sectionstates that only 14 (22%) of the participants had PSA levels more than 4 ng/mL. The median PSA ofthe patients was 2.15 which is also well within normal ranges for the lowest age groups used forPSA cut offs. This disparity between results and the discussion section needs to be clarified.Authors noted a statistically significant drop in the PSA levels at baseline and at 3 months. Further,94% of patients had more than 25% drop which was the predefined significant change.The predefined significant change occurred. However, the baseline PSA itself was not high. Theauthors need to discuss this in their limitations. It is possible that the 25% reduction considered byauthors may not be the “minimal clinically important difference”. The other possibility is that theseeffects have been affected by the low baseline prostate involvement that was presupposed as 80%for sample size calculation.Response: We thank Dr Basheer for drawing our attention to this important aspect of interpretation of serum PSA levels in our study population. We would like to clarify that we stated that serum PSA levels were elevated in most men with febrile UTI, based on the observation that almost 94% of men had a significant fall in serum PSA values at 3 months compared to their baseline value. Following Dr Basheer’s suggestion, we have incorporated age-specific cut offs in the revised manuscript to interpret the PSA values at baseline (Please see last paragraph under ‘Study procedure’ in Version2). It is true that only 38% of patients had a baseline PSA value above the upper limit of their age-specific reference age. We use the 97.5th percentile value as the upper limit of reference range. However, serum PSA values in apparently normal healthy men in a specific age-group could show considerable variability within the reference range. For example, in the age-group 50-59 years the minimum value was 0.06 ng/mL, while the maximum was 5.9 ng/mL. Thus, even if the baseline value was within the reference range, a dynamic fall on follow-up would suggest that the baseline was elevated for a given patient.Nevertheless, we agree that the statement “We found that most men with fUTI requiring hospitalization had elevated serum PSA levels” could be confusing, and hence we have modified this in Version 2. We have now modified it as “We found that most men with fUTI requiring hospitalization had significant decrease in serum PSA levels at 3 months follow-up indicating an elevated baseline value, and nearly a half of them had a decrease in prostatic volume on follow-up.”As suggested by Dr Basheer, we checked for a linear relationship between age and baseline PSA levels by doing a simple linear regression. However, we did not find a relationship between age and PSA levels (coefficient = 0.017; P = 0.486). Although it is well known that PSA levels increase with age, we did not find such a relationship. Most probably, infection-induced changes in PSA distort and overshadow any such underlying relationship in our dataset. Similar to our findings, Ulleryd et al (reference 3) also did not find a correlation between age and serum PSA levels during episode of fUTI in men.We hope Dr Basheer finds the modifications satisfactory, and we once again express our gratitude for your suggestions. We would definitely address any other ensuing concerns regarding our manuscript.Thank you." } ] } ]	1	https://f1000research.com/articles/9-617
https://f1000research.com/articles/9-607/v1	15 Jun 20	{ "type": "Research Article", "title": "Further investigation of gateway effects using the PATH study", "authors": [ "Peter N Lee", "John S Fry", "John S Fry" ], "abstract": "Background: Interest exists in whether youth e-cigarette use (“vaping”) increases risk of initiating cigarette smoking. Using Waves 1 and 2 of the US PATH study we reported that adjustment for vaping propensity using Wave 1 variables explained about 80% of the unadjusted relationship. Here we use data from Waves 1 to 3 to avoid over-adjustment if Wave 1 vaping affected variables recorded then. Methods: Our main analysis M1 concerned Wave 2 never smokers who never vaped by Wave 1, linking Wave 2 vaping to Wave 3 smoking initiation, adjusting for Wave 1 predictors. We conducted sensitivity analyses that: excluded Wave 1 other tobacco product users; included other product use as an extra predictor; or considered propensity for smoking or any tobacco use, rather than vaping. We also conducted analyses that: adjusted for propensity as derived originally; ignored Wave 1 data; used exact age (not previously available) as a confounder rather than grouped age; attempted residual confounding adjustment by modifying predictor values using data recorded later; or considered interactions with age. Results: In M1, adjustment removed about half the excess OR (i.e. OR–1), the unadjusted OR, 5.60 (95% CI 4.52-6.93), becoming 3.37 (2.65-4.28), 3.11 (2.47-3.92) or 3.27 (2.57-4.16), depending whether adjustment was for propensity as a continuous variable, as quintiles, or for the variables making up the propensity score. Many factors had little effect: using grouped or exact age; considering other products; including interactions; or using predictors of smoking or tobacco use rather than vaping. The clearest conclusion was that analyses avoiding over-adjustment explained about half the excess OR, whereas analyses subject to over-adjustment explained about 80%. Conclusions: Although much of the unadjusted gateway effect results from confounding, we provide stronger evidence than previously of some causal effect of vaping, though some doubts still remain about the completeness of adjustment.", "keywords": [ "Cigarettes", "Confounding", "Over-adjustment", "E-cigarettes", "Gateway effects", "Modelling", "Propensity score" ], "content": "Abbreviations\n\nCI, confidence interval; OR, odds ratio; PATH, Population Assessment of Tobacco and Health.\n\n\nIntroduction\n\nIn youths, use of e-cigarettes (“vaping”) has increased considerably in recent years in many countries (e.g. (Barrington-Trimis et al., 2016; Best et al., 2016; Miech et al., 2019)). It is generally recognized that vaping significantly reduces exposure to harmful constituents compared to smoking (National Academies of Sciences Engineering and Medicine, 2018), so one might expect risks from vaping to be much lower (Nutt et al., 2014). However, there are concerns about the rise in vaping. The concern of interest here is the possibility that vaping may encourage some individuals to start smoking who would otherwise not have done so, often referred to as the “gateway” effect. The concern that vaping may act as a gateway into smoking was originally brought sharply into focus by a 2017 meta-analysis (Soneji et al., 2017) which combined data from nine cohort studies in young people in the US which related previous vaping to later smoking initiation. It reported that, among never-smokers at baseline, ever vaping at baseline strongly predicted initiating smoking in the next 6 to 18 months, with an odds ratio (OR) of 3.62 (95% confidence interval (CI) 2.42-5.41) after adjusting for various factors predictive of initiation. Similarly past 30-day vaping at baseline also predicted later 30-day cigarette use (OR 4.25, 95% CI 2.52-7.37).\n\nWe have previously published two papers relating to the gateway effect. Our first paper (Lee et al., 2018) considered various general issues. It made a number of relevant points:\n\nThe studies that reported that vaping significantly predicts initiation of smoking after adjusting for various other predictors, used sets of predictors that were generally quite incomplete.\n\nResidual confounding arising from the predictors being inaccurately measured was not taken account of in any of the studies.\n\nAdjusting more precisely may have reduced the association substantially.\n\nAny true gateway effect would only alter smoking prevalence modestly.\n\nIn youths in the US and UK in 2014–2016 smoking prevalence declined more rapidly than the preceding trend would predict, contrary to what might expect if any large gateway effect existed.\n\nEven given the existence of some gateway effect, the introduction of e-cigarettes would still likely reduce smoking-related mortality.\n\nOur second paper (Lee & Fry, 2019) described results of our own analyses, based on data from Waves 1 and 2 of the Population Assessment of Tobacco and Health (PATH) study, a nationally representative longitudinal cohort study in the United States of tobacco use and how it affects the health of people. Wave 1 was conducted from 12 September 2013 to 15 December 2014, with Wave 2 the first annual follow-up. For each Wave, data are available separately for Youths (aged 12–17 years) and Adults (aged 18+ years), the Youth data including some information from the parents. Publicly available data files include extensive information on use of various types of tobacco products and on a range of variables linked to initiation of tobacco. Note that where youths become 18 between successive Waves of the survey, their data will be available in the Adult data rather than the Youth data. Also, additional youths who were under 12 at the time of Wave 1 are added into the Youth data when they reach the age of 12 at a subsequent Wave.\n\nIn our main analyses we included youths who had never smoked cigarettes by Wave 1, and had data on smoking initiation by Wave 2. We constructed a propensity score for ever e-cigarette use using variables recorded at Wave 1 and found that adjustment reduced the unadjusted OR markedly, from 5.70 (95% CI 4.33-7.50) to 2.48 (1.85-3.31), 2.47 (1.79-3.42) or 1.85 (1.35-2.53), whether adjustment was made using quintiles of the propensity score, using propensity as a continuous variable, or using each variable making up the score. In sensitivity analyses we confirmed that adjustment explained most of the apparent gateway effect.\n\nAlthough we found that confounding was a major factor, explaining most of the observed gateway effect, we were particularly concerned about the possibility of over-adjustment, if taking up e-cigarettes had affected the values of some of the Wave 1 predictor variables considered. At the time, we noted that the possibility of over-adjustment could be avoided using data from Waves 1, 2 and 3 of the PATH study, by relating initiation of cigarette smoking at Wave 3 to vaping at Wave 2, restricting attention to those who, at Wave 1, had never vaped, and using propensity indicators recorded at Wave 1 linked to uptake of e-cigarettes by Wave 2.\n\nHere we describe the results of extensive analyses conducted based on Waves 1, 2 and 3, which not only include the main analyses envisaged at the time of our earlier paper (Lee & Fry, 2019), but also a variety of sensitivity and alternative analyses.\n\n\nMethods\n\nSome aspects of the analyses described here are the same as those described earlier (Lee & Fry, 2019) and are not presented again here. The selection of demographic and other predictor variables is the same as before, except that in some analyses we use exact age (12, 13, 14, 15, 16 and 17), which could now be estimated from the age group (12–14, 15–17) at the three Waves and the Wave when youths became adults (18+) for the first time. Use of the person-level weights provided in the PATH study database is as before, as is the process by which a sequence of logistic regression analyses is used to develop the shorter list of demographic variables to be used in forming the propensity scores.\n\nOur main analysis M1 is based on those with data at Waves 1, 2 and 3 who had never smoked cigarettes by Wave 2 and had never used e-cigarettes by Wave 1. This analysis predicts Wave 3 ever smoking from Wave 2 ever e-product use, with adjustment based on Wave 1 predictors used to derive a propensity index for taking up e-products between Waves 1 and 2, and exact age being used in preference to grouped age. Note that, whereas in Wave 1 questions in PATH related only to e-cigarette use, in Waves 2 and 3 questions related to ever e-product use, which also included use of e-cigars, e-pipes and e-hookahs.\n\nAssociated with main analysis M1 are four sensitivity analyses (S1 to S4) which are otherwise similar, except that:\n\nS1. Those who had ever used other tobacco products at Wave 1 are excluded;\n\nS2. Ever use of other tobacco products at Wave 1 is included as an additional predictor variable;\n\nS3. The analysis is based on a propensity score for ever cigarette smoking rather than for ever vaping; or\n\nS4. The analysis is based on a propensity score for ever use of any tobacco product rather than for ever vaping.\n\nNote that in our original paper (Lee & Fry, 2019) we also presented results of a further sensitivity analysis, based on linking current vaping to current smoking. This was not repeated here as numbers of new current smokers in current vapers were very low.\n\nMain analysis M2 is similar to M1, except that analysis adjusts for the propensity index as originally derived (Lee & Fry, 2019), based on 12 variables recorded at Wave 1. Alternative versions of M2 substitute exact age rather than grouped age in deriving the propensity index, and/or included Wave 1 vapers in the analysis.\n\nMain analysis M3 adjusts for a propensity index derived by linking Wave 2 predictors to Wave 2 e-product use. This is a replicate of the analysis conducted originally (Lee & Fry, 2019), but using a different period of taking up cigarettes. Data for Wave 1 were ignored, except that where the data for a characteristic was “ever in last 12 months”, Wave 1 data were used to define “ever”. An alternative version of M3 replaces grouped age by exact age in deriving the propensity index.\n\nApart from analyses linking Wave 2 e-product use to additional cigarette smoking at Wave 3 in those who had never smoked at Wave 2, two additional analyses (A1 and A2) were also conducted.\n\nAdditional analysis A1 relates e-cigarette use at Wave 1 to cigarette smoking at Wave 2 as in our earlier publication (Lee & Fry, 2019), but based on individuals who provided data at all three Waves. One version of this uses the same 12 variables as before to develop the propensity index, the other replaces grouped age by exact age. The OR from this analysis can be combined with that reported for main analysis M2 to give a combined estimate of the gateway effect for Wave 1 to 2 initiation and Wave 2 to 3 initiation based on the same set of variables determined at Wave 1.\n\nAdditional analysis A2 ignores Wave 2 data and relates e-cigarette use at Wave 1 to cigarette smoking at Wave 3 using the same 12 variables as before, but replacing grouped age by exact age.\n\nConsideration of residual confounding was also taken into account for three of the analyses described above (M1, M3, A1), all involving exact age. In each case, the list of predictor variables was unaltered from that used originally, but the values of the predictor variables and of the propensity index were revised based on data available at all three Waves. For age, individual year of age at Wave 1 was used, while gender and Hispanic origin did not change between Waves. For the other variables used to form the propensity index, we used all the available data, generally choosing the response most associated with increased e-cigarette use where response varied between Waves (see Additional File Table 1, Extended data, for further details (Lee, 2020)).\n\nFor analyses M1, M3 and A1, alternative versions were also run in which the number of variables adjusted for was increased by also including interactions of age with each of the other three predictors most strongly linked to the relevant gateway effect.\n\nRelevant data were transferred for analysis to a ROELEE database, and analysed using the ROELEE program (Release 59, Build 49). All these analyses could be run using the GLM Package and the Step Function from the R Program (https://www.r-project.org/).\n\n\nResults\n\nInitial analyses linked exact age, four other demographic variables (gender, Hispanic origin, race and census region) and 60 other selected predictor variables to ever e-product use at Wave 2 in those who had not smoked or used e-cigarettes at Wave 1. A propensity index based on 16 variables was derived using the three step process described earlier (Lee & Fry, 2019). Additional File Table 2 (see Extended data (Lee, 2020)) shows the steps at which different variables were eliminated from consideration, while Table 1 gives the fitted equation for the propensity index.\n\nNote: The model is based on 8058 youths with data on all 16 predictors who neither smoked nor used e-cigarettes at Wave 1.\n\na The variables are shown in order of their inclusion into the model.\n\nb The OR is per unit of the graded variable which represents decreasing curiosity.\n\nc The OR is per unit of the graded variable which represents decreasing likelihood.\n\nd The OR is per unit of the graded variable which represents decreasing likelihood, with those originally entered as missing because they thought that they would not smoke a cigarette in the next year scored as “definitely not” (Level 4).\n\nAs shown in Table 2, adjustment for propensity removed about half the excess OR (i.e. OR−1), the unadjusted OR of 5.60 (95% CI 4.52-6.93) reducing to either 3.37 (2.65-4.28) or 3.11 (2.47-3.92), depending on whether adjustment was as a continuous variable or as quintiles. A similar reduction in the OR, to 3.27 (2.57-4.16), was achieved by adjusting for the 16 variables individually. It can also be seen that, for the first seven variables adjusted for, the adjusted OR decreased steadily, to 3.25. Further adjustment had little or no effect, with introducing additional variables sometimes slightly increasing the estimated OR and sometimes slightly decreasing it.\n\nNotes: The table shows the effects of adjustment based on the Wave 1 predictors used to derive a propensity index for taking up e-products between Wave 1 and 2. The analyses are based on those with data at Waves 1, 2 and 3 who had never smoked cigarettes by Wave 2 and had never used e-cigarettes by Wave 1. Between Waves 2 and 3261/7367 (3.54%) of never users of e-products at Wave 2 took up smoking, while 148/893 (16.57%) of ever users did so. For individuals who were 16-17 at Wave 1, adult data were used to determine e-product use and cigarette smoking at later Waves. The table includes the results of a stepwise regression based on successively including the most significant adjustment variables, given that ever e-product use at Wave 2 was included in the model.\n\nFour sensitivity analyses of M1 were carried out, fuller details being given in Table 3 to Table 6 of the Additional File (see Extended data (Lee, 2020)).\n\nCompared to M1, S1 excluded those who had ever used products other than cigarettes or e-cigarettes at Wave 1, both in the construction of the propensity index and in estimating the gateway effect. Whereas M1 involved 8260 youths, of which 409 initiated smoking between Waves 2 and 3, S1 involved 7945, of which 359 took up smoking. The propensity index developed for S1 involved all the 16 variables shown in Table 2, except for “Number of times seen Movie 4” and “Think you will try a cigarette soon”. Here, the pattern of results is similar to that for Table 2, with the unadjusted OR of 5.66 (95% CI 4.49-7.13) reducing to either 3.45 (2.67–4.46), 3.24 (2.53–4.15), or 3.23 (2.49–4.18), depending on whether adjustment was made for propensity as a continuous variable, propensity as quintiles, or all the 14 variables individually.\n\nCompared to M1, the only difference for S2 was that ever smoked other tobacco products at Wave 1 was added to the 16 variables used in M1 to make up the propensity score, and was forced into the regression models. Starting with the same unadjusted OR as M1, the adjusted ORs were very similar; 3.37 (2.64–4.29), 3.07 (2.44-3.87) and 3.20 (2.50-4.08), after adjustment for propensity (continuous), propensity (quintiles), or all the individual variables.\n\nWhereas M1 (and S1 and S2) adjusted for variables found to be predictive of initiating e-product use at Wave 2, S3 adjusted for variables predictive of cigarette smoking. Here, the final model included 27 variables. The unadjusted OR of 5.65 (95% CI 4.55-7.01) slightly differed from that in M1 as the individuals considered had to have non-missing data on 27 variables rather than 16. However, the overall effect of adjustment was again similar, with the OR reducing to 3.28 (2.56-4.22) after adjustment for all 27 variables. As for M1, adjustment for the first four variables had the most effect. Adjustment for the first seven variables reduced the OR to 3.26 (2.57-4.13), similar to the OR after adjustment for all 27. Propensity adjustment was not carried out in S3.\n\nCompared to M1, S4 adjusted for variables predictive of take-up of any tobacco product between Waves 1 and 2. Here, the propensity index was based on 18 variables, with the unadjusted OR of 5.74 (4.55-7.23) reducing to 3.31 (95% CI 2.56-4.28), 3.19 (2.48-4.09), or 3.21 (2.47-4.18), after adjustment for propensity (continuous), propensity (quintiles), or all the individual variables. Adjustment for all 18 variables had a similar effect to adjustment for the most important 10 variables, where the OR was 3.20 (2.47-4.14).\n\nHere, instead of deriving the Wave 1 predictors linked to uptake of e-cigarettes between Waves 1 and 2, analysis M2 uses the same set of Wave 1 predictors used in our earlier work (Lee & Fry, 2019), the results being shown in Table 3. Here, the unadjusted OR of 5.74 (95% CI 4.62-7.13) reduced to 3.54 (2.81-4.45) after adjustment for propensity as quintiles and to 3.45 (2.72-4.37) after adjusting for the individual variables. While adjustment here removed about half the excess OR, the reduction was less, to 4.53 (3.62-5.68), after adjustment for propensity as a continuous variable. The reductions were similar if exact age rather than age group was included in the list of variables. Here, the unadjusted OR was reduced to 3.51 (2.79-4.41) after adjustment for propensity as quintiles, 4.59 (3.66-5.74) after adjustment for propensity as a continuous variable, and 3.39 (2.67-4.30) after adjustment for the individual variables.\n\nNotes: The table shows the effects of adjustment based on the same Wave 1 predictors as used in our original paper (Lee & Fry, 2019). The analyses are based on those with data at Waves 1, 2 and 3 who had never smoked cigarettes by Wave 2 and had never used e-cigarettes by Wave 1. Between Waves 2 and 3, 249/7133 (3.49%) of never users of e-products at Wave 2 took up smoking, while 146/880 (16.59%) of ever users did so. For individuals who were 16-17 at Wave 1, adult data were used to determine e-product use and cigarette smoking at later Waves. The table includes the results of a stepwise regression based on successively including the most significant adjustment variables, given that ever e-product use at Wave 2 was included in the model.\n\nSimilar analyses were also run that did not exclude those who had used e-cigarettes by Wave 1. This increased the number of ever e-product users who took up smoking from 146 to 201, and slightly increased the unadjusted OR to 5.95 (4.89-7.23). However, the pattern of decline following adjustment was quite similar. For example, the OR adjusted for the individual variables reduced to 3.31 (2.65-4.12) using grouped age and to 3.26 (2.62-4.06) using exact age.\n\nAs noted in the Methods section, M3 is essentially a replicate of our earlier work (Lee & Fry, 2019), but using a different period of introduction of cigarettes. The propensity score developed was based on 18 variables, using age group or exact age as alternatives. The results, shown in Table 4, indicate that, as earlier (Lee & Fry, 2019), a large proportion of the unadjusted association can be explained by adjustment. The largest proportion was explained by adjusting for the 18 variables making up the propensity score, with the unadjusted OR of 6.70 (95% CI 5.40-8.32) reducing to 2.25 (1.74-2.91) or 2.75 (1.75-2.93) depending on whether the list of variables included age range or exact age. However, most of this reduction could be explained by adjustment for propensity.\n\nNotes: The table shows the effects of adjustment based on Wave 2 predictors linked to use of e-products in Wave 2. The analyses are based on those with data at Waves 2 and 3 ignoring data from Wave 1. Between Waves 2 and 3, 228/8233 (2.77%) of never users of e-products at Wave 2 took up smoking, while 145/949 (15.28%) of ever users did so. For individuals who were 17 at Wave 2, adult data were used to determine cigarette smoking at Wave 3. The table includes the results of a stepwise regression based on successively including the most significant adjustment variables, given that ever e-product use at Wave 2 was included in the model. The first set of ORs is based on a model including age group, while the second is based on a model including exact age.\n\nCombining the Wave 2 to 3 results shown in Table 4 with the Wave 1 to 2 results reported earlier (Lee & Fry, 2019) by fixed-effect meta-analysis gives an unadjusted OR of 6.30 (5.31-7.46), which is reduced to 2.65 (2.24-3.18), 2.53 (2.07-3.10) or 2.08 (1.70-2.54) depending on whether adjustment is for propensity (quintiles), propensity (continuous) or all the variables making up the propensity score. This represents reductions in the excess OR of, respectively, 68.9%, 71.1% or 79.8%.\n\nTable 5 summarizes the main results of these analyses and compares them with those reported earlier (Lee & Fry, 2019). While the original analyses were based on 9423 youths, 421 of whom initiated smoking, the new analyses were based on 8700 youths, 389 of whom initiated smoking. As can be seen, the results in the original analysis, based on grouped age, were similar to those from the new analyses, whether grouped or exact age was used.\n\nNotes: Each set of ORs is based on those who had never smoked cigarettes by Wave 1. The first analysis is as summarized in Table 1. The last two analyses only exclude those without data at Wave 3.\n\nThe results from analysis A1 for grouped age may theoretically be combined with those from analysis M2 shown in Table 3, as they both use the Wave 1 predictors from our original paper (Lee & Fry, 2019), with exact age replacing grouped age, and are both based on individuals with data at all three Waves. However, as illustrated by the results adjusted for all 12 variables, where the ORs are 3.45 (95% CI 2.72-4.37) from Table 3 and 1.97 (1.42-2.73) from Table 5, these estimates are heterogeneous (p<0.001), providing a random-effects combined estimate of 2.64 (1.52-4.57).\n\nThis analysis is similar to that reported originally (Lee & Fry, 2019) but relates to a longer follow-up period, and uses exact rather than grouped age. The results of this analysis, shown in Table 6, are quite similar to those shown in Table 5. Again, an unadjusted OR is markedly reduced by adjusting for propensity, whether as quintiles or as a continuous variable, and is further reduced by adjusting for all the 12 individual variables considered.\n\nNotes: The table shows the effects of adjustment based on the same Wave 1 predictors as used in our original paper (Lee & Fry, 2019) but replacing age range by exact age. The set of ORs is based on those with data at Waves 1, 2 and 3 who had never smoked cigarettes by Wave 1. Between Waves 1 and 3, 716/8334 (8.59%) of never users of e-cigarettes at Wave 1 took up smoking, while 123/366 (33.61%) of ever users did so. The table includes the results of a stepwise regression based on successively including the most significant adjustment variables, given that ever e-product use at Wave 1 was included in the model.\n\nTable 7 summarizes the main results shown in Table 2 for main analysis M1, which make no allowance for residual confounding, and compares them with the results of an analysis using the same list of predictor variables, but with values modified in an attempt to adjust for residual confounding. As can be seen, markedly more of the unadjusted association was explained when allowance for residual confounding was made, with the adjusted ORs in the range 2.36 to 2.46 when allowance was made, compared with 3.11 to 3.37 when it was not. Note that the unadjusted ORs in the two sets of results vary slightly, as missing values in some individuals in the original analyses were replaced by estimates taken from other Waves.\n\nNotes: The “no allowance” results correspond to those in Table 6.\n\nThe analyses are based on those with data at Waves 1, 2 and 3 who had never smoked cigarettes by Wave 2 and had never used e-cigarettes by Wave 1. Between Waves 2 and 3 261/7367 (3.54%) of never users of e-products at Wave 2 took up smoking, while 148/893 (16.57%) of ever users did so in the population considered in the “no allowance” analyses The corresponding figures in the “allowance” analyses were 267/7682 (3.48%) and 150/915 (16.39%). For individuals who were 16-17 at Wave 1, adult data were used to determine e-product use and cigarette smoking at later Waves. The table includes the results of a stepwise regression based on successively including the most significant adjustment variables, given that ever e-product use at Wave 2 was included in the model.\n\nWhile allowance for residual confounding has quite a marked effect for analysis M1, the analysis which avoided the possibility of over-adjustment, it did not for analyses M3 and A2, which did not avoid this possibility. Detailed results are shown in Table 7 and Table 8 in the Additional File (see Extended data (Lee, 2020)).\n\na % excess explained =100*(ORu – ORA) / (ORu–1) where ORu is the unadjusted OR, and ORA is the adjusted OR.\n\nb P as Q = propensity as quintiles.\n\nc P as C = propensity as a continuous variable.\n\nVersions of analyses M1, M3 and A1 were also seen, in which the number of variables adjusted for was extended by also including interactions of age with each of the other three predictors most strongly linked to the gateway effect. For analysis M1, allowance for these interactions had virtually no effect, the original estimate of 3.27 (95% CI 2.57-4.16) shown in Table 2 without including interactions changing to 3.26 (2.55-4.15) when interactions were included in the model. For analysis M3, the estimate changed only from 2.27 (1.75-2.93) to 2.35 (1.81-3.05), while for analysis A1, it changed from 1.98 (1.43-2.75) to 2.06 (1.48-2.88).\n\nTable 8 summarizes the results from 18 of the analyses described above, expressing the extent to which adjustment explained the unadjusted OR using the statistic 100 x (ORU – ORA) / (ORU – 1) where ORU is the unadjusted OR, and ORA is the adjusted OR. The most obvious impression from the table is that the results largely fall into two groups.\n\nResults from the original analysis and for analyses M3, A1 and A2 (rows A, K to O, and Q to R of Table 8) all show that as much as about 80% of the unadjusted excess OR can be explained by adjustment for the full set of variables in the model, with somewhat less, typically about 70%, explained using propensity as quintiles or as a continuous variable.\n\nIn contrast, results from virtually all of analyses M1 and M2 (rows B to K) show that only about 50% of the unadjusted excess OR can be explained by adjustment for the full set of variables, with propensity as quintiles giving generally similar results.\n\nThe difference between these two groups is that the first set of results are subject to the problem of over-adjustment, with the values of the predictors used possibly having been affected by having used e-cigarettes. This is mainly so where the baseline Wave was Wave 1, but was also true for analysis M3 where Wave 1 data were essentially ignored. In contrast, the second set of results avoided over-adjustment by considering follow-up from Wave 2 to 3, with predictors based on Wave 1 data in youths who had never used e-cigarettes. However, in this second set of results the variables used were not as up-to-date as in the first analyses.\n\nThe variant analysis of M1, allowing for residual confounding (row P), gives an intermediate result, with about 70% of the excess risk being explained, whether by the full set of variables or by propensity. This analysis, however, does not avoid the problem of over-adjustment as it incorporates some information from Waves where individuals were already using e-cigarettes.\n\nIt is clear from Table 8 that many of the variables studied had little effect on the pattern of results. These included use of grouped or exact age, taking into account use of other products, and using predictors of cigarette smoking or any tobacco use rather than predictors of e-cigarette use.\n\nTwo other conclusions may be drawn from Table 8. One is that adjustment for propensity as quintiles or as a continuous variable generally gives very similar results, with the exception of analysis M2 and its variants, where propensity as a continuous variable explained substantially less of the unadjusted excess OR. Inspection of the detailed modelling results showed that, whereas in other analyses, the logarithm of the OR increased fairly linearly with quintiles of propensity, in the case of analysis M2 and its variants it did not. Thus, in M1 for example, the log ORs by quintile were 0, 0.73, 1.11, 1.66 and 2.52, while in M2 they were 0, 0.21, 0.96, 1.51 and 2.19, with very little rise between quintiles 1 and 2.\n\nThe other is that adjustment for the first six variables in the model generally explained a very substantial part of the unadjusted excess OR explained by the full set. Though this was not true for analysis M2, it was still true that adjustment for the last eight or nine variables explained far less of the excess OR than did the first eight or nine.\n\n\nDiscussion\n\nIn our publication based on Waves 1 and 2 (Lee & Fry, 2019) our analyses showed that an unadjusted estimate of the gateway effect 5.70 (85% CI 4.33-7.50) could be considerably reduced by adjustment, to 1.59 (1.14-2.20) in the most striking case. Because of the marked reduction in the OR following adjustment, and the possibility of incomplete control for confounding we regarded it as “unclear whether prior vaping actually increases uptake of cigarette smoking”. However, we did note the possibility of over-adjustment, with vaping at Wave 1 possibly having affected the recorded values of some of the variables used for adjustment.\n\nAt that time we noted that this possibility of over-adjustment could be addressed in analyses relating initiation of cigarette smoking at Wave 3 to vaping at Wave 2, restricting attention to those youths who, at Wave 1, had never vaped, and using adjustment variables recorded at Wave 1. This we have done in the analyses reported here, and our major finding is that adjustment reduced the excess risk far less, by only about 50% rather than about 80%, in our main analysis M1.\n\nWhile these results more strongly support the existence of a true gateway effect of taking up vaping, there must still remain doubt about its magnitude. One reason is that predictors recorded a year before the baseline may not fully account for the characteristics of the youth at the start of follow-up. A second reason is that, although the PATH study records data on a whole range of possibly relevant characteristics, there may be some relevant predictors not considered. A third reason is that the answers to some of the questions may have been inaccurately measured. We have attempted to address this problem of residual confounding by amending values of predictors recorded at Wave 1 to take into account data recorded at later Waves. However, this problem re-introduces the problem of over-adjustment as Wave 2 and 3 values may have been affected by vaping. Theoretically, one could use data from Waves 1 to 4, using data for Waves 1 and 2 from youths who have never vaped to produce more accurate estimates of the predictors to use for a study of gateway effects between Waves 3 and 4. But this would add to the problem of using predictors recorded some time before follow-up.\n\nSince the time that we published our earlier analysis (Lee & Fry, 2019) and our paper on general considerations relating to vaping as a possible gateway into cigarette smoking (Lee et al., 2018) a number of other authors have presented evidence from prospective studies (Bold et al., 2018; Chien et al., 2019; Kinnunen et al., 2019; Morgenstern et al., 2018; Pénzes et al., 2018; Primack et al., 2018; Treur et al., 2018). The studies vary in the extent to which potential confounding variables have been adjusted for, with large OR estimates tending to be reported in studies with more limited control. Thus, a study in the Netherlands (Treur et al., 2018), which adjusted only for sex, age education and a single indicator of propensity to smoke, reported an OR of 11.90 (95% CI 3.36-42.11) for the relationship between ever use of e-cigarettes with nicotine and initiation of cigarette smoking during follow-up. Also, a study in the US (Bold et al., 2018), which adjusted only for demographic variables and use of other tobacco products, reported ORs of 7.08 (2.34-21.42) and 3.87 (1.86-2.06) depending on the follow-up period studied, while another US study (Pénzes et al., 2018), with limited control for confounding variables, reported an OR of 3.57 (1.96-6.45). Apart from a US study (Primack et al., 2018) ,which reported an OR of 6.8 (1.7-28.3), following adjustment for ten covariates independently associated with initiation of smoking, most of the other studies that appear to have better control for confounding gave lower estimates. These included a study in Taiwan (Chien et al., 2019), which reported an OR of 2.14 (1.66-2.75), a study in Germany (Morgenstern et al., 2018), which reported an OR of 2.18 (1.65-2.87) and a study in Finland (Kinnunen et al., 2019), which reported that adjustment reduced the OR from 11.52 (4.91-26.56) to 2.92 (1.09-7.85). Notably, a study in Great Britain (East et al., 2018) reported an OR of 11.89 (3.56-39.72) estimated using the usual logistic method, but a reduced value of 1.34 (1.05-1.72) using causal mediation analysis.\n\nGenerally our results are consistent with the literature in confirming that a substantial proportion, but not all, of the observed association between e-cigarette use and subsequent initiation of cigarette smoking can be explained by adjustment for factors linked to susceptibility to tobacco. However, large cohort studies with high quality, accurate, data on a wide range of predictive factors recorded at regular intervals will be needed to gain better insight into the magnitude of any true causal effect of vaping. The PATH study with its multiple Waves and comprehensive questionnaire should prove more and more useful in the future.\n\nThere are, in theory, various effects of e-cigarettes (Lee et al., 2018). Beneficial effects occur when individuals who would have continued to smoke take up vaping instead, and when vaping helps smokers to quit or reduce cigarette consumption. Adverse effects, apart from when vaping encourages individuals to start smoking, would occur if smokers who intended to quit switch instead to vaping, or if smokers add vaping to their usual consumption of cigarettes. When trying to estimate the health impact of e-cigarettes, one must consider all these effects.\n\nBy using data from three Waves of the PATH study, the analyses of the gateway effect reported here improve on those reported earlier (Lee & Fry, 2019) based on the first two Waves by allowing potential confounding variables to be determined at a time before vaping started. Whereas the earlier analyses suggested that the adjustment for confounding explained about 80% of the unadjusted relationship between vaping and subsequent initiation of smoking, our current analyses suggest that adjustment explains only about 50%. This provides stronger evidence of a true effect of vaping, although doubt still remains about its true magnitude for reasons discussed.\n\n\nData availability\n\nNational Addiction & HIV Data Archive Program: Population Assessment of Tobacco and Health (PATH) Study [United States] Public-Use Files (ICPSR 36498). https://doi.org/10.3886/ICPSR36498.v9 (United States Department of Health and Human Services (USDHHS), 2019).\n\nThe data are available under the Terms of Use as set out by ICPSR, which can be accessed when users start the process of downloading the data.\n\nOpen Science Framework: Further investigation of gateway effects using the PATH study https://doi.org/10.17605/OSF.IO/7ECQH (Lee, 2020).\n\nThis project contains the following extended data files:\n\nGateway paper for F1000 Research_Additional file.docx\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank Esther Afolalu for assistance in acquiring the data from the PATH study, and Zheng Sponsiello-Wang and Christelle Chrea for providing technical comments at various stages. We also thank Jan Hamling for assistance in running the analyses, and Yvonne Cooper and Diana Morris for typing the various drafts of the paper.\n\n\nReferences\n\nBarrington-Trimis JL, Urman R, Leventhal AM, et al.: E-cigarettes, cigarettes, and the prevalence of adolescent tobacco use. Pediatrics. 2016; 138(2): e20153983. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBest C, van der Sluijs W, Haseen F, et al.: Does exposure to cigarette brands increase the likelihood of adolescent e-cigarette use? A cross-sectional study. BMJ Open. 2016; 6(2): e008734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBold KW, Kong G, Camenga DR, et al.: Trajectories of e-cigarette and conventional cigarette use among youth. Pediatrics. 2018; 141(1): e20171832. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChien YN, Gao W, Sanna M, et al.: Electronic cigarette use and smoking initiation in Taiwan: Evidence from the First Prospective Study in Asia. Int J Environ Res Public Health. 2019; 16(7): 1145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEast K, Hitchman SC, Bakolis I, et al.: The association between smoking and electronic cigarette use in a cohort of young people. J Adolesc Health. 2018; 62(5): 539–547. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKinnunen JM, Ollila H, Minkkinen J, et al.: Nicotine matters in predicting subsequent smoking after e-cigarette experimentation: A longitudinal study among Finnish adolescents. Drug Alcohol Depend. 2019; 201: 182–187. PubMed Abstract \| Publisher Full Text\n\nLee P: Further Investigation of Gateway Effects Using the PATH Study.OSF. 2020. https://www.doi.org/10.17605/OSF.IO/7ECQH\n\nLee P, Fry J: Investigating gateway effects using the PATH study [version 2; peer review: 2 approved]. F1000Res. 2019; 8: 264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee PN, Coombs KJ, Afolalu EF: Considerations related to vaping as a possible gateway into cigarette smoking: an analytical review [version 3; peer review: 2 approved]. F1000Res. 2018; 7: 1915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiech R, Johnston L, O'Malley PM, et al.: Trends in adolescent vaping, 2017-2019. N Engl J Med. 2019; 381(15): 1490–1491. PubMed Abstract \| Publisher Full Text\n\nMorgenstern M, Nies A, Goecke M, et al.: E-Cigarettes and the use of conventional cigarettes. Dtsch Arztebl Int. 2018; 115(14): 243–248. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Academies of Sciences Engineering and Medicine: Public health consequences of e-cigarettes. The National Academies Press, Washington DC. 2018. Publisher Full Text\n\nNutt DJ, Phillips LD, Balfour D, et al.: Estimating the harms of nicotine-containing products using the MCDA approach. Eur Addict Res. 2014; 20(5): 218–225. PubMed Abstract \| Publisher Full Text\n\nPénzes M, Foley KL, Nadasan V, et al.: Bidirectional associations of e-cigarette, conventional cigarette and waterpipe experimentation among adolescents: A cross-lagged model. Addict Behav. 2018; 80: 59–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrimack BA, Shensa A, Sidani JE, et al.: Initiation of traditional cigarette smoking after electronic cigarette use among tobacco-naive US young adults. Am J Med. 2018; 131(4): 443.e1–443.e9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoneji S, Barrington-Trimis JL, Wills TA, et al.: Association between initial use of e-cigarettes and subsequent cigarette smoking among adolescents and young adults: A systematic review and meta-analysis. JAMA Pediatr. 2017; 171(8): 788–797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreur JL, Rozema AD, Mathijssen JJP, et al.: E-cigarette and waterpipe use in two adolescent cohorts: cross-sectional and longitudinal associations with conventional cigarette smoking. Eur J Epidemiol. 2018; 33(3): 323–334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUnited States Department of Health and Human Services (USDHHS): Population assessment of tobacco and health (PATH) Study [United States] Public-Use Files (ICPSR 36498-V9). 2019. Publisher Full Text" }	[ { "id": "66520", "date": "22 Jul 2020", "name": "James Sargent", "expertise": [ "Reviewer Expertise Adolescent substance use." ], "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 thoughtful analysis of PATH data to determine an unbiased estimate of the relation between initial e-cigarette use among never cigarette smokers and subsequent cigarette smoking. I particularly like the idea of using W1 predictors of W2 e-cigarette onset to parcel out the over adjustment that could occur if these variables are assessed at the same time. I also liked the multitude of sensitivity analyses that showed it doesn't really matter, for example, how propensity scores are modeled. I see no major weaknesses. However I have a few suggestions.\nIt might not be unreasonable to have a statistician review the analysis.\n\nThis is a complete case analysis. Given that there are missing data for each individual variable and that there is loss to follow up, the authors need to convince us with some sort of sensitivity analysis that the results are not largely affected by attrition bias.\n\nThe literature review makes it seem like these are the only authors who have published on gateway effects using PATH data. They need to cite other PATH papers, point out weaknesses in them, and help us understand why this publication is worthy of attention. One worthy of particular attention used a propensity score analysis similar to these authors' W1-W2 analysis1\n\nOne limitation not mentioned is that cigarette smoking onset does not make addicted cigarette smoker. This needs to be mentioned as a limitation.\n\nThe authors miss some of the many studies that examined the relation between initial use of e-cigarettes and subsequent cigarette smoking. They could fill in that gap by mentioning and citing a meta-analysis conducted by Khouja in Tobacco Control that identified 17 prospective studies2. It is worth comparing their best estimate with the combined estimates presented in that meta analysis.\n\nFinally, given that there have been so many prospective studies, and all have pointed to a gateway effect, it seems reasonable to conclude that there is one, that is, that use of these devices independently increases risk for subsequent use of cigarettes. I realize that we could continue to quibble about the effect size, but this study does a good job of convincing us that the relative risk is real and that it is substantial, around 3. It seems like it might be an opportunity to also help us understand the population significance of the finding. The authors could do that with this population-based sample (which includes weights) by determining what proportion of the observed cigarette initiation is attributable to the gateway effect using attributable risk methods (risk difference as opposed to risk ratio). They could use the weights to determine the number of new cigarette initiators there were in the US that year attributable to e-cigarette exposure. This would be a real and novel contribution that would help investigators compare the public health consequences to youth with the public health consequences resulting from increased smoking cessation.\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?\nI cannot comment. A qualified statistician is required.\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": [ { "c_id": "6070", "date": "09 Nov 2020", "name": "Peter Lee", "role": "Author Response", "response": "Reply to comments made by James Sargent We thank the reviewer for the time he has spent and the useful comments made. Our replies to the points made are given in bold face type, making it clear where we have amended the original version of the paper. Note that the changes made to the paper are also intended to answer the points made by Shu Xu, the other reviewer. We hope that our answers and the changes to the paper will allow the revision to be approved. Approved With Reservations This is a thoughtful analysis of PATH data to determine an unbiased estimate of the relation between initial e-cigarette use among never cigarette smokers and subsequent cigarette smoking. I particularly like the idea of using W1 predictors of W2 e-cigarette onset to parcel out the over adjustment that could occur if these variables are assessed at the same time. I also liked the multitude of sensitivity analyses that showed it doesn't really matter, for example, how propensity scores are modeled. I see no major weaknesses. However I have a few suggestions. It might not be unreasonable to have a statistician review the analysis. Both the authors of this paper are experienced statisticians, as is Shu Xu, the other reviewer. This is a complete case analysis. Given that there are missing data for each individual variable and that there is loss to follow up, the authors need to convince us with some sort of sensitivity analysis that the results are not largely affected by attrition bias. We have added a new paragraph into the discussion, starting “Other issues are...” and hope this meets the reviewer’s point. The literature review makes it seem like these are the only authors who have published on gateway effects using PATH data. They need to cite other PATH papers, point out weaknesses in them, and help us understand why this publication is worthy of attention. One worthy of particular attention used a propensity score analysis similar to these authors' W1-W2 analysis1. We have added a new paragraph in the discussion, after the one referring to other studies on the gateway issue, to consider other studies using PATH data, including the Watkins study on which we had commented previously in our 2019 paper. One limitation not mentioned is that cigarette smoking onset does not make addicted cigarette smoker. This needs to be mentioned as a limitation. At the end of the paragraph in the discussion starting “Generally our consistent” we have made the point that some of those recorded as taking up smoking at Wave 3 may only have taken it up for a short while, a limitation that can be answered better in analyses based also on data from later Waves. The authors miss some of the many studies that examined the relation between initial use of e-cigarettes and subsequent cigarette smoking. They could fill in that gap by mentioning and citing a meta-analysis conducted by Khouja in Tobacco Control that identified 17 prospective studies2. It is worth comparing their best estimate with the combined estimates presented in that meta analysis. We are not sure why the reviewer thought we were not citing other studies. The first paragraph of the introduction refers to the meta-analysis of Soneji et al. which considered nine studies, while the second paragraph of the introduction refers to our 2018 paper which includes a detailed commentary on 15 studies. Also the fourth paragraph of the discussion refers to quite a number of recent studies. However, we have now made it clear in the paragraph summarizing conclusions from our 2018 paper that it considered 15 cohort studies that have reported unadjusted and adjusted estimates of the gateway effect, nine considered in the 2017 meta-analysis by Soneji et al. and six additional studies. We have also added a paragraph in the introduction mentioning the recent review by Khouja et al. that the reviewer referred to. Finally, given that there have been so many prospective studies, and all have pointed to a gateway effect, it seems reasonable to conclude that there is one, that is, that use of these devices independently increases risk for subsequent use of cigarettes. I realize that we could continue to quibble about the effect size, but this study does a good job of convincing us that the relative risk is real and that it is substantial, around 3. It seems like it might be an opportunity to also help us understand the population significance of the finding. The authors could do that with this population-based sample (which includes weights) by determining what proportion of the observed cigarette initiation is attributable to the gateway effect using attributable risk methods (risk difference as opposed to risk ratio). They could use the weights to determine the number of new cigarette initiators there were in the US that year attributable to e-cigarette exposure. This would be a real and novel contribution that would help investigators compare the public health consequences to youth with the public health consequences resulting from increased smoking cessation. In order to illustrate the population effect we have added a new paragraph in the discussion (starting “A question of interest is..”) which estimates the percentage of new smokers associated with exposure to e-cigarettes as about 23%. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required. See comment above. Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes References 1. Stanton C, Bansal-Travers M, Johnson A, Sharma E, et al.: Longitudinal e-Cigarette and Cigarette Use Among US Youth in the PATH Study (2013–2015). JNCI: Journal of the National Cancer Institute. 2019; 111 (10): 1088-1096 Publisher Full Text 2. Khouja JN, Suddell SF, Peters SE, Taylor AE, et al.: Is e-cigarette use in non-smoking young adults associated with later smoking? A systematic review and meta-analysis.Tob Control. 2020. PubMed Abstract \| Publisher Full Text I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We hope that we have answered the reviewer’s reservations adequately." } ] }, { "id": "71761", "date": "13 Oct 2020", "name": "Shu Xu", "expertise": [ "Reviewer Expertise Longitudinal data analysis", "propensity score methods", "missing data method", "tobacco research." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors examined the association between youth prior e-cigarette use and increased risk of subsequent cigarette smoking using the Waves 1 – 3 data from the PATH study. This work is an extension of their previous studies which were published in Lee et al. (2018) and Lee and Fry (2019), the latter was based on the Waves 1 and 2 data from the PATH study. This study is interesting because the authors conducted three main analyses studying the association between e-cigarette use and subsequent cigarette smoking along with sensitivity analyses. This review emphasized the statistical methodology and results reporting. A few major concerns are below.\nI feel the readability of this paper would be improved if authors could (1) focus on what is the limitation of the previous articles, (2) clearly state what are the new analyses about based on what has been done previously, and (3) state why versions of M1, M2, M3 were conducted and the logic behind them. The authors need to provide a full picture of the study design and analytical plan of the current study. In case some details are overlapped with previous articles when referred to the previous article, authors need to at least summarize the details instead of releasing no specific information.\n\nThe main concern of the previous study is about the possibility of “over-adjustment,” and the extent to which the association between prior e-cigarette use and subsequent cigarette initiation has been “over-adjusted.” It would be critical to evaluate whether covariate balance was sufficient when propensity scores had been considered in the current analyses. Without covariate balance, the results of the current study may be considered unreliable. Thus, detailed results such as (a) propensity score distribution by e-cigarette exposure groups and (b) comparison of the extent of covariate imbalance are desired.\n\nIn the Methods section, authors need to clearly state how the missing values were treated in analyses of the current study. This also involved how authors treated the missing values when selecting covariates of versions of M1, M2, and M3. The results of the current study could be misleading if only participants with complete data were considered.\n\nIt was unclear to me why to study the continuous age and grouped age and compare the difference. It seems like continuous age provided an exact measure however grouped age did not. Putting participants into categories is rarely defensible unless authors provide further justification. It is also unclear to me why only interactions with age (no other covariates, for example, race) were considered.\nMinor concerns are below.\nIn tables, in addition to individuals who were 16-17 at Wave 1, adult data were used. Please clarify, for those who were 15-16 at Wave 1 (those who were 18+ at Wave 3), whether adult data were also used in this study?\n\nThe abstract was very confusing. It failed to provide an overview of the study. For example, a clear introduction of the methods and results of M1 and have been presented. This information regarding M2 and M3 were not clearly reported.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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": [ { "c_id": "6071", "date": "09 Nov 2020", "name": "Peter Lee", "role": "Author Response", "response": "Reply to comments made by Shu Xu We thank the reviewer for the time he has spent and the useful comments made. Our replies to the points made are given in bold face type, making it clear where we have amended the original version of the paper. Note that the changes made to the paper are also intended to answer the points made by James Sargent, the other reviewer. We hope that our answers and the changes to the paper will allow the revision to be approved. Approved With Reservations The authors examined the association between youth prior e-cigarette use and increased risk of subsequent cigarette smoking using the Waves 1 – 3 data from the PATH study. This work is an extension of their previous studies which were published in Lee et al. (2018) and Lee and Fry (2019), the latter was based on the Waves 1 and 2 data from the PATH study. This study is interesting because the authors conducted three main analyses studying the association between e-cigarette use and subsequent cigarette smoking along with sensitivity analyses. This review emphasized the statistical methodology and results reporting. A few major concerns are below. I feel the readability of this paper would be improved if authors could (1) focus on what is the limitation of the previous articles, (2) clearly state what are the new analyses about based on what has been done previously, and (3) state why versions of M1, M2, M3 were conducted and the logic behind them. The authors need to provide a full picture of the study design and analytical plan of the current study. In case some details are overlapped with previous articles when referred to the previous article, authors need to at least summarize the details instead of releasing no specific information. The three paragraphs of the discussion starting “Our second paper..” describe in some detail the analyses we had previously conducted using data from Waves 1 and 2 only, what the main results of these analyses were, and the fact that the estimates were open to the possibility of over-adjustment if taking up e-cigarettes had affected the values of some of the Wave 1 predictor variables considered. It also makes it clear that our earlier paper described how this possibility could be avoided by using data from Waves 1, 2 and 3. We have now amended the final paragraph of the discussion to make it clear that analysis M1 in the current paper was that envisaged in our earlier paper, and that this was the main objective of our work. In the methods section, there was already some comment on why we had conducted the other main analyses, the sensitivity analyses and the alternative analyses, but this has now been extended in various places to make it clearer. Where details of our analyses are the same as those in our earlier analyses, it seems needlessly duplicative to repeat these details in the current paper, and is not the usual thing to do in such a situation. The main concern of the previous study is about the possibility of “over-adjustment,” and the extent to which the association between prior e-cigarette use and subsequent cigarette initiation has been “over-adjusted.” It would be critical to evaluate whether covariate balance was sufficient when propensity scores had been considered in the current analyses. Without covariate balance, the results of the current study may be considered unreliable. Thus, detailed results such as (a) propensity score distribution by e-cigarette exposure groups and (b) comparison of the extent of covariate imbalance are desired. Our latest paper has removed the possibility of over-adjustment in our previous work by the use of propensity indicators based on data recorded at Wave 1 in those who, at that time, had never vaped. The reviewer questions whether covariate balance is sufficient after the propensity scores are taken into account. This has been investigated in Tables 2, 3 and 4 for the three main analyses in turn by considering whether adjustment for the individual variables making up the propensity index materially affected the estimated gateway effect. The effect was generally quite small, suggesting that reasonable balance had been achieved. We think that including the additional material suggested by the reviewer would add little other than extra complexity. We also note that our previous paper did not include such material and was approved by the reviewers who considered it. In the Methods section, authors need to clearly state how the missing values were treated in analyses of the current study. This also involved how authors treated the missing values when selecting covariates of versions of M1, M2, and M3. The results of the current study could be misleading if only participants with complete data were considered. As we note in the first sentence of the methods section “Some aspects of the analyses described here are the same as those described earlier (Lee & Fry, 2019) are not presented again here.” In that paper we made it clear that all the logistic regression analyses used “required individuals with complete data on all variables”, and that the various stages in developing propensity scores used “groups of conceptually-related variables, with missing values likely to be on the same individuals”. We prefer not to repeat the description of this part of the methodology in the current paper. However, in the new paragraph we have added into the discussion (starting “Other issues are...”), we have addressed your point that basing the analysis only on complete data might be misleading. This point is similar to one raised by another reviewer. We hope you find this satisfactory. It was unclear to me why to study the continuous age and grouped age and compare the difference. It seems like continuous age provided an exact measure however grouped age did not. Putting participants into categories is rarely defensible unless authors provide further justification. As regards age, the 2019 paper we had published based only on Wave 1 and 2 subdivided individuals into ages 12-14 and 15-17 as the data were only available in that form. Assuming that the Waves were conducted a year apart (which they approximately were) we could infer that those who were 12-14 at Wave 1 and 15-17 at Wave 2 were 14 at Wave 1 (and 15 at Wave 2), and that those who were 15-17 at Wave 1 and adults at Wave 2 were 17 at Wave 1 (and 18 at Wave 2). However we could not estimate the exact age of those who were 12, 13, 15 or 16 at Wave 1. The position changed in the analyses using Wave 3 as well, as we could define those who were 12-14 throughout as 12 at Wave 1, those who were 12-14 at Waves 1 and 2 and 15-17 at Wave 2 as 13 at Wave 1 and so on. While it would be preferable to use exact age throughout in some ways, here we were carrying out further analyses using the propensity index developed in the 2019 paper which included a term based on grouped age. As the paper presents the main analyses using both grouped age and exact age, and the results were much the same, there is no real problem. It is also unclear to me why only interactions with age (no other covariates, for example, race) were considered. On the basis that age had a major effect on the rate of e-cigarette use and on uptake of smoking, we included interactions of age with the three predictors most strongly linked to the relevant gateway effect. As this had essentially no effect on the estimates of the gateway effect, we felt that looking at further interactions would not be worthwhile. Race was not a predictor that was included in the propensity index, so it seemed highly unlikely that including interactions with it would have had any major effect. It would of course have been theoretically possible to consider many more predictors, including interactions of each predictor with each other predictor, higher order interactions, or quadratic or cubic terms in some predictors, but one has to stop somewhere. However in the third paragraph of the discussion we have changed “there may be some relevant predictors not considered” to “there may be some relevant predictors or interactions of predictors not considered.” Minor concerns are below. In tables, in addition to individuals who were 16-17 at Wave 1, adult data were used. Please clarify, for those who were 15-16 at Wave 1 (those who were 18+ at Wave 3), whether adult data were also used in this study? Those who were 17 at Wave 1 would have been 18 at Wave 2 so adult data would have been used. Similarly, those who were 16 at Wave 1 would have been 18 at Wave 3 so adult data would again have been used. However, those who were 15 at Wave 1 would not have been adults at Wave 3, so adult data were irrelevant. To avoid confusion we have changed age ranges like “16-17” to “16 or 17” in the various places they occurred in the paper. The abstract was very confusing. It failed to provide an overview of the study. For example, a clear introduction of the methods and results of M1 and have been presented. This information regarding M2 and M3 were not clearly reported. We are constrained by the 300 word limit for the abstract, but have modified the abstract (particularly the methods section) to try to make things clearer. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We hope that we have answered the reviewer’s reservations adequately." } ] } ]	1	https://f1000research.com/articles/9-607
https://f1000research.com/articles/9-765/v1	23 Jul 20	{ "type": "Systematic Review", "title": "Estimating the impact of pneumococcal conjugate vaccines on childhood pneumonia in sub-Saharan Africa: A systematic review", "authors": [ "Chukwuemeka Onwuchekwa", "Bassey Edem", "Victor Williams", "Emmanuel Oga", "Victor Williams", "Emmanuel Oga" ], "abstract": "Background: This study aimed to summarise the evidence on the impact of routine administration of 10-valent and 13-valent pneumococcal conjugate vaccines on pneumonia in children under five years of age in sub-Saharan Africa. Methods: A systematic search of the literature was conducted including primary research reporting on the impact of 10- or 13-valent pneumococcal vaccines on childhood pneumonia in a sub-Saharan African country. Case-control, cohort, pre-post and time-series study designs were eligible for inclusion. Thematic narrative synthesis was carried out to summarise the findings. Results: Eight records were included in the final analysis, 6 records were pre-post or time-series studies, 1 was a case-control study and 1 report combined pre-post and case-control studies. Vaccine impact on clinical pneumonia measured as percentage reduction in risk (%RR) was mostly non-significant. The reduction in risk was more consistent in radiological and pneumococcal pneumonia. Conclusions: Evidence of the positive impact of routine infant pneumococcal vaccination on clinical pneumonia incidence in sub-Saharan Africa is inconclusive. Ongoing surveillance and further research is required to establish the long term trend in pneumonia epidemiology and aetiology after PCV introduction. PROSPERO registration: CRD42019142369 30/09/19", "keywords": [ "Pneumonia", "Streptococcus pneumoniae", "child", "sub-Saharan" ], "content": "Introduction\n\nPneumonia is one of the leading causes of childhood deaths globally, particularly in sub-Saharan Africa1. Annually over a 100 million cases of pneumonia are reported in children less than five years of age, mostly in poor countries in Africa and Asia2–4.\n\nStreptococcus pneumoniae is the major cause of childhood pneumonia deaths and is the leading cause of vaccine-preventable child deaths globally5–7. Pneumococcal pneumonia causes between 1 and 4 million episodes of pneumonia in Africa yearly8. There are currently 97 known serotypes of S. pneumoniae, characterised by the polysaccharide capsule antigen9. The 97 serotypes differ in carriage potential and propensity to cause invasive disease including pneumonia, otitis media and meningitis10,11, with the 13 most common serotypes accounting for 70 – 75% of invasive pneumococcal disease globally8,11,12.\n\nPneumococcal conjugate vaccines (PCV) have been licensed since 2000; previous polysaccharide-based vaccines were found to have poor immunogenicity in children8. An initial 7-valent PCV included serotypes 4, 6B, 9V, 14, 18C, 19F and 23F providing cover against 67% of disease-causing serotypes. The 10-valent and 13-valent PCV include in addition to the 7-valent serotypes 1, 5 and 7F; and 1, 3, 5, 7F, 19A, and 6A. The 10-valent vaccine covers 70% - 84% while the 13-valent vaccine covers about 74% - 88% % of invasive disease-causing serotypes8,13,14. Sub-Saharan African countries like South-Africa and The Gambia introduced the 7-valent PCV into routine infant vaccination schedule in 200915,16. Many countries in sub-Saharan Africa through the support of Gavi, the vaccine alliance, have incorporated the 10- or 13-valent PCV into their expanded program of immunization (EPI) schedules. PCV vaccination has been associated with a significant decline in invasive pneumococcal disease incidence globally and at individual country level9,15,16. While there is evidence of effectiveness against invasive pneumococcal disease, the impact of vaccination on clinical and radiological pneumonia remains unclear16.\n\nThis review aims to summarise the existing evidence on the impact of routine administration of pneumococcal conjugate vaccines on clinical pneumonia, radiological pneumonia and pneumococcal pneumonia in children under five years of age in sub-Saharan Africa.\n\n\nMethods\n\nThe systematic review protocol was developed in accordance with the PRISMA-P guidelines17, and registered with PROSPERO on 30 September 2019 (CRD42019142369).\n\nWe included primary, individual and population-based studies conducted in sub-Saharan Africa evaluating 10-valent or 13-valent PCV impact in children published in English since 1 January 2010. This time was chosen because the earliest countries in this region to introduce PCV into routine infant vaccine schedule did so in 2009. The eligibility criteria are detailed in Table 1 below.\n\nStudies with invasive pneumococcal disease (IPD) as outcome were considered for inclusion if pneumonia cases were reported separately.\n\nThe study designs eligible for inclusion were pre-post quasi-experimental, interrupted time series, Cohort, and Case-control studies. For pre-post studies and interrupted time series, we included only studies where the final outcome assessment was made at least 3 years after vaccine introduction.\n\nWe conducted a search of peer-reviewed and grey literature relating to the study question. PubMed search was first conducted on 16 July 2019, with final search on 31 July 2019. Scopus search was conducted on 20 Jul 2019, Embase (Ovid) was searched on the 29 Jul 2019. Other peer-review sources include Africa-Wide Information (29 July 2019) and African Index Medicus (24 July 2019). Grey literature sources include OpenGrey (20 July 2019), and ProQuest Dissertation & theses global (20 July 2019), London School of Hygiene and Tropical Medicine research online (22 July 2019) and University of Edinburgh library (28 July 2019).\n\nThe search strategy combined the key concepts of the research question and was based on the PICOS framework. The three components or concepts were: population of interest (children below 5 years of age), intervention being investigated (pneumococcal conjugate vaccine) and the outcome of interest (pneumonia). The Boolean operators AND, and OR were used to combine the search concepts.\n\nFurther details of the database search strategy and date of searches can be found as extended data18.\n\nTwo members of the review team conducted the database screening independently. Reading through the titles and abstracts of the search results we identified records to be included in the full-text screening based on the eligibility criteria. Records for which there was uncertainty or disagreement about eligibility during the title and abstract screening were included for full-text screening. The second stage of the screening involved reading the full-text of the records to determine if they were eligible for inclusion. Finally, we searched through the references of eligible papers for other relevant publications that could be included in the review. The PRISMA flow diagram for the study selection procedure is shown in Figure 1 in the result section.\n\nThe following information was collected for each included record: year of publication, study location and country, study setting (hospital-based or population-based), study design, study aim (to assess impact or effectiveness), data sources (clinical or laboratory), study population description, HIV status of participants, type of PCV in current use (PCV 10 or PCV13), year PCV7 introduced, year PCV10 or PCV13 introduced, reported coverage during PCV10 or PCV13 period, baseline and post-vaccination periods (for pre-post and interrupted time-series), outcome definition, outcome measure and confidence interval if reported. The information was collected directly into an extraction form in Excel by one member of the review team and crosschecked by a second member19. Disagreement was resolved by consensus after discussion with another member of the review team. No additional information was sought from investigators or authors.\n\nWe assessed the quality of case-control studies assessing vaccine effectiveness, we used the National Heart, Lung and Blood Institute Quality assessment tool for case-control studies. We adapted the National Heart, Lung and Blood Institute Quality Assessment Tool for Before-After studies with No Control Group to assess quality in pre-post and interrupted time series analyses20. The study quality assessment tools are available at https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools and available as extended data21.\n\nThe primary outcomes of interest in this review were clinical pneumonia and radiological pneumonia. The secondary outcome was pneumococcal pneumonia. In pre-post and interrupted time-series studies comparing outcome incidence before and after PCV introduction, measures were presented as percentage reduction in incidence and incidence ratios. Where possible incidence ratios were converted to percentage reduction in incidence: % reduction in risk = (1 – aRR) X 100%; where aRR is the adjusted Risk/Rate ratio for post- and pre-vaccination periods22. In case-control studies we presented adjusted vaccine effectiveness (aVE) as reported by the authors. When adjusted odd Ratios (aOR) were presented we estimated aVE as: adjusted vaccine effectiveness = (1 – aOR) X 100%19; where aOR = adjusted odd ratio. All calculations were made in Microsoft Excel 2016, and graphical presentations were performed in Stata 1323.\n\nPlanned quantitative synthesis could not be conducted in this review due to variation in included studies. We therefore, present a narrative synthesis of the impact of PCV on childhood clinical pneumonia, radiological pneumonia and pneumococcal pneumonia.\n\n\nResults\n\nThe database search produced 3581 distinct titles, from which we identified 34 relevant records for the full-text review. We excluded a further 26 records after full-text assessment. Eight records were included in the review as shown in Figure 1 below.\n\nFour of the included studies were conducted in South Africa (23 – 26), two were conducted in Kenya24,25, and one each from Rwanda26, and The Gambia27. There were 7 studies with pre-post or interrupted time-series design and two case-control studies (Mackenzie et al. included report on a case-control study). Most of the study locations had 7-valent PCV introduced before transitioning to the 13-valent PCV except in Kenya where the 10-valent PCV was used without a 7-valent PCV period. Table 2 below shows the characteristics of articles included in the review.\n\nNA: Not applicable, * Reported along with the pre-post study.\n\nOne of the seven population-based studies were considered to be of poor quality with significant risk of bias. None of the population-based studies were blinded hence there is the possibility of bias in the findings. The two case-control reports were graded as having good quality. Details on the study quality assessment are available as extended data21.\n\nThere was variation in the definition of clinical and radiological pneumonia, and in the method of identification of pneumococcal pneumonia among the included studies. Some studies applied the WHO standards for identification of radiological pneumonia and clinical pneumonia34,25,27. One study based pneumonia diagnosis on ICD-10 coding from clinical notes35. Studies reporting on pneumococcal pneumonia were based on culture with occasional confirmation by polymerase chain reaction (PCR). Table 3 below summarises the findings of the included studies.\n\nBased on WHO clinical classification of severe and very severe pneumonia, * pneumonia hospitalization based on ICD-10 coding, ¶ 50% credible interval based on Bayesian methods (negative values indicate an increase in incidence), € Identification based on polymerase chain reaction, ± pneumonia classified based on WHO criteria or based on abnormal CXR plus C-reactive protein >40mg/L.\n\nImpact on clinical pneumonia. Four population-based studies measured the impact of 10- or 13-valent vaccine on clinical pneumonia with inconsistent findings. Silaba et al. reported a decline in hospitalization for severe or very severe pneumonia based on WHO clinical definition25. Mackenzie et al. showed no significant decline in clinical pneumonia incidence in all under-five age groups three years after 13-valent PCV introduction27. Solomon et al. estimated a significant decline in pneumonia hospitalizations in infants and children between 24 – 59 months based on Bayesian methods. This effect was also found among HIV infected and HIV uninfected children35. However, these figures were estimated around a 50% credible interval. Figure 2 graphically displays the reported reduction in clinical pneumonia incidence.\n\nImpact on radiological pneumonia. Two impact studies evaluated radiological pneumonia as outcomes25,27. Mackenzie et al. reported a decline in WHO defined radiological pneumonia in all age groups with decline in the range of 22 – 29%. This was most pronounced in the 12 to 23-month age group. Silaba et al. also reported a 48% decline in radiological pneumonia in the entire under-five population. A similar age-related trend was observed, with children between 12 to 23-month age experienced the largest reduction in radiological pneumonia25.\n\nThe case-control study reported by Mackenzie et al. using community controls showed a vaccine effectiveness of about 28%, however, this did not reach statistical significance. Mahdi et al. reported adjusted vaccine effectiveness measures using both community and hospital controls34. Vaccine effectiveness was significant with community controls (aVE 32.1%, 95% CI 4.6% - 51.6%), but not with hospital controls (aVE 20%, 95% CI -9.3% – 41.6%).\n\nImpact on pneumococcal pneumonia. All three studies that reported on pneumococcal pneumonia were based on microbiological diagnosis with or without PCR confirmation29,36,24. Included studies consistently showed decline in cases of pneumococcal pneumonia after vaccine introduction irrespective of method of pneumococcal identification. Figure 3 shows the reduction in pneumococcal pneumonia incidence reported from included studies.\n\n\nDiscussion\n\nThis review set out to answer the question: has the introduction of pneumococcal conjugate vaccine resulted in a decline in childhood pneumonia? From our review, we can summarise that the population impact of pneumococcal vaccination on pneumonia depends largely on how pneumonia is classified. We observed overall that when the outcome is clinical pneumonia, the impact tends to be modest at best. We see this in the reports by Mackenzie et al., Silaba et al., and Solomon et al. However, it is important to note that the severity of pneumonia differed in these studies. Mackenzie et al. evaluated clinical pneumonia at the population level and showed results that were not statistically significant in all age groups. Silaba et al., however, looked at clinical pneumonia hospitalizations (i.e. severe or very severe pneumonia) and showed significant difference in incidence after vaccine introduction. We also see a subtle decline in hospitalizations for clinical pneumonia based on ICD-10 coding. Clinical pneumonia is a non-specific outcome and therefore may not be ideal for evaluating disease-specific vaccine impact. There was a more positive but still modest impact observed with radiological pneumonia as an outcome. This is likely because radiological pneumonia is more specific for pneumococcal disease. Overall, there was a consistent decline in pneumococcal pneumonia cases in the post-vaccine period in reported impact studies29,36,24. Two studies that reported on vaccine effect in HIV infected children showed that pneumococcal vaccine had similar impact as with HIV uninfected children29,35.\n\nThe overall trend in the findings from this review is similar to reports from other parts of the world. Meta-analyses from other regions have found modest decline in clinical and radiological pneumonia hospitalizations. A meta-analysis using global data from 12 pre-post and time series studies produced a pooled reduction in hospitalization rates for clinical pneumonia of 17% and 9% in the under 24 months and 24 – 59 months’ age groups. However, they reported a more significant decline in hospitalization rates due to radiological pneumonia of 31% and 24% in the same age groups37. A randomised placebo-controlled trial on 9-valent vaccine showed vaccine effectiveness of 37% based on per-protocol population22. Overall, it appears that pneumococcal vaccination has its greatest impact in preventing the more severe forms of pneumonia for which children are likely to be hospitalised. This is probably due to the finding that bacterial pathogens are more likely to cause severe pneumonia28. The minimal impact on clinical pneumonia might suggest an increase in other forms of pneumonia or may be due to serotype replacement30–32. We observed an age-related trend in vaccine effect, with the 12 to 23-month age group experiencing the greatest benefit. This might be due in part to a greater proportion of children under 12 months having not completed their vaccination schedules; and potentially waning immunity in the older age groups33.\n\nThis review has some limitations that have to be considered when interpreting our findings. First was the inconsistency in the definition of pneumonia outcomes between studies, which made combining the impact measured between studies impractical. While some studies used comparable methods for outcome ascertainment, this was not consistent across studies. The WHO standardised definition of clinical and radiological pneumonia is markedly helpful in this situation as it ensures consistency across studies38. Studies conducted in locations with a functional health and demographic surveillance system like in the Upper River region of The Gambia and in Kilifi, in Kenya, were particularly robust as they combined consistent pneumonia surveillance methods with up-to-date population information24,27. Some studies relied on routine clinical data which is usually of variable quality35,26.\n\nIt is also important to note that while we set out to evaluate the impact of 10-valent and 13-valent vaccines, all of the study locations except in Kenya had a period of 7-valent vaccine use. Therefore, is it impossible to separate the effect of the 7-valent from the 10- and 13-valent vaccines since the 7-valent PCV might have changed the pre-existing disease trend.\n\nLike all time-trend studies - including pre-post studies, phenomena such as regression to the mean, seasonality, trend, and history bias have to be considered in the analysis. By including a control outcome and conducting sensitivity analysis, some of the included studies considered the impact of history bias and trend on their results39.\n\nPublication bias is a potential limitation of this review. However, due to the small number of reports in each outcome category, we did not formally assess for publication bias using a funnel plot40. We are therefore unable to comment on publication bias in this review. Finally, due to limited resources we were unable to include studies published in other languages; hence, language bias cannot be ruled out in our review.\n\n\nConclusion\n\nTo the best of our knowledge this is the first systematic attempt at summarising the impact and effectiveness of routine pneumococcal vaccination on childhood pneumonia from this region. The 10- and 13-valent PCV use as part of infant immunization is effective in preventing the more severe forms of childhood pneumonia. There appears to be a smaller effect on clinical pneumonia especially when all severity spectra are included. There is the need for consistency in pneumonia definition for the purpose of disease surveillance and the WHO clinical definitions provide an appropriate option ensuring ease of implementation and reproducibility. One major issue encountered was that few studies had applied comparable pneumonia definitions in estimating disease burden prior to pneumococcal vaccine introduction hence making trend analysis difficult. There is the need for the generation of updated information on the causes of pneumonia in this region in this era of extensive pneumococcal vaccine use. Ongoing surveillance is needed to investigate the long-term trend in childhood pneumonia in the PCV era in sub-Saharan Africa.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nThis project contains the following extended data:\n\nFigshare: Detailed search https://doi.org/10.6084/m9.figshare.12656309.v218\n\nFigshare: Data extraction form https://doi.org/10.6084/m9.figshare.1260826419\n\nFigshare: Quality assessment tool https://doi.org/10.6084/m9.figshare.1260826421\n\nFigshare: PRISMA checklist for ‘Estimating the impact of pneumococcal conjugate vaccines on childhood pneumonia in sub-Saharan Africa: A systematic review’. https://doi.org/10.6084/m9.figshare.12608672.v241\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgment\n\nThe authors acknowledge the invaluable input of Dr Kristien Verdonck in reviewing the initial manuscript and guiding interpretation and presentation of the review findings. A previous version of this article is available from bioRxiv42.\n\n\nReferences\n\nBlack RE, Morris SS, Bryce J: Where and why are 10 million children dying every year? Lancet. Elsevier; 2003 [cited 2016 Jul 16]; 361(9376): 2226–34. PubMed Abstract \| Publisher Full Text\n\nHowie SRC, Murdoch DR: Global childhood pneumonia: the good news, the bad news, and the way ahead. Lancet Glob Health. 2019; 7(1): e4–e5. PubMed Abstract \| Publisher Full Text\n\nNair H, Simões EAF, Rudan I, et al.: Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: A systematic analysis. Lancet. 2013; 381(9875): 1380. Publisher Full Text \| Free Full Text\n\nMcAllister DA, Liu L, Shi T, et al.: Global, regional, and national estimates of pneumonia morbidity and mortality in children younger than 5 years between 2000 and 2015: a systematic analysis. Lancet Glob Heal. 2019; 7(1): e47–e57. 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PubMed Abstract \| Publisher Full Text\n\nKadioglu A, Weiser JN, Paton JC, et al.: The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol. 2008; 6(4): 288–301. PubMed Abstract \| Publisher Full Text\n\nBogaert D, De Groot R, Hermans PWM: Streptococcus pneumoniae colonisation: The key to pneumococcal disease. Lancet Infect Dis. 2004; 4(3): 144–54. PubMed Abstract \| Publisher Full Text\n\nNgocho JS, Magoma B, Olomi GA, et al.: Effectiveness of pneumococcal conjugate vaccines against invasive pneumococcal disease among children under five years of age in Africa: A systematic review. PLoS One. 2019; 14(2): e0212295. 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. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOnwuchekwa C, Edem BE, Oga EA, et al.: Detailed search. figshare. Dataset. 2020; http://www.doi.org/10.6084/m9.figshare.12656309.v2\n\nOnwuchekwa C, Edem BE, Oga EA, et al.: Data extraction form.xlsx. figshare. Dataset. 2020; http://www.doi.org/10.6084/m9.figshare.12608198.v1\n\nInstitute NHL, B: Study Quality Assessment Tools-NHLBI. NIH. 2016. Reference Source\n\nOnwuchekwa C: Quality assessment tool. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12608264.v1\n\nCutts FT, Zaman SMA, Enwere G, et al.: Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: Randomised, double-blind, placebo-controlled trial. Lancet. 2005; 365(9465): 1139–46. PubMed Abstract \| Publisher Full Text\n\nStataCorp: Stata Statistical Software: Release 13. College Station, TX: StataCorp LP. 2013.\n\nHammitt LL, Etyang AO, Morpeth SC, et al.: Effect of ten-valent pneumococcal conjugate vaccine on invasive pneumococcal disease and nasopharyngeal carriage in Kenya: a longitudinal surveillance study. Lancet. [Internet]. 2019 [cited 2019 Jul 22]; 393(10186): 2146–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilaba M, Ooko M, Bottomley C, et al.: Effect of 10-valent pneumococcal conjugate vaccine on the incidence of radiologically-confirmed pneumonia and clinically-defined pneumonia in Kenyan children: an interrupted time-series analysis. Lancet Glob Heal. [Internet]. 2019; 7(3): e337–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGatera M, Uwimana J, Manzi E, et al.: Use of administrative records to assess pneumococcal conjugate vaccine impact on pediatric meningitis and pneumonia hospitalizations in Rwanda. Vaccine. [Internet]. 2016 [cited 2019 Jul 16]; 34(44): 5321–8. PubMed Abstract \| Publisher Full Text\n\nMackenzie GA, Hill PC, Sahito SM, et al.: Impact of the introduction of pneumococcal conjugate vaccination on pneumonia in The Gambia: population-based surveillance and case-control studies. Lancet Infect Dis. [Internet]. 2017 [cited 2019 Jul 20]; 17(9): 965–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO’Brien KL, Baggett HC, Brooks WA, et al.: Causes of severe pneumonia requiring hospital admission in children without HIV infection from Africa and Asia: the PERCH multi-country case-control study. Lancet. 2019; 394(10200): 757–779. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVon Mollendorf C, Tempia S, Von Gottberg A, et al.: Estimated severe pneumococcal disease cases and deaths before and after pneumococcal conjugate vaccine introduction in children younger than 5 years of age in South Africa. PLoS One. 2017; 12(7): e0179905. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadhi S: Clinical spectrum and complications of childhood pneumonia in the era of bacterial conjugate vaccines. Int J Infect Dis. 2014; 21(SUPPLEMENT 1): 25. Publisher Full Text\n\nKwambana-Adams B, Hanson B, Worwui A, et al.: Rapid replacement by non-vaccine pneumococcal serotypes may mitigate the impact of the pneumococcal conjugate vaccine on nasopharyngeal bacterial ecology. Sci Rep. 2017; 7(1): 8127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeinberger D, Malley R, Lipsitch M: Serotype replacement in disease after pneumococcal vaccination. Lancet. 2011; 378(9807): 1962–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerical AC, Harris D, Dela Cruz CS, et al.: Pneumococcal vaccination strategies. An update and perspective. Ann Am Thorac Soc. 2016; 13(6): 933–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMadhi SA, Groome MJ, Zar HJ, et al.: Effectiveness of pneumococcal conjugate vaccine against presumed bacterial pneumonia hospitalisation in HIV-uninfected South African children: A case-control study. Thorax. 2015 [cited 2019 Jul 16]; 70(12): 1149–55. PubMed Abstract \| Publisher Full Text\n\nIzu A, Solomon F, Nzenze SA, et al.: Pneumococcal conjugate vaccines and hospitalization of children for pneumonia: a time-series analysis, South Africa, 2006–2014. Bull World Health Organ. [Internet]. 2017 [cited 2019 Jul 16]; 95(9): 618–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTempia S, Wolter N, Cohen C, et al.: Assessing the impact of pneumococcal conjugate vaccines on invasive pneumococcal disease using polymerase chain reaction-based surveillance: An experience from South Africa. BMC Infect Dis. [Internet]. 2015 [cited 2019 Jul 16]; 15(1): 450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlicino C, Paganino C, Orsi A, et al.: The impact of 10-valent and 13-valent pneumococcal conjugate vaccines on hospitalization for pneumonia in children: A systematic review and meta-analysis. Vaccine. 2017; 35(43): 5776–5785. PubMed Abstract \| Publisher Full Text\n\nCherian T, Mulholland EK, Carlin JB, et al.: Standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies. Bull World Health Organ. 2005; 83(5): 353–9. PubMed Abstract \| Free Full Text\n\nBernal JL, Cummins S, Gasparrini A: Interrupted time series regression for the evaluation of public health interventions: A tutorial. Int J Epidemiol. 2017; 46(1): 348–355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLau J, Ioannidis JP, Terrin N, et al.: The case of the misleading funnel plot. BMJ. 2006; 333(7568): 597–600. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOnwuchekwa C, Edem BE, Oga EA, et al.: PRISMA checklist. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12608672.v2\n\nChukwuemeka O, Bassey EE, Williams V, et al.: Estimating the impact of pneumococcal conjugate vaccines on childhood pneumonia in sub-Saharan Africa: A systematic review. bioRxiv. 865154. 2019. Publisher Full Text" }	[ { "id": "67910", "date": "06 Oct 2020", "name": "Blandina Mmbaga", "expertise": [ "Reviewer Expertise Paediatric infectious disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this manuscript. The author works on the important topic taking into account pneumonia still remains as one of the major causes of childhood morbidity and mortality. Assessing the impact of vaccines while differentiating clinical, radiological outcome support the evidence on the vaccine impact.\n\nThe manuscript is written in good English language.\nThe methodology is clear on how the review was conducted and how they reached final articles for inclusion.\n\nNo major specific comments and would recommend the manuscript acceptance.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [ { "c_id": "6008", "date": "06 Oct 2020", "name": "chukwuemeka Onwuchekwa", "role": "Author Response", "response": "Thank you for the favourable review." } ] }, { "id": "73233", "date": "02 Nov 2020", "name": "Dodi Safari", "expertise": [ "Reviewer Expertise Medical microbiology" ], "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 systematic review with the title “Estimating the impact of pneumococcal conjugate vaccines on childhood pneumonia in sub-Saharan Africa”, the authors showed evidence of the impact of pneumococcal conjugate vaccines (PCV10 and PCV13) in Kenya, Gambia, Rwanda, and South Africa is inconclusive based on clinical pneumonia incidence.\nThe authors may need to elaborate how the impact of the first introduction of PCV7 vaccine on pneumonia incidence in this systematic review since most of the study location had PCV7 introduced before PCV10/PCV13, except in Kenya (page 5).\n\nThe authors need to add the information on the immunization doses and schedule in each location and to discuss further how these differences had an impact on the outcomes of PCV10 and PCV13 vaccination.\n\nThe authors need to discuss further the health and economic status in each location that can affect the outcomes of PCV10 and PCV13 vaccination.\n\n4. Please add the new update of pneumococcal polysaccharide capsule (Introduction part): https://mbio.asm.org/content/11/3/e00937-201\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6091", "date": "09 Nov 2020", "name": "chukwuemeka Onwuchekwa", "role": "Author Response", "response": "Thank you for reviewing our work and for the constructive comments and suggestions. We have carefully considered your comments and address them below: 1. As stated in our original submission: “it impossible to separate the effect of the 7-valent from the 10- and 13-valent vaccines since the 7-valent PCV might have influenced the pre-existing disease trend”. We have gone further to stress that separating the effect of the 7-valent from subsequent 13-valent vaccination is further compounded by the short time lapse between the two vaccines (2009 and 2011). We therefore added the statement: “However, because of the short time lapse between the 7-valent and 10- or 13-valent vaccine roll-out in these countries, it is unlikely that a significant change in pneumonia trend would be demonstrable” 2. Thank you for drawing our attention to the influence of dosing schedule, this comment was very helpful. We have included the dosing schedule used in each country as described by the authors in Table 2 (Description of included studies). In the introduction section, we have also included a sentence describing the current vaccine schedule: “The vaccines are usually administered as three doses in early infancy (3 + 0 schedule), or two doses in early infancy plus a booster in late infancy (2 + 1 schedule)” While there is evidence that pneumococcal vaccine schedules which include a booster (e.g. 2 + 1 or 3 + 1 schedules) are associated with a more robust immune response, it has not been shown that these schedules have any effect of carriage or indeed pneumonia incidence(1). We have therefore not explored this further as we considered this to be outside the boundaries of our review, and we have too few studies to make any type of comparison. 3. Thank you for this comment. We agree with your statement about the influence of Health and economic situation of the populations and how this could have influence childhood pneumonia and indeed uptake of vaccination. We also indicate here that the uptake of vaccination in all the reports were above 90% in the relevant population. Furthermore, the population based impact studies compared populations before and after vaccine roll-out, with a relatively short time lapse between the baseline and post-vaccination periods. It is our belief that socioeconomic variables are unlikely to have changed significantly at population-level, and is likely to have minimal influence on the observed findings. More importantly, most of the population based studies (time-series and before-after studies) included a control group or control condition, which would allow for assessment of the influence of historical bias (including changes in socioeconomic conditions)(2) 4, Thank you for drawing our attention to the most recent information of pneumococcal serotypes. We have noted this comment and made changes to the manuscript to reflect this, also included is the reference you suggested. References 1. Whitney CG, Goldblatt D, O’Brien KL. Dosing schedules for pneumococcal conjugate vaccine: considerations for policy makers. Pediatr Infect Dis J. 2014 Jan;33 Suppl 2:S172-81. 2. Bernal JL, Cummins S, Gasparrini A. The use of controls in interrupted time series studies of public health interventions. Int J Epidemiol. 2018;47(6):2082–93." } ] } ]	1	https://f1000research.com/articles/9-765
https://f1000research.com/articles/9-1306/v1	09 Nov 20	{ "type": "Research Article", "title": "The effects of touch-screen technology usage on hand skills among preschool children: a case-control study", "authors": [ "Ahmad Zamir Che Daud", "Nurul Afiq'ah Aman", "Chi-Wen Chien", "Jenni Judd", "Nurul Afiq'ah Aman", "Chi-Wen Chien", "Jenni Judd" ], "abstract": "Background: Little is known on how time spent on touch-screen technology affects the hand skills development of preschool children. This study aimed to investigate the effects of touch-screen technology usage on hand skills among preschool children. Methods: Case-control design was employed to compare the hand skills of children who were engaged in touch-screen technology. A total of 128 participants aged between five and six years old who attended preschool were recruited and divided into two groups: high usage touch-screen technology (HUTSTG) and, low usage touch-screen technology (LUTSTG). Children's Hand Skills ability Questionnaire (CHSQ) and Assessment of Children's Hand Skills (ACHS) were used to evaluate the children's hand skills. Results: There were significant differences in the hand skills of preschool children between HUTSTG and LUTSTG. Results showed that preschool children in LUTSTG had better hand skills in all domains of CHSQ (p≤0.001) and ACHS (p<0.001) as compared to HUTSTG. Conclusion: Frequent use of touch-screen technology might cause disadvantages to the development of hand skills among preschool children.", "keywords": [ "Child Behaviour", "Child Development", "Hand", "Motor Skills", "Screen Time" ], "content": "Introduction\n\nThe proliferation of electronic technology in recent years has played a significant role in people's everyday life. As these technology usages become more crucial in everyday life, the prevalence of the possession of devices among teens has become very high in numbers and is escalating among younger children1. Touch-screen technology usage among children had increased rapidly from 8%t to 40% in two years2. Time spent using touch-screen technology has multiplied three times from 5 hours per day in 2011 to 15 hours per day in 20131.\n\nInstead of engaging and exploring their surroundings, children tend to spend most of their time playing and interacting with touch-screen technology3. Children use touch-screen technology for various functions such as watching videos, playing games and listening to songs4. Touch-screen technology has altered the way children play, interact and learn due to the interactive application of touch-screen technology that offers some entertainment and attractions5. Interactive technology becomes an issue as motor development is dependent on children's motor experience6. As children manipulate objects in their surroundings using their hands and fingers, it involves motor coordination, joint stability, muscle strength, visual perception and touch-ability. However, when using touch-screen technology, the involvement of motor coordination, muscle strength and dexterity are relatively low when compared to activities such as drawing, handwriting or playing with objects and toys7. Touch-screen technology usage reduces the need to use hand skills such as grasping, in-hand manipulation and reaching as it only involves necessary fingers movements such as tapping, pressing, zooming and double-tapping8.\n\nFrequent engagement in touch-screen technology reduces children's chances to be involved in physical activities and may hinder the development of mature hand skills9. One study reported that there was an improvement in manual dexterity, fine motor integration, and fine motor precision in typically developing preschool children who did not use any touch-screen technology for 24 weeks compared to the children who frequently engaged with touch-screen technology7. This finding supports the assumption that frequent use of touch-screen technology without specific purposes, such as playing games and watching videos, might limit hand skill development of children.\n\nHowever, the usage of this technology has been claimed to bring advantages, especially to those who learn kinesthetically through touch, movement and gesture10. There is a positive correlation between touch-screen technology usage and hand skills ability11,12. A previous study reported that children who actively engaged in touch-screen technology developed hand skills ability earlier suggesting that the use of touch-screen technology may improve the efficiency of hand skills8. Therefore the engagement with touch-screen technology could be considered as a healthy exercise for growing children4. With the use of fingers to operate touch-screen technology, children have no risk of injury as compared to playing outside. Children would gain better knowledge of their hands and finger use, and become more efficient in a short period. Despite the positive association between touch-screen technology usage and hand skills, little is known on how the time spent using this technology may affect development of hand skills of preschool children. In the event where touch-screen technology has turned into an imperative method of play, analyzing its impact on the hand skills ability of children is exceptionally important. Therefore, this study explored the effects of time spent using touch-screen technology and hand skills among preschool children.\n\nChildren's age and gender, parents' level of education, their number of siblings, and family socioeconomic status need to also be considered as these factors that may affect children's hand skills other than touch-screen technology usage alone. In particular, family plays a significant role in early childhood development years. Older siblings might be a model for the development of motor skills in a younger child, and the number of siblings may have a correlation with hand skills of children13,14. Mother's educational level may have an association with a higher level of cognitive and higher earnings, which leads to better opportunities for children's development and exploration15. Socioeconomic status has also been found to be a predictor to the performance of hand skills among children16,17. Children that had better socioeconomic status accomplished better hand skills as compared to children with low socioeconomic status due to early school years that allowed children to engage in various activities at school18. There are a few studies that report the association between gender and hand skills. One study reported a likely low association between gender and hand skills19. Girls accomplished better in manual dexterity skills, especially in paper-based and pencil-based activities as compared to boys20–22. Age, was also reported as a predictor for the development of hand skills and was more noticeable among younger children23,24. A recent study found that age plays a significant role in hand skill development25. Since these personal and environmental factors might influence the hand skills of children, this study also aimed to examine the interaction of each element on hand skills.\n\nThe main research question this study addressed was: what are the effects of touch-screen technology usage on hand skills of pre-school children?\n\n\nMethods\n\nA case-control study design was employed to investigate the hand skills between high usage touch-screen technology (HUTSTG) and low usage touch-screen technology (LUTSTG) groups. This observational study design allows the researchers to investigate the status of exposure of touch-screen technology without any interventions on hand skills26. The study design was able to determine whether low or high exposure of touch-screen technology is associated with the outcomes (hand skills)27. Demographic characteristics, hand skills ability and frequency of touch-screen technology usage data were collected from August to September 2019.\n\nParticipants were divided into two groups based on their touch-screen technology usage in accordance with Malaysian Dietary Guidelines, which recommends screen time among preschool children should be less than two hours per day28. Children who used touch-screen technology more than two hours per day were allocated to the HUTSTG group (case), and if they did not, they were assigned to the LUTSTG group (control). The guideline for screen time was developed to promote natural development and a life balance between sleep, sedentary behavior and physical activity among children in a 24-hour period28,29. Touch-screen technology usage included all screen-based activities using a tablet or smart phone such as playing games, using mobile applications and watching videos. Touch-screen technology usage was reported by parents or caregivers in the number of minutes. Recruitment of participants was from the Ministry of Education (MOE) preschools in Southern Region of Malaysia.\n\nPurposive sampling was used to recruit the participants. Participants were then randomly selected by using computer generated random number in order to reduce the possibility of the human bias in cases selection. Inclusion criteria for HUTSTG group were typically developing children: (i) aged 5–6 years old; (ii) attending the MOE preschool; (iii) engaging in touch-screen technology (either phone or tablet) for more than 120 minutes per day, according to parent-report; and (iv) parents are also able to participate in this study to complete the CHSQ. While for LUTSTG, the inclusion criteria were similar for HUTSTG except than the children engaging in touch-screen technology (either phone or tablet) for less than 120 minutes per day according to the parent report. Preschool children that had been diagnosed with diseases or disorders associated with developmental delays based on parents' report were excluded from this study for both groups.\n\nSample size calculation was calculated using the G*Power 3.1 software, with power set at 0.80 to detect the medium-effect size (d = 0.50) and α = 0.05 with allocation ratio N2/N1 of 1. Therefore, a total of 128 participants with 64 children in each group were recruited to achieve adequate power.\n\nInitially, parents of preschool children were asked to complete the Children's Hand Skills ability Questionnaire (CHSQ) that was used to evaluate the uses of the hands by the children aged 2–12 years old while performing activities of the daily life from the perspectives of the parents or the caregivers30. This CHSQ assessment comprises of 21 activities of the hand skills that are divided into three domains of leisure and play (8 activities), school/education (8 activities), and Activities of Daily Living (ADL) (6 activities). Likert scale with three-level was used in the CHSQ that are; 1 (extremely difficult), 2 (difficult) and 3 (no difficulty) indicating the difficulty level while performing the 21 activities. The \"not applicable\" was marked if the child does not perform the activity in the last three months. CHSQ assessment can be used as a companion assessment to know the range of difficulties in performing hand skill activities before evaluating the children's hand skills using Assessment of Children's Hand Skills (ACHS)31. CHSQ is reported to have a sufficient person-response validity, and satisfactory internal and external construct validity to capture children's' manual ability in the domain of ADL, leisure and play; and school/education30. Average scores for each domain in CHSQ were calculated to determine the difficulty level of the child while engaging in leisure and play, school/education and ADL activities.\n\nA performance-based observation was then conducted with all participants using ACHS. This naturalistic observational assessment was used in this present study to assess the actual performance of the child's hand skills in a real-life context when engaging in several types of daily activities in everyday settings32. This standardized assessment comprised of 5 categories of hand skills items; (1) hand skills without interacting with objects (manual gesture, body contact); (2) \"arm-hand\" use object-related hand skills (reaching, turning, carrying, throwing, catching, moving, stabilizing); (3) \"adaptive skilled hand use\" object-related hand skills (grasping, holding, in-hand manipulating, releasing, isolated finger movements); (4) \"bimanual use\" object-related hand skills (transferring, using both hands simultaneously, using both hands cooperatively); and (5) general quality of hand skills (accuracy, pace, movement quality). These hand skills items for each activity are rated on a 6-point rating scales; a score of 6 indicates very effective hand skill performance, while a score of 1 indicates very ineffective hand skill performance. Two or three activities that were scored 1 (extremely difficult) or 2 (difficult) by parents in the CHSQ were observed. Observations and scoring were done while children performed the selected activities for 20–30 minutes at their preschools. Evidence for the content, construct, discriminant and convergent validity of ACHS were demonstrated, indicating that this assessment can be used with confidence to measure children's real-life hand skill performance31,33,34. ACHS is also reported to have acceptable intra-rater, inter-rater and test-retest reliability to quantify children's hand skills use32,33. Composite scores of ACHS were computed to determine the overall hand skills performance of preschool children while doing all the selected activities.\n\nChi-square and Fisher‘s exact test was conducted to determine whether differences in demographic characteristics of participants existed between HUTSTG and LUTSTG groups. Independent T-test was used in order to investigate any significant differences in hand skills ability among preschool children between HUTSTG and LUTSTG groups. Two-way analysis of variance (ANOVA) was conducted in order to explore the effects and interactions of personal and environmental factors (age of the preschool children, gender of the preschool children, number of siblings, household income status, mother‘s age, mother‘s educational level) on hand skills ability between HUTSTG and LUTSTG groups. Lastly, Pearson’s correlation coefficients was computed in order to investigate the relationship between the children‘s hand skills ability based on parents‘ reported questionnaire, CHSQ and performance-based observation, ACHS. Missing data was treated using expectation maximization.\n\nThe ethics review committee of Universiti Teknologi MARA (UiTM) (REC/223/19) approved the study. Permissions from the Ministry of Education (KPM.600-3/2/3-eras-3447) and Johor State Education Department (JPNJ.PP.600-1/1/2Jld.2-42) were also obtained as this study involved the preschool children under the MOE. Before the preschool children participated in the study, the procedures were explained to the parents, and their written consent for their child and themselves to participate was given.\n\n\nResults\n\nThe HUTSTG and LUTSTG groups each consisted of 64 preschool children in which the gender (male and female), and age (five years and six years) were balanced between the groups (Table 1). There were no significant differences in all demographic characteristics of the participants between HUTSTG and LUTSTG groups.\n\naFisher's Exact Test; bChi-Square\n\nResults showed that there were significant differences for all domains in CHSQ between LUTSTG and HUTSTG groups. Table 2 shows the results of independent t-tests for both groups using the average scores of each domain in CHSQ. There was also a significant difference of the hand skill performance of ACHS between LUTSTG (M = 2.702, SD = 1.808) and HUTSTG (M = 0.956, SD = 1.266) groups with t (126) = 6.329, p = 0.000.\n\nTwo-way ANOVA was conducted to examine the factors that may affect hand skills based on the performance-based evaluation, ACHS. Table 3 shows that there were non-significant interactions between touch-screen technology usage and all the factors (gender, age, number of siblings, mother's age, mother's educational level and household income). The effects of touch-screen technology usage on hand skills (ACHS) were not influenced by those factors, and the hand skills of preschool children were influenced solely by touch-screen technology usage.\n\nTwo-way ANOVA was also conducted to examine the factors that may affect hand skills based on parent's reported questionnaire, CHSQ. Table 3 demonstrates that there were no significant interactions between touch-screen technology usages. All factors such as gender, age, number of siblings, mother's age, mother's educational level, and household income did not influence hand skills; in contrast, the effects of touch-screen technology usage on hand skills for all domains in the CHSQ (Play and Leisure, School/Education and ADL) did.\n\nCHSQ represented the parents' reported hand skills and ACHS represented performance-based observation of hand skills. The correlation between these two measures was studied. There was a non-significant correlation between ACHS score and the play/leisure domain of CHSQ, r = 0.082, p = 0.358. However, there was a very weak positive correlation between ACHS score and school/education domain of CHSQ (r=0.231, p=0.009). Similarly, there was a weak positive correlation, r=0.187, p=0.035 (Table 4) between ACHS score and activities of daily living (ADL) domain.\n\n\nDiscussion\n\nThis study aimed to examine the effects of touch-screen technology usage on hand skills ability of preschool children and proposed to explore the factors that might influence the hand skills of the children. Both parents' reported questionnaire (CHSQ) and performance-based evaluation (ACHS) showed significantly better hand skills in the LUTSTG group compared with the HUTSTG group. The results suggest that children that are engaged with touch-screen technology for more than two hours per day have less adequate hand skills as compared to children who use touch-screen technology for less than two hours per day. The results were aligned with the assumption that frequent usage of touch-screen technology might limit the development of hand skills among preschool children7. As presumed by 35, traditional games such as puzzles, board games and construction blocks have been replaced by the use of touch-screen technology. Movements required when using touch-screen technology are different from those involved when playing traditional games, doing most activities of daily living and completing school tasks, and reduces the children's experiences in manipulating and handling objects in real life3,9, which may explain why children in the LUTSTG group have better hand skills.\n\nThe social context in which children live and are being raised, including the socioeconomic status of the family, mother's educational level and the number of siblings influenced the development of children13–15,17,18. Our study demonstrated no significant interaction effects between all personal and environmental factors (such as gender, age, number of siblings, mother's age, mother's educational level and household income) on hand skills for both HUTSTG and LUTSTG groups. Touch screen usage alone influenced the hand skills of preschool children in this study.\n\nThe parent-reported evaluation should be complemented alongside a real-life observational evaluation to elucidate the most precise evaluation32. In this study, data from parents' reported questionnaire (CHSQ) was correlated with the data from the performance-based evaluation (ACHS). This study found that there was a positive correlation between the school/education and activities of daily living (ADL) domain of CHSQ and ACHS. It showed that school activities (writing, copying, coloring and drawing) and ADL activities (drinking, eating and washing hands) are the most common activities done in school and home environment. Parents' reported questionnaire on their children's hand skills (i.e., the results of CHSQ) is in agreement with the performance-based evaluation while children are engaged in school tasks and ADL activities in the preschool. However, the correlations were weak, and to evaluate the hand skills of children it is better to have a performance-based evaluation rather than parents' reported evaluation. For the leisure domain of CHSQ, there is no correlation between the domain and the ACHS score and showed a contradiction between the leisure activities observed by parents in the home environment with the performance-based evaluation done in the school environment. School is not a familiar place for children to frequently participate in leisure and play activities such as playing blocks, card games and stringing beads.\n\nThere were a few limitations found in this study. First, this study relied on parents' reports for the time spent by their children using touch-screen technology. Second, this study did not take into consideration the age of children when they first started using touch-screen technology. Early use of touch-screen technology might have effects on the hand skills of children. Thirdly, this study did not investigate other areas of development that might relate to the impact of touch-screen technology usage, for instance, the development of visual perception, language and cognitive functions of children. Use of touch-screen technology might be useful for improving visual perceptions, language or cognitive functions of children. Still, it showed otherwise in the hand skills of children.\n\nFuture study needs to obtain further information on children's use of touch-screen technology not just from parents but also their caregivers in their childcare centres. Future study also warrants the effects of touch-screen technology usage on the development of visual perceptions and cognitive functions.\n\n\nConclusion\n\nPositive and negative effects of touch-screen technology have been debated. Many previous studies have investigated the effects of touch-screen technology on several aspects of childhood development. The advantages may sometimes overshadow the disadvantages of this technology, however, they must be considered. Results of this study suggest that there are differences in hand skills development between HUTSTG and LUTSTG groups and those children with frequent use of touch-screen technology may have difficulties with their hand skills when performing daily life activities.\n\n\nData availability\n\nThe underlying data related to this article has been previously published in a data article by the authors: https://doi.org/10.1016/j.dib.2020.10635836. The data article contains the following data:\n\n1) Raw data for analysis results of the effects of touch-screen technology usage on hand skills.\n\n- Spreadsheet (16kb):\n\n2) Raw scores and the calculated average for CHSQ assessment.\n\n- Spreadsheet (41kb):\n\n3) Raw scores for each item of ACHS assessment.\n\n- Spreadsheet (34kb):\n\n4) Computed composite scores of ACHS assessment.\n\n- Spreadsheet (24kb):\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to thank the pre-school children involved in this study and their parents, teachers and principals for their cooperation and assistance.\n\n\nReferences\n\nSigdel S: Technology and Learning Capacity of Children: A Positive Impact of Technology in Early Childhood. 2017. Reference Source\n\nAlghamdi Y: Negative Effects of Technology on Children of Today. Oakl Univ. 2016. 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Data Br. 2020." }	[ { "id": "74574", "date": "15 Dec 2020", "name": "Kounosuke Tomori", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe effects of touch-screen technology usage on hand skills among preschool children: a case-control study\nAbstract: Background:\n\n“Little is known on how time spent on touch-screen technology affects the hand skills development of preschool children.” You have frequently cited the literature that the negative effect of touch-screen technology and you have a hypothesis in the manuscript. So, I think you should show it in the background (I have been thought the group in high-intensity use the technology has good hand skills until reading the result!).\n\nIntroduction:\nAdvancing technology is very fast. In the first paragraph, you showed the literature to spend time using the touch-screen tool. But this data looks old a little bit. Please update this. (if possible)\n\nThe comprehensive literature review is good, but it is unclear what focus on your study. The sentence of research interesting & novelty needs to be added to the introduction section. What is the difference between your study from previous studies?\n\nMethod:\nDoes the difference between HUTSTG and LUTSTG groups spend time using the technology devise over 120 min or not?\n\nIf possible, please explain how to decide the effect size (d=0.5).\n\nWho and how did data collection for CHSQ and ACHS?\n\nPlease explain the interpretation of the score of CHSQ and ACHS. Is a high score better or not?\n\nData analysis:\nYou conducted the data analysis using two-way ANOVA. This analysis is not wrong, but still unclear what factor is effective in the result. Multivariate analysis is frequently applied to the case-control design. If the Logistic regression analysis is applied, the result shows an odds ratio, and the factors are comparable. Please re-consider the data analysis, if you agree with this.\n\nDiscussion:\nI think the discussion is well written. It may be more interesting if the data regarding traditional play or daily activities which affect to development of hand skill is collected. It is still unclear what factor was decreased instead of using the time the touch-screen device.\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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "100534", "date": "09 Dec 2021", "name": "Nerdy Nerdy", "expertise": [ "Reviewer Expertise Pharmacology", "Medicinal Chemistry", "Pharmaceutical Chemistry", "Pharmaceutical Science", "Social Sciences", "Health Science", "Public Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\nAdequate to describe the introduction, but please also be fair by stating acceptable negative side of gadget to children.\n\nPlease clearly state the novelty of the research.\n\nMethodology: Not enough to describe how to correlate the hand skill with the usage.\n\nResults and Discussion: Have been well enough to present the data and adequate literature to support the results.\n\nSuggestion: Please attached the similarity check report.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\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/9-1306
https://f1000research.com/articles/9-1302/v1	05 Nov 20	{ "type": "Research Article", "title": "A simulation of a medical ventilator with a realistic lungs model", "authors": [ "Tamir Yeshurun", "Yoav Bar David", "Alon Herman", "Stav Bar-Sheshet", "Ronen Zilberman", "Gil Bachar", "Alexander Liberzon", "Gideon Segev", "Tamir Yeshurun", "Yoav Bar David", "Alon Herman", "Stav Bar-Sheshet", "Ronen Zilberman", "Gil Bachar", "Alexander Liberzon" ], "abstract": "Background: The outbreak of COVID-19 pandemic highlighted the necessity for accessible and affordable medical ventilators for healthcare providers. To meet this challenge, researchers and engineers world-wide have embarked on an effort to design simple medical ventilators that can be easily distributed. This study provides a simulation model of a simple one-sensor controlled, medical ventilator system including a realistic lungs model and the synchronization between a patient breathing and the ventilator. This model can assist in the design and optimization of these newly developed systems. Methods: The model simulates the ventilator system suggested and built by the “Manshema” team which employs a positive-pressure controlled system, with air and oxygen inputs from a hospital external gas supply. The model was constructed using Simscape™ (MathWorks®) and guidelines for building an equivalent model in OpenModelica software are suggested. The model implements an autonomously breathing, realistic lung model, and was calibrated against the ventilator prototype, accurately simulating the ventilator operation. Results: The model allows studying the expected gas flow and pressure in the patient’s lungs, testing various control schemes and their synchronization with the patient’s breathing. The model components, inputs, and outputs are described, an example for a simple, positive end expiratory pressure control mode is given, and the synchronization with healthy and ARDS patients is analyzed. Conclusions: We provide a simulator of a medical ventilation including realistic, autonomously breathing lungs model. The simulator allows testing different control schemes for the ventilator and its synchronization with a breathing patient. Implementation of this model may assist in efforts to develop simple and accessible medical ventilators to meet the global demand.", "keywords": [ "COVID-19", "Mechanical Ventilator", "Simulation" ], "content": "Introduction\n\nOne of the positive COVID-19 consequences is a great social gathering of creators, scientists and engineers to assist the worldwide pandemic effort, including the design of custom-made open source ventilators1. The review of Pearce (2020) covers about 160 publications and links to websites that provide computer-assisted design (CAD) models, construction and installation instructions and bills of materials. It is probably not covering hundreds of other projects that are not published yet or could not pass the strict definitions of the open-source ventilator of the author.\n\nManshema is an emergency ventilation machine and was created during the Assuta COVID-19 Hackathon Sprint by the group comprising engineers, medical doctors, and scientists. The Manshema Ventilator (MV) was designed to assist in the ventilation of patients who are capable of autonomous breathing yet require assistance to maintain a sufficient positive end expiratory pressure (PEEP) and blood oxygen saturation levels.\n\nOne of the major drawbacks of the custom-made open-source ventilator designs is that these are created in a very short time and do not allow detailed analysis of their performance, quality assurance and thus regulatory approval. One of the key points is a lack of proper set of mathematical models that describe the performance of a specific ventilator due to a large variety of the parts, sensors and components used in its creation. This study is addressing this gap by creating a detailed mathematical model and a simulator of a realistic lung ventilation, carefully calibrated and tuned specifically to the MV design, parts and sensors. The simulation will provide the design team the opportunity to design the next version, to extend the ventilator capabilities and to assure its performance corresponding to the specific patient condition. Furthermore, the simulation provides a template for a large variety of open source designs, such as Ambu bag ventilators or linear actuator ventilators, and may eventually lead to a closed loop, feedback-back control at the level of commercial regulatory approved mechanical ventilators.\n\n\nMethods and materials\n\nThe MV consists of an input branch which mixes air and oxygen from the hospital reservoirs and feeds it into the patient mask, and an expiratory output branch which is opened or closed by the control system. Figure 1a shows a schematic illustration of the MV. The input compressed air and oxygen are supplied by the hospital central reservoirs. The flow from each of the reservoirs is controlled with a flow control valve. After the flow control valves, the gases flow through two similar pipes, mix and flow through the main pipe and mask pipe towards the breathing mask. A pressure relief valve marked Popoff is located between the Main pipe and the Mask pipe. This pressure relief is set to mechanically control the maximal pressure in the system and avoid over-pressuring the lungs. The expiratory air flows through a directional check valve which does not permit breathing the exhaled gasses. After the check valve, the outlet pipe leads to the expiratory flow control system which opens and closes the expiratory pipe flow path using an ON/OFF solenoid valve. The outlet of the control system is connected to a pressure relief valve that is set according to the required PEEP valve. Figure 1b shows a CAD drawing of the complete MV system, and Figure 1c shows a CAD drawing of the main components of the MV. Figure 1d is a photograph of the main components of the MV prototype.\n\n(a) A schematic description of the Manshema ventilator. The pressure measurements and control system are not shown. (b) A CAD drawing of the full MV, (c) a CAD drawing of the MV main components, (d) a photograph of the main components of the MV prototype. PEEP, positive end expiratory pressure; CAD, computer-assisted design; MV, Manshema ventilator.\n\nIn order to reduce the system costs to minimum, the MV operates with a single pressure sensor and a solenoid valve which controls the gas flow out of the system. The minimum and maximum pressure in the system are controlled with a hysteresis control scheme. This control strategy is realized with a pressure sensor located near the inlet to the solenoid valve and a relay which sets the state of the solenoid valve according to the measured pressure. In its initial state, the solenoid valve is closed directing the input air to the patient allowing the pressure to build up. When the patient exhales, the pressure at the expiratory pipe increases rapidly and after it reaches the expiratory positive airway pressure (EPAP), the relay opens the solenoid valve, the exhaled air is removed, and the pressure starts to drop. Once the patient starts to inhale, air is removed from the pipes and the pressure drops. When the pressure reaches the inspiratory positive airway pressure (IPAP) the solenoid valve closes, and the breathing cycle continues.\n\nThe MV model was built with MathWorks® Simulink® Simscape™ Gas system toolbox. The outline for the model followed the MathWorks® “Medical Ventilator with Lung Model” example and was modified to describe the MV design, control system as well as an autonomously breathing patient. The model source files, along with an elaborated description of model parameters, variables, Simulink® block parameters and their values can be found under the data availability section as well as in the model OSF webpage2.\n\nFigure 2 shows the block diagram of the model and Figure 3 shows a block diagram of the control system. The lungs are modeled as a translational mechanical converter that is coupled to a spring, a damper, and a force source. The spring and damper model the mechanical compliance and resistance of the lungs3 and force source models the muscle induced pressure4 which is a result of the patient autonomous breathing (patients that cannot breath autonomously can be modeled by replacing the variable muscle pressure term with a constant pressure). The pressure induced by the muscles contraction and relaxation, Pmus, is realized with exponential functions as described by Fresnel et al.4:\n\nWhere T1 is the time period for muscles contraction in every breathing cycle and Ttot is the breathing cycle length. τc and τr are the contraction and relaxation time constants, respectively, and Pmax is the maximum pressure that can be induced by the muscles. All the parameters in Equation (1) can be easily derived from the mouth occlusion pressure, P0.1 and the breathing frequency, fv as described in 4. A block diagram of the lungs branch is shown in Figure 4.\n\nModel calibration. The model was calibrated against the MV prototype in two steps. First, the parameters of the PEEP and Popoff pressure relief valves were calibrated by comparing the model output to the measured output when the lungs port was blocked. In this set of experiments, the pressure at the inlet to the solenoid control valve was measured as a function of the total input gas flow rate when the solenoid valve was open and when it was closed. The experiment was repeated for PEEP values of 2, 5 and 10 cmH2O. For each of the pressure relief valves in the model the set pressure differential and the maximum valve open area were tuned to provide the best fit to the measured data. Figure 5 shows the modeled and measured pressure as a function of the input gas flow rate and PEEP values of 2, 5 and 10 cmH2O.\n\nThe modeled and measured pressure as a function of the input gas flow rate and PEEP values of 2, 5 and 10 cmH2O. PEEP, positive end expiratory pressure.\n\nNext, the model calibration was tested by comparing model results to the output of the MV prototype when it was connected to an IMTMedical Easylung test lung. In order to minimize the effects of the popoff and PEEP valves, the total input gas flow rate was set to 10 L/min and the PEEP was set to 2 cmH2O. To probe the transient response of the MV, the solenoid valve was opened and closed in intervals of 3.5 seconds. Under these conditions, pressure drops across the different parts of the system are low, the Popoff valve remains closed throughout the experiment and the pressure in the system is determined mostly by the mechanical lung parameters. This provides optimal conditions for calibrating the mechanical lungs’ compliance and resistance in the model. Figure 6 shows the measured and modeled pressure (a) and gas flow (b). The gray regions in the figure are time periods in which the solenoid valve is closed. The fitted spring and damper constants for the lungs model are 148.5 N/m and 40 N/(m/s), respectively. When the solenoid valve is closed, air is directed into the test lung, increasing the pressure in it. Then, when the solenoid valve is reopened, the air in the test lung along with air from the reservoirs flow out of the system through the PEEP pressure relief valve. The MV model was able to capture the measured transient response of the system well. Particularly, the modeled gas flow follows the measured values closely.\n\nThe measured and modeled pressure (a) and gas flow (b) in a mechanical lung experiment. The PEEP was set to 2cmH2O and the total input gas flow was set to 10 L/min. The regions with the gray background are time periods in which the solenoid valve was closed. The fitted spring and damper constants for the lungs model are 148.5 N/m and 40 N/(m/s), respectively. PEEP, positive end expiratory pressure.\n\nOnce the lungs parameters were found, the model predictions were compared to experimental measurements at input flow rates of 20L/min and 30 L/min and a PEEP of 5 cmH2O, which better represent the operating conditions of the MV. Figure 7 shows the modeled and measured pressure and flow rates with input gas flow rates of 20 L/min (Figure 7 a and b) and 30 L/min (Figure 7 c and d). The regions with the gray background in Figure 7 are the time periods in which the solenoid valve was closed. Unlike the lung calibration experiments in which the Popoff valve remains constantly closed, at a flow rate of 20 L/min the Popoff valve opens when the pressure rises above 13 cmH2O and closes back when it drops below 8 cmH2O. This transition, which is not instantaneous, is not captured well in the simulation, resulting in a deviation between the measured and modeled responses. Nevertheless, the simulation was able to predict fairly well the minimum and maximum pressure in the system, which are most important for its safe operation. In a similar manner, there is a deviation between the measured and modeled flow in the system when the input gas flow rate. The overestimation of the calculated air flow under higher input flow rates may be a result of leakages in the system that are not considered in the model.\n\nThe measured and modeled pressure (a,c) and gas flow (b,d) in a mechanical lung experiment. The PEEP was set to 5cmH2O and the total input gas flow was set to 20 L/min (a,b) and 30 L/min (c,d). PEEP, positive end expiratory pressure.\n\nOpen source implementation. The model can be implemented using similar or equivalent components in the open source OpenModelica software utilizing Modelica.Fluid and Modelica.OpenHydraulics libraries. In Table 1, we detail equivalent or most similar components, together with component names that could be used in construction of an equivalent model.\n\n\nResults\n\nAfter the model is calibrated and tested, we turn to simulate the ventilation of autonomously breathing patients. We start by simulating the ventilation of a healthy person which is embodied by a breathing rate of 15 breaths per minute5, and an occlusion pressure of 4 cmH2O4. The simulated lungs spring and damper constants are as in Figure 6. Figure 8 a, b and c show the simulated pressure, flow and tidal volume, respectively. Positive flow denotes gas flow out of the system, for example when air is inhaled into the lungs. The gas flow is negative when gas flows into the MV, for example when the patient is exhaling. The pink regions in the figure are the periods in which the patient is trying to inhale and the solenoid valve is closed, the light blue regions are the periods in which the solenoid valve is open and the patient is trying to exhale, the purple regions are periods where the patient is attempting to inhale while the solenoid valve is open and white areas are periods in which the patient is attempting to exhale while the solenoid valve is closed. The color coding for the patient breathing state and the state of the solenoid valve is summarized in Table 2. As the patient begins to inhale (purple region), the lungs expand, air flows into the lungs, and the pressure in the expiratory pipe drops. Once the pressure reaches the IPAP, the solenoid valve closes (pink region) and the input gas is inhaled by the patient, resulting in a nearly constant gas flow at a rate that is determined by the flow regulating valves at the inputs to the system. The pressure in the lungs decreases at the beginning of this stage as the lungs are still expanding and then it increases slowly as the lungs fill with air. When the patient attempts to exhale the pressure increases rapidly in the system. At first the solenoid valve is still closed and gas flows into the patient’s lungs (white region), but once the EPAP is reached, the solenoid valve opens and the air flows out of the MV through the expiratory pipe (light blue region).\n\nThe calculated pressure (a) flow (b) and tidal volume (c) time evolution in the lungs and at the control system solenoid valve. The patient breathing rate was taken to be 15 breaths per minute, the occlusion pressure is 4 cmH2O, IPAP was set to 5 cmH2O and the EPAP was set to 13 cmH2O. The color coding is as described in Table 2.\n\nFigure 9 shows the time evolution of the pressure, air flow and tidal volume simulating the ventilation of an acute respiratory distress syndrome (ARDS) patient. The patient breathing rate was taken to be 20 breaths per minute5, the occlusion pressure is 6.65 cmH2O6, IPAP was set to 5 cmH2O and the EPAP was set to 13 cmH2O5. The color coding is as described in Table 2. It can be easily seen that MV output ventilation of the ARDS patient is very similar to that shown in Figure 8. The slight decrease in tidal volume is a result of the higher breathing rate which is recommended for patients with ARDS5. The low variance in the MV output with respect to the patient condition is a result of the fairly constant gas flow upon inhalation. This may be an advantage since it allows simple tuning of the ventilation parameters according the guidelines for specific respiratory syndromes.\n\nThe calculated pressure (a) flow (b) and tidal volume (c) time evolution in the lungs and at the control system solenoid valve. The patient breathing rate was taken to be 20 breaths per minute, the occlusion pressure is 6.65 cmH2O, IPAP was set to 5 cmH2O and the EPAP was set to 13 cmH2O. The color coding is as described in Table 2.\n\nPerfect synchronization between the MV and the patient is obtained if the solenoid valve opens and closes exactly when the patient attempts to inhale and exhale. Thus, the synchronization level of the MV can be optimized by attempting to minimize the white and purple regions in the output plot.\n\n\nConclusions\n\nIn this study we provide a solution for the majority of the custom-designed ventilators created around the world in a response to the COVID-19 crisis. The simulator can lead to the opportunity to assure quality of the designed machines versus the digital twin model, analyze human response as compared to the realistic lung model and enable future, regulatory approved designs.\n\n\nData availability\n\nOpen Science Framework: A simulation of a controlled medical ventilator with a realistic lungs model. https://doi.org/10.17605/OSF.IO/XJKC82.\n\nThis project contains the following underlying data:\n\nPeep and Popoff calibration (data underlying Figure 5):\n\nBaseWorkspace.mat (Base parameters data, Simulink® model initial function callback)\n\nload_ventilator_variables_PEEP_2.m (Calibration of set PEEP 2 cmH2O experiment, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation (PEEP and Popoff) and control valve settings)\n\nload_ventilator_variables_PEEP_5.m (Calibration of set PEEP 5 cmH2O experiment, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation (PEEP and Popoff) and control valve settings.)\n\nload_ventilator_variables_PEEP_10.m (Calibration of set PEEP 10 cmH2O experiment, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation (PEEP and Popoff) and control valve settings)\n\nPlot_ventilator_output_PEEP_2.m (Output results plotting script, Simulink® model stop function callback)\n\nPlot_ventilator_output_PEEP_5.m (Output results plotting script, Simulink® model stop function callback)\n\nPlot_ventilator_output_PEEP_10.m (Output results plotting script, Simulink® model stop function callback)\n\nManshema_mechanical_lung_Plugged_lungs_exp_PEEP_2.slx (Calibration of set PEEP 2 cmH2O experiment, Simulink® simulation model file. Output pressure and flow prediction, underlying Figure 5)\n\nManshema_mechanical_lung_Plugged_lungs_exp_PEEP_5.slx (Calibration of set PEEP 5 cmH2O experiment, Simulink® simulation model file. Output pressure and flow prediction, underlying Figure 5)\n\nManshema_mechanical_lung_Plugged_lungs_exp_PEEP_10.slx (Calibration of set PEEP 10 cmH2O experiment, Simulink® simulation model file. Output pressure and flow prediction, underlying Figure 5)\n\nRC and C values calibration (underlying Figure 6)\n\nBaseWorkspace.mat (Base parameters data, Simulink® model initial function callback)\n\nflow-10-peep-2-lung.csv (Flow measurement data with set peep 2 cmH2O and flow set 10 lpm at Easylung lung)\n\nflow-10-peep-2-out.csv (Flow measurement data with set peep 2 cmH2O and flow set 10 lpm at expiratory output)\n\nload_ventilator_variables_full_exp_10Lpmin_PEEP_2.m (Calibration of RC and C values with set PEEP 2 cmH2O experiment, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation and control valve settings)\n\nplot_ventilator_output_full_exp_10Lpmin_PEEP_2.m (Output results plotting script, Simulink® model stop function callback)\n\nManshema_mechanical_lung_timed_10Lpmin_PEEP_2.slx (Calibration of RC and C values for lungs model, at set PEEP 2 cmH2O experiment with 10 lpm, Simulink® simulation model file, raw pressure and flow prediction underlying Figure 6)\n\nModel - MV prototype Comparison (underlying Figure 7)\n\n∘ Lungs comparison 20Lpm, underlying Figure 7a and 7b\n\n▪ BaseWorkspace.mat\n\nBase parameters data, Simulink® model initial function callback.\n\n▪ flow-20-peep-5-lung.csv\n\nFlow measurement data with set peep 5 cmH2O and flow set 20 lpm at Easylung lung.\n\n▪ flow-20-peep-5-out.csv\n\nFlow measurement data with set peep 5 cmH2O and flow set 20 lpm at expiratory output.\n\n▪ load_ventilator_variables_full_exp_20Lpmin_PEEP_5.m\n\nModel and experiment comparison with set PEEP 5 cmH2O experiment, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation and control valve settings.\n\n▪ plot_ventilator_output_full_exp_20Lpmin_PEEP_5.m\n\nOutput results plotting script, Simulink® model stop function callback.\n\n▪ Manshema_mechanical_lung_timed_20Lpmin_PEEP_5.slx\n\nModel and experiment comparison at set PEEP 5 cmH2O experiment with 20 lpm, Simulink® simulation model file, raw pressure and flow prediction underlying Figures 7a and 7b.\n\n∘ Lungs comparison 30Lpm (underlying Figure 7c and 7d)\n\n▪ BaseWorkspace.mat\n\nBase parameters data, Simulink® model initial function callback.\n\n▪ flow-30-peep-5-lung.csv\n\nFlow measurement data with set peep 5 cmH2O and flow set 30 lpm at Easylung lung.\n\n▪ flow-30-peep-5-out.csv\n\nFlow measurement data with set peep 5 cmH2O and flow set 30 lpm at expiratory output.\n\n▪ load_ventilator_variables_full_exp_30Lpmin_PEEP_5.m\n\nModel and experiment comparison with set PEEP 5 cmH2O experiment, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation and control valve settings.\n\n▪ plot_ventilator_output_full_exp_30Lpmin_PEEP_5.m\n\nOutput results plotting script, Simulink® model stop function callback.\n\n▪ Manshema_mechanical_lung_timed_30Lpmin_PEEP_5.slx\n\nModel and experiment comparison at set PEEP 5 cmH2O experiment with 30 lpm, Simulink® simulation model file, raw pressure and flow prediction underlying Figures 7c and 7d.\n\nHealthy Patient model testing (underlying Figure 2, 3, 4 and 8)\n\n∘ BaseWorkspace.mat\n\nBase parameters data, Simulink® model initial function callback.\n\n∘ load_variables_Healthy.m\n\nHealthy patient lungs and breathing model simulation, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation (PEEP and Popoff) and control valve settings.\n\n∘ plot_output_Healthy.m\n\nHealthy patient lungs and breathing model simulation output results plotting script, Simulink® model stop function callback, underlying Figure 8.\n\n∘ Manshema_20Lpmin_PEEP_5_simulated_lung.slx\n\nHealthy patient lungs and breathing Simulink® simulation model file, blocks model, underlying Figures 2, 3, 4 and raw pressure, flow and volume prediction underlying Figure 8.\n\nARDS patient model testing (underlying Figure 9)\n\n∘ BaseWorkspace.mat\n\nBase parameters data, Simulink® model initial function callback.\n\n∘ load_variables_ARDS.m\n\nARDS patient lungs and breathing model simulation, variables calculation script, Simulink® model initial function callback. Input of simulated respiration, input gas flow, pressure regulation (PEEP and Popoff) and control valve settings.\n\n∘ plot_output_ARDS.m\n\nARDS patient lungs and breathing model simulation output results plotting script, Simulink® model stop function callback, underlying Figure 9.\n\n∘ Manshema_20Lpmin_PEEP_5_ARDS.slx\n\nARDS patient lungs and breathing Simulink® simulation model file, raw pressure, flow and volume prediction underlying Figure 9.\n\nOpen Science Framework: A simulation of a controlled medical ventilator with a realistic lungs model. https://doi.org/10.17605/OSF.IO/XJKC82.\n\nThis project contains the following extended data:\n\n- Manshema_Sim_Parameters.csv (Table of input parameters and variables calculations for healthy and ARDS patients’ simulations)\n\n- Manshema_Blocks_Properties.csv (Extended table of Simulink® blocks properties input parameters for all simulations)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors wish to thank to all the people involved in the discussions and contributors to the brainstorming group originated at the Faculty of Engineering, Tel Aviv University: Nati Ben Hamo, Prof. Yoram Reich, Baruch Meierovich, Demitry Aronovsky, Yigal Edrey, and Danny Berko and all the members of the Manshema team: Mordechai Halfon, Dr. Elad Grozovsky, Roi Darnell, Ivry Shapira and Omri Mizrahi.\n\n\nReferences\n\nPearce JM: A review of open source ventilators for COVID-19 and future pandemics [version 2; peer review: 3 approved]. F1000Res. 2020; 9: 218. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSegev G, Liberzon A: A simulation of a controlled medical ventilator with a realistic lungs model. 2020. http://www.doi.org/10.17605/OSF.IO/XJKC8\n\nOtis AB, Fenn WO, Rahn H: Mechanics of breathing in man. J Appl Physiol. 1950; 2(11): 592–607. PubMed Abstract \| Publisher Full Text\n\nFresnel E, Muir JF, Letellier C: Realistic human muscle pressure for driving a mechanical lung. EPJ Nonlinear Biomed Phys. 2014; 2(1): 1–18. Publisher Full Text\n\nSiegel MD, Hyzy RC: Ventilator management strategies for adults with acute respiratory distress syndrome. UpToDate. 2019; 3: 1–42. Reference Source\n\nHerrera M, Blasco J, Venegas J, et al.: Mouth occlusion pressure (P0.1) in acute respiratory failure. Intensive Care Med. 1985; 11(3): 134–139. PubMed Abstract \| Publisher Full Text" }	[ { "id": "79574", "date": "09 Mar 2021", "name": "Dan Stieper Karbing", "expertise": [ "Reviewer Expertise Modeling of pulmonary physiology. Decision support for mechanical ventilation management." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents an emergency ventilator as well as a software model for simulating mechanical ventilation and testing the design of mechanical ventilators. It is a worthy endeavor indeed. However, the manuscript in its current form suffers from having to both document ventilator and simulator design and provide evaluation results.\n\nGeneral comments:\nModel calibration: Were the experiments repeated with different valves to check reproducibility between valves for this calibration?\n\nWhat were the parameters of the easylung test lung, e.g. resistance and compliance? There is some discrepancy between simulated and measured values of pressure and flow, in particular for model predictions (fig 7), which would expectedly be aggravated and more serious in severely ill patients potentially leading to falsely accepting a dangerous design.\n\nPlease consider sharing table 1 instead as an electronic supplement and provide tables with the model parameters used for simulating patient cases.\n\nDefinition of good agreement between model simulations and measurements is currently done 'by eye'. The evaluation would be strengthened by a quantitative evaluation and criterion of good agreement, e.g. by Bland-Altman plots.\n\nPlease provide model parameters for simulation of healthy and ARDS patient examples. Flows, pressures and volumes appear very similar for identical ventilator settings. This would not be the case for a real ARDS patient. The simulation therefore does not appear realistic in its current form.\n\nPatient-ventilator synchrony covers a variety of situations. Figures 8 and 9 do not provide an evaluation comprehensive enough to document that the simulator can be used to test ventilator functioning in relation to this phenomenon.\n\nThe presented results do not, to a convincing degree, support the current unambiguous conclusion. A more comprehensive evaluation is required to support that the simulator can indeed secure sufficient quality of ventilator designs.\n\nSpecific comments:\nFigure 5: Only 1 red curve but varying points - due to simulated values at closed solenoid value being the same but measured values varying?\n\nPlease use different symbols for the measured values at the three different PEEP levels. It is difficult to see in some instances if a point belong to the PEEP 2, 5 or 10 experiment.\n\nPg 7, 2nd col, ln 12-14: I did not understand this sentence, please consider revising it.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\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? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1302
https://f1000research.com/articles/9-1301/v1	05 Nov 20	{ "type": "Brief Report", "title": "Unusual occurrence of rare Geastrum species with an abnormal stoma development found in Madurai, Tamil Nadu, India", "authors": [ "Veluswamy Karthikeyan" ], "abstract": "Unusual occurrence of Geastrum species fungi, belonging to the class Basidiomycetes, found in riverbank regions of Madurai, Tamil Nadu, India. Its detailed illustrations and morphological characterization analysis are reported in this study.", "keywords": [ "Basidiomycets", "Geastrum and Characterization" ], "content": "Introduction\n\nThe species of the genus Geastrum, classified under order Geastrales, is rare and the species are red listed (Piętka & Kujawa in 2012; Wojewoda et al., 2006). Although in the distant past they were recorded in several locations worldwide, sightings of this attractive earthstar in plains are now highly limited due to anthropogenic activity. Kirk et al. (2008) and Perez (2009) described 50 species of Geastrum in a dictionary of fungi. Zamora et al. (2014) described 100 to 120 species of Geastrum based on field and herbarium studies.\n\nThere have been many reports about the pluralistic distribution of Geastrum species in Western Ghats by Karun & Sridhar, 2014, in Central India by Verma et al. (2018) and in Tamil Nadu by Meenakshi & Selvam (2020). Geastrum species share morphology with Myriostoma species, as reported by Alexov et al. (2012); Esqueda (2009); Outcoumit et al. (2009) and Pawłowski & Adamska (2008).\n\nMany mycologists classify Myriostoma in between Geastraceae and Astraeaceae. However, currently it is classified under Geastraceae (Dring, 1973; Sunhede, 1989). Using rDNA and other genetic markers, its molecular taxonomic position within Geastraceae was confirmed, and it is closely associated with the genus of Geastrum (Hosaka et al., 2006; Krüger et al., 2001).\n\nThese inedible fungi are widely distributed in Africa, Asia, North and South America, and Europe, where they grow in humus-rich forests. Geastrum are rather rare fungi; they have been characterized as critically endangered, red listed and threatened in many European countries like Poland, Montenegro, Bulgaria, Slovakia, Sweden, Switzerland and Czech Republic (Wojewoda et al., 2006 and Piętka & Kujawa, 2012). Consistent and periodical surveys of macrofungi result in identification of new fungi. The present objective of this work is to report the Geastrum species found in the riverbank canal of Madurai, Tamil Nadu, India with an abnormal stoma development.\n\n\nMethods\n\nThe specimens were collected from our college campus: Thiagarajar college, Teppakulam, Madurai, Tamil Nadu, India. The soil texture of the collection site is highly humus and moist, with shady regions provided by large trees in the nearby area. The collected specimens were photographed, subcultured in potato dextrose medium (pH 6.5) and preserved in formalin bottles (10%).\n\nCollected specimens were dried using a glass desiccator and analysed morphologically and anatomically. Morphological characters were observed using a stereomicroscope (Labline, model number 3923061230; 20X & 40X with flat field) and measurements were taken with a ruler. Microscopic features (e.g., basidiospores, capillitium, hyphae of peridium) were studied under light microscopy at high magnification (40X). Lacto-phenol (0.1%) mixed with trypan blue was used to study the gleba. Microscopic images were taken using Camera Lucida (HLP-3 Model). Standard specimen identification described by Smith (1963) was followed for the anatomical and morphological features based microscopic examination.\n\n\nResults and discussion\n\nThe specimen (TMC 1001) has been deposited in the collection of Gasteroid fungi at the Department of Botany, Thiagarajar college, Teppakulam, Madurai, Tamil Nadu, India.\n\nThe fruiting body size measures 40 mm in width by 20 mm in height, is solitary, and consists of an upper stalked spore case and radiating rays below (Figure 1 and Figure 2). The exoperidium has 10–12 rays, which are revolute, thin, united in the base and free at the tips, and triple layered, with a blackish brown mycelial layer (Figure 3). The tips curve towards the basal position. The peridium is bilayered. The endoperidium body spore case is greyish brown, up to 19 mm in diameter, with many stalks attached. The spore case opens by many pores through which spores escape.\n\nThe fungus has brown basidispores with a globose and verrucose structure. Brown eucapillitium threads are thin walled, straight, aseptate and up to 3μm in diameter. The exoperdium is composed of hyaline, aseptate to rarely septate, unbranched, thick walled, tightly packed hyphae, up to 4μm in diameter, wall thickness up to 1.7 μm. The endoperidium is unbranched, aseptate, brown in colour, with tightly packed hyphae measuring up to 3μm in diameter.\n\nAlthough these fungi are cosmopolitan in distribution, due to the periodical environmental disruption nowadays, occurrence of these fungi is highly limited. According to Signalarter (2000) and Benkert (2003), the Geastrum species serve as indicators of specific habitats in need of conservation.\n\nA) Geastrum in field. B and C) Geastrum size measurement.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nI grateful to our beloved management of Thiagarajar College for the permission to carry out this study. I deeply acknowledge to all my students of I-M.Sc., Botany for their valuable help during collection of this rare fungi.\n\n\nReferences\n\nAlexov R, Vassilev D, Nedelev P, et al.: New Records of seven rare and noteworthy basidiomycetes from Bulgaria. Trakia Journal of Sciences. 2012; 10(2): 10–6.\n\nBenkert D: Berlin und die Mark Brandenburg-ein Paradies für Erdsterne (Geastrales). Verhandlungen des Botanischen Vereins von Berlin und Brandenburg. 2003; 136: 231–268.\n\nDring DM: The Fungi: An Advanced Treatise (Eds. G.C. Ainsworth, FK. Sparrow and AS. Sussman) IV B. pp.451-78 Academic Press. 1973; 451–78.\n\nEsqueda M, Sánchez A, Rivera M, et al.: First records of gasteroid fungi from the Ajos–Bavispe National Forest Reserve and Wildlife Refuge, Sonora, México Revista Mexicana de Micologia. 2009; 30: 19–29.\n\nHosaka K, Bates ST, Beever RE, et al.: Molecular phylogenetics of the gomphoid-phalloid fungi with an establishment of the new subclass Phallomycetidae and two new orders. Mycologia. 2006; 98(6): 949–59. PubMed Abstract \| Publisher Full Text\n\nKarun NC, Sridhar KR: Geasters in the Western Ghats and west coast of India. Acta Mycol. 2014; 49(2): 207–219. Publisher Full Text\n\nKirk PM, Cannon PF, Minter DW, et al.: Ainsworth and Bisby’s dictionary of the Fungi. 10th ed. Wallingford: CABI; 2008. Reference Source\n\nKrüger D, Binder M, Fischer M, et al.: \"The Lycoperdales. A molecular approach to the systematics of some gasteroid mushrooms\". Mycologia. 2001; 93(5): 947–57. Publisher Full Text\n\nMeenakshi R, Selvam K: Diversity And Molecular Identification Of Selected Wood Degrading Fungi From Chitteri Hills, Eastern Ghats of Tamilnadu, India. International Journal of Scientific & Technology Research. 2020; 9: 02.\n\nOutcoumit A, Touhami AO, Douira A: Bulletin Mycologique et Botanique Dauphine-Savoie. 2009; 49(192): 41–6. Reference Source\n\nPawłowski B, Adamska E: Nowe,najliczniejsze w Polsce stanowisko gwiazdy wieloporowatej Myriostoma coliforme (Dicks.) Corda w Toruniu\" [A new locality of Myriostoma coliforme (Dicks.) Corda in Toruń with the most abundant occurrence of this species in Poland] Chrońmy Przyrodę Ojczysta (in Polish). 2008; 64(2): 70–6. Reference Source\n\nPerez EF: O gênero Geastrum Pers. (Phallomycetidae, Basidiomycota) em Algumas Áreas De MataAtlántica E Caatinga No Rio Grande Do Norte, Brasil [MSc thesis]. Natal: Universidade Federal Do RioGrande Do Norte; 2009. Reference Source\n\nPiętka J, Kujawa A: A new location for Geastrum lageniforme Vittad. in Poland. Pol J Environ Stud. 2012; 21: 1791–1795. Reference Source\n\nSignalarter NJ: Indikatorer på skyddsvärd skog. Flora över kryptogamer. Jönköping: Skogsstyrelsen; 2000.\n\nSmith AH: The Mushroom hunter’s field guide. University of Michigan Press, Annarbor. 1963; 67. Reference Source\n\nSunhede S: Geastraceae (Basidiomycotina): Morphology, ecology end systematics with special emphasis on the North European species, Fungiflora. 1989. Reference Source\n\nVerma RK, Pandro V, Raj D, et al.: Diversity of macro-fungi in Central India-XVII: Geastrum fimbriatum and Geastrum triplex. Tropical Forest Research Institute, Jabalpur, MP, India. 2018; 5–10. Reference Source\n\nWojewoda W, Ławrynowicz M: Red list of the macrofungi in Poland. In: Mirek Z, Zarzycki K, Wojewoda W, Szeląg Z, editors. Red list plants and fungi in Poland. Karków: W Szafer Institute of Botany, Polish Academy of Sciences; 2006; 53–70.\n\nZamora JC, Calogne FD, Hosaka K, et al.: Systematics of the genus Geastrum (Fungi: Basidiomycota) revisited. Taxon. 2014; 63: 477–497. Publisher Full Text\n\nZamora JC, Calogne FD, Martín MP: Combining morphological and phylogenetic analyses to unravel systematics in Geastrum sect. Schmidelia. Mycologia. 2014; 106(6): 1199–1211. PubMed Abstract \| Publisher Full Text" }	[ { "id": "83859", "date": "04 May 2021", "name": "Julieth Sousa", "expertise": [ "Reviewer Expertise Taxonomy", "Sistematics", "Mycology." ], "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 presence of more than one peristome and more than one stalk are reported by Sunhede, S. 1989. Geastraceae (Basidiomycotina). Morphology, ecology and systematics with special emphasis on the North European species. Synopsis Fungorum 1: 1-534.\nThe description need improvements:\nAdd details about Peristome. Add spores measurements. Add photos of microstructures.\nPlease add some discussions/conclusions.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\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? Partly", "responses": [ { "c_id": "6643", "date": "06 May 2021", "name": "veluswamy karthikeyan", "role": "Author Response", "response": "As per the reviewer, we accept the revision required. We here describe the: 1. Peristome size and structure peristome are up to 3μm in diameter. 2. The spore print was not taken at that time we are unable to determine the spore size." } ] }, { "id": "123904", "date": "27 Apr 2022", "name": "Shwet Kamal", "expertise": [ "Reviewer Expertise Mushroom biology", "Genetics and breeding", "Production technology", "etc." ], "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\n1. Geastrum species are commonly found in India so this is not a rare genus in India.\n\n2. There is no mention about the species. Also there is no information about the sequences. The species may be sequenced to get the correct identification of species.\n\n3. No mention about the abnormal stoma development as mentioned in the title of the manuscript. Also, 'Stoma' is not a term in Geastrum glossary.\n\n4. Only photographs of the specimen are given in the manuscript. Nothing has been given on microscopic images or camera lucida drawings.\n5. The techniques of preservation as a herbarium specimen is not detailed.\n\nOverall the manuscript is poorly written as no detailed data and methods are provided.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1301
https://f1000research.com/articles/9-1300/v1	05 Nov 20	{ "type": "Research Article", "title": "The ameliorative effect of atorvastatin on serum testosterone and testicular oxidant/antioxidant system of HFD-fed male albino rats", "authors": [ "Mohammed Esmail", "Mohammed Kandeil", "Ali Mahmoud El-Zanaty", "Mohammed Abdel-Gabbar", "Mohammed Esmail", "Mohammed Kandeil", "Ali Mahmoud El-Zanaty" ], "abstract": "Background: There is a mutual effect between central obesity and low total serum testosterone. Moreover, oxidative stress acts as a bridge between obesity and its complications. Taken together, we aimed to evaluate whether atorvastatin (AS), a cholesterol-lowering drug, has protective potential against high fat diet (HFD)-induced low fertility, which was exemplified in serum testosterone determination. Moreover, we aimed to deduce a putative mechanism of action through evaluation of the testicular oxidant/antioxidant system. Methods: Adult male albino Wistar rats (Rattus norvegicus albinus) were divided into three groups: 1) normal control group, rats were fed a normal diet for four weeks; 2) HFD group, rats were fed an HFD for four weeks; and 3) AS group, rats were fed an HFD and 5 mg/kg/day atorvastatin for the last two weeks of the experiment. Serum atherogenic index, testosterone, and thyroid stimulating hormone were estimated. Moreover, testicular reduced glutathione and malondialdehyde contents, as well as glutathione-S-transferase, superoxide dismutase, and glutathione reductase activities were also determined. The statistical differences were analyzed using analysis of variance (ANOVA). Results: AS ameliorated the increased level of serum atherogenic index induced by an HFD, as well as testicular malonaldehyde and reduced glutathione levels. On the other hand, AS increased the depleted level and activity of serum testosterone and testicular glutathione reductase, respectively, induced by HFD. Conclusion: The ameliorative effect of AS on the deteriorated level of total serum testosterone induced by HFD might partially be due to oxidant/antioxidant disturbance. Further studies should be carried out to evaluate mTOR pathway contribution, which could enable researchers to deduce drugs targeting members of the oxidant/antioxidant system and/or mTOR pathway to ameliorate putative HFD-induced low fertility.", "keywords": [ "HFD", "atorvastatin", "atherogenic index", "testosterone", "antioxidants", "MDA", "testis", "male rats" ], "content": "Abbreviations\n\nAX, atherogenic index; HDL-c, cholesterol of high-density lipoprotein; AS, atorvastatin; HFD, high fat diet; CN, normal control group; MDA, malondialdehyde; ELFA, enzyme-linked fluorescent assay; ROS, reactive oxygen species; GSH, reduced form of glutathione; SOD, superoxide dismutase; GST, glutathione-S transferase; TG, triacylglycerol; GR, glutathione reductase; TSH, thyroid stimulating hormone\n\n\nIntroduction\n\nObesity or overweight is related to an elevated risk of many medical problems such as depression and infertility. In those that are obese or overweight, hyperlipidemia can increase the cholesterol content of platelets, endothelial cells, smooth muscle cells, neutrophils and polymorphonuclear leucocytes and may be a source of reactive oxygen species (ROS)1. Moreover, increased free fatty acidemia and triglyceridemia, which are consequences of high fat diet (HFD) supplementation, are implicated in obesity complications2,3.\n\nThe WHO has reported that approximately 1.9 billion people are overweight and 650 million people are obese worldwide4. Moreover, defective sperm quality constitutes 30% of infertile couples, who represent around 16% of the worldwide population5. Interestingly, no precise cause can be elucidated for primary or secondary infertility in around 25% of couples6. Although literature connecting semen parameters and obesity are contradictory, it is thought that there is a mutual effect between low serum testosterone and central obesity7. It has been reported that an increased glycerol level, which is HFD-induced, might lead to a leaky blood-testis barrier, disrupting the homeostasis of the tubular fluid and promoting the apoptosis of germ cells8.\n\nIt has also been suggested that oxidative stress acts as a bridge between obesity and its complications9. Dyslipidemia-generated elevated ROS and/or inflammatory substances are considered to be causes of metabolic disorders such as infertility10,11. High ROS levels may render testicular cells ineffective at maintaining a convenient environment for spermatogenesis12. Hence, decreasing dyslipidemia may improve HFD-induced poor fertility and atorvastatin (AS), which is a cholesterol-lowering drug, may be beneficial in this way.\n\n\nMethods\n\nAll animal handling was in accordance with the instructions and guidelines of the Experimental Animal Ethics Committee, Faculty of Science, Beni-Suef University, Egypt (Approval Number: BSU/FS/2020/18).\n\nA total of 30 apparently healthy adult male albino Wistar rats that initially weighed approximately 85 g were used in the beginning of this study to overcome any sudden death or diseases to rats. Later, we excluded seven rats because they did not achieve serum atherogenic index (AX) > 0.24, required for inclusion. Two rats died after sub-allocation of HFD-fed rats. Table 1 shows the final number of rats used and their allocation. The animals were purchased from Helwan University, Cairo, Egypt and then housed in the Biochemistry Department, Faculty of Science at Beni-Suef University. The rats were acclimatized for one week to exclude any intercurrent infection. Rats were kept in polypropylene cages with well aerated stainless-steel covers in a controlled environment that was maintained under a 12-hour light/dark cycle, a temperature of 24°C (±3°C) and 50–70% humidity. The rats were supplied with a standard diet and had free access to tap water. All efforts were made to reduce the number and suffering of the rats under investigation. These efforts included: 1) continuous checking of animal house air conditioning and continuous and careful supply of drinking water; 2) refreshing the animal house with ordinary air daily for 30 mins; 3) cleaning the cages and the animal house; 4) using the effective anesthesia dose; 5) carrying out euthanasia of animals far away from the other alive rats; 6) carefully disposing of euthanized animals far away from the other alive rats.\n\nCN, normal control; HFD, high fat diet; AS, atorvastatin.\n\nNormal standard food was purchased from Mecca Factory, Beni-Suef, Egypt. The high fat diet consists of the normal rat chow mixed with 15% palm oil and 1% cholesterol in accordance with Matos et al., with some modifications13. Compositions of normal standard and high fat foods are illustrated in Table 2. AS was purchased from Egyptian International Pharmaceutical Industries Co (EIPCO) 10th of Ramadan City, Egypt. All other chemicals were of analytical grade and were obtained from standard commercial suppliers.\n\nRats were divided into three groups as shown in Table 1. Selection of animals was carried out haphazardly without performing any prior randomization method.\n\n1. Normal control group (CN): Rats were fed a normal diet, ad libitum, for four weeks; the whole period of the experiment. Rats were also given the equivalent volume of corn oil, as a vehicle, by oral gavage daily for the last two weeks.\n\n2. HFD group: Rats were fed an HFD, ad libitum, for four weeks of the experiment, in addition to equivalent volume of corn oil by oral gavage daily for the last two weeks.\n\n3. AS group: Rats in this group were fed a high fat diet for four weeks and treated orally with a dose of 5 mg/kg/day atorvastatin suspended in corn oil for the last two weeks of the experiment. The dose was double of that used by Oktem et al.14.\n\nThe 30 rats were allocated to six cages; five rats/each cage. On the first day, we selected a cage for commencing treatment randomly, then, every day we selected the starting cage serially. Selection of an animal inside each cage was arbitrary. The oral gavage started at 10 AM until nearly 12 AM. Rats treatments were carried out at animal house in Faculty of Science. The route of AS administration was by oral gavage, which is easy and suitable for rats.\n\nThe first author was aware of most stages of the experiments. Most of the authors and technicians contributed to animal euthanasia and collection of specimens. Therefore, blinding was not achieved during allocation nor treating stages; however, it was achieved during assessment and data analysis stages.\n\nThe dose was adjusted every week based on the changes in body weight (b. w.) to ensure a constant dose per kg b. w. of rats over the entire period of study. After two weeks of the onset of the experiment, rats were fasted overnight (10–12 h) and blood samples were collected from the lateral tail vein for screening of the AX according to the formula: log10 (TG/HDL-c). Rats that exhibited AX less than 0.24 were excluded. This criterion was defined before any data was collected. Rats with AX more than 0.24 were allocated to the HFD group and AS-administered group for an extra two weeks. At the end of the experiment, the rats were deprived of food overnight and anaesthetized by inhalation with diethyl ether. Blood samples were collected from the jugular vein and then the rats were killed by sudden cervical decapitation while they were anaesthetized.\n\nImmediately after weighing the testes, each testis was homogenized in 5 ml cold buffer (100mM potassium phosphate, PH 7.0, containing 2mM EDTA) per gram of tissue. For the biochemical analysis of antioxidants, including reduced glutathione (GSH) levels were determined according to the method of Beutler et al.15. Moreover, glutathione-S-transferase (GST), superoxide dismutase (SOD), and glutathione reductase (GR) activities were also determined16–18. Besides that, determination of malondialdehyde (MDA) level, as an indicator for lipid peroxidation, was also estimated according to Ohkawa et al.19.\n\nDetermination of testicular GSH content. The protein content of the testicular sample was precipitated by adding an equal volume of 4 % sulphosalicylic acid. After centrifugation, 0.5 ml of the supernatant was added to 4.5 ml of 0.1 mM Bis-(3-carboxy-4-nitrophenyl)-disulfide reagent in 0.1 M phosphate buffer, pH 8. The color intensity was determined spectrophotometrically at 412 nm.\n\nDetermination of testicular GST activity. After addition of glutathione (0.5 mM), and sodium phosphate buffer (0.1 M), pH 7.3, to the tissue sample, preincubation was carried out for 5 min at 37°C. Then, 1-chloro-2,4-dinitrobenzene (CDNB, 0.5 mM) was added to the incubation mixture. Thereafter, trichloroacetic acid solution (33%) was added and the mixture was centrifuged. The CDNB conjugate was measured in the supernatant at 340 nm. The quantity of enzyme that enhanced the formation of 1µmol of CDNB conjugate/mg protein/min was considered as one unit of enzyme activity. The molar extinction coefficient was 9.6 mM−1 cm−1.\n\nDetermination of testicular SOD activity. The reaction mixture contained the sample, tris/EDTA buffer, 12.1%, pH 8, and pyrogallol 10 mM. SOD prevents pyrogallol auto-oxidation by removing superoxide anions. As described, 0.1 buffer was mixed with 1 ml tissue sample, then 0.05 ml pyrogallol was added. The absorbency was read at 0 and 10 mins after the addition of pyrogallol. One unit of enzyme activity is the amount of enzyme that resist a 50% change in extinction per one min. The modification used to determine SOD activity was the use of pyrogallol instead of cytochrome c. ΔA/min was calculated as follows:\n\nEnzyme activity (U/ml) =\n\n[ΔA/min (sample)]/ [ΔA/min (control) × 50 %]\n\nThen U/ml was converted to U/g tissue.\n\nDetermination of testicular MDA content. 1 ml of tissue homogenate was mixed with 2 ml of trichloroacetic acid (TCA, 7.5%) and then centrifuged. 2 ml of the formed supernatant was mixed with thiobarpeturic acid (TBA, 0.7 %) and boiled for 10 mins. The color intensity of the reactants (TBARS) was measured at 532 nm. An extinction coefficient of 156000 M−1 cm−1 was used for calculation.\n\nSerum triglyceride levels were determined according to the method of Tietz20. Total serum cholesterol and HDL-cholesterol concentrations were detected according to the procedure of Allain et al.21. Serum AX was calculated according to the equation [AX= Log(TG/HDL-C)]22. AX is a useful measure of response to pharmacological intervention. Total serum testosterone and thyroid stimulating hormone (TSH) were estimated by enzyme-linked fluorescent assay (ELFA).\n\nDetermination of serum triglyceride concentration. Serum triglyceride concentration was measured using the Randox triglycerides assay (Cat No. TR 213). The kit included reagent 2 which contained 4-chlorophenol (6 mmol/l), magnesium acetate (5 mmol/l), and tris buffer (50 mmol/l). Reagent 3 contained 4-amino antipyrine (1.0 mmol/l), ATP (1.0 mmol/l), lipases (≥100 U/ml), glyceolkinase (≥120 U/l), glycerol-3-phosphate oxidase (≥2.5 U/ml), and peroxidase (≥4.0 U/ml). Moreover, the working solution was performed by mixing the contents of reagent 3 with 30 ml of reagent 2. On the other hand, reagent 1 contained triglyceride (200 mg/dl) as a standard. As described, 10 μl of the serum or standard was added to 1 ml of the working reagent and then incubated for 5 min at 37°C. Thereafter, the absorbance of sample (Asample) and standard (Astandard) against reagent blank were measured at 456 nm. Triglycerides level (mg/dl) = (Asample/Astandard) × 200.\n\nDetermination of serum cholesterol concentration. Serum cholesterol concentration was measured using the Randox Cholesterol assay (Cat No. CH 201). The cholesterol kit included reagent 1 which contained cholesterol (200 mg/dl) as a standard, while reagent 2 contained 3,5dichloro-2-hydroxybenzene sulfonic acid (4mmol/l), and tris buffer (50mmol/l) detergent (0.2 %). Reagent 3 contained cholesterol esterase (≥160 U/l), cholesterol oxidase (≥ 120 U/l), peroxidase (≥2000 U/l), and 4-aminoantipyrine (0.45 mmol/l). The working solution was prepared by mixing reagent 3 with 30 ml of reagent 2. The procedure for determining of serum total cholesterol level was similar to that of triglyceride determination, except the absorbance measurement was at 500 nm.\n\nDetermination of serum HDL-cholesterol concentration. Serum HDL-cholesterol concentration was measured using the Randox HDL-cholesterol assay (Cat No. CH 203). Briefly, 50 μl of phosphtungestic acid (13.9 mol/l) was added to 500 μl of sample to precipitate LDL, VLDL and chylomicron fractions in the presence of magnesium ions, allowed to stand for 10 min, centrifuged for 10 min at 4000 rpm. Then, the supernatant was collected to determine the cholesterol content as described before. HDL-cholesterol concentration was calculated as follows:\n\nHDL-cholesterol concentration (mg/dl) = Asample × 180.\n\nDetermination of serum total testosterone level. Serum total testosterone level was measured using the VIDAS® Testosterone II kit (Cat No. 414320, bioMérieux, France). The antibodies used were monoclonal anti-testosterone antibodies produced in mice. All assay steps were performed automatically by the instrument. The strip consisted of 10 wells covered with a labeled foil seal. The last well of each strip was a cuvette in which the fluorometric reading was performed. The wells in the center section of the strip contained the various reagents required for the assay.\n\nThe wells reagents included: well-1 (the sample well), well-2 (Conjugate): phosphate buffer, bovine albumin, alkaline phosphatase-labeled anti-testosterone antibody, and preservative (300 μL), and well-3 (Pre-treatment solution): phosphate buffer, bovine albumin, dissociation agent, preservative (600 μL). Additionally, wells-4, -5, and -6 were empty, while wells-7, -8, and -9 were (wash buffer) contained tris, surfactant, and preservative (600 μL). Well-10 (reading cuvette with substrate) contained 4-Methyl-umbelliferyl-phosphate (0.6 mmol/L), diethanolamine (DEA, 0.62 mol/L or 6.6%, pH 9.2, and 1 g/L sodium azide 300 μL).\n\nBriefly, the procedure aimed, firstly, to isolate testosterone from the carrier proteins in the sample by using the pre-treatment solution. The pre-treated sample was transferred to a well containing a testosterone antibody (conjugate) labelled with alkaline phosphatase. For the anti-testosterone-specific antibody sites, the antigen in the sample and the testosterone antigen attached to the SPR ® inner wall. During the washing steps, unbound pieces were removed. The substrate (4-Methylumbelliferyl phosphate) was cycled in and out of the SPR ® during the final detection stage. The hydrolysis of this substrate was catalyzed by the conjugate enzyme into a fluorescent product (4-Methylumbelliferone), whose fluorescence was measured at 450 nm. The fluorescence intensity was inversely proportional to the testosterone concentration present in the sample. The results were automatically determined by the instrument at the end of the assay with regard to the calibration curve stored in memory.\n\nDetermination of serum TSH level. Serum TSH level was measured using the VIDAS® TSH kit (Cat No. 30400, bioMérieux, France). The protocol was like that of total testosterone determination except we used mouse monoclonal anti-TSH antibodies instead of anti-testosterone antibodies.\n\nData are expressed as mean ± SE. The statistical differences between continuous variables was analyzed using analysis of variance (ANOVA). Statistical analysis was performed using SPSS v.24 (SPSS Inc., released 2007, SPSS for Windows, version 16.0, Chicago, IL). Values with p < 0.05 were considered significant.\n\n\nResults\n\nData resulting from our experiment were deposited in Harvard Dataverse23. Supplementing rats with an HFD produced a significant increase (p < 0.05) of 31% in serum AX relative to CN. However, administering AS ameliorated this effect by significantly decreasing serum AX by 18% relative to the HFD-fed group. HFD caused a profound decrease of 62% in total serum testosterone, while AS alleviated this negative effect by increasing the total serum testosterone level by 92% (p < 0.05). However, neither HFD nor AS showed a significant effect on serum TSH at p ≥ 0.05 (Table 3).\n\nValues are expressed as means ± SE.\n\nMeans that have different superscript letters are significantly different at P ≤ 0.05.\n\n% changes were calculated by comparing HFD means with CN means and AS means with HFD means.\n\nCN, normal control; HFD, high fat diet; AS, atorvastatin.\n\nRegarding MDA, HFD exerted a tremendous increase in testicular levels of MDA of 113%. AS exhibited an amelioration of this effect, causing a 54% decrease in MDA levels relative to the HFD group (p < 0.05). Testicular GSH content was profoundly increased by HFD supplementation by 87%, while AS supplementation caused a 30% decrease in GSH content (p < 0.05) (Table 4).\n\nValues are expressed as means ± SE.\n\nMeans that have different superscript letters are significantly different at P ≤ 0.05.\n\n% changes were calculated by comparing HFD means with CN means and AS means with HFD means.\n\nCN, normal control; HFD, high fat diet; AS, atorvastatin.\n\nGR activity was decreased by 42% by HFD but increased by 112% by AS (p < 0.05). There were moderate increases of 14% and 11% in SOD and GST activities, respectively, after HFD supplementation. However, a decrease of 4% was observed in SOD activity after AS administration (p < 0.05), while AS produced an insignificant change in GST activity relative to the HFD group (p ≥ 0.05) (Table 5).\n\nValues are expressed as means ± SE.\n\nMeans that have different superscript letters are significantly different at P ≤ 0.05.\n\n% changes were calculated by comparing HFD means with CN means and AS means with HFD means.\n\nCN, normal control; HFD, high fat diet; AS, atorvastatin.\n\n\nDiscussion\n\nLipid profile amelioration is considered to be an important target in order to foster β-oxidation of fatty acids, otherwise fatty acids would be oriented towards the esterification pathway, leading to accumulation of TAG and LDL cholesterol24. Hence, statins such as AS can help in this trend. They can up-regulate the LDL receptor and reduce cholesterol absorption and biosynthesis, an effect that leads to rapid clearance of LDL particles from circulation25.\n\nStatins were thought to improve sperm parameters regarding dyslipidemia. However, Pons-Rejraji et al. counteracted this finding, as statins might induce local inflammation or oxidative stress, with persistent effects on prostatic and epididymal secretion26. In agreement with this, some evidence has shown that statins might be detrimental to testosterone production27 and might induce impotence28. Other studies indicated that patients undergoing statin therapy to lower serum cholesterol levels have neither androgen deterioration29 nor impotence30 due to sterol regulatory element binding protein (SREBP)/Srd5a2 system alteration31. The different pharmacokinetic or physicochemical properties of various statins may contribute considerably to total serum testosterone levels.\n\nTo share in this debate, we introduced an HFD to rats and treated them with AS. In our experiment, an HFD profoundly decreased serum testosterone, which was counteracted by supplementing with AS, although serum levels were not elevated to that of the normal control. It was previously reported that the decrease of total serum testosterone in obese men is due to increased serum estrogen as a result of increased aromatase activity32.\n\nThe detrimental effects of HFD may be mediated, in our study, via an elevated testicular malondialdehyde level, which is a marker of oxidative stress. However, the ameliorative effect of AS may be partially explicated by lowering serum AX and testicular malondialdehyde levels. Surprisingly, HFD markedly elevated testicular glutathione level and GR activity, which may be a kind of compensatory mechanism. The substantial contribution of the oxidant/antioxidant system in the deterioration effects of HFD on fertility was obvious.\n\nAdditionally, cholesterol synthesis is not completely inhibited in peripheral tissues in statin-treated patients33. Therefore, enough cholesterol is available in gonadal and adrenal cells to maintain steroid hormone synthesis; this in line with our data.\n\nAccumulated data suggest mTOR as a promising mechanism for explaining HFD-caused detrimental effects on fertility. In one study, mTOR and p70s6k were weakly expressed in the HFD group, which may be reversed by statin34.\n\nOur data enables researchers to deduce drugs targeting members of the oxidant/antioxidant system and/or mTOR pathway to ameliorate putative HFD-induced infertility.\n\n\nData availability\n\nHarvard Dataverse: Replication Data \"The Ameliorative Effect of Atorvastatin on Fertility and Testicular Oxidant/Antioxidant System of HFD-Fed Male Albino Rats\". https://doi.org/10.7910/DVN/9LEX1O23.\n\nThis project contains the following underlying data:\n\nStat (AS).docx (statistics report of data)\n\nOutcomes (AS).docx (raw GSH, GR, SOD, and GST and MDA levels, as well as serum total testosterone and TSH levels for all 21 rats)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank technical staff members of Biochemistry Department, Faculty of Science, Beni-Suef University for their substantial aid while performing this experiment.\n\n\nReferences\n\nHensley K, Robinson KA, Gabbita SP, et al.: Reactive oxygen species, cell signaling, and cell injury. 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PubMed Abstract \| Publisher Full Text\n\nAbdel-Gabbar M, Esmail M, Kandeil M, et al.: Replication Data \"The Ameliorative Effect of Atorvastatin on Fertility and Testicular Oxidant/Antioxidant System of HFD-Fed Male Albino Rats\". Harvard Dataverse. V5. 2020. http://www.doi.org/10.7910/DVN/9LEX1O\n\nBrault M, Ray J, Gomez YH, et al.: Statin treatment and new-onset diabetes: a review of proposed mechanisms. Metabolism. 2014; 63(6): 735–745. PubMed Abstract \| Publisher Full Text\n\nAl-Naqeep G, Ismail M, Allaudin Z: Regulation of low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression by thymoquinone-rich fraction and thymoquinone in HepG2 cells. J Nutrigenet Nutrigenomics. 2009; 2(4–5): 163–172. PubMed Abstract \| Publisher Full Text\n\nPons-Rejraji H, Brugnon F, Sion B, et al.: Evaluation of atorvastatin efficacy and toxicity on spermatozoa, accessory glands and gonadal hormones of healthy men: a pilot prospective clinical trial. Reprod Biol Endocrinol. 2014; 12: 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchooling CM, Au Yeung SL, Freeman G, et al.: The effect of statins on testosterone in men and women, a systematic review and meta-analysis of randomized controlled trials. BMC Med. 2013; 11: 57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSolomon H, Samarasinghe YP, Feher MD, et al.: Erectile dysfunction and statin treatment in high cardiovascular risk patients. Int J Clin Pract. 2006; 60(2): 141–5. PubMed Abstract \| Publisher Full Text\n\nCoogan PF, Rosenberg L, Strom BL: Statin use and the risk of 10 cancers. Epidemiology. 2007; 18(2): 213–219. PubMed Abstract \| Publisher Full Text\n\nHerrmann HC, Levine LA, Macaluso J Jr, et al.: Can atorvastatin improve the response to sildenafil in men with erectile dysfunction not initially responsive to sildenafil? Hypothesis and pilot trial results. J Sex Med. 2006; 3(2): 303–8. PubMed Abstract \| Publisher Full Text\n\nSeo YK, Zhu B, Jeon TI, et al.: Regulation of steroid 5-alpha reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin. Exp Cell Res. 2009; 315(18): 3133–3139. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen PG: Obesity in men: the hypogonadal-estrogen receptor relationship and its effect on glucose homeostasis. Med Hypotheses. 2008; 70(2): 358–360. PubMed Abstract \| Publisher Full Text\n\nGermershausen JI, Hunt VM, Bostedor RG, et al.: Tissue selectivity of the cholesterol lowering agents lovastatin, simvastatin and pravastatin in rats in vivo. Biochem Biophys Res Commun. 1989; 158(3): 667–675. PubMed Abstract \| Publisher Full Text\n\nCui X, Long C, Zhu J, et al.: Protective Effects of Fluvastatin on Reproductive Function in Obese Male Rats Induced by High-Fat Diet through Enhanced Signaling of mTOR. Cell Physiol Biochem. 2017; 41(2): 598–608. PubMed Abstract \| Publisher Full Text" }	[ { "id": "75299", "date": "25 Jan 2021", "name": "Ahmed A. Mousa", "expertise": [ "Reviewer Expertise Oxidative stress and andrology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors have done a good effort in this work and they concluded that AS has ameliorative effect on the deteriorated level of total serum testosterone induced by HFD might partially be due to oxidant/antioxidant disturbance.\nMajor comments:\nWould you have a reference for induction of obesity in just for 4 weeks with palm oil and cholesterol?\n\nWhat was the final weight of rats at the end of experiment?\n\nWhy did authors not provide any study on assessment of sperm parameters?\n\nEIPCO Commercial HFD, what is the purpose for it in markets or is it only for research purposes?\n\nWhy did authors not formulate HFD by themselves?\n\nPlease add name and model of spectrophotometer and ELISA plate reader.\n\nMinor comment\nAdd the full name of mTOR in abstract section and abbreviations section.\nIn Methods: delete All efforts were made to reduce the number and suffering of the rats under investigation. These efforts included: 1) continuous checking of animal house air conditioning and continuous and careful supply of drinking water; 2) refreshing the animal house with ordinary air daily for 30 mins; 3) cleaning the cages and the animal house; 4) using the effective anesthesia dose; 5) carrying out euthanasia of animals far away from the other alive rats; 6) carefully disposing of euthanized animals far away from the other alive rats.\nThe first author was aware of most stages of the experiments. Most of the authors and technicians contributed to animal euthanasia and collection of specimens. Therefore, blinding was not achieved during allocation nor treating stages; however, it was achieved during assessment and data analysis stages.\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": [] }, { "id": "77679", "date": "10 Feb 2021", "name": "Mostafa Waly", "expertise": [ "Reviewer Expertise Biochemistry and oxidative Stress" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article and the theme of the reported research will add needed knowledge in the field.\nI have few minor comments to optimize the quality of the manuscript as follow:\nPlease provide the weight gain of the experimental animals throughout the duration of the experiment. I would recommend a figure as an illustration.\n\nThe statistical analysis, One Way ANOVA, should be followed by post analysis; for example Tukey's test. This will provide significant power for the reported results.\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/9-1300
https://f1000research.com/articles/9-1297/v1	04 Nov 20	{ "type": "Research Article", "title": "Synthesis and characterization of chitosan nanoparticles of Achillea millefolium L. and their activities", "authors": [ "Dolly Kain", "Suresh Kumar", "Dolly Kain" ], "abstract": "Background: Achillea millefolium L. is an herbal aromatic plant of family Asteraceae reported to have various medicinal activities in the literature. The current study evaluated the potential of chitosan nanoparticles of A. millefolium as an effective strategy for targeted treatment of bacterial diseases and urolithiasis. Methods: A. millefolium was collected from Poonch, Jammu and Kashmir, and its inflorescence extracted in water by maceration. Chitosan nanoparticles of A. millefolium (AMCSNPs) were prepared by ionic gelation method using 0.1% chitosan, different concentrations of the cross-linking agent sodium tripolyphosphate (STPP; 0.5%, 1%, 1.5%, 2%) and different concentrations of A. millefolium extract (0.5%, 1%, 1.5%, 2%). Characterization of AMCSNPs was done using UV-Vis spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy, dynamic light scattering (DLS) and transmission electron microscopy (TEM). Antibacterial screening of AMCSNPs was performed by well-diffusion method. Antiurolithiatic screening of AMCSNPs was done by nucleation and aggregation assay. Results: The best chitosan nanoparticles of A. millefolium (AMCSNPs) were obtained with 0.1% chitosan, 1% STPP and 20% A. millefolium. These AMCSNPs showed maximum zone of inhibition of 30±0.5 mm using the well-diffusion method against both Bacillus subtilis (Gram-positive) and Pseudomonas aeruginosa (Gram-negative) and maximum antiurolithiatic activity with 68% inhibition shown at aggregation stage. Conclusions: The current study suggests that AMCSNPs are an excellent strategy for targeted drug delivery for treatment of bacterial diseases and urolithiasis.", "keywords": [ "Achillea millefolium L.", "AMCSNPs", "Drug delivery", "Targeted treatment", "Antibacterial", "Antiurolithiatic" ], "content": "Introduction\n\nNatural biopolymers are attractive products of living organisms as they serve a number of different applications for human health due to their biodegradability, such as vaccine delivery, drug development, and food preservatives1. Chitosan (CS) is a natural biopolymer and a derivative of chitin. It is obtained from different sources of chitin and differs on the basis of its degree of deacetylation2. In the last few years, CS nanoparticles (CSNPs) have drawn much attention due to their biodegradability, biocompatibility, quantum size effects, large surface to volume ratios, and their simple and inexpensive production3–5. Different biological activities of CSNPs have been reported, such as antimicrobial, antioxidant, anticancer6, drug delivery, tissue engineering, carbon nanotube, food preservative, and purification of water7. CSNPs successfully used in drug delivery for treatment of various diseases, including ocular drug delivery8, per-oral drug delivery9, nasal drug delivery10, pulmonary drug delivery11, mucosal drug delivery12, gene delivery13, buccal drug delivery14, vaccine delivery15, vaginal drug delivery16, and cancer therapy17 have been reported. Achillea millefolium is a perennial herbal aromatic plant belonging to the family Asteraceae with characteristically finely divided leaves and inflorescence in corymbose cluster. It has been reported to have different medicinal activities, including antibacterial and diuretic18,19. The current study was designed to evaluate the potential of CSNPs of A. millefolium (AMCSNPs) as an effective alternative of targeted drug delivery and treatment of various diseases, including bacterial infections specifically urolithiasis.\n\n\nMethods\n\nA. millefolium was collected from Pathanteer Village, Mendhar Tehsil, Poonch District(Jammu and Kashmir, India; GPS coordinates 33° 39’ 40” N- 74° 11’ 11” E) and identified by Raw Materials Herbarium and Museum (RHMD), National Institute of Science Communication and Information Resources (NISCAIR), Pusa with reference IDNISCAIR/RHMD/consult/2018/3293-94. 5g powder of inflorescence of A. millefolium was extracted in 50 ml of water by maceration at 90° C using water bath.\n\nCrude extract of the plant was evaporated using rotary evaporator (Khera KI- 102), which resulted in the semi solid form of extract. This was then weighed and dissolved in a known amount of solvent for making a stock concentration of the plant extract. Different concentrations were made by serial dilution.\n\nStrains of Bacillus subtilis (MTCC 441) and Pseudomonas aeruginosa (MTCC 1688) were procured from MTCC Chandigarh. All the chemicals used (chitosan, acetic acid, sodium tripolyphosphate (STPP), Tween-80, calcium chloride, sodium oxalate, tris buffer and NaCl) were of good quality and purchased from Fisher Scientific International, Inc.\n\nCSNPs were prepared by ionic gelation method. 10ml 0.1% chitosan solution was made in 1% acetic acid with different percentages of the cross-linking agent STPP (0.5%, 1%, 1.5% and 2%). 5ml of STPP was added drop wise to the chitosan-acetic acid solution, which was magnetically stirred at room temperature. An opalescent color was observed, and stirring was continued for 60 min.\n\nTo obtain AMCSNPs, variable concentrations of plant extract (5%, 10%, 15% and 20%) were added to the 10 ml chitosan solution by magnetic stirring prior to adding the 5 ml STPP drop wise. This solution was stirred for a further 2 h followed by centrifugation at 10000g for 10 min and then the AMCSNPs were washed three times with distilled water. The pH of the nanoparticles was maintained at 4.8, and 12 drops of 1% Tween-80 was used to prevent agglomeration.\n\nPercentage encapsulation efficiency of each concentration of extract was determined using the following formula20–22:\n\nEncapsulation efficiency (%) = (total amount – free amount/total amount) 100.\n\nUV-Vis spectroscopy using SPUV-1000 spectrophotometer attached to Mwave professional software 2.0 (or any software used to obtain the UV- Vis absorption spectra) and spectrum between 200 nm-700 nm was obtained for determining the main absorbing region. FTIR (Fourier Transform-Infrared) Spectroscopy using spectrometer (Brukers) in the range of 1000 cm-1–3500 cm-1 to identify the peaks of main functional groups, DLS (Dynamic Light Scattering) in the range between 0 nm to 1000 nm using zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) at room temperature for particle size distribution and TEM (Transmission electron microscopy) at an accelerating voltage of 200 kV using Tecnai G2 30U-twin kV Ultra-twin microscope to study the morphology.\n\nPrimary culture of bacteria was obtained from lyophilized culture by inoculating in LB broth, which was incubated in an incubator shaker at 120 rpm and 37°C for 12–16 h. Pure colonies of each bacterium were obtained from primary culture by streak plate method using LB agar plates, which were inoculated in LB broth, incubated in an incubator shaker at 120 rpm 37°C for 12–16 h. Absorption of bacterial culture was adjusted to 0.1±0.02 at 600nm using SP-UV1000 spectrophotometer to reach the concentration of 108 CFU/ml for final use, which is equal to 0.5 McFarland standards, as previously performed in the literature23–25 to obtain a similar concentration of each bacterium. Each reading was taken thrice.\n\nAntibacterial screening of AMCSNPs was done using well-diffusion method31. 1.5% LB agar plates were used and a 5mm cork-borer made four wells in each plate. 20 µl of B. subtilis and P. aeruginosa culture was added to the plates and spread using a glass spreader. 100 µl of AMCSNPs was poured in the wells. Plates were sealed with parafilm, incubated at 28° C for 12–16 h and the zone of inhibition (ZOI) recorded.\n\nNucleation and aggregation assays were used to determine the antiurolithiatic potential of AMCSNPs.\n\nNucleation assay: The method of Hennequin et al.27 was used with some minor modifications. Solutions of calcium chloride and sodium oxalate were prepared at a final concentration of 3mmol/l and 0.5mmol/l, respectively, in a buffer containing Tris 0.05mol/l and NaCl 0.15mol/l at pH 5.5. A total of 1.9 ml of calcium chloride solution mixed with 200 µl of AMCSNPs was incubated for 30 minutes in a 37° C water bath. Crystallization was started by adding 1.9 ml of sodium oxalate solution. Equal volume of water has used for a control instead of AMCSNPs. The optical density of the solution was recorded at 620 nm for 420 sec using spectrophotometer SPUV- 1000.\n\n% Inhibition = {(Abs. Control- Abs. Sample)/ Abs. Control} 100\n\nAggregation assay: The method of Hess et al.28 was used with some minor modifications. `Seed' CaOx monohydrate (COM) crystals were prepared by mixing calcium chloride and sodium oxalate at 50mmol/l. Both the solutions were equilibrated in a 60° C water bath for 1h and then cooled at 37° C overnight. The crystals were harvested by centrifugation at 10000g and then evaporated at 37° C. COM crystals were used at a final concentration of 0.8 mg/ml, buffered with Tris 0.05 mol/l and NaCl 0.15 mol/l at pH 5.7. A total of 1 ml of AMCSNPs were added in a test tube to 3 ml COM crystal solution and incubated at 37°C. Equal volume of water was used for a control instead of AMCSNPs. Absorption at 620 nm was recorded at different time intervals (30 min, 60 min, 90 min, and 120 min).\n\n% Inhibition = {(Slope Control- Slope Sample)/ Slope Control} * 100\n\nStatistical analysis was performed using Microsoft Excel 2007. One-way ANOVA was used followed by t-test to determine the significant difference of antibacterial activity between different samples and regression analysis was used to plot graphs of nucleation and aggregation assays.\n\n\nResults\n\nOn addition of STPP to chitosan solution, an opalescent color was observed, which indicates the formation of CSNPs. Different concentrations of STPP (0.5%, 1%, 1.5%, and 2%) were used for nanoparticle preparation and 1.0% STPP was found to be most suitable with sharpest peak shown by UV spectroscopy, indicating the most CSNPs made (Figure 1). Therefore, 1.0% STPP was used to obtain AMCSNPs. Different percentages of A. millefolium water extract (5%, 10%, 15% and 20%) in 0.1% of chitosan solution were used to make AMCSNPs and excellent loading efficiency was observed, i.e. 94%, 94.7%, 94.7%, and 95.2% for 5%, 10%, 15% and 20% A. millefolium respectively. A standard graph for absorbance of A. millefolium extract at 417 nm (maximum absorption verses concentration of extract) was obtained. The amount of loaded extract was determined using the standard graph as a decrease in the absorption values of the supernatant of AMCSNPs indicated the loading of extract of the nanoparticles. Loading efficiency was calculated using the above encapsulation efficiency formula for each concentration. Hence 20% AMCSNPs have been used for further analysis.\n\nA broad absorption band between 200 to 300 nm was shown for the UV spectrum of AMCSNPs (Figure 2). FTIR spectra of CS showed peaks at 3324.15, 2153.28, 1638.72 and 1279.29; FTIR spectra of CSNPs showed peaks at 3317.48, 2139.29 and 1638.46; and FTIR spectrum of AMCSNPs showed peaks at 3281.73, 2163.36 and 1636.78 (Figure 3). DLS revealed the size range of nanoparticles with Z average of 118nm having characteristic peaks at 10 nm, 122 nm and 712 nm, and highest intensity was recorded at size 10nm (Figure 4). TEM was used to study the morphology of the nanoparticles, which revealed a spherical shape with smooth surface. TEM also revealed the size of AMCSNPs: <100 nm with smallest size of 4.15 nm (Figure 5).\n\nFourier Transform-Infrared graph of chitosan (A), chitosan nanoparticles, (B) and Achillea millefolium chitosan nanoparticles (C).\n\nAMCSNPs exhibited excellent antibacterial activity against both Gram-positive B. subtilis and Gram-negative P. aeruginosa. AMCSNPs showed three-times the increase in antibacterial activity as compared with A. millefolium extract only (control); ZOI increased from 10 mm to 30mm against both B. subtilis and P. aeruginosa with a statistically significant difference between A. millefolium, CSNPs and AMCSNPs (Figure 6).\n\nAMCSNPs showed significant antiurolithiatic activity with 68% inhibition in the aggregation assay and 51.26% inhibition in the nucleation assay as compared to 55.132% and 9.09% inhibition by A. millefolium extract (control). In the nucleation assay, the % inhibition is nearly equal in the case of A. millefolium and AMCSNPs, but CSNPs did not show any inhibition. In the aggregation assay there is a significant increase in % inhibition with 9.09%, 63.63% and 68% inhibition by A. millefolium, CSNPs and AMCSPs, respectively, (Figure 7).\n\nAntiurolithiatic activity of Achillea millefolium chitosan nanoparticles (AMCSNP’s) as shown by a nucleation assay (A) and aggregation assay (B). AM= A. millefolium, CSNP’s= chitosan nanoparticles.\n\n\nDiscussion\n\nFor characterization of nanoparticles, different techniques used in the literature including UV- Vis spectroscopy, FTIR spectroscopy, DLS and TEM29,30. In our study, a UV-Vis absorption band for AMCSNPs of 200-300 nm indicates the presence of a CO group in the CSNPs, as reported by Vaezifer et al.31. A shift of FTIR peaks from 3317.48, 2139.29 and 1638.46 for CSNPs to 3281.73, 2163.36 and 1636.78 for AMCSNPs indicates that the loading of A. millefolium into the CSNPs, as reported by Khan et al.32. Our DLS results are comparable to the average size of CSNPs reported in literature, i.e. 189 and 197 nm by Khan et al.32, 216 nm by Agarwal et al.33, and size range of 135–729 nm by Rasaee et al.34 and 6.5-1331.2 nm by Iswanti et al.35. TEM of AMCSNPs in our study revealed a spherical shape with a smooth surface, which was also reported by Da Silva et al.21.\n\nChitosan is a positively charged macromolecule, which interacts with the negatively charged microbial membrane, and results in the breakage of intracellular components. Chitosan acts as a chelating agent and limits toxin production and microbial growth36–38. Antibacterial screening of Ocimum basilicum CSNPs against E. coli and Bacillus vallismortis have been reported by Rasaee et al.34, chitosan-tripolyphosphate nanoparticles against Staphylococcus aureus and P. aeruginosa have been reported by Bangum et al.39, and against phytopathogens of tomato Xanthomonas and Erwinia strains by Oh et al.29. Gallic acid-chitosan conjugates have been reported to inhibit the formation of calcium oxalate crystals by Queiroz et al.40. Antiurolithiatic activity of Aerva lanata chitosan nanoparticles at 0.8 µg/ml concentration through nucleation assay have been reported by Chandirika et al.41 and Tridax procumbens by Chandirika et al.42. In our study, AMCSNPs showed excellent antibacterial activity against both B. subtilis and P. aeruginosa, and significant antiurolithiatic activity at aggregation stage of urolithiasis.\n\n\nData availability\n\nFigshare: Chitosan nanoparticles of Achillea millefolium L., https://doi.org/10.6084/m9.figshare.12936293.v343.\n\nThis project contains the following underlying data:\n\nOutput files of chitosan nanoparticles with different concentrations of STPP\n\nRaw data for Figures 1, 2 and 4\n\nUnedited and uncropped FT-IR graphs and TEM images of AMCSNPs\n\nZOI of antibacterial activity of AMCSNPs\n\nAbsorption values of antiurolithiatic activity AMCSNPs\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors are thankful to the Principal, Dr. Manoj K. Khanna, Ramjas College, Prof. S.B. Babbar, Head, Prof. K.S. Rao, and Prof. Veena Agrawal, Department of Botany, University of Delhi, Delhi for providing necessary facilities and encouragement during the course of investigation.\n\n\nReferences\n\nGomes LP, Paschoalin VMF, Del Aguila EM: Chitosan nanoparticles: production, physicochemical characteristics and nutraceutical applications. Rev Virtual Quim. 2017; 9(1): 387–409. Reference Source\n\nShukla SK, Mishra AK, Arotiba OA, et al.: Chitosan-based nanomaterials: a state-of-the-art review. Int J Biol Macromol. 2013; 59: 46–58. PubMed Abstract \| Publisher Full Text\n\nOchekpe NA, Olorunfemi PO, Ngwuluka NC: Nanotechnology and drug delivery part 2: nanostructures for drug delivery. Tropical J Pharmaceut Res. 2009; 8: 275. Publisher Full Text\n\nHe X, Hwang HM: Nanotechnology in food science: Functionality, applicability, and safety assessment. J Food Drug Anal. 2016; 24(4): 671–681. 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PubMed Abstract \| Publisher Full Text\n\nHess B, Nakagawa Y, Coe FL: Inhibition of calcium oxalate monohydrate crystal aggregation by urine proteins. Am J Physiol. 1989; 257(1 Pt 2): 99–106. PubMed Abstract \| Publisher Full Text\n\nOH JW, Chun Se C, Chandrasekaran M: Preparation and In Vitro Characterization of Chitosan Nanoparticles and Their Broad- Spectrum Antifungal Action Compared to antibacterial activities against phytopathogens of tomato. Agronomy. 2019; 9(1): 21. Publisher Full Text\n\nArya A, Kumar S, Suryavanshi A, et al.: Biosynthesis of metallic nanoparticles from medicinal plants: A review. J Nanosci Technol. 2019; 5(5): 827–831. Publisher Full Text\n\nVaezifar S, Razavi S, Golozar MA, et al.: Effects of some parameters on particle size distribution of chitosan nanoparticles prepared by ionic gelation method. J Clust sci. 2013; 24: 891–903. Publisher Full Text\n\nKhan MA, Zafaryad M, Mehdi SH, et al.: Characterization and anti-proliferative activity of curcumin loaded chitosan nanoparticles in cervical cancer. Int J Biol Macromol. 2016; 93(Pt A): 242–253. PubMed Abstract \| Publisher Full Text\n\nAgarwal M, Agarwal MK, Shrivastav N, et al.: Preparation of chitosan nanoparticles and their in-vitro characterization. Int J Life Sci Scienti Res. 2018; 4(2): 1713–1720. Publisher Full Text\n\nRasaee I, Ghannadnia M, Honari H: Antibacterial properties of biologically formed chitosan nanoparticles using aqueous leaf extract of Ocimumbasilicum. Nanomed J. 2016; 3(4): 240–247. Reference Source\n\nIswanti FC, Nurulita I, Djauzi S, et al.: Preparation, characterization and evaluation of chitosan- based nanoparticles as CpG ODN carriers. Biotechnol Biotechnol Equip. 2019; 33(1): 390–396. Publisher Full Text\n\nLeuba JL, Stossel P: Chitosan and other polyamines: Antifungal activity and interaction with biological membranes. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChandirika JU, Lakshmi PS, Mathiazhagan A, et al.: Design and development of chitosan nanoparticles in target novel drug delivery systems for Urolithiasis. European J Biomed Pharm Sci. 2015; 2(4): 528–537. Reference Source\n\nChandirika JU, Sindhu R, Selvakumar S, et al.: Herbal extract encapsulated in chitosan nanoparticles: a novel strategy for the treatment of Urolithiasis. Indo American J Pharm Sci. 2018; 5(3): 1955–1961. Publisher Full Text\n\nKain D, Kumar S: Chitosan nanoparticles of Achillea millefolium L. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12936293.v3" }	[ { "id": "83420", "date": "12 May 2021", "name": "Alok Arun", "expertise": [ "Reviewer Expertise Plant Biotechnology", "Genomics", "Molecular Genetics", "Bioinformatics" ], "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 assessed the chitosan nanoparticles isolated from a herbal aromatic plant Achillea millefolium (here after referred as AMCSNPs) using ionic gelation method after applying various concentrations of plant extract. Further, the authors measured the antiurolithatic and antibacterial activity using two bacterial species and consequently propose AMCSNPs to be an alternative strategy for targeted delivery.\nThe research seems to be original and will definitely contribute to broadening the fundamental knowledge about AMCSNPs. However, the authors have potential to make this paper stronger by including few minor details/corrections:\n1. Why was only inflorescence used for extract isolation? Providing an explanation can highlight the significant of tissue selection for chitosan nanoparticles isolation.\n2. Besides herbarium, did others use any other method to ascertain the identity of plant species used in the study?\n3. Authors mention the use of Hess et al., for performing the aggregation assay. There is a brief mention of minor modifications in the protocol. Can they highlight the specific modifications that was used in the protocol?\n4. The TEM images are not very clear. Can the authors provide the magnification used for these images?\n5. The sentence in discussion, \"A shift of FTIR peaks from 3317.48, 2139.29 and 1638.46 for CSNPs to 3281.73, 2163.36 and 1636.78 for AMCSNPs indicates that the loading of A. millefolium into the CSNPs, as reported by Khan et al.\" is not clear. Can authors rephrase the sentence and provide clarity?\n\n6. The paper will greatly benefit from an image of the plant and the tissue that was used in the study.\n7. The study design may have included one more tissue (for example leaf, stem etc) from the plant for comparative purposes.\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? Partly\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "82696", "date": "24 May 2021", "name": "Dipjyoti Chakraborty", "expertise": [ "Reviewer Expertise Stress Biology", "Secondary Metabolite Biotechnology", "Proteomics" ], "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 used crude extracts of Achillea millefolium. The possible constituents of the inflorescence used especially with emphasis on secondary metabolites should be discussed with previous references.\nConsidering seasonal/low availability of flowers, how do the authors justify using inflorescence? Why not any other plant part?\nJustify extraction in water at 90°C, are there any previous reports? What was the duration of extraction?\nWhy were only Bacillus subtilis, and Pseudomonas aeruginosa used for the study?\nThe UV spectrum shows absorbance beyond 1 in the Y-axis (Figure-1). It is generally not recommended to have Abs above 1 as it gives erroneous results and the sample is suitably diluted. The authors may consider.\nSince the spectrum shows very low or no absorbance other than the uv region, authors may provide another graph - maybe in the inset showing the spectrum only in the UV region for better signature spectrum.\nFigure 2 should also be looked into on similar lines.\nThe authors have used the \"well-diffusion method\" for antibacterial screening and thus have not been able to calculate MIC. What was the concentration of extract used for the Zone inhibition study and whether a range of concentration was tried?\nDiscussion section needs a few lines on the possible constituents of A. millefolium extract and its implication on the formation of the nanoparticles and or the bioactivity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": [ { "c_id": "6741", "date": "01 Jun 2021", "name": "Suresh Kumar", "role": "Author Response", "response": "I am thankful to the reviewer for critical analysis of work. The point wise response is as follows: Two reasons for use of Inflorescence, firstly its diuretic uses confirmed by the traditional healers of the collection site. Secondly, crude extract of inflorescence has shown maximum antiurolithiatic activity which was determined in my previous study. The temperature of extraction was used as per literature which gives best result at – 10 to 20 ºC than B.P. of solvent used). Duration of extraction was 48 hrs. Both bacteria are reported as causal agent for several human diseases/ infections (urinary tract infections, soft tissue infections, bacteremia, gastrointestinal infections, respiratory system infections) and also it cover the two main categories of gram negative (P. aeruginosa) and positive bacteria (B. subtilis). The sharp peak at particular wavelength indicates the formation of nanoparticles; further dilution will not cover the entire spectrum and absorption pattern. The antibacterial activity of nanoparticles was determined prepared by 1 mg/ml extract and compared with the same crude extract to determine the enhancement in the activity. GC-MS of crude extract of inflorescence of A. millefolium reveals the presence of different bioactive compounds including Lupeol, Stigmasterol, alpha.-amyrin, and gamma.-sitosterol." } ] } ]	1	https://f1000research.com/articles/9-1297
https://f1000research.com/articles/9-370/v1	15 May 20	{ "type": "Study Protocol", "title": "Early childhood bilingualism: effects on brain structure and function", "authors": [ "Sezgi Goksan", "Froso Argyri", "Jonathan D. Clayden", "Frederique Liegeois", "Li Wei", "Jonathan D. Clayden", "Frederique Liegeois", "Li Wei" ], "abstract": "Growing up in a bilingual environment is becoming increasingly common. Yet, we know little about how this enriched language environment influences the connectivity of children’s brains. Behavioural research in children and adults has shown that bilingualism experience may boost executive control (EC) skills, such as inhibitory control and attention. Moreover, increased structural and functional (resting-state) connectivity in language-related and EC-related brain networks is associated with increased executive control in bilingual adults. However, how bilingualism factors alter brain connectivity early in brain development remains poorly understood. We will combine standardised tests of attention with structural and resting-state functional magnetic resonance imaging (MRI) in bilingual children. This study will allow us to address an important field of inquiry within linguistics and developmental cognitive neuroscience by examining the following questions: Does bilingual experience modulate connectivity in language-related and EC-related networks in children? Do differences in resting-state brain connectivity correlate with differences in EC skills (specifically attention skills)? How do bilingualism-related factors, such as age of exposure to two languages, language usage and proficiency, modulate brain connectivity? We will collect structural and functional MRI, and quantitative measures of EC and language skills from two groups of English-Greek bilingual children - 20 simultaneous bilinguals (exposure to both languages from birth) and 20 successive bilinguals (exposure to English between the ages of 3 and 5 years) - and 20 English monolingual children, 8-10 years old. We will compare connectivity measures and attention skills between monolinguals and bilinguals to examine the effects of bilingual exposure. We will also examine to what extent bilingualism factors predict brain connectivity in EC and language networks. Overall, we hypothesize that connectivity and EC will be enhanced in bilingual children compared to monolingual children, and each outcome will be modulated by age of exposure to two languages and by bilingual language usage.", "keywords": [ "Bilingual", "children", "brain", "MRI", "language", "executive control." ], "content": "Introduction\n\nEarly life experiences shape brain and cognitive development1,2. The study of bilingualism provides a unique model to examine the neural changes linked to early experiences, i.e. whether there are early bilingual language experience effects on the brain, since two languages can be acquired from birth or one language can be acquired from birth and a second language later in life3–5. Despite there being a greater number of multilingual adults in Europe compared with monolinguals6, the effect of early bilingual language exposure on brain structure and function is still poorly understood4. Crucially, most research has focused on adults (see 7 for a review), after decades of bilingualism exposure, alongside other experiences, making it increasingly difficult to associate changes in brain networks with specific experience-related factors.\n\nOur study addresses the following question: Are brain connectivity changes in bilingual children mainly driven by (i) brain maturation stage at age of exposure to two languages, or by (ii) experience-related factors, for example, frequency of use of both languages?\n\nA wide body of research suggests that bilingualism confers advantages in executive control (EC), i.e. the ability to control attention, to inhibit distractions and to shift between goals (e.g. 8, but see 9 for an opposing view). It is hypothesized that this “bilingual advantage” is linked to the ongoing need to manage two language systems, for which EC is required3. Brain networks involved in supporting or engaging EC include the fronto-parietal control network (FCN), salience network (SLN) and default mode network (DMN)10, used for attending, switching/inhibitory control and disengaging in response to external stimuli, respectively11–14. In short, early bilingualism in combination with regular practice of two languages may be associated with better performance on some EC tasks, as well as increased connectivity between EC-related brain networks, because of increased time spent using and controlling two language systems15,16.\n\nAlthough there are a growing number of functional magnetic resonance imaging (MRI) studies investigating the impact of bilingualism on brain activity during executive function tasks (for example 17,18, in adults and 19 in children), there are very few studies on the effects of bilingualism on resting brain networks known to be related to executive function20. Specifically, few studies have asked whether bilingual experience modifies resting-state brain networks, such as (i) the FCN (which includes dorsolateral frontal regions and the inferior/superior parietal lobules), (ii) the SLN (which includes the anterior insula and the dorsal anterior cingulate gyrus), and (iii) the DMN (which includes the posterior cingulate gyrus, the ventromedial prefrontal cortex (vmPFC) and the inferior parietal lobule/angular gyri)10,20–27. An investigation addressing this question in bilingual adults found there was greater negative correlation between the vmPFC (part of the DMN) and the dorsolateral prefrontal cortex (part of FCN) in simultaneous vs. sequential bilinguals27. Moreover the stronger this interaction, the quicker bilinguals responded during “interference suppression” trials in a cognitive control (i.e. Simon) task27. Of note, no monolingual adult control group was included in the study. Our study will allow us to disentangle brain alterations in domain-general (EC) vs. in language-specific networks in bilingual and monolingual children.\n\nAdditionally, bilingualism factors such as age of exposure to two languages, language usage and proficiency have been shown to impact differences in functional brain activity and connectivity28,29. One study has reported that earlier age of acquisition of two languages was associated with stronger functional connectivity between the left inferior frontal gyrus pars triangularis (IFGpt, i.e. Broca’s area, BA 45) and its right homolog and with the right inferior parietal lobule (IPL, part of the FCN)4. A more recent study also found the same relationship in the bilateral IFG in a different group of highly proficient bilingual adults30. Furthermore, the authors observed that greater ‘diversity of language use’ (i.e. an environment in which both languages are commonly utilised and segregated use of each language is not routine) was positively correlated with functional connectivity between cortical and subcortical brain regions (namely between the anterior cingulate cortex and left putamen, and the left caudate and bilateral superior temporal gyrus (STG))30. Altogether, there is emerging evidence that bilingualism factors influence brain functional connectivity, yet little is known about how these connectivity alterations are related to EC performance.\n\nThere is also growing evidence that bilingualism alters the grey and white matter structure in the brain. For example, in adult studies, greater volume and grey matter density are found in regions associated with language, such as the bilateral IFG, IPL, anterior cingulate cortex, caudate and putamen31–35 However, a recent study in pre-school children (aged between 3 and 5 years) found evidence of greater functional connectivity in bilinguals than monolinguals but no structural differences in the IFG, thus suggesting that structural changes may only appear after prolonged exposure to two languages36.\n\nDiffusion-weighted MRI studies have revealed white matter alterations associated with bilingualism (for example, see 37 for a recent review), as measured by indices related to structural connectivity such as Fractional Anisotropy (FA)38. Increased FA in language-specific white matter tracts, such as the inferior fronto-occipital fasciculus (IFOF) has been reported in both adults39 and children (aged between 8 and 11 years old)40. Mohades and colleagues (2012) showed that simultaneous bilingual children (who had exposure to both language before 3 years old) had greater FA within the IFOF compared to successive bilingual children (where age of exposure to two languages was between 3 and 5 years)40. As most MRI studies have focused on adults, it is difficult to disentangle the effects of early environmental changes from those of decades of exposure to bilingualism and other experiences.\n\nIn order to disentangle the effects of bilingualism factors and maturational factors on connectivity, we will recruit bilingual children with either early or later (3 to 5 years) age of onset of exposure to two languages and extensively characterise their linguistic experience.\n\nThe effects of bilingualism on brain structural and functional connectivity can be observed as soon as most EC skills are well developed. We will recruit bilingual children aged 8 – 10 years old because this is before the time of significant brain changes related to puberty, yet an age when EC skills are well developed41.\n\n\nObjectives\n\nOur overall aim is to investigate whether bilingual experience alters EC- and language-related brain networks in children using advanced MRI methods.\n\nSpecifically, we will\n\n(i) examine whether structural and functional connectivity at rest (‘connectivity’ hereafter) differs between two groups of bilingual children and one group of monolingual children;\n\n(ii) identify how age of onset of exposure to two languages, language usage, and proficiency in two languages modulates brain connectivity.\n\n(1) Bilinguals vs. Monolinguals. Early bilingualism will affect connectivity in specific executive control (EC) networks that have been implicated in the ability to engage EC. Both bilingual groups will have stronger functional connectivity within the FCN and DMN networks than monolinguals (as seen for adults in 10).\n\n(2) Simultaneous vs. Successive Bilinguals. The effect of bilingualism on language and EC connectivity will be moderated by age of onset of exposure to two languages. Specifically, the bilingual simultaneous group will show enhanced FCN connectivity relative to the successive bilingual group (as seen for adults in 4).\n\n(3) Within the bilingual groups, language usage and proficiency will moderate the effect of bilingualism on (i) the language network and (ii) the EC network involved in inhibition, i.e. the SLN14, as seen behaviourally15. Therefore, we predict a positive correlation between proficiency/usage of both languages and connectivity within the SLN network, and within the language network, across bilingual participants.\n\n\nProtocol\n\nInclusion criteria. Three groups of typically developing monolingual and bilingual children aged between 8 and 10 years of age will be recruited.\n\nGroup 1: English monolingual children, who have exclusively been exposed to English at home since birth (i.e. they are born to English-speaking parents).\n\nGroup 2: English-Greek simultaneous bilingual children, exposed to these two languages from birth.\n\nGroup 3: English-Greek successive bilinguals, where exposure to Greek was from birth and exposure to English was between the ages of 3 and 5 years (children are born in Greece and arrived in the UK between the ages of 3 and 5 years).\n\nExclusion criteria. Children are excluded from the study if they (a) have had regular exposure from a young age to other languages other than English and Greek; (b) if their Greek-speaking parents were born and/or have lived in the UK for most of their lives, and did not migrate from Greece to live in the UK during adulthood; (c) are not in mainstream schooling; (d) have any contraindications for MRI (e.g. have metal implants or braces); (e) have history of hearing impairment and/or have been diagnosed with language learning difficulties; (f) have a confirmed or suspected diagnosis of developmental conditions (e.g. ADHD, autism, dyslexia), neurological conditions (e.g. epilepsy, cerebral palsy), severe chronic medical condition or being born extremely premature (earlier than 33 weeks gestational age); or (g) have non-verbal intelligence below 70 (assessed using Raven’s Coloured Progressive Matrices, see Methodology section for more detail).\n\nThe research project has been approved by the UCL Institute of Education Research Ethics Committee (approval number: 1080). Prior to participation, researchers will obtain written informed consent from one parent/guardian and written informed assent from each child participant.\n\nProcedure. Standardised tests are used to measure the children’s linguistic and non-linguistic abilities. The bilingual children’s parents are also asked to complete a questionnaire recording detailed information about their child’s exposure to each language. Furthermore, all children are invited to take part in an MRI brain scanning session during which structural (anatomical and diffusion-weighted) and functional (resting-state) images are acquired.\n\nBackground measures: non-verbal ability and verbal working memory. The Raven’s Coloured Progressive Matrices (CPM) test is used to assess the non-verbal ability of the child participants (suitable for use with children aged 4 to 11 years42). During the task each child is asked to complete a puzzle by choosing the correct missing piece from six options. A total of 36 puzzles are presented so that each participant can obtain a maximum score of 36 (one point for each puzzle). Raw CPM scores are then converted to an age-appropriate normalised score (standard score). Standard scores have mean = 100 and standard deviation (SD) = 1542. The lowest and highest scores a child can get are <60 or >140 respectively. No significant gender differences were found in a large, normative sample reported by Raven et al., and distributed by Pearson Inc. (Girls: mean = 101.44, SD = 15.74, Boys: mean = 99.31, SD = 15.242).\n\nVerbal working memory is assessed using subtests that involve repeating series of numbers forwards and backwards (CELF-4 UK43). The use of this task is advantageous because it involves minimal linguistic/lexical demand, as children only need to be familiar with digits 1 to 9. It is also suitable for children aged between 5 and 16 years old. Sequences are presented until the child fails to recall two consecutive sequences. The maximum possible raw score for each task is 16 and 14. Standard scores range from 1 to 19 (mean = 10, standard deviation = 3, CELF-4 UK43).\n\nExpressive vocabulary. The Raven’s Crichton Vocabulary Scales (CVS) test is used to assess verbal ability (suitable for use with children aged 4 to 11 years42). The CVS is a standardised expressive vocabulary test designed for use with the CPM. It assesses the knowledge of words and is constructed to cover as closely as possible the same age range of intellectual development as the CPM. During the CVS, children are asked to describe the meaning of words in a list (80 words). Both English and Greek vocabulary are assessed for bilinguals and only in English for monolinguals. Total raw vocabulary scores are then converted to standard scores (mean = 100, SD = 15). No significant gender differences were found within two large, normative samples, distributed by Pearson Inc. (English – Girls: mean = 101.05, SD = 15.14, Boys: mean = 99.92, SD = 15.47; Greek – Girls: mean = 66.5, SD = 43.6, Boys: mean = 67.0, SD = 41.842).\n\nExecutive control. We also record performance on EC skills, focusing on the selective, sustained and switching components of attention. These are assessed using four subtests from a standardized computer-based test (TEA-Ch 244).\n\n(1) Attention switching is measured in a subtest that involves sorting four objects according to two defined categories (bags and shoes are sorted for either their colour or whether they pair with a hand or feet).\n\n(2) Selective attention is measured via a subtest of target detection amongst distractors.\n\nSustained attention is measured by the final two tasks;\n\n(3) Children have to detect target sounds (dog barks) while ignoring distractors (other animal noises) and they must concentrate in order to do this successfully for 15 trials (the final score is weighted for accuracy on all trials).\n\n(4) Each child’s reaction time (RT) is measured during a 5 minute test (pressing a button in response to the appearance of a blue blob on the screen).\n\nThe composite scores per subtest are calculated as follows: (1) mean RT for switch trials (only correct responses used to calculate the mean), (2) average number of correct responses in two 30 s trials, (3) mean RT weighted for accuracy, and (4) mean RT. The computer-based TEA-Ch 2 assessment produces a PDF output with composite scores and standard scores (either population-based or education-referenced; mean = 10, standard deviation = 3) for each subtest. Standard scores range from 1 – 19.\n\nInhibitory control in the form of inhibition of a prepotent response is likely to be invoked during the switching attention task. During this task, participants must ignore goal-irrelevant stimuli (for example whether the object is red or blue) in order to produce a goal-relevant response (i.e. whether the object pairs with a hand or feet). It is of note that the rules in this task are presented in blocks of 5 and the mean reaction time is measured as the average response to the ‘switch trials’ only, i.e. the first response in each block where the rule has switched from either a red/blue decision to a hand/feet decision or vice versa.\n\nParental questionnaire on history of language exposure and biographical information. Detailed information on the bilingual children’s exposure to the two languages is collected via a structured parental questionnaire (ALEQ Heritage developed by Daskalaki et al., 201945, based on the Alberta Language Environment Questionnaire (ALEQ)46). The questionnaire includes questions about family demographics, age and length of exposure to the two languages, the child’s and the parents’ place of birth, language use among the bilingual child and family members in the home, other contexts of bilingual exposure and use (e.g. extracurricular and literacy activities), time of relocation to the UK, and parents’ education levels. Information about the socioeconomic status of the family will be calculated based on years of maternal education.\n\nThe original ALEQ was designed to measure the current English language use (input and output) in the bilingual child’s environment. The adaptation we use (ALEQ Heritage), however, measures the current Greek language use, and thus the 5-point Likert rating scale has been adapted accordingly. For each question in ALEQ Heritage, the scale is as follows: 0-English always/Greek never, 1-English usually/Greek seldom, 2-English 50 %/Greek 50 %, 3-English seldom/Greek usually, 4-English never/Greek always.\n\nFor the purposes of the current study we will extract the following outcome measure(s):\n\nLanguage use at home\n\nLanguage use at home is calculated by taking the average of two values: (i) Greek/English input and (ii) Greek/English output. Input and output are generated by inserting the Likert scale scores (in response to selected questions) in to the following formula: sum of scores) / (number of scores x 4) (see page 7 of Paradis’ ALEQ). Input is measured using questions about how frequently family members speak Greek/English to the child using the 5-point Likert scale discussed above, i.e. from 0 (English always/Greek never) to 4 (English never/Greek always). Output is measured using questions that explore the frequency with which the child speaks Greek/English to family members at home (on the same 0 - 4 scale). Greek input and output scores are then averaged to produce a score for language use at home, ranging from 0 to 1. This score provides a quantitative measure of the amount of English/Greek input and output the child receives and directs to family members. Higher language use scores (> 0.5) indicate a higher relative use of Greek language at home, whereas lower scores (< 0.5) indicate a higher relative use of English.\n\nRichness score\n\nEnglish and Greek richness scores range from 0 to 1. Each score is a proportion (out of 32) representing English/Greek language use during a range of activities both inside and outside the home. Activities include how frequently English and Greek are used with friends, during literacy activities (e.g. reading books, online language games, etc), and during other extra-curricular activities (e.g. music lessons, sports, etc) on a weekly basis. The calculation of richness scores in the adapted version of the questionnaire that we use differs from the original ALEQ because it includes more questions. The key differences are highlighted here. Firstly, Questions 28 and 31a have been combined and the focus is on how much formal Greek education the child receives. This combined question is included in the calculation of both English and Greek richness scores (rather than only being included for the ‘mother tongue’ in the original ALEQ, thus adding 4 extra points to the English score). In question 30 more activities have been added such as ‘use of Skype’, ‘use of mobile phone’, therefore Greek and English each have a maximum score of 14. In addition, question 32 has been repeated and can be included in the final richness score two further times (4 points per question). This captures more contexts in which children socialize with friends or relatives. Therefore, the total denominator is currently 32 for both English and Greek vs. 16 and 20 respectively in the original ALEQ, (see page 10 of Paradis’ ALEQ).\n\nCombined Greek score\n\nA combined score will be calculated and used to denote Greek language use (range 0 – 2). The score will be generated by adding together each child’s scores for language use at home and Greek richness. The combined score will more accurately represent each child’s exposure to and use of Greek in various contexts both inside and outside the home.\n\nMagnetic Resonance Imaging (MRI) scans. Children are scanned on a 3T Magnetom Prisma scanner (Siemens). The MRI scanner is specifically designed to be a child-friendly environment with images of fish on the walls. The scanner room is also equipped with a television screen that can be used to play a movie for children during the acquisition of structural images.\n\nTo prepare children for the scanner environment we use a booklet designed for children having an MRI scan at Great Ormond Street Hospital. In addition we create a mock scanning session before each scan where children practice laying still in a toy fabric tunnel while listening to scanner noises. Children can also choose whether to have their parent accompany them and remain in the scanner room for the duration of the scan. Each scanning session lasts approximately 45 minutes. This includes the time taken to set up and settle each participant followed by collection of the structural, diffusion and functional resting state images (total acquisition time approximately 22 min).\n\nData are acquired using a 64 channel head coil. A high-resolution magnetisation prepared rapid gradient echo (MPRAGE) T1-weighted 3D image is acquired for anatomical reference per participant using the following parameters (repetition time (TR) = 2300 ms, echo time (TE) = 2.74 ms, inversion time = 909 ms, flip angle (FA) = 8°, acquisition time (TA) = 5 min 21 s, field of view (FOV) = 256 × 256 mm, matrix size = 256 × 256, 240 slices, 1 mm isotropic voxels, single shot, slice acquisition = ascending, parallel acquisition technique (PAT) = GRAPPA). 3D diffusion-weighted MRI scans are collected using a single-shot multi-shell diffusion MRI sequence. The images are encoded along 60 independent directions with b-values of 1000 and 2200 s/mm2 (TR = 3050 ms, TE = 60 ms, FA = 90°, TA = 7 min 16 s, FOV = 220 × 220 mm, matrix size = 110 × 110, voxel size = 2 × 2 × 2 mm, number of contiguous axial slices = 66, slice thickness = 2mm, distance factor = 10 %, slice acquisition = interleaved, PAT = GRAPPA, multi-band acceleration factor = 2, fat suppression = on). We also acquire 13 b0 images and one reverse phase encoded b0.\n\nResting-state functional MRI (rs-fMRI) are acquired at the end of the scanning protocol, so children have become comfortable with the scanning environment. During the rs-fMRI scan children are asked to fixate their gaze on a cross. This method has been shown to increase reliability in connectivity metrics47. The acquisition parameters for the resting scan are as follows: T2* BOLD-weighted, GRE, EPI readout, FA = 75°, TE = 26 ms, TR = 1240 ms, TA = 6 min 18 s, number of measurements = 300, FOV = 200 mm, multi-band 2, 80×80 in-plane matrix size, 2.5 mm isotropic voxels, 40 slices, slice thickness = 2.5 mm, distance factor = 20 %, slice acquisition = interleaved, fat suppression = on, and with a single-band reference (SBref) image acquired at the start. A field map is also acquired (GRE, 2D, dual echo TE1/TE2 = 10/12.46 ms, TR = 1020 ms, TA = 2 min 47 s, FA = 90°, FOV = 200 mm, matrix size = 80 × 80, voxel size = 2.5 × 2.5 mm, 40 slices, slice thickness = 2.5 mm, distance factor = 20 %, slice acquisition = interleaved).\n\nAll data are pseudonymised. High-resolution MRI data has visibly identifiable features of the face removed. Data collected from paediatric participants often requires additional, bespoke consideration; in particular, such data can often contain more motion effects than normal. Therefore, all brain imaging data have visual quality checks to assess the presence of motion artefacts. For the structural and functional data automated quality control descriptors are generated (MRI quality control (MRIQC48)). For the diffusion weighted images, eddy quality control is used (eddy_qc49). The resulting descriptors will be used to detect excessive motion in relation to the rest of the data. Specific pre-processing pipelines (i.e. a series of analysis steps) are subsequently implemented for the structural, diffusion and resting-state functional MRI images (see Figure 1 and Figure 2, and see https://github.com/sgoksan/paed_mri_preprocessing for detailed code).\n\nA summary of the data processing tools used to prepare the (A) T1 weighted image and (B) diffusion weighted image for further analysis. Blue arrows denote pre-processing steps using freely available online tools (named within dark grey boxes). All pictures are of 3 dimensional images from one participant, and represent examples of input and output data files. As part of process (A), each high resolution T1 weighted structural image has (i) non-brain tissue removed using BET, (ii) spatial intensity variations (RF field inhomogeneity) corrected using FAST and (iii) brain tissue types automatically labelled to one of either grey matter, white matter or cerebrospinal fluid (CSF) using FAST. Blue = grey matter, red = white matter, green = CSF. High resolution structural images are shown in greyscale. Note: this figure does not contain all input files and options required for each tool. For a comprehensive list of inputs, see the scripts that accompany this analysis50.\n\nAs most software has been developed for use with adult data, some aspects of this analysis pipeline have been tailored for a paediatric cohort. These steps included (1) manual editing of fieldmap masks (see Figure 2 and Figure 3), (2) use of a paediatric template brain for registration of structural images (left-right symmetric, created by Fonov et al., using structural images from 112 children, aged 7 – 11 years51, see Figure 2 and Figure 4), (3) modification of inputs to ICA-AROMA in order to accept a paediatric template and related masks.\n\nImages from one participant represent examples of input and output data files. Blue arrows denote pre-processing steps using freely available online tools (named within dark grey boxes). Pictures with round corners represent 3 dimensional images, while a series of pictures represents 4 dimensional functional data. FEAT was run using the graphical user interface (rather than by running a script), therefore additional required options are detailed within the main text. Subscript numbers highlight steps that were modified in relation to a typical adult pipeline (see Figure 3 for (1) and Figure 4 for (2)).\n\n(A) Automated mask generated using BET (shown in yellow). The automated methods used to mask the fieldmap magnitude image were producing sub-optimal results (highlighted by red arrows). Fieldmap image (blue-red-yellow image) generated using the sub-optimal mask would leave distortion-susceptible brain regions without appropriate distortion correction (e.g. red arrow in frontal lobe region). (B) Mask following manual editing (yellow) and the subsequent fieldmap (blue-red-yellow) with appropriate coverage of distortion-susceptible regions.\n\n(A) Adult 1mm MNI Template Brain which is commonly used as the standard space image for functional MRI analysis (available as part of FSL). (B) A symmetric paediatric template brain that has been created by Fonov et al., 201151, using structural images from 112 children, aged 7 – 11 years. Use of an age-appropriate template is important in paediatric studies due to important differences between the brains of adults and children. For example, children have less developed frontal lobes, thinner corpus callosum and smaller ventricles.\n\nMRI data will be analysed using a combination of MRI analysis tools within FMRIB Software Library (FSL) version 6.0.252–54 and MRTrix355.\n\nStructural T1. Initial steps on the structural T1-weighted image include brain extraction (i.e. removal of non-brain tissue from the image) using FMRIB’s Brain Extraction Tool (BET56,57), correction of RF inhomogeneity (spatial intensity variations), and segmentation of the different brain tissue types in the T1-weighted structural images using FMRIB’s Automated Segmentation Tool (FAST58) (see Figure 1A).\n\nDiffusion weighted data. For the diffusion weighted structural images, corrections for susceptibility-induced distortions, eddy currents and movements of the head are performed using TOPUP and EDDY (part of FSL)52,59–61 (see Figure 1B).\n\nResting-state functional data. For the functional resting state data, FEAT (Version 6.0062) will be used to run motion correction of the functional data using MCFLIRT63, distortion correction using FUGUE, brain extraction using BET64 and grand mean scaling62. The following images will be prepared for input into FEAT: (1) reference image for motion correction of the functional data and (2) fieldmap and fieldmap magnitude images. A single-band reference (SBref, acquired at the start of the functional scan) will be bias corrected using FAST and then used as an alternative reference image for motion correction. The fieldmap magnitude image will initially have non-brain matter removed using BET and will then be manually edited (see Figure 3). Subsequently, the fsl_prepare_fieldmap function will be used to create a fieldmap image (for an example see Figure 3). FEAT will also register the SBref to the T1 weighted structural image using FMRIB’s linear image registration tool (FLIRT, rigid-body and boundary-based registration)63,65,66. FLIRT and FSL’s non-linear image registration tool (FNIRT) are then used to register each participant’s structural T1 weighted image to a Paediatric Template image54 (see Figure 4 for example of Paediatric Template). MELODIC (model-free fMRI analysis using probabilistic independent component analysis) will decompose functional data into spatially independent components67, which will subsequently automatically be labelled as signal (i.e. not movement) or noise (i.e. motion or physiological artefact) using ICA-AROMA (Automatic Removal of Motion Artifacts68). ICA components that depict physiological noise or movement are automatically removed (see Figure 5 and Figure 6). Lastly the data is high pass temporal filtered at 0.01 Hz (100 s period).\n\n(A) Images show the location of a chosen voxel (green crosshair), within the left inferior frontal gyrus (pars triangularis). (B) Time series plots showing the change in BOLD signal within the specified voxel for raw data (grey line) and final denoised and temporal filtered data (blue line). All time series are demeaned. Units of change in BOLD are arbitrary scanner units. (C) Plot showing the change in head position (left-right) in millimetres. Changes in head position align with large changes in BOLD signal, which are successfully minimised through denoising (via the use of ICA-AROMA).\n\nUsing MELODIC67, each individual participant’s 4 dimensional fMRI data is decomposed in to independent spatial and temporal components. ICA-AROMA is then used to (i) automatically identify “movement” components and (ii) remove them from the data68. (A) Examples of three separate components labelled as “movement” and subsequently removed from the data. (B) Examples of three separate components that were not labelled as movement and therefore remain within the data.\n\nWe will compare the brain structural (mean fractional anisotrophy (FA) from DWI-derived tracts of interest) and functional (mean correlation) connectivity in specific networks involved in language and executive control across our three study groups (i.e. simultaneous bilingual, sequential bilingual and monolingual). We will investigate the specificity of differences by also comparing connectivity indices within two networks not hypothesized to be influenced by bilingualism (visual and motor).\n\nTractography reconstruction. Diffusion data will be prepared for tractography analysis by estimating fibre orientations using multi-shell, multi-tissue, constrained spherical deconvolution69–71. Fibre orientation distributions will then be used to calculate mean FA per voxel, as well as run anatomically constrained tractography (ACT), generating streamlines for each tract of interest72. The mean FA per tract (weighted by the number of streamlines in each voxel) will then be extracted per participant. The five tracts of interest are two pathways involved in language, one involved in executive control and two control pathways: (a) arcuate fasciculus (dorsal language pathway), (b) inferior fronto-occipital fasciculus (ventral language pathway), (c) fronto-parietal tract (d) optic radiation tract (e) corticospinal tract.\n\nResting-state network connectivity. In order to investigate functional connectivity, we will extract mean blood oxygen level dependent (BOLD) responses from regions of interest (ROI) in selected resting-state networks. The BOLD responses (i.e. measurements of changes in BOLD signal collected every 1.24 s for the duration of the functional scan) will be calculated from each pre-defined region of interest (ROI), detailed below. Measurements are collected per voxel and will be averaged across the ROI to create a mean BOLD response across time. Each mean response will be correlated with those within the same network to produce a network-specific correlation matrix per participant. The bilateral ROIs for each network will be as follows:\n\n(a) language network: the IFG and posterior STG\n\n(b) executive control networks:\n\n(i) FCN: middle frontal gyrus (part of the dorsolateral prefrontal cortex) and inferior/superior parietal lobule,\n\n(ii) SLN: anterior insula and anterior cingulate gyrus,\n\n(iii) DMN: posterior cingulate gyrus, vmPFC, and inferior parietal lobule/angular gyrus,\n\n(iv) subcortical control regions: putamen and caudate,\n\n(c) visual network: primary visual cortex and lateral geniculate nucleus in the thalamus\n\n(d) motor network: the bilateral precentral gyrus (primary motor cortex region) and ventral lateral thalamic nucleus for functional connectivity.\n\nWe will use the following statistical methods to test our hypotheses.\n\n(1) To test whether early bilingualism will affect connectivity in EC networks, we will use graph analysis to calculate the global network efficiency within EC networks and compare this across the three groups.\n\n(2) To test whether age of exposure to two languages influences the mean functional connectivity within language and EC-related networks, we will calculate the average of all absolute connectivity values across all the edges within each network and compare this between the three groups using analyses of covariance.\n\n(3) To test whether age of exposure to two languages, Greek language usage and proficiency influences connectivity within language and EC-related networks we will compare the two bilingual groups on both behavioural measures of attention and of brain connectivity (i.e. mean FA and mean functional connectivity) within EC networks. We will use general linear models to examine how much unique variance in connectivity indices is explained by age of exposure to two languages, Greek language usage, and proficiency.\n\nAll analyses will include measures of non-verbal ability (CPM scores), working memory (scaled score for total forward and backward digit span) and age at MRI as covariates, where appropriate.\n\nAll software that will be used for analysis of the data is open access and therefore freely available online.\n\n\nPlans for dissemination\n\nProject findings will initially be disseminated as an open access preprint publication. This will be followed by publication as an original research article in a peer-reviewed journal. We will also share key findings and their implications with educators, policy makers and the wider public. Finally, dissemination to non-academic audiences will be done via public engagement events to schools and parents’ networks through UCL BiLingo (Dr Froso Argyri and Prof Li Wei are Co-founders of this service).\n\nPermission is requested from each parent/guardian in order to make their child’s anonymised data available online. Where open MRI data is a requirement for publication we will make consented, anonymised data available at that time, otherwise we aim to publish the anonymised MRI imaging data (for which permission was obtained from parents, including for images used in this protocol) on OpenNeuro at the end of the project. MRI data will be organised using the Brain Imaging Data Structure (BIDS) framework.\n\n\nStudy status\n\nThis project is ongoing and we are actively recruiting. Brain imaging and behavioural data has currently been collected from 48 participants.\n\n\nConclusion\n\nThis study will quantify how bilingualism and maturational factors impact executive control and brain connectivity in the child brain. We will examine the effect of bilingualism on both domain specific (language) and domain general (EC) networks. As a result, our findings will shed light on the early effects of enriched linguistic environment on brain maturation.\n\n\nData availability\n\nNo data is associated with this article.\n\nCustom written scripts that will be used to analyse each set of data are available: https://github.com/sgoksan/paed_mri_preprocessing\n\nArchived scripts as at time of publication: http://doi.org/10.5281/zenodo.377881150\n\nLicense: MIT License", "appendix": "Acknowledgements\n\nWe would like to thank Mrs Tina Banks and her team of research radiographers at Great Ormond Street Hospital for providing incredible support and expertise to the project. We also thank Professor Chris Clark for providing integral support and access to the MRI scanner. Finally, we are grateful to all the participants and parents who have volunteered and taken part in this research study. All research at Great Ormond Street Hospital NHS Foundation Trust and the UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre.\n\n\nReferences\n\nNeville H, Bavelier D: Human brain plasticity: evidence from sensory deprivation and altered language experience. Prog Brain Res. 2002; 138: 177–88. PubMed Abstract \| Publisher Full Text\n\nJohnson MH: Interactive specialization: a domain-general framework for human functional brain development? Dev Cogn Neurosci. 2011; 1(1): 7–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBialystok E: The bilingual adaptation: How minds accommodate experience. Psychol Bull. 2017; 143(3): 233–262. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "63542", "date": "27 May 2020", "name": "Christos Pliatsikas", "expertise": [ "Reviewer Expertise Bilingualism", "experience-dependent neuroplasticity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 present the research protocol of a well thought through and designed ongoing study on the effects of bilingualism on the brain structure and function of children. This study fills an important gap in the young but expanding literature on the effects of bilingualism on cognition and the brain, by expanding it to a severely under-researched age-range (young children)- crucially, the study is administering comprehensive behavioural and MRI batteries which will help uncover whether and how brain differences between bilingual and monolingual children translate to differences in behaviour.\n\nI found the rationale and objectives of the study clearly described, and the study design is very appropriate, although the use of the entire behavioural battery could have also been addressed with appropriate hypotheses, in order to further strengthen the study. In terms of methods, all three main testing components (demographic and language backgrounds, behavioural experiments tapping language and executive functions and the MRI battery), are appropriate, contemporary, and described in full detail allowing for replication of the study. The adaptations of the MRI data analyses to make them appropriate for a paediatric sample is of particular importance here. Finally, with respect to data analysis, it is not clear to me whether the authors were planning to look at grey matter measures (esp. since their T1 images have sufficient resolution) with methods included in FSL, such as whole-brain VBM and subcortical vertex analysis (FIRST). I would strongly advise that, as it will result in a much needed multi-modal investigation of the developing bilingual brain (e.g. see https://psyarxiv.com/kjq6m for a preprint of our recent work looking at both grey and white matter structure of the developing brain, but not brain function at rest (Pliatsikas et al., 20201). In all, this is a well-designed study and its outputs will be more than welcome in the field.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5597", "date": "16 Jun 2020", "name": "Sezgi Goksan", "role": "Author Response", "response": "Dr Christos Pliatsikas, On behalf of the authors of this manuscript, we would like to thank you for your invaluable feedback on our project. Unfortunately, due to the coronavirus pandemic, all staff employed on this project grant are being placed on furlough. Upon our return, we endeavour to respond to your comments and we intend to provide an updated version of the manuscript alongside our response. We hope you can forgive the delay and we look forward to continuing this discussion. Kind regards, Dr Sezgi Goksan." }, { "c_id": "6066", "date": "04 Nov 2020", "name": "Sezgi Goksan", "role": "Author Response", "response": "Dear Dr Christos Pliatsikas, Thank you for taking the time to review our manuscript and for your valuable feedback on our project. We would like to address two of your comments: “I found the rationale and objectives of the study clearly described, and the study design is very appropriate, although the use of the entire behavioural battery could have also been addressed with appropriate hypotheses, in order to further strengthen the study.” With regards to reporting specific hypotheses related to our executive control tests, given the contradictory evidence regarding a ‘bilingual advantage’ in executive control in adult studies (for example see (1,2) for reviews in favour and (3-5) for reviews arguing against), and the few studies investigating EC in childhood (see reviews from (6,7)), there is insufficient evidence for us to formulate a specific hypothesis. Instead, we intend to explore the behavioural data alongside the brain imaging data. In doing so, we hope to meaningfully contribute this debate. Furthermore, for our background measures, namely the tests of non-verbal fluid intelligence and verbal working memory, we have included new sentences elaborating on the use of these measures in the current literature (see ‘Background measures’ within the Methodology section in our revised manuscript). Added text, Methodology section, Background measures: “Fluid intelligence, working memory and controlled attention are related yet separable constructs (8-10). We have therefore chosen to collect measures of non-verbal fluid intelligence and verbal working memory as background variables, as in previous studies that have investigated executive control skills in bilingual children and adults (11-15). Based on Engle et al.’s theory, individual differences in working memory capacity and general intelligence will impact a child’s innate ability to control their attention (9). Moreover, it has been observed that greater resting-state connectivity of the precuneus/posterior cingulate with other regions of the default mode network, is positively correlated with working memory performance in adults (16). Therefore, we aim to account for individual variability related to measures of executive control by including scores for working memory and general intelligence as confounding factors in our connectivity analyses.” “with respect to data analysis, it is not clear to me whether the authors were planning to look at grey matter measures”. In this protocol we have chosen to focus specifically on measures of structural and functional connectivity in children, as this is the novel aspect of our study. We have therefore not described plans within for analysis of the cortical structures. However, given that we are collecting high-resolution structural images, this is certainly an avenue that can be explored further in the future as a complementary analysis project in which we would aim to replicate and extend results in the existing published literature (17,18). We are committed to sharing further analysis plans via additional scripts for the pre-processing and analysis of structural data on our github page: https://github.com/sgoksan/paed_mri_preprocessing References 1. Adesope OO, Lavin T, Thompson T, Ungerleider C. A Systematic Review and Meta-Analysis of the Cognitive Correlates of Bilingualism:. Review of Educational Research. [Online] SAGE PublicationsSage CA: Los Angeles, CA; 2010;80(2): 207–245. Available from: doi:10.3102/0034654310368803 2. Bialystok E. Reshaping the mind: the benefits of bilingualism. Canadian journal of experimental psychology = Revue canadienne de psychologie experimentale. [Online] 2011;65(4): 229–235. Available from: doi:10.1037/a0025406 3. Hilchey MD, Klein RM. Are there bilingual advantages on nonlinguistic interference tasks? Implications for the plasticity of executive control processes. Psychonomic Bulletin & Review. [Online] Springer-Verlag; 2011;18(4): 625–658. Available from: doi:10.3758/s13423-011-0116-7 4. Lehtonen M, Soveri A, Laine A, Järvenpää J, de Bruin A, Antfolk J. Is bilingualism associated with enhanced executive functioning in adults? A meta-analytic review. Psychological bulletin. [Online] 2018;144(4): 394–425. Available from: doi:10.1037/bul0000142 5. Donnelly S, Brooks PJ, Homer BD. Is there a bilingual advantage on interference-control tasks? A multiverse meta-analysis of global reaction time and interference cost. Psychonomic Bulletin & Review. [Online] Springer US; 2019;26(4): 1122–1147. Available from: doi:10.3758/s13423-019-01567-z 6. Barac R, Bialystok E, Castro DC, Sanchez M. The Cognitive Development of Young Dual Language Learners: A Critical Review. Early childhood research quarterly. [Online] 2014;29(4): 699–714. Available from: doi:10.1016/j.ecresq.2014.02.003 7. Ware AT, Kirkovski M, Lum JAG. Meta-Analysis Reveals a Bilingual Advantage That Is Dependent on Task and Age. Frontiers in Psychology. [Online] Frontiers Media SA; 2020;11: 2076. Available from: doi:10.3389/fpsyg.2020.01458 8. Baddeley AD, Hitch G. Working Memory. Psychology of Learning and Motivation. [Online] Academic Press; 1974;8: 47–89. Available from: doi:10.1016/S0079-7421(08)60452-1 9. Engle RW, Kane MJ, Tuholski SW. Individual differences in working memory capacity and what they tell us about controlled attention, general fluid intelligence, and functions of the prefrontal cortex. 1999. Available from: doi:10.1017/CBO9781139174909.007 10. Miyake A, Friedman NP. The Nature and Organization of Individual Differences in Executive Functions: Four General Conclusions. Current Directions in Psychological Science. [Online] SAGE PublicationsSage CA: Los Angeles, CA; 2012;21(1): 8–14. Available from: doi:10.1177/0963721411429458 11. Namazi M, Thordardottir E. A working memory, not bilingual advantage, in controlled attention. International Journal of Bilingual Education and Bilingualism. [Online] 3rd ed. Taylor & Francis Group; 2010;13(5): 597–616. Available from: doi:10.1080/13670050.2010.488288 12. Bonifacci P, Giombini L, Bellocchi S, Contento S. Speed of processing, anticipation, inhibition and working memory in bilinguals. Developmental science. [Online] John Wiley & Sons, Ltd; 2011;14(2): 256–269. Available from: doi:10.1111/j.1467-7687.2010.00974.x 13. Engel de Abreu PMJ. Working memory in multilingual children: is there a bilingual effect? Memory (Hove, England). [Online] 2nd ed. Taylor & Francis Group; 2011;19(5): 529–537. Available from: doi:10.1080/09658211.2011.590504 14. D'Souza AA, Moradzadeh L, Wiseheart M. Musical training, bilingualism, and executive function: working memory and inhibitory control. Cognitive research: principles and implications. [Online] 2nd ed. SpringerOpen; 2018;3(1): 11–18. Available from: doi:10.1186/s41235-018-0095-6 15. Antón E, Carreiras M, Duñabeitia JA. The impact of bilingualism on executive functions and working memory in young adults. Athanasopoulos P (ed.) Public Library of Science ONE. [Online] Public Library of Science; 2019;14(2): e0206770. Available from: doi:10.1371/journal.pone.0206770 16. Sala-Llonch R, Peña-Gómez C, Arenaza-Urquijo EM, Vidal-Piñeiro D, Bargalló N, Junqué C, et al. Brain connectivity during resting state and subsequent working memory task predicts behavioural performance. Cortex; a journal devoted to the study of the nervous system and behavior. [Online] 2012;48(9): 1187–1196. Available from: doi:10.1016/j.cortex.2011.07.006 17. Archila-Suerte P, Woods EA, Chiarello C, Hernandez AE. Neuroanatomical profiles of bilingual children. Developmental science. [Online] 2018;21(5): e12654. Available from: doi:10.1111/desc.12654 18. Thieba C, Long X, Dewey D, Lebel C. Young children in different linguistic environments: A multimodal neuroimaging study of the inferior frontal gyrus. Brain and cognition. [Online] 2019;134: 71–79. Available from: doi:10.1016/j.bandc.2018.05.009" } ] }, { "id": "63537", "date": "29 May 2020", "name": "Manuel Blesa", "expertise": [ "Reviewer Expertise Neonatal imaging", "diffusion MRI", "structural connectome", "functional MRI", "machine learning and statistical modelling." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 the presented study is very complete and it will provide meaningful information about how bilingualism alters brain connectivity in early brain development. The presented analysis plan is appropriate to answer the questions they are addressing. The scripts (already uploaded to GitHub) will be an important resource for anyone interested to work with these (or similar) datasets, and the availability of the datasets would be an important contribution to the community, as it will be useful for other researchers investigating the mechanisms that change with bilingualism during early brain development.\n\nThe study is very well designed, however, I have some questions/suggestions regarding the processing of the data and the statistical analysis. The FA is generally a good biomarker for brain development, however, it is very difficult to interpret.1 As the acquired multi-shell diffusion data permits it, did the authors consider calculating also NODDI maps?2 In addition, did the authors consider adding some preprocessing steps like denoising or removal of Gibbs rings artifacts to the diffusion pipeline?3,4\nFinally, the authors could provide more details on how they plan to control for confounding effects. One important confounder to take into account is gestational age at birth: in fact, some of the children were born between 33 and 37 weeks, and are therefore late preterm. It is well known that preterm babies have small differences in brain structure compared with term-born babies at term equivalent age, and this should be accounted for.5 Then they mention that socioeconomic status will be measured on the basis of maternal education, but they don’t specify how this variable will be used in their statistical models. Another potential confounder worth considering is brain size, as it correlates to general intelligence6 and it has a known effect on functional connectivity estimates (see e.g. Hänggi et al.7). Finally, there is a large literature on sex differences in language proficiency in childhood and brain development (see Etchell et al.8 for a systematic review). Although the authors report that there were no significant sex differences measured in normative samples for some of the scores they adopt, it would be worth clarifying the reasons for excluding this factor from the analysis.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5598", "date": "16 Jun 2020", "name": "Sezgi Goksan", "role": "Author Response", "response": "Dr Manuel Blesa Cábez and Dr Paola Galdi, On behalf of the authors of this manuscript, we would like to thank you both for your invaluable feedback on our project and in particular for your comments and suggestions regarding our preprocessing and statistical analysis plan. Unfortunately, due to the coronavirus pandemic, all staff employed on this project grant are being placed on furlough. Upon our return, we endeavour to respond to your comments and we intend to provide an updated version of the manuscript alongside our response. We hope you can forgive the delay and we look forward to continuing this discussion. Kind regards,Dr Sezgi Goksan" }, { "c_id": "6067", "date": "04 Nov 2020", "name": "Sezgi Goksan", "role": "Author Response", "response": "Dear Dr Manuel Blesa Cábez and Dr Paola Galdi, Thank you for taking the time to review our manuscript. We would like to thank you both for your valuable feedback on our project and specifically, for your comments and suggestions with regards to our pre-processing and statistical analysis plan. We would like to address three points: “The FA is generally a good biomarker for brain development, however, it is very difficult to interpret.1 As the acquired multi-shell diffusion data permits it, did the authors consider calculating also NODDI maps?2” Thank you for suggesting this additional measure. While the calculation of NODDI maps may provide complementary information regarding the underlying neurite morphology, at present we do not have any specific hypotheses with regards to how intracellular or extracellular neurite volumes would change in the bilingual brain. Similarly, we also do not report any specific hypothesis related to mean diffusivity or radial diffusivity. Given this, we have carefully considered all aforementioned measures and chosen not to include them in our statistical analysis plan. “did the authors consider adding some pre-processing steps like denoising or removal of Gibbs rings artifacts to the diffusion pipeline?” In order to address potential participant movement artifacts within our data, we run the latest version of Eddy (a tool within FSL), which simultaneously corrects for eddy currents and gross subject movement (1-3), thus providing some denoising our data. Additional steps such as noise removal and Gibbs ringing removal are reported to be beneficial when applied prior to running EDDY (4). While such artefacts have, thus far, not been observed in the visual inspection of our diffusion data, we agree that it is important that we assess the data when finalised and investigate whether further denoising and Gibbs removal improve the quality of the pre-processing. We have acknowledged this by adding new sentences outlining this intention to our revised manuscript (see ‘Diffusion weighted data’ within the MRI data analysis section). Moreover, we are committed to updating our publicly available scripts in future in order to reflect the final pre-processing pipeline. Added text, MRI data analysis section, Diffusion weighted data: “In addition, given our paediatric cohort, the final set of diffusion images will be carefully assessed to establish whether further denoising and removal of Gibbs ringing artefacts are required. These steps have been recommended for use in adult studies (4), and are available steps within MRTrix3 (5,6).” “provide more details on how they plan to control for confounding effects” For this point we have specifically addressed each of the confounding variables that have been highlighted by the reviewers. Namely, (i) gestational age at birth, (ii) socio-economic status, (iii) brain size and the relationship with general intelligence and (iv) gender. Firstly, we confirm that we are recording this information from each of our participants. We agree that the influence of these variables on our brain imaging data should be carefully considered and our aim is to balance these variables as best we can within each of our groups. Furthermore, we intend to examine the relationships between all of these measures in order to make an informed decision about whether to include each variable in our analysis of the brain imaging data. Gestational age: we collect each child’s gestational age at birth via verbal report from the parent(s). At present none of our participants were born preterm, however, as mentioned we are including all children born > 33 weeks gestational age (GA) in our study. The majority of longitudinal studies investigating brain-derived measures and cognitive outcomes in preterm-born children have focussed on children born < 33 weeks GA (for example (7-10)). More recently there has been evidence suggesting that children born late preterm (i.e. 33-36 weeks GA) have higher risk of developing poor cognitive outcomes compared to their term-born peers (11). In addition, as you mention, Thompson and colleagues reported that children born late preterm had (i) larger CSF volumes and (ii) lower FA when compared to a group of term-born infants (12). However, it is not known whether such differences persist later in childhood in late preterm-born children. Therefore, we aim to assess whether the data from any preterm-born children requires individual consideration when running our final analysis. If so, we will include gestational age at birth as a confound in our analysis. Socio-economic status (SES): SES impacts neurocognitive development, thereby influencing executive functions such as attention skills as well as measures of brain structure and function (13-16). In order to compare our groups fairly, we intend to match our groups for SES. As part of the Alberta Language Environment Questionnaire (17), number of years of maternal education is recorded as a measure of SES for our participants (see details of Parental Questionnaire in Methodology section). We have not specified any criteria related to SES as part of our recruitment, however in order to recruit groups from similar backgrounds we are signposting throughout our university, at local Greek schools (that provide classes at the weekend) and through inviting children and parents to mention our study to other friends at their schools. Through this approach we hope to minimise differences in SES between groups. Brain size and general intelligence: Brain size is an important factor to consider as it is related to general intelligence and brain-derived measures. This relationship has also been reported in children (18). One of our background measures, the outcome score from a Coloured Progressive Matrices (CPM) task, is included as a measure of general intelligence. We are also able to calculate a measure of brain size by extracting total cortical volume from each participant’s structural scan. This will allow us to examine the relationship between these two variables in our sample. We will include CPM scores or total cortical volume as a confound measure in our analyses of structural and functional connectivity in the brain. As such we intend to control for effects associated with general intelligence or brain size. Gender: Etchell et al., argue that sex differences may impact brain development and cognitive outcomes (19). Therefore, it is our intention to balance our groups for this factor. This is an additional goal of our current, ongoing recruitment and we have thus far been able to recruit relatively gender-balanced groups. Moreover, we will examine gender-related effects and use gender as a covariate where appropriate. Added text (bold), Statistical plan section: “We will examine our data for effects related to gender, socio-economic status and gestational age at birth. We will subsequently make an informed decision regarding whether to include these factors as covariates in our connectivity analyses. Moreover, all analyses will include measures of brain size (total cortical volume) or non-verbal ability (CPM scores), working memory (scaled score for total forward and backward digit span) and age at MRI as covariates, where appropriate.” References: 1. Andersson JLR, Sotiropoulos SN. An integrated approach to correction for off-resonance effects and subject movement in diffusion MR imaging. NeuroImage. 2016;125: 1063–1078. 2. Andersson JLR, Graham MS, Zsoldos E, Sotiropoulos SN. Incorporating outlier detection and replacement into a non-parametric framework for movement and distortion correction of diffusion MR images. NeuroImage. [Online] 2016;141: 556–572. Available from: doi:10.1016/j.neuroimage.2016.06.058 3. Graham MS, Drobnjak I, Zhang H. Realistic simulation of artefacts in diffusion MRI for validating post-processing correction techniques. NeuroImage. [Online] 2016;125: 1079–1094. Available from: doi:10.1016/j.neuroimage.2015.11.006 4. Maximov II, Alnaes D, Westlye LT. Towards an optimised processing pipeline for diffusion magnetic resonance imaging data: Effects of artefact corrections on diffusion metrics and their age associations in UK Biobank. Human Brain Mapping. [Online] 2019;40(14): 4146–4162. Available from: doi:10.1002/hbm.24691 5. Cordero-Grande L, Christiaens D, Hutter J, Price AN, Hajnal JV. Complex diffusion-weighted image estimation via matrix recovery under general noise models. NeuroImage. [Online] 2019;200: 391–404. Available from: doi:10.1016/j.neuroimage.2019.06.039 6. Kellner E, Dhital B, Kiselev VG, Reisert M. Gibbs-ringing artifact removal based on local subvoxel-shifts. Magnetic Resonance in Medicine. [Online] 2016;76(5): 1574–1581. Available from: doi:10.1002/mrm.26054 7. Johnson S. Cognitive and behavioural outcomes following very preterm birth. Seminars in Fetal & Neonatal Medicine. [Online] W.B. Saunders; 2007;12(5): 363–373. Available from: doi:10.1016/j.siny.2007.05.004 8. Moore T, Hennessy EM, Myles J, Johnson SJ, Draper ES, Costeloe KL, et al. Neurological and developmental outcome in extremely preterm children born in England in 1995 and 2006: the EPICure studies. BMJ. [Online] British Medical Journal Publishing Group; 2012;345(dec04 3): e7961–e7961. Available from: doi:10.1136/bmj.e7961 9. Ancel P-Y, Goffinet F, Group ATE-2W, Kuhn P, Langer B, Matis J, et al. Survival and Morbidity of Preterm Children Born at 22 Through 34 Weeks’ Gestation in France in 2011: Results of the EPIPAGE-2 Cohort Study. JAMA Pediatrics. [Online] American Medical Association; 2015;169(3): 230–238. Available from: doi:10.1001/jamapediatrics.2014.3351 10. Linsell L, Johnson S, Wolke D, O’Reilly H, Morris JK, Kurinczuk JJ, et al. Cognitive trajectories from infancy to early adulthood following birth before 26 weeks of gestation: a prospective, population-based cohort study. Archives of Disease in Childhood. [Online] BMJ Publishing Group Ltd; 2018;103(4): 363–370. Available from: doi:10.1136/archdischild-2017-313414 11. Woythaler M. Neurodevelopmental outcomes of the late preterm infant. Seminars in Fetal & Neonatal Medicine. [Online] W.B. Saunders; 2019;24(1): 54–59. Available from: doi:10.1016/j.siny.2018.10.002 12. Thompson DK, Kelly CE, Chen J, Beare R, Alexander B, Seal ML, et al. Characterisation of brain volume and microstructure at term-equivalent age in infants born across the gestational age spectrum. NeuroImage. Clinical. [Online] Elsevier; 2019;21: 101630. Available from: doi:10.1016/j.nicl.2018.101630 13. Hackman DA, Farah MJ, Meaney MJ. Socioeconomic status and the brain: mechanistic insights from human and animal research. Nature Reviews Neuroscience. [Online] Nature Publishing Group; 2010;11(9): 651–659. Available from: doi:10.1038/nrn2897 14. Hackman DA, Gallop R, Evans GW, Farah MJ. Socioeconomic status and executive function: developmental trajectories and mediation. Developmental science. [Online] 2nd ed. John Wiley & Sons, Ltd; 2015;18(5): 686–702. Available from: doi:10.1111/desc.12246 15. Farah MJ. The Neuroscience of Socioeconomic Status: Correlates, Causes, and Consequences. Neuron. [Online] 2017;96(1): 56–71. Available from: doi:10.1016/j.neuron.2017.08.034 16. Lawson GM, Hook CJ, Farah MJ. A meta-analysis of the relationship between socioeconomic status and executive function performance among children. Developmental science. [Online] 2018;21(2). Available from: doi:10.1111/desc.12529 17. Paradis J. Individual differences in child English second language acquisition: Comparing child-internal and child-external factors. Hulk A, Marinis T (eds.) Linguistic Approaches to Bilingualism. [Online] John Benjamins; 2011;1(3): 213–237. Available from: doi:10.1075/lab.1.3.01par 18. Thompson DK, Matthews LG, Alexander B, Lee KJ, Kelly CE, Adamson CL, et al. Tracking regional brain growth up to age 13 in children born term and very preterm. Nature communications. [Online] Nature Publishing Group; 2020;11(1): 696–11. Available from: doi:10.1038/s41467-020-14334-9 19. Etchell A, Adhikari A, Weinberg LS, Choo AL, Garnett EO, Chow HM, et al. A systematic literature review of sex differences in childhood language and brain development. Neuropsychologia. [Online] 2018;114: 19–31. Available from: doi:10.1016/j.neuropsychologia.2018.04.011" } ] } ]	1	https://f1000research.com/articles/9-370
https://f1000research.com/articles/9-1296/v1	03 Nov 20	{ "type": "Data Note", "title": "The EU one-stop-shop collection of publicly available information on COVID-19 in vitro diagnostic medical devices", "authors": [ "Mauro Petrillo", "Maddalena Querci", "Olga Tkachenko", "Ioana-Raluca Siska", "Enrico Ben", "Alexandre Angers-Loustau", "Alessia Bogni", "Antonino Brunetto", "Marco Fabbri", "Linda Garlant", "Antoon Lievens", "Amalia Munoz", "Valentina Paracchini", "Danilo Pietretti", "Antonio Puertas-Gallardo", "Barbara Raffael", "Eleonora Sarno", "Virginie Tregoat", "Fabrizio Zaro", "Guy Van den Eede", "Maddalena Querci", "Olga Tkachenko", "Ioana-Raluca Siska", "Enrico Ben", "Alexandre Angers-Loustau", "Alessia Bogni", "Antonino Brunetto", "Marco Fabbri", "Linda Garlant", "Antoon Lievens", "Amalia Munoz", "Valentina Paracchini", "Danilo Pietretti", "Antonio Puertas-Gallardo", "Barbara Raffael", "Eleonora Sarno", "Virginie Tregoat", "Fabrizio Zaro", "Guy Van den Eede" ], "abstract": "The JRC COVID-19 In Vitro Diagnostic Devices and Test Methods Database, aimed to collect in a single place all publicly available information on performance of CE-marked in vitro diagnostic medical devices (IVDs) as well as in house laboratory-developed devices and related test methods for COVID-19, is here presented. The database, manually curated and regularly updated, has been developed as a follow-up to the Communication from the European Commission “Guidelines on in vitro diagnostic tests and their performance” of 15 April 2020 and is freely accessible at https://covid-19-diagnostics.jrc.ec.europa.eu/.", "keywords": [ "In-vitro diagnostics", "covid-19", "sars-cov-2", "detection method" ], "content": "Introduction\n\nThe Communication from the Commission “Guidelines on in vitro diagnostic tests and their performance”1, published on 15 April 2020, states the following under the Further Actions Needed section: “The Commission, supported by the ECDC, health technology assessment experts and in vitro diagnostics competent authorities, will assist Member States with a centralised overview of available information on test performance and act as a single point of contact for management of this information. Taking stock of the state of the art on a regular basis will support Member States’ informed decisions on national testing strategies, as well as support the continuous development of devices by manufacturers.”\n\nAs an initial step in collecting performance information of devices and in house methods, to address the above need, European Commission services (Directorate-General for Health and Food Safety [DG SANTE], Directorate-General Joint Research Centre [DG JRC], Directorate-General for Research and Innovation [DG RTD]), together with the European Centre for Disease Prevention and Control (ECDC), several experts from in vitro diagnostics competent authorities and from the European Network for Health Technology Assessment (EUnetHTA)1, published the working document \"Current performance of COVID-19 test methods and devices and proposed performance criteria\"2 on 16 April 2020.\n\nThe JRC capitalised on its expertise in knowledge management to conduct the literature review as part of this work and, as a follow-up action to the need identified in the Communication, committed to make the information broadly accessible and to update the compilation as new data become available.\n\nThe outcome of these actions is the JRC COVID-19 In Vitro Diagnostic Devices and Test Methods Database presented here, a single place collection of all publicly available information on performance of CE-marked in vitro diagnostic medical devices (IVDs) as well as in house laboratory-developed devices and related test methods for COVID-19. The database is freely accessible at https://covid-19-diagnostics.jrc.ec.europa.eu/.\n\n\nMethods\n\nThe initial information is gathered following the strategy for documentation on test methods and devices indicated in Section 3 \"Methodology used\" of the Current performance of COVID-19 test methods and devices and proposed performance criteria - Working document of Commission services2.\n\nThe retrieved information is manually verified and curated. In particular:\n\n• For CE-marked devices, only the information that the manufacturer has chosen to make publicly available is included in the database. Full information on the manufacturer’s performance evaluation of the device is contained in the technical documentation required by EU legislation3. As manufacturer technical documentation is usually not publicly available and is therefore not included, a form is available to stakeholders to update and integrate the information: manufacturers are invited to submit performance information on new devices, which are not yet listed in the database, or to provide data not available to the authors at the time of the last update. The submitted information, once verified against the source provider, is taken into consideration for updating the database.\n\n• Performance details (as retrieved from manufacturers’ web pages) are provided only for devices commercially available with CE-IVD mark. Products labelled as for ‘research use only’ or ‘under development’ as well as products fulfilling regulatory frameworks other than the one in place in the EU are listed for information only. The correctness of information, such as performance data of the listed devices, has not been confirmed by checking raw experimental data or full technical documentation of the manufacturer nor by own laboratory verification or by any clinical validation studies.\n\n• A team of JRC experts regularly takes care of updating the Scientific Literature section, which includes scientific articles reporting about the use and performances of devices and related test methods. This section regards also the so-called in house2 or laboratory developed devices, which are used in healthcare institutions but are not commercially available. These are not included in the list of devices, as they are not easily identifiable by name, but scientific publications on such devices and related test methods are included in the section.\n\n• Information on publicly available results of validation studies and on publications by national/regional health technology assessment bodies and Joint Action EUnetHTA4 is included. These reports are produced in response to requests from national authorities and support national policy and decision-making on testing for COVID-19.\n\nIn silico simulations of NAAT methods, on available high-quality and full length sequenced SARS-CoV-2 genomes (provided as collaboration with GISAID5) are performed by using ThermonucleotideBLAST6. The tool is run on each assembled genome with default parameters, except for the following ones: -e=30, -E=20, --max-gap=4, --max-mismatch=4. Putative not redundant target amplicons are then extracted to build the final target dataset that is added to the database.\n\nAt the time of writing, high-quality (i.e. <1% Ns and <0.05% unique amino acid mutations) and full length (i.e. >29,000 bp) sequenced SARS-CoV-2 genomes have been manually downloaded from the GISAID website. To simplify the sharing of information and the related analysis, a formal agreement is under finalisation between GISAID and JRC.\n\n\nResults\n\nThe database is structured in a way aimed to:\n\n• facilitate sharing of information among researchers, in line with the principle of FAIR sharing;\n\n• link the devices, and their features, to scientific articles that reported their use and performance, in order to have a clear, transparent and fully open data source where users can look at and make the right choice in the selection of the device or method to use;\n\n• verify and monitor if natural occurring SARS-CoV-2 mutations give rise to \"false negative\".\n\nData records are currently organised and available in three main sections:\n\n1. COVID-19 in vitro diagnostic medical devices - This section gives access to publicly available in vitro diagnostic medical devices for COVID-19 and is being updated periodically. Additional performance (as retrieved from manufacturers’ web pages) is provided only for devices or kits commercially available with CE-IVD mark.\n\nDirect link to this dataset page, where data can be downloaded in CSV format, is: https://covid-19-diagnostics.jrc.ec.europa.eu/devices.\n\n2. Scientific literature on COVID-19 test methods and devices - This section allows browsing on performance of test methods and devices for COVID-19 diagnostics retrieved from selected scientific articles and is being updated periodically.\n\nDirect link to this dataset page, where data can be downloaded in CSV format, is: https://covid-19-diagnostics.jrc.ec.europa.eu/literature.\n\n3. SARS-CoV-2 target regions - This section is an inventory of PCR-based nucleic acid amplification tests (NAATs) used by laboratories and in silico simulations of these assays on available high-quality and full length sequenced SARS-CoV-2 genomes (in collaboration with GISAID5). The latter are pre-computed values corresponding to the extent of matching of the primers and probes from NAAT database methods against high-quality, full length genomic sequences, which are made available by GISAID to enable this analysis.\n\nIn addition, the database has pointers to the EUnetHTA publications repository. EUnetHTA Joint Action 3 is the scientific and technical component of EU cooperation on HTA. It builds on years of long standing collaboration between HTA agencies, financially supported by the EU since early 2000. The current Joint Action (2016 – 2021) is co-funded by the EU Health Programme and includes government appointed organisations and a large number of relevant regional agencies and not-for-profit organisations that produce or contribute to HTA in Europe (i.e. 86 organisations from 26 Member States plus Norway, Serbia, Switzerland, Ukraine and United Kingdom). Responding to the needs of policy makers and public health authorities for researched, timely and reliable information, EUnetHTA has launched its COVID-19-related repository of publications and outputs. The repository gathers publications by EUnetHTA and by HTA organisations on testing methods and devices, but also on treatment options, and other public health measures relevant to COVID-19. Direct link to this page is: https://covid-19-diagnostics.jrc.ec.europa.eu/eunethta.\n\nFinally, a Submit your device section provides forms for manufacturers and NAAT developers to submit information on devices not yet listed in the database or to provide performance data not available to the authors at the time of the last update. The submitted information, once verified against the source provider, is taken into consideration for updating the database.\n\nAt the time of writing, the database includes 869 devices and 382 selected articles, easily linked to each other, as shown in Figure 1. Considering the rapidly evolving situation in relation to the development and commercialisation of diagnostic devices for COVID-19, the completeness of the information is limited to the time of the last update as indicated in the database for each individual item. For this reason, manufacturers are invited to submit and update the information through the corresponding \"Submit your device\" section's form.\n\nThis graph visualises the relations (grey lines) among devices (coloured according to the corresponding detection principle) and the scientific articles (black spots) in which they are mentioned. The large “corona” near the centre and the group near the top right corner are clustered around two documents that refer to 1729 and 2310 devices, respectively. It is quite evident that publications are specialised with respect to devices detection principle (i.e. the used technology): few publications (only three) mention different detection principles' devices. Please note that devices without references are not represented. The size of the symbol representing the device is proportional to the corresponding number of references (min 1, max 8).\n\nWith respect to NAAT methods, currently eight NAAT methods are available in the database: seven are from the World Health Organization (WHO) support to COVID-19 and are in-house PCR protocols assays posted online on the WHO website7 while one was developed by the JRC in the context of the production of EURM-019, a universal positive control material to be used in the testing of SARS-CoV-2 by RT-PCR (described in reference 8). The possibility to evaluate whether variations occurring overtime in the viral genomes can affect PCR-based detection methods is fundamental to guarantee reliability of the detection. The aims of the SARS-CoV-2 target regions section in the database are therefore both to detect potential target regions of the NAAT methods and to highlight possible differences with the expected reference sequence that might affect the performance of NAATs. As of 01 May 2020, about 10% of the retrieved genomes were found to be not detectable in silico by at least one of the eight NAAT methods, as shown in Figure 2. As an example, a case of variation potentially affecting NAAT methods detectability is shown in Figure 3.\n\nAs of 01 May 2020, more than 16,000 high quality (<1% Ns and < 0.05% unique amino acid mutations) and full length (> 29,000 bp) viral genomes were made available by GISAID5. By performing in silico PCR simulations with eight NAAT methods, the graph shows that about 10% of the 16,000 selected genomes (cumulative numbers) were found to be not detectable in silico by at least one of the eight NAAT methods. Blue area represents the (cumulative) number of genomes in silico detected by all eight NAAT methods; red area represents the (cumulative) number of those not detected by at least one NAAT method.\n\nAs of 01 May 2020, more than 16,000 high quality (<1% Ns and < 0.05% unique amino acid mutations) and full length (> 29,000 bp) viral genomes were made available by GISAID5. An example of identified genomic variations that can affect NAAT methods target detectability is here reported and it regards the first method developed by the US Centers for Disease Control and Prevention and supported by WHO in 7. 1) On the top, the target sequence (in red the left primer, in green right primer, and in azure the probe annealing region) that has been found in several genomes. With respect to the expected reference target, a variation (C/T transition, marked by *) is present at the beginning of the probe annealing region that can potentially affect the detection method performance. 2) Below, the table with details about the genomes where this variation of the target sequence has been found: the two columns with # indicate the number of genomes with such a sequence, aggregated by region and country, respectively. In the table, the dates of first and last appearance of the variation are reported for each country, in order to provide information on the \"spread\" of the variation.\n\nAs for devices, considering the rapidly evolving situation in relation to COVID-19, NAAT methods developers are invited to submit updated information or new developed ones through the corresponding \"Submit your device\" section's form.\n\n\nDiscussion\n\nAccording to our knowledge, it is the first time that a repository provides and links information on COVID-19 in vitro diagnostic medical devices and the scientific articles testing and reporting their performance.\n\nTo date, more than 100 requests, either for addition of new in vitro diagnostic medical devices or for updating the information, have been received through the forms complied by manufacturers, as demonstration of the strong need of having a structured data sharing point for this kind of information. However, it is important to highlight that this resource does NOT represent a list of devices approved or authorised for use either by the European Commission or by Member States’ national authorities. There is no central approval system for in vitro diagnostic medical devices in the EU. The currently applicable legislation for placing COVID-19 diagnostic devices on the market in the EU is Directive 98/79/EC on in vitro diagnostic medical devices3. Under the current Directive3, for COVID-19 devices that are designed for professional use, the manufacturer may affix the CE-mark to the product after having ensured the compliance of the device with the Directive and drawn up a declaration of conformity. For COVID-19 devices that are designed for use by lay persons (self-tests), the manufacturer must also apply to a third party body called a notified body that will do additional verification and issue a certificate. As a consequence, the JRC should not be deemed responsible for the validity of such data.\n\nAs recently reported by Guglielmi12, “even once a test is working beautifully in the lab, it still faces an arduous journey to mass usage. The first challenge is to verify performance, because quality can vary”. We believe that the here presented database contributes to the required prompt handling of the on-going public health COVID-19 situation. It makes sharing information easier and helps medical professionals, scientists and laboratories understand and properly assess COVID-19 medical devices quickly. The overview of the market may also help manufacturers develop and improve their own devices, in line with the current EU legislation.\n\n\nData availability\n\nThe database is freely accessible at https://covid-19-diagnostics.jrc.ec.europa.eu/. CSVs of the ‘COVID-19 in vitro diagnostic medical devices’ and ‘Scientific literature on COVID-19 test methods and devices’ databases at the time of publication have been archived on Zenodo repository (see below). However, the database is updated very frequently, and the latest data can be downloaded in these sections (described in Results) as CSVs.\n\nZenodo: JRC COVID-19 In Vitro Diagnostic Devices and Test Methods Database. http://doi.org/10.5281/zenodo.411760113.\n\nThis project contains the following underlying data:\n\n- covid-19-methods.csv (CSV of COVID-19 in vitro diagnostic medical devices database at time of publication)\n\n- covid-19-literature.csv (CSV of Scientific literature on COVID-19 test methods and devices database at time of publication)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nThe ‘SARS-Cov2 target regions’ database is enabled using third party data from GISAID, subject to GISAID’s Terms and Conditions. The database is freely accessible at https://covid-19-diagnostics.jrc.ec.europa.eu/amplicons and GISAID data are available directly from GISAID at https://www.gisaid.org/5. Access to GISAID data requires registration and agreement to the conditions for use at https://www.gisaid.org/registration/register/.\n\n\nCode availability\n\nCode used to perform scientific literature mining is freely available at https://github.com/ec-jrc/JRC_COV19_IVD-DEVs-TEMs_DB.\n\nArchived code at time of publication: https://doi.org/10.5281/zenodo.408460014.\n\nLicense: BSD-3-Clause", "appendix": "Acknowledgements\n\nWe gratefully acknowledge the Authors and the Originating laboratories where the clinical specimen or virus isolate was first obtained and the Submitting laboratories, where sequence data have been generated and submitted to GISAID, on which this research is based.\n\nWe acknowledge all the manufacturers and repository website owners of the devices that have been analysed; all the authors, publishers and literature repositories of the papers that have been analysed.\n\n\nFootnotes\n\n1 EUnetHTA (EUropean network for Health Technology Assessment) Joint Action 3 (2016 – 2021) is the scientific and technical component of EU cooperation on HTA. The Joint Action is co-funded by the EU Health Programme and includes government appointed organisations and a large number of relevant regional agencies and not-for-profit organisations that produce or contribute to HTA in Europe.\n\n2 The in house devices are those referred to in Art.1(5) of Directive 98/79/EC: “devices manufactured and used only within the same health institution and on the premises of their manufacture or used on premises in the immediate vicinity without having been transferred to another legal entity”. They are exempt from requirements of Directive 98/79/EC but may be subject to national law.\n\n3 From 26 May 2022, the Directive will be replaced by Regulation (EU)2017/746 on in vitro diagnostic medical devices11. The Regulation lays down a transitional period starting on the date of its entry into force (May-2017) during which the conformity of in vitro diagnostic medical devices can be assessed either under the Regulation or under the Directive. More information on the regulatory framework, together with a number of guidance documents, may be found on the Commission’s dedicated medical devices website https://ec.europa.eu/health/md_sector/overview_en.\n\n\nReferences\n\nCommunication from the Commission Guidelines on COVID-19 in vitro diagnostic tests and their performance 2020/C 122 I/01C/2020/2391. Reference Source\n\nCurrent performance of COVID-19 test methods and devices and proposed performance criteria - Working document of Commission services. Reference Source\n\nRegulation (EU) 2017/746 of the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical devices and repealing directive 98/79/EC and Commission Decision 2010/227/EU, OJ L 117, 5.5. 2017; 176–332. Reference Source\n\nSee https://eunethta.eu/covid-19-diagnostics/\n\nShu Y, McCauley J: GISAID: Global initiative on sharing all influenza data - from vision to reality. Euro Surveill. 2017; 22(13): 30494. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGans JD, Wolinsky M: Improved assay-dependent searching of nucleic acid sequence databases. Nucleic Acids Res. 2008; 36(12): e74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAt time of writing available at https://www.who.int/publications/m/item/molecular-assays-to-diagnose-covid-19-summary-table-of-available-protocols\n\nThe method is available at https://crm.jrc.ec.europa.eu/p/EURM-019\n\nGonzalez JM, Shelton JW, Diaz-Vallejo M, et al.: Immunological assays for SARS-CoV-2: an analysis of available commercial tests to measure antigen and antibodies. 2020. Publisher Full Text\n\nCheng MP, Papenburg J, Desjardins M, et al.: Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus 2: A Narrative Review. Ann Intern Med. 2020; 172(11): 726–734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDirective 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices, OJ L 331, 7.12. 1998; 1–37. Reference Source\n\nGuglielmi G: The explosion of new coronavirus tests that could help to end the pandemic. Nature. 2020; 583(7817): 506–509. PubMed Abstract \| Publisher Full Text\n\nPetrillo M: JRC COVID-19 In Vitro Diagnostic Devices and Test Methods Database (Version v1) [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4117601\n\nGallardo AP, gabbaan: ec-jrc/JRC_COV19_IVD-DEVs-TEMs_DB: First release of JRC_COVID-19_Literature_search (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.4084600" }	[ { "id": "75984", "date": "06 Jan 2021", "name": "Angelo Facchiano", "expertise": [ "Reviewer Expertise Bioinformatics", "biochemistry", "molecular biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presents a significant work aimed to collect publicly available information on test methods for COVID-19. Due to the amount of devices and methods available, the database described offers a useful tool to be addressed in the field. I find of special interest the example on genomic variations that can affect detectability. The manuscript is well presented and written. I find in the manuscript only minor points to be addressed:\nAs a reader, I would find in the article an example of the output obtained when the database is searched.\n\nI would not find explanation of abbreviations in the abstract if not really used there.\n\nabbreviations should be explained at the first appearance (check for NAAT; IVD - if removed from abstract, FAIR - explain or add a reference; and so on).\n\nunder keywords, check upper/lower case.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "75227", "date": "01 Feb 2021", "name": "Christopher Viljoen", "expertise": [ "Reviewer Expertise Human Molecular Biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Abstract would be strengthened with an introductory sentence regarding context to COVID-19 and the need for such information.\n\nThe Introduction needs a short contextualized introduction to the need for this type of information in a COVID-19 pandemic.\n\nThe Introduction could also indicate how the Commission has responded to the need for the information as indicated above.\n\nThe paragraph on COVID-19 mutations and test methods needs to include some context to the problem.\n\nThe Discussion - 1st paragraph - should indicate \"in vitro diagnostic medical devices and assays\".\n\nThe article would benefit from some concluding comments reiterating the importance of having access to the information presented in the database.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1296
https://f1000research.com/articles/9-1288/v1	30 Oct 20	{ "type": "Study Protocol", "title": "Long-term health effects of antipyretic drug use in the ageing population: protocol for a systematic review", "authors": [ "Seema Mahesh", "Esther van der Werf", "Mahesh Mallappa", "George Vithoulkas", "Nai Ming Lai", "Esther van der Werf", "Mahesh Mallappa", "George Vithoulkas", "Nai Ming Lai" ], "abstract": "Background: Fever is suppressed with drugs due to discomfort and risk of organ damage. However, there is some compelling evidence for the benefits of fever. The elderly are a special population in this regard as they have a blunted fever response. The benefit-harm balance of antipyretic use in this population is unclear.\n\nThis study aims to provide the synthesized best evidence regarding long-term health effects of antipyretic treatment in the elderly during infections, investigating the onset/worsening of common chronic diseases, for e.g., thyroid disorders, connective tissue diseases and chronic obstructive pulmonary disease/asthma. Methods: A systematic review will be performed to establish the best evidence available regarding antipyretic treatment in the elderly, searching databases such as Medline, Embase and Cochrane CENTRAL from their inception till date for all types of studies. Studies that consider the drugs in analgesic role will be excluded. The search will be reported following the ‘Preferred Reporting Items for Systematic Reviews and Meta-Analyses’ (PRISMA) guidelines. Randomized control trials, quasi experimental studies, observational studies, case series and reports will be included. The primary outcome measure being onset/worsening of chronic inflammatory diseases. Other outcomes include relief of symptoms, length of hospital stay, patient satisfaction, mortality, blood/immune parameters indicative of morbidity and complications of the infection. Risk of biases in randomized studies will be assessed through the Cochrane risk of bias tool. For other study types, appropriate tools such as CASP/QUIPS/Cochrane non-randomised studies tool will be used. Meta-analysis will be conducted on the Cochrane RevMan software and where pooling of data is not possible, a narrative synthesis will be performed. Overall certainty of evidence will be assessed through the GRADE approach. Discussion: The study aims to provide evidence regarding benefit-harm balance of antipyretic use in the elderly population to inform clinical practice and future research. Systematic review registration: PROSPERO CRD42020160854", "keywords": [ "antipyretic", "long term", "elderly", "older adults", "aged", "ageing", "health effects" ], "content": "Introduction\n\nThe leading cause for mortality has steadily shifted from communicable to non-communicable diseases over the past few decades. There has been a 14% increase in mortality due to non-communicable diseases since 1980, indicating a change in the global health scenario1. Currently, the global burden of disease estimate shows that 73.4% of global deaths were from non-communicable disease causes2. This is especially true of the ageing population3,4.\n\nResearch has shown that one of the chief mechanisms in the development of non-communicable (chronic) diseases is chronic low-grade inflammation, which the body is not able to resolve completely5. Acute inflammation, a defence mechanism, when it fails to resolve, seems to perpetuate as chronic inflammation5,6. Vithoulkas and Carlino7 have posited that repeatedly suppressing acute inflammatory diseases/fevers, through antipyretic/anti-inflammatory/antibiotic drugs, may lead to lack of efficient acute inflammatory response, resulting in a low-grade subacute inflammation. This may eventually turn into chronic inflammation. The immune system then loses the ability to mount an efficient acute inflammatory response. Here we find a plausible explanation for the elderly being unable to raise a high temperature; the process of aging involving chronic inflammation8. This process is probably much slower in those endowed with very healthy and long lives, preserving their efficient acute inflammatory response8. Vithoulkas and Carlino7 propose that among the causes for the change in the global health scenario, from being predominantly infectious to chronic inflammatory diseases, the way acute inflammatory diseases are being over treated is also one. Fever, a highly regulated and essential physiological response, serves as an immediate booster of immune surveillance during infections. The febrile range of temperature acts on the various cell types involved in the tightly orchestrated acute inflammatory response9. Chronic inflammation, on the other hand, has been found to be perpetuated by failure to resolve acute inflammation10.\n\nThe elderly are known to have a blunted febrile response11, due to decreased production of endogenous pyrogens, immunosenescence and hypo nutritional status8,12,13. Such a blunted fever response is known to be associated with increased mortality, indicating the importance of a robust febrile response in infections14,15.\n\nWe must, however, also consider that high fever is proven to be dangerous in those whose cardiorespiratory reserve is compromised – for example as may happen in ischemic stroke and cardiac arrest. In these people therapeutic hypothermia is practiced, showing that hyperthermia and even normothermia is detrimental at times16,17. Fever suppression has long been shown to increase pathogen activity in the body18. Whereas onset of fever has been shown to be beneficial and in some terminal cases even increase survival possibilities in the host19. Lack of fever and leukocytosis led to increased mortality in community acquired pneumonia in the elderly, and another study has demonstrated that introducing acetaminophen, the common drug for fever, did not help reduce the intensive care unit admission days in the elderly with infection20,21. On the other hand, there is evidence showing the benefit of reducing fever, such as averting febrile convulsion in children, which may result in brain injuries and neuropsychiatric alterations22,23 and also in the elderly where the extra metabolic cost and the danger of organic damage from fever exists due to debilitating co-morbidities22,24,25. Thus, while there is logic behind suppressing fever as it can be detrimental, it is not entirely clear in what way the presence of fever is beneficial or in which situation harmful to the body26,27. Whether to treat or not treat fever, especially in adults, has remained a debate with no consistent recommendation based on sound evidence28. In view of the widespread global use of antipyretics, it is imperative to evaluate the best current evidence on the benefit-harm balance of antipyretic use for fever suppression.\n\nThis systematic review aims to synthesize evidence on the effects of antipyretic drugs in fever suppression during infection in the elderly, especially in the long-term.\n\nThis review will not explore the use of antipyretics for acute pain or other acute inflammatory conditions in the absence of fever.\n\nWhat is the long-term effect of antipyretic treatment in the elderly with fever from infectious aetiology?\n\n\nMethods\n\nThis study will be a systematic review and meta-analysis (where applicable), to assess the long-term effects of antipyretic fever treatment during infections in the elderly. The focus of the study will primarily be on the long-term health effects concerning the onset of worsening of chronic inflammatory diseases common in elderly populations, such as thyroid disorders, connective tissue disorders and asthma. Besides, other outcomes from antipyretic treatment such as effect on fever symptoms, mortality, length of hospital stay and patient satisfaction.\n\nA protocol for this systematic review and meta-analysis is registered with PROSPERO (International Prospective Register for Systematic Reviews; CRD42020160854) and the PRISMA-P checklist is provided as extended data29. Any changes made thereafter will be updated on the PROSPERO database and reported in the final paper.\n\nPopulation. As per the World Health Organization (WHO) classification30, people aged 60 years and above with fever during infection will be included (studies will be included if the age definition is according to the definition of WHO; studies where the sample population was at least 70% comprising of the elderly or providing a subgroup analysis for this age group will also be considered). The population will include both healthy elderly individuals and those with any disease diagnosis.\n\nStudies involving elderly people who are on regular analgesics/antipyretics for their chronic complaints will be excluded from the review.\n\nIntervention. We will include any study that includes treatment with any form of antipyretic for fever during infections in the elderly. Though many antipyretics are also analgesics, the role sought here is that of antipyresis and the analgesic role will not be included. Studies that do not clarify the intended antipyretic/analgesic role will not be included\n\nComparator. We will include any study that compares the antipyretic drugs to any other treatment, placebo or no treatment.\n\nOutcomes\n\n(a) Primary outcomes:\n\nLong term health effects from antipyretic treatment. Long-term in this context implies effect that lasts for over 3 months from the episode of fever treatment. Onset of most common chronic immune mediated/inflammatory diseases in the elderly viz.,\n\ni) Thyroid disorders: Autoimmune disorders of the thyroid gland – autoimmune thyroiditis; hyperthyroidism e.g., Grave’s disease [E05 category of International Classification of Diseases 10 (ICD 10)]31 and hypothyroidism, e.g., Hashimoto’s thyroiditis (E06.3 category of ICD 10)31;\n\nii) Connective tissue disorders: Disorders involving the musculoskeletal system as elaborated in ICD 10 classification categories M02.8 – M03, M05 – 07, M10 – 12.4, M15 – 19, M30 – 36 (except the juvenile forms) M43, M60.1, M60.8 – 61.1, M61.5 and M61.931;\n\niii) Chronic obstructive pulmonary disease/asthma: disorders involving the conditions as under ICD 10 classification category J40 - J4731;\n\n(b) Secondary outcomes:\n\nThe effect of antipyresis during infection on:\n\ni) Fever suppression: We will extract data from the studies that provide effect on temperature and discomfort associated with it during infection with antipyretic drugs.\n\nii) Length of stay at hospital: The studies that measure the number of days of hospitalization during infections and the effect of antipyresis will be considered and this specific data will be extracted from them.\n\niii) Patient satisfaction: If studies assess level of satisfaction in patients from treatment with antipyretics against no such treatment or placebo (usually done on predetermined scales of measurement), we will extract this data.\n\niv) Mortality: The data from studies that measure the mortality score with antipyresis during infection and compare with no treatment or placebo will be recorded.\n\nv) Effect on blood/immune parameters indicative of morbidity (platelet count, erythrocyte sedimentation rate, C-reactive protein (CRP)/high sensitivity CRP and liver enzymes): Many studies measure the difference in immune and blood parameters during infection with and without antipyretic treatment. This is especially relevant in infections which have a great impact on the blood parameters such as dengue. Such data will be assessed for this outcome.\n\nvi) Effect on further complications of the infection: If evidence exists on the influence of antipyretic treatment on preventing or facilitating complications then such studies will be analysed for their data.\n\nSubgroup analysis: since the population in consideration for this study does not discriminate the different states of health at the outset, a subgroup analysis of healthy elderly, those with common chronic diseases and those with severe immune impairment/frail elderly, will be made in each of the above category if such data may be delineated.\n\nStudy types. Randomized controlled trials, quasi experimental studies, non-randomized control trials, before and after studies, observational studies (retrospective and prospective), case control studies, and case reports and series will all be included, and the data will be pooled if possible.\n\nSearch strategies for the respective databases will be designed to identify and collate studies over different databases, viz. PubMed, EMBASE, Cumulative Index to Nursing and Allied Health Literature (CINAHL), PsycINFO, Elsevier ClinicalKey, ScienceDirect, Cochrane Library, Clinicaltrials.gov and the World Health Organization (WHO) and International Clinical Trials Registry Platform (ICTRP). They will be searched from the inception of their records till the time of final analysis for this study. The search will include all studies that have reported fever treatment in the elderly during infections with regard to any of the said outcomes, comparing with no treatment/placebo/any other treatment.\n\nThe search will be conducted using Medical Subject Headings (MeSH) for PubMed and Emtree and keywords for EMBASE and other keywords denoting a combination of intervention (antipyretic, paracetamol, acetaminophen, placebo etc.) and population (older adults, aged, ageing, elderly etc.) for other databases. We will search only published studies and no grey literature will be included. A search strategy drafted for MEDLINE search is included as extended data29.\n\nA restriction will be made to obtain human studies only, but no restriction will be laid on language. An attempt to understand the non-English articles will be made through the Google translate application and if unable to understand despite this, a native speaker will be sought for help. The articles that are extracted will be screened for relevant referenced papers for those that may not be available through database search. Full text screened articles that are excluded will be done so with explanation for the exclusion.\n\nThe process of screening will be depicted in a Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) flowchart.\n\nThe data will be collected using special data extraction forms predesigned for each type of study. Data will be collected as per the following categories from each paper extracted from the search:\n\nJournal name; title; authors; year of publication; funding sources.\n\nType of study; study setting; sampling size; control and interventions arms; type of infection; type of intervention; details of intervention delivery - dose, combination etc; blinding\n\nMean age; range of age; gender distribution; inclusion and exclusion criteria; health status\n\nOutcomes assessed; follow-up period; participant attrition; statistical analysis population (Intention to treat/per protocol); statistical analysis; conclusions.\n\nThe relevant statistical comparisons and measures will be extracted from these studies for each of the outcomes.\n\nSpecific exclusion criteria: studies dealing with effect of the said drugs in the role of analgesics with elderly population taking them on a regular basis will be excluded. Studies that make no distinction on the role of the drug as analgesic or antipyretic will be excluded.\n\nMissing data. Study authors will be contacted for any missing data and a reminder will be sent. On failure of response from authors, we will continue with the analysis of available studies as they are. Any missing data will be recorded as such and the whole sample (intention to treat) will be considered for analysis.\n\nTwo investigators will independently review the studies for their eligibility and will also review the data collected as described above. Any disagreement or discrepancies between them will be appealed to the third reviewer whose decision shall settle the disagreement. The data will be summarized in a table showing the number of studies and their respective demographic, intervention and outcome measures. Mean, median, range and standard deviation will be calculated for continuous measures.\n\nFor the included randomized control trials, The Cochrane Bias tool 2.032 assesses results of a study for bias through five measuring domains: selection, performance, detection, attrition, reporting. The biases are ranked according to the level of risk they pose - low/high/unclear. The quality of evidence will have to be agreed upon by both investigators who will independently assess them; any disagreements will be resolved by the third reviewer.\n\nFor other study types, such as non-randomized studies, observational studies etc., tools such as the Cochrane tool for non-randomized studies, Critical Appraisal Skills Program (CASP) (Nadelson & Nadelson, 2014) and Quality in Prognostic Studies (QUIPS) (Hayden, van der Windt, Cartwright, Côté & Bombardier, 2013) tool will be used.\n\nWe will perform meta-analysis, where applicable, using Review Manager 5.3 (RevMan 2014)33, with a random-effect model, following the recommendations of Cochrane. Where possible, our primary data analyses will follow the intention-to-treat principle: namely, the original number of participants allocated to each study arm will be used as the denominator in subsequent analyses. We will synthesize, for the sake of meta-analysis, the study results in the form of risk ratios, risk differences, number of patients needed to be treated (Benefit), number of patients needed to be treated (Harm) and weighted mean differences with their respective 95% CIs, as detailed under measures of treatment effect.\n\nStudies that cannot yield these data will be excluded from the meta-analysis, and a narrative synthesis will be carried out for such studies according to the narrative synthesis framework of Cochrane consumers and communication group34\n\nInitially, all antipyretics, regardless of types, routes and duration will be grouped together. This is in order to find the overall effect and the degree of consistency of the findings between studies. If sufficient data are available, subgroup analysis will be carried out with respect to type, dose and duration of antipyretics. For example, dosage may be assessed under subgroups of standard dose/low dose/high dose; the route may be assessed under parenteral/oral/rectal suppositories; types may be assessed under non-steroidal anti-inflammatory drugs/acetaminophen/salicylates/others.\n\nThe overall certainty of evidence will be assessed using the GRADE approach (Grading of Recommendation, Assessment, Development and Evaluation)35 and it will be categorized as high, moderate, low or very low. The certainty will be rated by default as ‘high’ if randomized controlled trials are the basis of the evidence, with downgrade of the certainty of evidence upon the identification of at least a serious concern on the following aspects: risk of bias, inconsistency, indirectness, imprecision and publication bias. On the other hand, the default rating for non-randomized studies is ‘low’, with possibility of upgrading the certainty of evidence based on the consideration of large effects, dose-response relationship and the presence of plausible confounders to the observed effects\n\nThe authors intend to publish the completed review in a peer reviewed journal.\n\n\nStudy status\n\nInitial search has been made and studies have been downloaded for further screening and extraction\n\n\nDiscussion\n\nThe question here is whether the elderly benefit from antipyretic treatment in the long term or if it causes damage. While the physician decides based on the immediate requirement of relieving the patient, it will help to know the outcome of such practice, especially if it can be avoided in cases where such treatment may be dispensed with. Most antipyretics, such as acetaminophen, act on the cyclo-oxygenase pathways and inhibit prostaglandin-E2, thus inhibiting fever. The mechanism may inadvertently be causing other long term effects36.\n\nThe authors are not aware of any other systematic review addressing this particular question.\n\nThis study also aims to highlight gaps in clinical research on the long-term effects of antipyretics for aged patients that is used during infections.\n\n\nData availability\n\nNo data is associated with this article.\n\nFigshare: Extended data_SeemaMahesh, https://doi.org/10.6084/m9.figshare.13116458.v129.\n\nThis project contains the following extended data:\n\n- Draft search strategy for Medline.\n\n- Data extraction forms templates for different types of studies\n\nFigshare: PRISMA-P checklist for ‘Long-term health effects of antipyretic drug use in the ageing population: protocol for a systematic review’, https://doi.org/10.6084/m9.figshare.13116458.v129.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nGBD 2015 Mortality and Causes of Death Collaborators: Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388(10053): 1459–544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGBD 2017 Causes of Death Collaborators: Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 2018; 392(10159): 1736–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng L, Li P, Wang X, et al.: Distribution and determinants of non communicable diseases among elderly Uyghur ethnic group in Xinjiang, China. PLoS One. 2014; 9(8): e105536. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin K, Simpkins JW, Ji X, et al.: The Critical Need to Promote Research of Aging and Aging-related Diseases to Improve Health and Longevity of the Elderly Population. Aging Dis. 2014; 6(1): 1–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarnig C, Bezema T, Calder PC, et al.: Activation of Resolution Pathways to Prevent and Fight Chronic Inflammation: Lessons From Asthma and Inflammatory Bowel Disease. Front Immunol. 2019; 10: 1699. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSugimoto MA, Sousa LP, Pinho V, et al.: Resolution of Inflammation: What Controls Its Onset? Front Immunol. 2016; 7: 160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVithoulkas G, Carlino S: The \"continuum\" of a unified theory of diseases. Med Sci Monit. 2010; 16(2): SR7–15. PubMed Abstract\n\nGinaldi L, Loreto MF, Corsi MP, et al.: Immunosenescence and infectious diseases. Microbes Infect. 2001; 3(10): 851–7. PubMed Abstract \| Publisher Full Text\n\nFisher DT, Vardam TD, Muhitch JB, et al.: Fine-tuning immune surveillance by fever-range thermal stress. Immunol Res. 2010; 46(1–3): 177–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSugimoto MA, Vago JP, Perretti M, et al.: Mediators of the Resolution of the Inflammatory Response. Trends Immunol. 2019; 40(3): 212–27. PubMed Abstract \| Publisher Full Text\n\nFalsey AR, Baran A, Walsh EE: Should clinical case definitions of influenza in hospitalized older adults include fever? Influenza Other Respir Viruses. 2015; 9 Suppl 1(Suppl 1): 23–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorti G, Paradisi F: [Pathogenetic mechanisms responsible for producing a secondary immunodeficiency state]. J Chemother. 1994; 6 Suppl 3: 6–10. PubMed Abstract\n\nFranceschi C, Bonafe M, Valensin S, et al.: Inflamm-aging. An evolutionary perspective on immunosenescence. Ann N Y Acad Sci. 2000; 908(1): 244–54. PubMed Abstract \| Publisher Full Text\n\nNorman DC: Fever in the elderly. Clin Infect Dis. 2000; 31(1): 148–51. PubMed Abstract \| Publisher Full Text\n\nWiewel MA, Harmon MB, van Vught LA, et al.: Risk factors, host response and outcome of hypothermic sepsis. Crit Care. 2016; 20(1): 328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurisu K, Yenari MA: Therapeutic hypothermia for ischemic stroke; pathophysiology and future promise. Neuropharmacology. 2018; 134(Pt B): 302–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernard SA, Gray TW, Buist MD, et al.: Treatment of comatose survivors of out-of-hospital cardiac arrest with induced hypothermia. N Engl J Med. 2002; 346(8): 557–63. PubMed Abstract \| Publisher Full Text\n\nHusseini RH, Sweet C, Collie MH, et al.: Elevation of nasal viral levels by suppression of fever in ferrets infected with influenza viruses of differing virulence. J Infect Dis. 1982; 145(4): 520–4. PubMed Abstract \| Publisher Full Text\n\nJampel HD, Duff GW, Gershon RK, et al.: Fever and immunoregulation. III. Hyperthermia augments the primary in vitro humoral immune response. J Exp Med. 1983; 157(4): 1229–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhkee S, Srinath L, Ramirez J: Community-acquired pneumonia in the elderly: association of mortality with lack of fever and leukocytosis. South Med J. 1997; 90(3): 296–8. PubMed Abstract \| Publisher Full Text\n\nYoung P, Saxena M, Bellomo R, et al.: Acetaminophen for Fever in Critically Ill Patients with Suspected Infection. N Engl J Med. 2015; 373(23): 2215–24. PubMed Abstract \| Publisher Full Text\n\nDonoso A, Arriagada D: Fever and antipyretic therapy in the septic patient in the intensive care unit: an update. Bol Med Hosp Infant Mex. 2018; 75(4): 203–215. PubMed Abstract \| Publisher Full Text\n\nEl-Radhi AS: Febrile Seizures. Clinical Manual of Fever in Children. Springer; 2018; 179–92. Publisher Full Text\n\nLee JC, Cia CT, Lee NY, et al.: Causes of death among hospitalized adults with dengue fever in Tainan, 2015: emphasis on cardiac events and bacterial infections. bioRxiv. 2018; 507137. Publisher Full Text\n\nCarey JV: Literature review: should antipyretic therapies routinely be administered to patients with [corrected] fever? J Clin Nurs. 2010; 19(17–18): 2377–93. PubMed Abstract \| Publisher Full Text\n\nEgi M, Makino S, Mizobuchi S: Management of fever in critically ill patients with infection. J Emerg Crit Care Med. 2018; 2: 10. Publisher Full Text\n\nHobohm U: Fever therapy revisited. Br J Cancer. 2005; 92(3): 421–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLudwig J, McWhinnie H: Antipyretic drugs in patients with fever and infection: literature review. Br J Nurs. 2019; 28(10): 610–8. PubMed Abstract \| Publisher Full Text\n\nMahesh S, van der Werf E, Mallappa M, et al.: Extended data_SeemaMahesh. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.13116458.v1\n\nWorld Health Organization: Men Ageing And Health Achieving health across the life span 01WHO/NMH/ (e book). World Health Organization; 2001. Reference Source\n\nWorld Health Organization: ICD-10: international statistical classification of diseases and related health problems: tenth revision. 2nd ed. Geneva: World Health Organization; 2004. Reference Source\n\nHiggins JP, Altman DG, Gøtzsche PC, et al.: The Cochrane Collaboration's tool for assessing risk of bias in randomised trials. BMJ. 2011; 343: d5928. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCochrane Collaboration: Review Manager (RevMan) 5.3 [program]. 5.3. 5 (Build Date: 30/10/14 11: 54) version. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration. 2014.\n\nRyan R: Cochrane Consumers and Communication Review Group: data synthesis and analysis. Cochrane Consumers and Communication Review Group. 2013. Reference Source\n\nSchünemann H, Brożek J, Guyatt G, et al.: GRADE handbook for grading quality of evidence and strength of recommendations. Updated October 2013. The GRADE Working Group, 2013. 2013. Reference Source\n\nRajakariar R, Yaqoob MM, Gilroy DW: COX-2 in inflammation and resolution. Mol Interv. 2006; 6(4): 199–207. PubMed Abstract \| Publisher Full Text" }	[ { "id": "80627", "date": "22 Mar 2021", "name": "Sylwia Elżbieta Wrotek", "expertise": [ "Reviewer Expertise Fever", "inflammation", "immunity", "antipyretics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol describes methods how to investigate the effect of antipyretic drugs on long-term health of elderly people. Until now the effect of antipyretics was a subject of a few meta-analyses. The novelty of this study is the selection of population of elderly people. The authors will search a variety of databases to collect relative information on antipyretic drugs used in elderly. The study methods are appropriate and authors are going to use best practice standards for systematic review. Many tools to assess the risk of bias will be involved. Meta-analysis will be conducted and the GRADE approach for evaluating the quality of evidence will be used. In general this proposal is well planned and this research will be interesting and important. In my opinion sufficient details are provided to allow the researchers to follow.\nThis protocol however can be improved and especially the background should be developed in order to describe the issues more precisely.\n1. In Introduction:\nThe hypothesis that early inhibition of acute inflammation may lead to subacute or chronic state is commonly known, therefore more references are needed than only paper #7.\n\nAuthors state that a blunted febrile response may be associated with increased mortality. In fact this issue is still under discussion. It is noteworthy that in some studies NSAIDs had no effect on the course of the disease or even had beneficial effects. Please, include this information in Introduction.\n\nReference #18 concerns ferrets. Other reference that concerns humans should be provided.\n\nOne of the most common mistakes in many articles about fever is treating hyperthermia as equal to fever. Unlike hyperthermia, fever is controlled elevation of body temperature due to the upward resetting of the hypothalamic thermostat. Therefore, instead of reference #19 about the effect of hyperthermia on isolated cells, please provide other paper about fever.\n2. In Research question: Authors are going to investigate infectious fever. Which factor/s will be taken into account to differentiate infectious fever from non-infectious fever?\n3. In Methods/Search strategy: Please provide a list of key words that will be asked precisely. Adding key words such as Coxibs, NSAIDS, acetylsalicylic acid (ASA) should be considered.\n4. In Methods/ Study eligibility criteria: The exclusion criteria should be described more in detail. The research is targeted on the population who take antipyretics solely for fever. Taking NSAIDs due to its analgesic properties and for anti-plateled therapy should not be analysed.\nIn my opinion after minor revision of this protocol this study will make an important contribution to the knowledge about geriatrics and hopefully will finally answer the intriguing question whether taking antipyretics can modify recovery of patients.\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6482", "date": "22 Mar 2021", "name": "Seema Mahesh", "role": "Author Response", "response": "Thank you Dr Wrotek for your valuable suggestions. We will revise the paper accordingly and publish Thanking you Seema Mahesh Corresponding author" } ] }, { "id": "128427", "date": "28 Apr 2022", "name": "Atakan Yilmaz", "expertise": [ "Reviewer Expertise Emergency Medicine" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 study to determine the long-term health effects of antipyretic use in the elderly population. In terms of clinical treatment strategies, there may be differences between the young population and the elderly population in many issues. The elderly population is a special group for which many issues need to be studied. I will be eagerly awaiting the results of the study.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "8164", "date": "28 Apr 2022", "name": "Seema Mahesh", "role": "Author Response", "response": "Thank you very much for the review. The revision is now complete and I am awaiting it's publication. I will update the link here once it is published. Dr Seema Mahesh" } ] } ]	1	https://f1000research.com/articles/9-1288
https://f1000research.com/articles/9-984/v1	13 Aug 20	{ "type": "Systematic Review", "title": "Effects of stretching exercises on human gait: a systematic review and meta-analysis", "authors": [ "Thomas Vialleron", "Arnaud Delafontaine", "Sebastien Ditcharles", "Paul Fourcade", "Eric Yiou", "Thomas Vialleron", "Sebastien Ditcharles", "Paul Fourcade", "Eric Yiou" ], "abstract": "Background: Stretching is commonly used in physical therapy as a rehabilitation tool to improve range of motion and motor function. However, is stretching an efficient method to improve gait, and if so, for which patient category? Methods: A systematic review of randomized and non-randomized controlled trials with meta-analysis was conducted using relevant databases. Every patient category and every type of stretching programs were included without multicomponent programs. Data were meta-analysed where possible. Estimates of effect sizes (reported as standard mean difference (SMD)) with their respective 95% confidence interval (95% CI) were reported for each outcome. The PEDro scale was used for the quality assessment. Results: Twelve studies were included in the analysis. Stretching improved gait performance as assessed by walking speed and stride length only in a study with a frail elderly population, with small effect sizes (both SMD= 0.49; 95% CI: 0.03, 0.96; PEDro score: 3/10). The total distance and the continuous walking distance of the six-minute walking test were also improved only in a study in an elderly population who had symptomatic peripheral artery disease, with large effect sizes (SMD= 1.56; 95% CI: 0.66, 2.45 and SMD= 3.05; 95% CI: 1.86, 4.23, respectively; PEDro score: 5/10). The results were conflicting in healthy older adults or no benefit was found for most of the performance, spatiotemporal, kinetic and angular related variables. Only one study (PEDro score: 6/10) showed improvements in stance phase duration (SMD=-1.92; 95% CI: -3.04, -0.81), swing phase duration (SMD=1.92; 95 CI: 0.81, 3.04), double support phase duration (SMD= -1.69; 95% CI: -2.76, -0.62) and step length (SMD=1.37; 95% CI: 0.36, 2.38) with large effect sizes. Conclusions: There is no strong evidence supporting the beneficial effect of using stretching to improve gait. Further randomized controlled trials are needed to understand the impact of stretching on human gait.", "keywords": [ "stretching", "gait", "performance", "balance", "physical therapy" ], "content": "Introduction\n\nGait is a highly complex motor skill that is classically considered as an integrative measure and a predictor of health in older adults (e.g. 1; cf. also 2 and 3 for recent research topics on this matter). The loss of gait or its alteration with pathological conditions are known to be related to mortality, especially in the elderly (e.g. 4,5), stressing the importance of addressing gait disorders in physiotherapy. Gait requires body propulsion and balance control for safe progression, two “subtasks” that require the coordination of multiple skeletal muscles and the integration of sensory information arising from the vestibular, visual and somatosensory systems6–8. As such, gait may expose populations with sensory or motor deficits to the risk of falling with serious consequences for health and autonomy. For these reasons, improving gait is a major aim in rehabilitation for most neurological/orthopaedic disorders, such as stroke or Parkinson’s disease, and for frail older adults. Various therapeutic methods have been used to improve gait, such as resistance training9, endurance training10, balance training11, whole body vibrations (for a complete review, see Fischer et al., 201912), multi-component exercise programs13 and stretching14.\n\nThe successful completion of numerous daily life activities is conditioned by the ability to move efficiently through a sufficient range of motion (ROM)15. Recent studies on gait initiation16–18 and seat-to-stand task19,20 showed that the experimental restriction of postural chain ROM induced by orthosis wear in young healthy adults led to instability and lower motor performance. It is well established that ROM significantly decreases with aging21–26 and more generally with reduced functional demand (e.g. sedentarity, immobilization, disease etc.)15. Consequently, stretching has become an important part of many sport and rehabilitation programs to maintain or improve ROM, reduce stiffness and promote physical activity. This method has been applied in older adults27,28, patients with stroke29, Parkinson’s disease30, multiple sclerosis31, plantar fasciitis32 and spastic paraplegia33, for example. In sport programs, the influence of stretching on motor performance remains an issue of debate, although recent reviews conclude that maximal muscle performance (e.g. force, power, jump height, reaction time, etc.) is impaired primarily immediately after long durations of stretch (>90 seconds)34,35. To date, no review has collected results on the relationship between stretching and locomotor performance in rehabilitation programs.\n\nHence, the purpose of this article is to analyse the effects of a stretching program on gait in each patient category by means of a systematic literature review and meta-analysis, comparing the gait outcomes of the intervention groups with the control groups. It will contribute to provide evidence-based practice from scientific data in order to integrate stretching in rehabilitation programs in a reasoned manner.\n\n\nMethods\n\nThe Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology was employed in this systematic review36. A completed PRISMA checklist was submitted to an online repository (Reporting guidelines).\n\nPubMed, Science Direct, Springer and Sage databases were used for a comprehensive systematic literature search for articles published prior to 28 April 2020 with no time limit. In addition, a manual search was conducted using the reference list of selected studies. The keywords used for the search strategy in PubMed were: “stretching” AND (gait OR walk). We included only articles published in English or French.\n\nThe selection procedure was conducted by two experts in rehabilitation (TV and AD). Disagreements were discussed with a third expert in a group until a mutual consensus was reached. First, a review was performed on all available titles obtained from the literature search with the selected keywords. All relevant or potentially relevant titles were included in the subsequent phase. Then, the abstracts were reviewed with all relevant or potential articles included in the following phase. Finally, full-text articles were reviewed to ensure that only relevant studies were included. In the same way, reference lists of all included articles were reviewed to possibly include articles through cross-referencing.\n\nWe included randomized controlled trials (RCT) and controlled clinical trials (CCT) published in peer-reviewed journals that aimed to explore the effects of stretching on gait parameters. We included all categories of subjects, all stretching techniques and different durations of treatment since standardized protocols are lacking in the purpose of the present study. Gait could be evaluated by functional tests, electromyographic (EMG) or biomechanical analysis. The following exclusion criteria were used: lack of gait assessment, non-application of muscle stretching, multimodal exercise programs, no control group, case report and review.\n\nData were extracted from the selected articles by one of the authors (TV). The extracted data were checked by another author (AD) and disagreements were resolved with a third (EY).\n\nThe following data were extracted for each selected article: (1) the names of the authors and the date of publication; (2) the number of subjects involved in the experiment with their characteristics and breakdown in each group; (3) stretching training details (in the following order: number of participants, stretching technique, muscle groups stretched, number of sets, duration of stretch, frequency, protocol duration); (4) control group details; and (5) the main outcomes related to gait with the main results. When information could not be provided, it was indicated by a “?”.\n\nThe PEDro scale was used to assess the risk of bias, and thus the methodological quality of the selected studies37. This scale was chosen for its ability to provide an overview of the external (criterion 1), internal (criteria 2–9) and statistical (criteria 9 and 10) validity of clinical trials. The scale is divided into 11 criteria, but the first is not calculated in the total score. The output of each criterion could be either “yes” (y), “no” (n) or “do not know” (?). A “y” was given a score of one point, while an “n” or “?” was assigned zero points. Studies with a total score of 5–10/10 (≥ 50%) were considered to be of high quality, and scores of 0–4/10 (<50%) of low quality38. Two evaluators independently assessed the quality of the included studies. In the event of disagreement, a group discussion was held with a third expert to reach a consensus.\n\nEstimates of effect sizes (comparing the intervention group and the control group) accompanied with a measure of statistical uncertainty (95% confidence interval [95% CI]) were calculated for each outcome when sufficient data were reported. Estimates of effect sizes were reported by standard mean difference (SMD) and their respective 95% CI. In this way, the magnitude of the overall effect can be quantified as trivial (<0.2), small (0.2–0.49), moderate (0.5–0.79) or large (≥0.8)39,40. When data were lacking to calculate estimates of effect sizes, exact p values were reported.\n\nWhen at least two studies used the same outcome, meta-analysis was performed, comparing the intervention groups with the control groups. When outcomes were identified in only one study, no meta-analysis could be performed but the effect of intervention was still calculated, reporting the estimate of effect size and its 95% confidence interval. Statistical analysis and figures (i.e. forest plot to facilitate the visualization of values) were produced using a random-effect model in Review Manager software (RevMan, v 5.3, Cochrane Collaboration, Oxford UK). A random-effect model was used to take into account heterogeneity between study effects. Statistical heterogeneity was calculated using the I2 and Cochrane Q statistic tests39. Statistical significance was set at p<0.05.\n\nThe strength of evidence of primary outcomes was established as described by Van Tulder et al. 200341 based on effect size estimates with a measure of statistical uncertainty (SMD; 95% CI), statistical heterogeneity (I2) when applicable (multiple studies) and risk of bias (PEDro scale). The level of evidence was considered strong with consistent findings among multiple high-quality RCT (at least two RCT with a PEDro score ≥5/10 that were statistically homogenous: I2 p≥0.05). The level of evidence was considered moderate with consistent findings among multiple low-quality RCT and/or CCT (two trials with a PEDro score <5/10 that were statistically homogenous) and/or one high quality RCT. The level of evidence was considered limited when only one low quality RCT and/or CCT was identified. The level of evidence was conflicting when there was inconsistency among multiple trials (I2 p < 0.05).\n\n\nResults\n\nA total of 821 titles were screened in the first search stage, one more was included through cross-referencing, and 671 were excluded because they did not concern our research question. Following exclusion, 150 studies were considered for an abstract review. A further 105 were excluded in this second stage because they did not meet the inclusion criteria. Finally, 45 full-text articles were assessed for eligibility with 33 not accepted (Figure 1).\n\nThus, 12 articles were ultimately included in this systematic review. Six studies evaluated the effects of stretching in healthy older adults14,42–46, one in a frail elderly population47, one study in an elderly population with stable symptomatic peripheral artery disease48, one in stroke patients49, one study in adults with limited ankle ROM associated with a history of lower limb overuses injury50, one study in healthy adults with limited ankle dorsiflexion range of motion51 and one in healthy young adults52. A summary of the studies selected is provided in Table 1, and their quality assessment is reported in Table 2. The results in different patient categories are reported below.\n\nSMD: standard mean difference, CI: confidence interval, 10MWT: 10-meter walk test, 6MWT: 6-minute walk test, ROM: range of motion, ?: information not provided.\n\nn: criterion not fulfilled; y: criterion fulfilled; ?: criterion not mentioned; total score: each item (except the first) contributes 1 point to the total score, yielding a PEDro scale score that can range from 0 to 10.\n\nHealthy older adults\n\nDescription of the studies and quality assessment\n\nSix studies examined the effects of stretching on healthy elderly subjects14,42–46. Regarding the characteristics of the subjects, the average sample size was 46.6±33.9 subjects (ranging from 1914 to 96 subjects44) and the mean age was 70.1±3.6 years (ranging from 65.4043 to 75.30 years14). Regarding the characteristics of the training programs, the average training duration was 8.6±2.7 weeks (ranging from 443 to 12 weeks45), with an average frequency of 8.3±6.2 sessions per week (ranging from 245 to 14 sessions42,44,46). The average number of sets per session was 4.5±2.8 sets (ranging from 246 to 10 sets14), with an average stretching time of 45.0±18.9 seconds (ranging from 1514 to 60 seconds43,45,46). Static stretching was provided in all studies. The muscle groups stretched were the hip flexors42–44,46, ankle plantar flexors14,42,43,45, ankle dorsiflexors45, hip extensors43, knee extensors and flexors45. There was great heterogeneity in gait outcomes. Angular variables during gait included peak hip extension42,44,46, ankle plantar flexion during gait44, ankle range of motion during gait43, anterior pelvis tilt43,46, knee range of motion43, pelvic rotation43, lateral pelvic tilt43 and hip range of motion43. Spatiotemporal variables were: gait speed42,43,46, stance and swing durations43, double support phases43, step length43 and stride length43,46. Kinetic variables were hip torque44 and ankle plantar flexion power44. Finally, two functional tests were used: the 10-meter walk test (10MWT)14 and the 6-minute walk test (6MWT)45. Regarding the quality of the studies, the average PEDro score was 4.6±1.6 and one study was identified as a non-randomized trial45. The range of score varied from 345,46 to 714.\n\nMeta-analyses\n\nFour meta-analysis were conducted for the following outcomes (Figure 2): gait speed, stride length, hip extension during gait and anterior pelvic tilt.\n\nComparisons between intervention and control groups for gait speed (A), stride length (B), hip extension (C) and anterior pelvic tilt (D) in healthy older adults.\n\nGait speed: For gait speed (Figure 2A), two studies were included in the meta-analysis42,43. One study was excluded because intervention and control groups were not similar at baseline46. Statistical analysis showed no significant difference between groups (SMD= 0.45; 95% CI: -1.15, 2.06), with heterogeneous results (I2=86%, p=0.007). Thus, the level of evidence was conflicting.\n\nStride length: For stride length (Figure 2B), two studies were included in the meta-analysis42,46. Statistical analysis showed no significant difference between groups (SMD= 0.22; 95% CI: -0.44, 0.88), with consistent results (I2=59%, p=0.12). Only one study was of high quality42, thus a moderate level of evidence supports the lack of beneficial effect of stretching to improve stride length in the elderly.\n\nHip extension: For hip extension during gait (kinematic data) (Figure 2C), three studies were included in the meta-analysis42,44,46. Statistical analysis showed no significant difference between groups (SMD= 0.20; 95% CI: -0.06, 0.47), with consistent results (I2=0%, p=0.99). Two studies were of high quality42,44, thus a strong level of evidence supports the lack of beneficial effect of stretching to improve hip range of motion during gait in the elderly.\n\nAnterior pelvic tilt: For anterior pelvic tilt (Figure 2D), three studies were included in the meta-analysis42,44,46. Statistical analysis showed no significant difference between groups (SMD= -0.70; 95% CI: -1.60, 0.21), with heterogeneous results (I2=87%, p<0.01). Thus, the level of evidence was conflicting.\n\nEffects of interventions in other outcomes\n\nFor the outcomes below, no meta-analysis could be performed because only one study was identified. Nevertheless, for each outcome, effect size estimates with a measure of statistical uncertainty (95% CI) were provided.\n\nAngular variables during gait initiation: The study of Christiansen et al. (2008)42 showed no significant difference between stretching and control groups for ankle dorsiflexion during gait (SMD=0.29; 95% CI: -0.36, 0.94) with a moderate level of confidence (PEDro score: 5/10). The study of Kerrigan et al. (2003)44 showed no significant difference between groups for ankle plantar flexion (SMD=-0.05; 95% CI: -0.45, 0.35), with a moderate level of confidence (PEDro score: 6/10). The study of Cristopoliski et al. (2009)43 showed no significant difference between groups for lateral pelvic tilt (SMD= 0.93; 95% CI: -0.02, 1.88) and knee range of motion (SMD= 0.23; 95% CI: -0.67, 1.12), with a moderate level of confidence (PEDro score: 6/10).\n\nKinetic variables: The study of Kerrigan et al. (2003)44 showed no significant difference between groups for hip torque (SMD= 0.35; 95% CI: -0.06, 0.75) and ankle plantar flexion power (SMD=0.00; 95% CI: -0.40, 0.40), with a moderate level of confidence (PEDro score: 6/10).\n\nSpatiotemporal variables: The study of Cristopoliski et al. (2009)43 showed no significant difference between groups for cycle duration (SMD= -0.24; 95% CI: -1.14, 0.66), heel contact velocity (SMD= -0.46; 95% CI: -1.37, 0.45) and toe clearance (SMD= 0.91; 95% CI: -0.04, 1.86). However, the study showed significant decreases with large effect sizes in stance phase duration (SMD=-1.92; 95% CI: -3.04, -0.81), double support phase duration (SMD= -1.69; 95% CI: -2.76, -0.62) in favour of the stretching group as compared to the control group. Additionally, the authors found significant increases with large effect sizes of swing phase duration (SMD=1.92; 95 CI: 0.81, 3.04) and step length (SMD=1.37; 95% CI: 0.36, 2.38) in favour of the stretching group as compared to the control group. The study obtained a PEDro score of 6/10, thus, the level of evidence for these outcomes was moderate.\n\nFunctional tests: The study of Gajdosik et al. (2005)14 showed no significant difference between groups for the 10MWT (SMD= -0.76; 95% CI= -1.70, 0.18), with a moderate level of confidence (PEDro score: 7/10). The study of Locks et al. (2012)45 showed no significant improvement of the 6MWT in favour of the stretching group as compared to the control group (SMD= -0.04; 95% CI:-0.86, 0.79) with a limited level of confidence (low quality CCT with a PEDro score of 3/10).\n\nFrail elderly\n\nDescription of the study and quality assessment\n\nThe study of Watt et al. 2011 examined the effects of stretching on frail elderly subjects47. Regarding the characteristics of the subjects, 74 subjects were included, and the mean age was 77.0±8.0 years. Regarding the characteristics of the training programs, the stretching program lasted ten weeks, with a frequency of 14 sessions per week (two sessions per day). Participants performed two sets per session, holding the stretch for 60 seconds (static stretching), alternating the right and left limb (four minutes in total). The muscle group stretched was the hip flexors. The outcomes were cadence (steps/minute), walking speed (meters/second), stride length (meters) peak hip extension (degree) and peak anterior pelvic tilt (degree). Regarding the quality assessment, the study was identified as RCT and had an average PEDro score of 3 (low level of evidence).\n\nEffects of intervention\n\nThe study of Watt et al. (2011) showed no significant difference between groups in angular variables, i.e. peak hip extension and anterior pelvic tilt (SMD= 0.22; 95% CI: -0.24, 0.68 and SMD= -0.05; 95% CI: -0.51, 0.41 respectively). There was also no significant difference for cadence (SMD= 0.13; 95% CI: -0.33, 0.59). However, the study showed significant improvements in favour of the stretching group with small effect sizes in some performance-related variables, i.e. walking speed and stride length (both SMD= 0.49; 95% CI: 0.03, 0.96).\n\nElderly with symptomatic peripheral artery disease\n\nDescription of the study and quality assessment\n\nThe study of Hotta et al. (2019) examined the effects of stretching in elderly with symptomatic peripheral artery disease48. Regarding the characteristics of the subjects, 13 subjects were included and the mean age was not mentioned. Regarding the characteristics of the training programs, the stretching program lasted four weeks, with a frequency of five sessions per week. Participants performed one set daily, holding the stretch for 30 minutes (static stretching with splints). The muscle group stretched was ankle plantar flexors. The gait outcome was 6MWT. Regarding the quality assessment, the study was identified as RCT and had an average PEDro score of 5 (moderate level of evidence).\n\nEffects of intervention\n\nThe study of Hotta et al. (2019) showed significant improvements in favour of the stretching group for both total walking distance and continuous walking distance with large effect sizes (SMD= 1.56; 95% CI: 0.66, 2.45 and SMD= 3.05; 95% CI: 1.86, 4.23 respectively).\n\nStroke\n\nDescription of the study and quality assessment\n\nThe study of Kim et al. (2013) examined the effects of stretching on stroke patients49. Only a static muscle stretching training group and control group were included in the analysis. Regarding the characteristics of the subjects, 24 subjects were included, and the mean age was 53.3±3.1 years. Regarding the characteristics of the training programs, the stretching program lasted six weeks, with a frequency of four sessions per week. Participants performed one set per session, holding the stretch for 20 minutes (static stretching). The muscle group stretched was ankle plantar flexors. The outcome was the sway of the centre of pressure during the stance phase. Regarding the quality assessment, the study was identified as CCT and had an average PEDro score of 3 (low level of evidence).\n\nEffects of intervention\n\nThe study of Kim et al. (2013) showed no significant difference between groups in the sway of the centre of pressure (SMD=0.75; 95% CI: -0.09, 1.58).\n\nYoung adults with limited ankle range of motion and a history of lower limb overuse injury\n\nDescription of the study and quality assessment\n\nThe study of Johanson et al. (2006) examined the effects of stretching on healthy adults with limited passive ankle-dorsiflexion range of motion (less than eight degrees) and a history of lower limb overuse injury50. Regarding the characteristics of the subjects, 19 subjects were included and the mean age was 30.3±9.8 years. Regarding the characteristics of the training programs, the stretching program lasted three weeks, with a frequency of two sessions per day. Participants performed five sets per session, holding the stretch for 30 seconds (static stretching). The muscle group stretched was ankle plantar flexors. The outcomes were ankle dorsiflexion and time-to-heel-off during the stance phase of gait. Regarding the quality assessment, the study was identified as RCT and had an average PEDro score of 5 (moderate level of evidence).\n\nEffects of intervention\n\nThe study of Johanson et al. (2006) showed no significant difference between groups in ankle dorsiflexion during gait in both the right and left ankle (SMD= 0.50; 95% CI: -0.42, 1.43 and SMD= 0.41; 95% CI: -0.52, 1.33 respectively). There was also no significant difference between groups for time-to-heel-off during the stance phase of gait in both the right and left ankle (SMD= -0.50; 95% CI: -1.43, 0.43 and SMD= -0.48; 95% CI: -1.41, 0.45 respectively).\n\nYoung adults with limited ankle range of motion\n\nDescription of the study and quality assessment\n\nThe study of Johanson et al. (2009) examined the effects of stretching on young adults with limited passive ankle-dorsiflexion range of motion (less than five degrees)51. Regarding the characteristics of the subjects, 16 subjects were included, and the mean age was 27.4±8.2 years. The characteristics of the training programs were the same as described above50. The muscle group stretched was the ankle plantar flexors. The outcomes were maximum ankle dorsiflexion, maximum knee extension and EMG amplitude of the gastrocnemius during the stance phase of gait. Regarding the quality assessment, the study was identified as RCT and had an average PEDro score of 6 (moderate level of evidence).\n\nEffects of intervention\n\nThe study of Johanson et al. (2009) showed no significant difference between groups in angular variables during gait, i.e. maximum ankle dorsiflexion and maximum knee extension (SMD= 0.53; 95% CI: -0.48, 1.53 and SMD= -0.07; 95% CI: -1.05, 0.91 respectively). There was also no significant difference between groups for EMG variables, i.e. medial and lateral gastrocnemius activity (SMD= 0.37; 95% CI: -0.62, 1.36 and SMD= 0.00; 95% CI=: -0.98, 0.98 respectively).\n\nHealthy young adults\n\nDescription of the study and quality assessment\n\nThe study of Godges et al. (1993) examined the effects of stretching on healthy young adults52. Only a static hip extension stretching group and control group were included in the analysis. Regarding the characteristics of the subjects, 16 subjects were included, and the mean age was 21.0±1.0 years. Regarding the characteristics of the training programs, the stretching program lasted three weeks, with a frequency of two sessions per week. Participants performed three sets per session, holding the stretch for two minutes (static stretching). The muscle group stretched was the hip flexors. The outcome was walking economy (ml/kg/min). Regarding the quality assessment, the study was identified as RCT and had an average PEDro score of 5 (moderate level of evidence).\n\nEffects of intervention\n\nThe study of Godges et al. (1993) showed no significant difference between groups in gait economy in terms of oxygen consumption (SMD= 0.83; 95% CI: -0.21, 1.87).\n\n\nDiscussion\n\nThe aim of this systematic review was to determine the effects of a stretching program on human gait by means of a systematic literature review and meta-analysis. Twelves studies were identified in six different patient categories. Statistical analyses showed no strong level of evidence supporting the beneficial effect of a stretching program to improve any gait outcome. The major issue in conducting meta-analyses and establishing strong level of evidences was the great heterogeneity in gait variables. The results obtained in the different patient categories are discussed in detail below.\n\nThe healthy older adult population was the most studied. Two muscle groups were systematically stretched in the six identified studies: hip flexors42–44,46,47 and ankle plantar flexors14,42,43. Hip flexor stiffness, associated with reduced hip extension during gait has been demonstrated in the elderly and may alter gait53,54. In the same way, decreased calf muscle length associated with restricted dorsiflexion range of motion is well documented in older adults26,55,56. A decreased ankle dorsiflexion ROM has been correlated with poorer balance test scores in the elderly57 and may contribute to an increased risk of falls58. All the studies included in the present analysis showed that specific stretching programs were efficient to improve passive range of motion of the targeted joints, but results are more heterogeneous regarding gait performance and dynamic ROM. This led to inconsistency in the results or the impossibility to conclude with a strong level of evidence that a stretching program improves gait in healthy older adults. Moreover, when improvement in ROM or gait performance occurred, it was not associated with a significant increase in dynamic hip extension or ankle dorsiflexion. Only trends toward increased dynamic ROM after stretching interventions were observed42,44,46. This observation was consistent in young adults.\n\nIn stroke patients, ankle plantar flexor stretching has been successfully used to improve ankle stiffness59–62. Decreased plantar flexors stiffness may have a beneficial effect on postural control during gait because triceps surae is known to play an important role during gait63–65 and an increase in muscle stiffness might alter synergistic muscle activities during human gait. However, only one non-randomized study49 was identified and included in the current systematic review. Other studies that used stretching in multicomponent programs66–68 or in control groups69,70 were identified but excluded because of the addition of resistance training or the lack of a control group. Nevertheless, it should be noted that some studies showed improvements between pre- and post-stretching conditions. Forrester et al. (2014) showed that both robotic ankle mobilizations and manual ankle stretching improved gait velocity in stroke patients at hospital discharge compared to baseline69. Similarly, Park et al. (2018) showed that both static ankle stretching and ankle mobilizations improved gait speed after four weeks of treatment compared to baseline70. Other authors showed that one week of immobilization in dorsiflexed position (casting) followed by one week of plantar flexor stretching and gait training improved gait performances in 10MWT and 6MWT66. Hence, these encouraging results suggest that further randomized controlled trials of good quality are needed to explore the ability of ankle stretching to improve gait parameters in stroke or in other neurological diseases exposing patients to joint stiffness, e.g. Parkinson’s disease71.\n\nIn healthy adults, the interest of practicing stretching to improve gait seems limited as they are assumed to have sufficient mobility for walking. Moreover, the included study involved athletic males52, a population that is known to be more flexible than inactive persons72. Even in young adults with limited ankle ROM, stretching did not improve dynamic dorsiflexion during gait50,51. Stretching programs in apparently healthy adults should be more indicated after a prolonged period of reduced functional demand (e.g. immobilization, sedentarity), when ROM is insufficient to practice a specific activity or when high levels of flexibility are required for sport performance (e.g. gymnastics or dance) and in sports that involve stretch-shortening cycles (e.g. basketball, volleyball)15.\n\n\nConclusion\n\nTwelve studies were identified, involving a total of 442 subjects. Despite some improvements in isolated studies, statistical analyses showed no strong level of evidence supporting the beneficial effect of using stretching alone to improve gait outcomes in rehabilitation programs. The major obstacle in conducting meta-analyses and establishing strong levels of evidence were the great heterogeneity in gait variables and the low quality of the included studies. Because the effects of stretching are not clear, further randomized controlled trials of good quality are needed to understand the impact of stretching on human gait. Currently, stretching is more recommended to maintain and improve ROM rather than improve gait parameters and should be integrated in multicomponent programs.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: PRISMA checklist and PRISMA flow diagram for ‘Effects of stretching exercises on human gait: a systematic review and meta-analysis’, https://doi.org/10.7910/DVN/N8ZXNB73.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nLeBrasseur NK: Gait as an Integrative Measure and Predictor of Health Across Species. J Gerontol A Biol Sci Med Sci. 2019; 74(9): 1411–1412. 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PubMed Abstract \| Publisher Full Text\n\nHotta K, Batchelor WB, Graven J, et al.: Daily Passive Muscle Stretching Improves Flow-Mediated Dilation of Popliteal Artery and 6-minute Walk Test in Elderly Patients with Stable Symptomatic Peripheral Artery Disease. Cardiovasc Revasc Med. 2019; 20(8): 642–648. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim TH, Yoon JS, Lee JH: The Effect of Ankle Joint Muscle Strengthening Training and Static Muscle Stretching Training on Stroke Patients’ C.O.P Sway Amplitude. J Phys Ther Sci. 2013; 25(12): 1613–1616. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohanson MA, Wooden M, Catlin PA, et al.: Effects of gastrocnemius stretching on ankle dorsiflexion and time-to heel-off during the stance phase of gait. Phys Ther Sport. 2006; 7(2): 93–100. Publisher Full Text\n\nJohanson MA, Cuda BJ, Koontz JE, et al.: Effect of stretching on ankle and knee angles and gastrocnemius activity during the stance phase of gait. J Sport Rehabil. 2009; 18(4): 521–534. PubMed Abstract \| Publisher Full Text\n\nGodges JJ, MacRae PG, Engelke KA: Effects of Exercise on Hip Range of Motion, Trunk Muscle Performance, and Gait Economy. Phys Ther. 1993; 73(7): 468–477. PubMed Abstract \| Publisher Full Text\n\nKerrigan DC, Lee LW, Collins JJ, et al.: Reduced hip extension during walking: healthy elderly and fallers versus young adults. Arch Phys Med Rehabil. 2001; 82(1): 26–30. PubMed Abstract \| Publisher Full Text\n\nKerrigan DC, Todd MK, Della Croce U, et al.: Biomechanical gait alterations independent of speed in the healthy elderly: evidence for specific limiting impairments. Arch Phys Med Rehabil. 1998; 79(3): 317–322. PubMed Abstract \| Publisher Full Text\n\nGajdosik RL, Linden VWD, Williams AK: Influence of Age on Length and Passive Elastic Stiffness Characteristics of the Calf Muscle-Tendon Unit of Women. Phys Ther. 1999; 79(9): 827–838. PubMed Abstract \| Publisher Full Text\n\nGajdosik RL, Vander Linden DW, McNair PJ, et al.: Slow passive stretch and release characteristics of the calf muscles of older women with limited dorsiflexion range of motion. Clin Biomech (Bristol, Avon). 2004; 19(4): 398–406. PubMed Abstract \| Publisher Full Text\n\nMecagni C, Smith JP, Roberts KE, et al.: Balance and ankle range of motion in community-dwelling women aged 64 to 87 years: a correlational study. Phys Ther. 2000; 80(10): 1004–1011. PubMed Abstract \| Publisher Full Text\n\nGehlsen GM, Whaley MH: Falls in the elderly: Part II Balance, strength, and flexibility. Arch Phys Med Rehabil. 1990; 71(10): 739–741. PubMed Abstract\n\nGao F, Ren Y, Roth EJ, et al.: Effects of repeated ankle stretching on calf muscle–tendon and ankle biomechanical properties in stroke survivors. Clin Biomech (Bristol, Avon). 2011; 26(5): 516–522. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoneine JL, Schieppati M, Gagey O, et al.: The Functional Role of the Triceps Surae Muscle during Human Locomotion. PLoS One. 2013; 8(1): e52943. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeptune RR, Kautz SA, Zajac FE: Contributions of the individual ankle plantar flexors to support, forward progression and swing initiation during walking. J Biomech. 2001; 34(11): 1387–1398. PubMed Abstract \| Publisher Full Text\n\nCarda S, Invernizzi M, Baricich A, et al.: Casting, taping or stretching after botulinum toxin type A for spastic equinus foot: a single-blind randomized trial on adult stroke patients. Clin Rehabil. 2011; 25(12): 1119–1127. PubMed Abstract \| Publisher Full Text\n\nGhasemi E, Khademi-Kalantari K, Khalkhali-Zavieh M, et al.: The effect of functional stretching exercises on functional outcomes in spastic stroke patients: A randomized controlled clinical trial. J Bodyw Mov Ther. 2018; 22(4): 1004–1012. PubMed Abstract \| Publisher Full Text\n\nMoore SA, Jakovljevic DG, Ford GA, et al.: Exercise Induces Peripheral Muscle But Not Cardiac Adaptations After Stroke: A Randomized Controlled Pilot Trial. Arch Phys Med Rehabil. 2016; 97(4): 596–603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nForrester LW, Roy A, Krywonis A, et al.: Modular Ankle Robotics Training in Early Sub-Acute Stroke: A Randomized Controlled Pilot Study. Neurorehabil Neural Repair. 2014; 28(7): 678–687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPark D, Lee JH, Kang TW, et al.: Four-week training involving ankle mobilization with movement versus static muscle stretching in patients with chronic stroke: a randomized controlled trial. Top Stroke Rehabil. 2019; 26(2): 81–6. PubMed Abstract \| Publisher Full Text\n\nRaza C, Anjum R, Shakeel NA: Parkinson’s disease: Mechanisms, translational models and management strategies. Life Sci. 2019; 226: 77–90. PubMed Abstract \| Publisher Full Text\n\nHaff G, Triplett NT, National Strength & Conditioning Association (U.S.), eds. Essentials of Strength Training and Conditioning. Fourth edition. Human Kinetics; 2016. Reference Source\n\nDelafontaine A: Effects of stretching exercises on human gait: A systematic review and meta-analysis. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/N8ZXNB" }	[ { "id": "69874", "date": "14 Sep 2020", "name": "Fabrice Mégrot", "expertise": [ "Reviewer Expertise motor control", "biomechanics", "neurosciences", "nonlinear dynamics", "clinical gait/movement analysis and gross motor function of children with cerebral palsy" ], "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\nAuthors propose a systematic review and meta-analysis to investigate the effects of stretching programs on gait and to determine how stretching might be valuable for rehabilitation. From 150 studies identified through systematic searches and critical appraisal of the literature, 12 articles were considered by the authors.\nThe various demographics of the participants in the reported studies implied a mixed range of age and aetiology, even if old persons, either with an asymptomatic or a pathological health status, were the most represented individuals.\nIn young adults, two studies by Johanson are reported. Is it not clear if both groups of young adults with limited range of motion were identical in both studies and had the same characteristics? If yes, merging the two studies should make the deal, if not please provide specification about the second group.\nOverall, the methodology is correct, and the meta-analysis well described. The results show weak effects of stretching on gait parameters.\nThe main issue arises from the various parameters which are considered in all the studies, both during gait initiation and straight walking (spatiotemporal parameters, kinematics, joint strength and dynamics, muscle activity, etc…). Therefore, in the results and the discussion, grouping the types of parameters according to their role in the process of walking would add value to this manuscript. The effects, demonstrated or not, have not all the same meaning depending on whether functional parameters or kinematics angles or even muscle strength and activity are considered.\n\nGait should be defined in a better way, especially how it is related to the different measures in terms of processes, even if there is anyway no real benefit demonstrated by stretching used as a unique therapy. Surprisingly, no article in children with cerebral palsy has been uncovered, while the clinical care of these children is mostly based on muscle stretching.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6054", "date": "30 Oct 2020", "name": "ARNAUD DELAFONTAINE", "role": "Author Response", "response": "Reviewer 1 :Dear ReviewerWe greatly you for scrutinizing our manuscript and for his relevant comments. We feel that the manuscript has improved. Comment 1. In young adults, two studies by Johanson are reported. Is it not clear if both groups of young adults with limited range of motion were identical in both studies and had the same characteristics? If yes, merging the two studies should make the deal, if not please provide specification about the second group.The participants in both studies were different. In the study of Johanson et al. (2006), the participants were healthy adults with limited passive ankle-dorsiflexion range of motion (less than 8 degrees) and a history of lower limb overuse injury. In the study Johanson et al. (2009), the characteristics of the stretching program were the same as in Joahnson et al. (2006), but the participants did not have any history of lower limb overuse injury. Reply: To make this difference clearer, precisions were added in the “included studies” and “results” sections:Please see L158-163 Thus, 12 articles were ultimately included in this systematic review. Six studies evaluated the effects of stretching in healthy older adults 23,51–55, one in a frail elderly population 56, one study in an elderly population with stable symptomatic peripheral artery disease57, one in stroke patients 58, one study in adults with limited ankle range of motion (less than 8 degrees) associated with a history of lower limb overuse injury 59, one study in healthy adults with limited ankle dorsiflexion range of motion (less than 5 degrees) 60 and one in healthy young adults 61Please see L285-288: “The study of Johanson et al. (2006) examined the effects of stretching on healthy adults with limited passive ankle-dorsiflexion range of motion (less than 8 degrees) and a history of lower limb overuse injury50. Regarding the characteristics of the subjects, 19 subjects were included and the mean age was 30.3±9.8 years.”Please see L302-306: “The study of Johanson et al. (2009) examined the effects of stretching on young adults with limited passive ankle-dorsiflexion range of motion (less than 5 degrees) 51. It is worth noticed that these participants were not the same than in the study of Johanson et al. (2006). In contrast, the characteristics of the training programs were the same as in Johanson et al. (2006)”. Comment 2. In the results and the discussion, grouping the types of parameters according to their role in the process of walking would add value to this manuscript. The effects, demonstrated or not, have not all the same meaning depending on whether functional parameters or kinematics angles or even muscle strength and activity are considered.Reply: In the results and the discussion, we chose to group the participants by patient categories because differences in ages and health parameters may result in different training effects. However, in each patient category, each variable was considered separately in an organized way. Comment 3. Gait should be defined in a better way, especially how it is related to the different measures in terms of processes, even if there is anyway no real benefit demonstrated by stretching used as a unique therapy.Reply: We agree. We add a whole paragraph at the beginning of the introduction section to specify how gait can be related to the different variables seen in the review:Please see L35-44: “Gait is the medical term used to describe the human whole body movement of walking1. Gait involves internal and external forces that act on the body to move the center of mass (COM) across a given distance2. It depends on many biomechanical features that can be observed during gait analysis such as center of mass shifts, joint range of motion, forces, muscle activity, joint moments, and joint powers3. Spatiotemporal features (e.g. velocity, step length, stride length, step with, step variability) and kinematics parameters (range of motion) can be observed subjectively with functional evaluations by clinicians(e.g. the Tinetti test4 or the timed up and go test5), but, it can be further objectified with biomechanical analysis in a laboratory2. Kinetics variables (the forces that cause the body to move) must be collected in a laboratory environment with force plates (e.g.6–9 for recent studies that used this technic)”. Comment 4. Surprisingly, no article in children with cerebral palsy has been uncovered, while the clinical care of these children is mostly based on muscle stretching.Reply: We agree. However, no article in children with cerebral palsy fitted our inclusion criteria during the systematic search of the literature. We add a whole paragraph in a specific section “limitations of the study”:Please see L397-406: Some patient categories were not included in the present review, although muscle stretching is commonly indicated in their clinical care to reduce spasticity82. This is for example the case for children with cerebral palsy83. In fact, we were able to identify studies in the literature focusing on the effects of stretching on gait in this population during the first phase of the present review, but the protocol of these studies combined stretching with another form of training (e.g.84,85) or there was no control group (e.g.86). These studies therefore did not fit with the inclusion criteria of the present systematic review and were consequently excluded. Now, it should be stressed that the effectiveness of static stretching to improve motor function in children with cerebral palsy is still controversial87, although some authors showed that functional stretching exercises may be effective to improve gait86. Further randomized controlled trials are needed to explore the impact of stretching on gait in this population. Best regards Arnaud Delafontaine on behalf of all the authors" } ] }, { "id": "69875", "date": "09 Oct 2020", "name": "Xu Wei", "expertise": [ "Reviewer Expertise Sports medicine", "osteoporosis", "osteosarcoma", "Spine surgery", "Scoliosis" ], "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 this article is to analyse the effects of a stretching program on gait in each patient category by means of a systematic literature review and meta-analysis, comparing the gait outcomes of the intervention groups with the control groups. This is a very interesting research direction and it has good innovation. But different ages and diseases have different gait results and different stretching training modes and intervention methods. How to eliminate the bias caused by these differences in meta-analysis and comparison?\n\nFour meta-analysis were conducted for the following outcomes (Figure 2): gait speed, stride length, hip extension during gait and anterior pelvic tilt. Why choose these four dimensions? Is there any theoretical basis?\n\nKinetic variables: The study of Kerrigan et al. (2003)44 showed no significant difference between groups for hip torque (SMD= 0.35; 95% CI: -0.06, 0.75) and ankle plantar flexion power (SMD=0.00; 95% CI: -0.40, 0.40), with a moderate level of confidence (PEDro score: 6/10).: It seems that gait analysis cannot directly measure muscle power. sEMG can only evaluate muscle recruitment signals to evaluate muscle fiber contraction. Therefore, how is ankle flexion power measured by gait analysis?\n\nTwelve studies were included in the analysis. Stretching improved gait performance as assessed by walking speed and stride length only in a study with a frail elderly population, with small effect sizes. There is no strong evidence supporting the beneficial effect of using stretching to improve gait. I think that since stretching can improve gait parameters for adults with special weakness, the effect of stretching on gait is significant, although it has little effect on healthy adults or young adults. Should it be explained? In general, this study made a meta-analysis on the effect of stretching on gait, which has good innovation. However, whether gait analysis has guiding significance for different groups of rehabilitation training is still controversial especially for healthy adults, so the research conclusion of this paper has practical significance in clinical guidance.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "6055", "date": "30 Oct 2020", "name": "ARNAUD DELAFONTAINE", "role": "Author Response", "response": "Dear ReviewerWe greatly you for scrutinizing our manuscript and for his relevant comments. We feel that the manuscript has improved.Comment 1. Different ages and diseases have different gait results and different stretching training modes and intervention methods. How to eliminate the bias caused by these differences in meta-analysis and comparison?Reply: We add some precisions in the discussion section (healthy older adults paragraph) to specify how we have limited the risk of bias:Please see L353-366: When data were meta-analyzed, we ensured that the groups and the training characteristics were similar to limit the risk of bias. This explains that a limited number of studies was included in the meta-analysis. It is worth noticing that the stretching technic was the same (i.e. static stretching), but that details of interventions varied across these studies. For example, both studies selected for the meta-analysis of gait speed included hip flexors and plantar flexors stretching, but, one study included hip extensor stretching52 whereas the other did not51. This difference may partially explain the heterogenous results in the meta-analysis (I2=86%, p=0.007). In the same way, the heterogenous results observed in the meta-analysis of anterior pelvic tilt (I2=87%, p<0.01) may be explained by the stretching of additional muscle groups (hip extensors and plantar flexors) in the study of Cristopoliski et al. (2009) compared to the other studies (in which only hip flexors were stretched)53,55. Nevertheless, heterogeneity in the results was not systematically observed between studies that used slight different protocols, as showed by the consistent results in the meta-analyses of stride length (I2=59%, p=0.12) and hip extension (I2=0%, p=0.99). Thus, we assume that we have limited the risk of bias in the meta-analyses. Comment 2. Four meta-analysis were conducted for the following outcomes (Figure 2): gait speed, stride length, hip extension during gait and anterior pelvic tilt. Why choose these four dimensions? Is there any theoretical basis?Reply: Only these four outcomes fitted with the inclusion criteria for meta-analysis. Meta-analyses were performed only when more than one trial was identified for each outcome. Additionally, to reduce the risk of bias, we ensured that the groups were similar. For example, in the study of Watt et al. (2011), the intervention group had a significantly higher gait speed than the control group, so the trial was excluded of the meta-analysis. Comment 3. Kinetic variables: The study of Kerrigan et al. (2003)44 showed no significant difference between groups for hip torque (SMD= 0.35; 95% CI: -0.06, 0.75) and ankle plantar flexion power (SMD=0.00; 95% CI: -0.40, 0.40), with a moderate level of confidence (PEDro score: 6/10).: It seems that gait analysis cannot directly measure muscle power. sEMG can only evaluate muscle recruitment signals to evaluate muscle fiber contraction. Therefore, how is ankle flexion power measured by gait analysis?Reply: In the study of Kerrigan et al. (2003), “joint torque and power calculations were based on the mass and inertial characteristics of each lower-extremity segment, the derived linear and angular velocities and accelerations of each lower-extremity segment, and the ground reaction force and joint center position estimates. Joint torques and powers were normalized for body weight and height and were reported as external in newton meters per kilogram meters (N.m/kg.m) and watts per kilogram meters (W/kg.m), respectively”.Unless special recommendation of the reviewer or the Editor, we feel it is not necessary to add how the ankle flexion power was measured in the study of Kerrigan et al. (2003). Comment 4. I think that since stretching can improve gait parameters for adults with special weakness, the effect of stretching on gait is significant, although it has little effect on healthy adults or young adults. Should it be explained? Reply: We add some precisions in the discussion section (young adults paragraph) to specify why stretching is less interesting to improve gait parameters in young healthy adults:Please see L387-390: In healthy adults, the interest of practicing stretching to improve gait seems limited as they are assumed to have sufficient mobility for walking. Moreover, the included study involved athletic males 61, a population that is known to be more flexible than inactive persons 81. Stretching should be more indicated when range of motion is limited24.Best regards Arnaud Delafontaine on behalf of all the authors" } ] } ]	1	https://f1000research.com/articles/9-984
https://f1000research.com/articles/9-1285/v1	30 Oct 20	{ "type": "Brief Report", "title": "Lethality model for COVID-19 based on social determinants of health: an approximation in 67 countries", "authors": [ "Fred G. Manrique-Abril", "Mario J. Pacheco-López", "Cristian F. Téllez-Piñerez", "Felipe Ortíz-Rico", "Juan D. Sarmiento-Páramo", "Mario J. Pacheco-López", "Cristian F. Téllez-Piñerez", "Felipe Ortíz-Rico", "Juan D. Sarmiento-Páramo" ], "abstract": "Background: Social, geographic, economic, demographic, and health factors were analysed to identify some social determinants related to case fatality of COVID-19 in 67 countries. Methods: A mixed generalized linear model with beta distribution with random intercept was used to estimate the effects of the explanatory variables on the lethality for COVID-19 in 67 countries. Results: The case fatality rate (CFR) was highest in the countries with the highest percentages of people over 60 years of age, the highest number of hospital beds,the highest mortalit yrate from diabetes, and the highest number of COVID-19 tests. Additional increases were seen based on literacy rates, health investment, death rate from cardiovascular disease, poverty rate, ratio of men, number of air passengers mobilized, number of days from the ﬁrst reported case to the start of quarantine, death rate from respiratory infections, and percentage of people living in urban areas. Conclusions: The statistical model used to predict lethality is novel because it allows the magnitude of the CFR to be analysed over a logistic model that classiﬁes countries considering the presence and absence of deaths. When considering a beta distribution with excess zeros, the model also allows countries without reported deaths due to COVID-19 at the analysed cut-off date to be included.", "keywords": [ "COVID-19 Pandemic", "Beta Regression", "Lethality", "social determinants." ], "content": "Introduction\n\nCOVID-19 is an infectious disease caused by the SARS-CoV-21,2 virus that originated in early December 2019 from the city of Wuhan, the capital of Hubei Province, in China3,4. Its virulence and reproducibility gave the virus a special character, and the WHO declared the disease a pandemic on March 11, 20205, due to the high number of infected people and deaths that it had caused around the world. In comparison, the 2003 outbreak of severe acute respiratory syndrome (SARS) had a fatality rate of about 10% (8,098 cases and 774 deaths), and Middle East respiratory syndrome (MERS) killed 34% of people with the disease between 2012 and 2019 (2,494 cases and 858 deaths)6.\n\nHowever, even though its death rate is minuscule, COVID-19 so far has caused more deaths (1871) than SARS and MERS combined (1632)6. As such, many epidemiologists, statisticians, mathematicians, and other researchers have focused on forecasting the number of new cases at the regional or global level4,5,7–9 and on detecting possible measures to decrease the probability of contagion and death in the most vulnerable populations3,10. Few studies can determine with sufficient statistical rigour which factors decrease the probability of death from COVID-19 with any level of statistical significance.\n\nThe SIR model is on the simplest compartmental models, and many models are derivatives of this basic form. The model consists of three compartments:\n\nS: The number of susceptible individuals. When a susceptible and an infectious individual come into \"infectious contact\", the susceptible individual contracts the disease and transitions to the infectious compartment. I: The number of infectious individuals. These are individuals who have been infected and are capable of infecting susceptible individuals. R for the number of removed (and immune) or deceased individuals. These are individuals who have been infected and have either recovered from the disease and entered the removed compartment, or died. It is assumed that the number of deaths is negligible with respect to the total population. This compartment may also be called \"recovered\" or \"resistant\". This model is reasonably predictive for infectious diseases that are transmitted from human to human.5. With these models, indicators of lethality are also calculated by dividing the number of deaths by the total number of those infected. This simple calculation is called the case fatality rate (CFR).\n\nIn practice, when a pandemic enters a territory, the susceptible often do not get sick, do not infect themselves, and do not die as established in the SIR model. Thus, one assumes that other factors are included in the SIR or susceptible, exposed, infected, recovered (SEIR) models (a compartmental deterministic model) that determine the infection and mortality. This would substantially modify the CFR, which is a measure of a disease’s severity and indicates the disease’s importance in terms of its ability to produce death. Given this order of ideas, lethality is calculated as CFR:\n\n\n\nWhere Nd=Number of deaths from a disease in a given period and Nc=Number of cases diagnosed with the same disease in the same period.\n\nSince each country has different characteristics in terms of the social determinants and the cases and deaths COVID-19 pandemic report is uniform, it is necessary to collect the available information to model the virus’s CFR and identify the factors that statistically increase or modify the risk of death. A model will give researchers a powerful tool to estimate the probability of death from COVID-19 and its causes will give researchers a powerful tool, alongside the existing ones for predicting the number of cases, for decision-making aimed at alleviating the consequences of deadly events. That is why, in this article, we propose to model the CFR for COVID-19 using a mixed beta model and adjust it for some social, geographic, economic, health and demographic variables as social determinants in the global context of the COVID-19 pandemic.\n\n\nMethods\n\nBased on the assumptions that the CFR by country can be affected by socioeconomic factors and by health and non-pharmaceutical measures, and that its behaviour may have varied since the start of the pandemic, a mixed beta11 regression model with random intercept was used to estimate the effects of the explanatory variables on the CFR for COVID-19 in 67 countries. A significance level of 10% was used to identify factors that had a statistically significant relationship with the COVID-19 CFR worldwide, see Table 1.\n\nData on the number of daily cases and deaths from COVID-19 were obtained from the Johns Hopkins University repository to construct the CFRs by country. The possible predictors of the CFR by country were obtained from the repositories of Population Pyramid, the Word Bank, the World Health Organization and the Topographic Mission Radar Shuttle, as well as from health policy, news and macro data. The seven-day corrected fatality rate was calculated nine times: on April 8, 15, 22 and 29; May 6, 13, 20 and 27; and June 3.\n\nThe parameterized beta probability density function, denoted by Y ∼ β(µ, ϕ), in terms of its mean (µ) and precision (ϕ) parameters,12, is given by\n\n\n\n0 < y < 1, with 0 < µ < 1,ϕ > 0, E(Y) = µ, Var(Y) = µ(1 − µ)/(1 + ϕ), and γ is as the Gamma function. For a random sample from Yi ∼ β(µ, ϕ), and assuming ϕ is constant, the beta regression model13 is specified by g(μi)=xiτβ=ηi, with β =(β1, β2, ..., βp)τ being a vector of the (p) unknown regression coefficients, xi = (xi1 ,..., xip)τ being a vector of (p) known covariates and ηi being a linear prediction. The model specification is completed by the choice of a link function, g(∗) : (0, 1) → IR. We adopt the Logit function g(µ) = log(µ/(1 − µ)). This model does not contemplate possible dependencies such as those induced by multiple measurements on the same observational unit or time.\n\nIncluding latent random effects on a grouped data structure is a parsimonious strategy, as compared to adding parameters to the fixed part of the model, while still accounting for nuisance effects.\n\nLet Yij denote an observation j = 1, . . . , ni within group i = 1, . . . , q and yi denote a ni -dimensional vector of measurements from the i-th group. Let bi be a q-dimensional vector of random effects, and assume the responses Yi j are conditionally independent with density\n\n\n\nand a link function g(μi)=xiτβ=ηi, vectors of known covariates xi j and zi j with dimensions (p) and (q), respectively, a (p) -dimensional vector of unknown regression parameters β and the precision parameter ϕ. The model specification is completed by bi\|Σ ∼ N(0, Σ) Gaussian random effects.\n\nA model was developed that accounted for the variables’ nature, which is bounded in the interval [0, 1]. A mixed beta model was then assumed to model the CFR, and a step procedure did what for the final selection. The statistical software R version 3.6.3 was used to fit the model, specifically the GAMLSS function. See 14 for more technical details.\n\n\nResults\n\nThe distribution of CFR by country is shown in Figure 1. For June 3, high CFR stand out in countries like Belgium, France, Mexico, England, Italy, Hungary, Holland and Sweden, as do the low values in Russia, Australia, New Zealand, Malaysia, Chile and Israel.\n\nAccording to the adjusted model (Figure 2), The variables with greater statistical significance in the model were three (60years, Health and Sex), the CFR increases inversely with health expenditure; and directly the CFR is higher where more people over 60 years of age live and there is a male population predominance.\n\nSo too does CFR increase, if the number of beds increases, if there are more diabetes mortality or greater death rate from cardiovascular diseases or death rate from respiratory infections. The CFR increase if there are a greater number of tests for SARS-CoV-2. The register lethality change with the literacy proportion in the country; the CFR increase with poverty, with a greater number of air passengers mobilised, number of days from the first case reported until the beginning of the quarantine, and percentage of people living in the urban area.\n\nAccording to the model, the CFR decreased in the countries with the low death rate from respiratory diseases, low obesity rate, low death rate from HIV/AIDS, low physicians per thousand inhabitants, low GDP in health care, low population density and a high number of nurses per thousand inhabitants.\n\n\nDiscussion\n\nEconomic Commission for Latin America and the Caribbean (ECLAC) studied the relationship between mortality in Latin American countries with some characteristics of their social development. A set of four social indicators (hospital beds per 1000 inhabitants, protein consumption, literacy and proportion of households with drinking water) have high linear correlations with life expectancy at birth (r = 0.94)15.\n\nThis global analysis of the socioeconomic determinants of COVID-19 mortality had several limitations. Countries had clear differences in their stage of historical development, with different modes of production being found. On the other hand, mortality was highly heterogeneous mortality between countries and in different populations within each country. Finally, the review of the available information indicates that the analysis is not systematic or complete, particularly for the most important analysis categories.\n\nNevertheless, the available data allow us to reach some important conclusions. For example, adjustment of the presented model allows factors related to COVID-19 case fatality to be identified. The methodology can be replicated with different cut-off dates, include more related factors or be used at sub-national levels.\n\nThe calculated case fatality differs from another model’s fatality proposed by researchers at the University Hospital of Lausanne and collaborators (15), who estimated a CFR of 3.59% as of March 1, 2020, considering that an adjustment must be made for the 14 previous days before death to coincide with the date of infection spread and infection, representing the maximum incubation period for the virus. The findings show that current figures may underestimate the potential threat of COVID-19 in symptomatic patients16.\n\n\nConclusions\n\nThe statistical model used to model fatalities is novel because it allows to analyse the magnitude of the fatality rate over a logistic model that classifies the countries considering the presence and absence of fatalities. Additionally, when considering a beta distribution with excess zeros, countries without reported deaths due to COVID-19 at the analysed cut-off date can be included. Finally, the variability obtained from the partial estimates made by the countries on different dates can be analysed.\n\nThe model’s results may have a high sensitivity to the data on reported case fatality from COVID-19, due to the sub-registry inherent to the measurement of infected people, because of the small number of tests carried out in some countries. However, the modelling strategy used is optimal for the type of information considered and can be replicated with new updates to the lethality measurement17.\n\n\nData availability\n\nThe population, cases, test number and deaths number by country were obtained from the repository Johns Hopkins University (JHU) at https://github.com/CSSEGISandData/COVID-19/tree/master/csse_covid_19_data.\n\nWorld Bank data were obtained using the following program: https://github.com/vincentarelbundock/WDI.\n\nR analysis code for this study is available at: https://github.com/DataStatistic/covid19model.\n\nArchived code at time of publication: http://doi.org/10.5281/zenodo.400700518.\n\nLicense: Creative Commons Attribution 4.0 International license.", "appendix": "References\n\nGorbalenya AE, Baker SC: The species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2. Nat Microbiol. 2020; 5(4): 536–544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu R, Zhao X, Li J, et al.: Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet. 2020; 395(10224): 565–574. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Q, Guan X, Wu P, et al.: Early transmission dynamics in wuhan, china, of novel coronavirus–infected pneumonia. N Engl J Med. 2020; 382(13): 1199–1207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi R, Pei S, Chen B, et al.: Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov-2). Science. 2020; 368(6490): 489–493. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManrique-Abril FG, Agudelo-Calderon CA, González-Chordá VM, et al.: SIR model of the COVID-19 pandemic in Colombia. Rev Salud Publica. 2020; 22(2): 1–9.\n\nMahase E: Coronavirus: covid-19 has killed more people than SARS and MERS combined, despite lower case fatality rate. BMJ. 2020; 368: m641. ISSN 1756-1833. PubMed Abstract \| Publisher Full Text\n\nGiuliani D, Dickson MM, Espa G, et al.: Modelling and predicting the spatiotemporal spread of Coronavirus disease 2019 (COVID-19) in Italy. arXiv Prepr. 2020. Reference Source\n\nGonzález-Jaramillo V, González-Jaramillo N, Gómez-Restrepo C, et al.: Impact of the COVID-19 pandemic on the Colombian population according to mitigation measures. Preliminary data from epidemiological models for the period March 18 to April 18, 2020. Rev Salud Pública. 2020; 22(2): 1–6. Reference Source\n\nPeng L, Yang W, Zhang D, et al.: Epidemic analysis of COVID-19 in China by dynamical modeling. arXiv Prepr. 2020. Reference Source\n\nFerguson NM, Laydon D, Nedjati-Gilani G, et al.: Impact of non-pharmaceutical interventions (NPIs) to reduce COVID-19 mortality and healthcare demand. Imperial Ac Uk. 2020; 1: 3–20. Reference Source\n\nOspina R, Ferrari SLP: A general class of zero-or-one inﬂated beta regression models. Comput Stat Data Anal. 2012; 56(6): 1609–1623. Publisher Full Text\n\nJorgensen B: Proper dispersion models (with discussion). Brazilian J Probab Stat. 1997; 11(2): 89–140. Reference Source\n\nFerrari SLP, Cribari-Neto F: Beta regression for modelling rates and proportions. J Appl Stat. 2004; 31(7): 799–815. Publisher Full Text\n\nRigby RA, Stasinopoulos DM, Lane PW: Generalized additive models for location, scale and shape. J R Stat Soc Ser C Appl Stat. 2005; 54(3): 507–554. Publisher Full Text\n\nRosas HB: Determinantes económicos y sociales de la mortalidad en América Latina. Rev Cuba Salud Publica. 2017; 43(2): 287–312. Reference Source\n\nBaud D, Qi X, Nielsen-Saines K, et al.: Real estimates of mortality following covid-19 infection. Lancet Infect Dis. 2020; 43(7): 773. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInfobae:¿Por qué no sabemos la verdadera tasa de mortalidad de COVID-19? 2020. Reference Source\n\nPacheco López M: DataStatistic/covid19model: First release COVID19 beta model. 2020. http://www.doi.org/10.5281/zenodo.4007005" }	[ { "id": "74024", "date": "13 Nov 2020", "name": "Ahmed M. Abbas", "expertise": [ "Reviewer Expertise Obstetrics and Gynecology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is interesting and the authors covered the aspects of the paper successfully. The research methods are appropriate and the results are presented in a clear manner. Discussion and conclusions are sufficient. Citations are updated and covering the topic. Tables are good. The article is very concise and informative.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "81713", "date": "26 Mar 2021", "name": "Hector Florez", "expertise": [ "Reviewer Expertise Bioinformatics", "data science", "computer science", "mathematics" ], "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 presents a mixed generalized linear model with beta distribution with a random intercept to estimate the effects of the explanatory variables on the CFR for COVID-19 in 67 countries. It is not clear why the authors concluded that a mixed generalized linear model with beta distribution with a random intercept is a proper model to perform the desired analysis. I suggest describing how the explanatory variables in table 1 were selected. I suggest extending the explanation of the equations presented in the subsection \"Beta mixed models\" Most variables have an effect around zero except for three variables (60years, Sex, Health). Based on this result, can someone interpret that just these three variables are worth to be analyzed? I am afraid that this result deserves a deeper discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "6938", "date": "28 Jul 2021", "name": "Fred Manrique-Abril", "role": "Author Response", "response": "It is not clear why the authors concluded that a mixed generalized linear model with beta distribution with a random intercept is a proper model to perform the desired analysis. Given that the response variable (Covid fatality rate) is observed for each country on more than one occasion, it is necessary to control the effect of the country in adjusting a model that explains said response variable. This requires a random intercept model. Most variables have an effect around zero except for three variables (60years, Sex, Health). Based on this result, can someone interpret that just these three variables are worth to be analyzed? I am afraid that this result deserves a deeper discussion. In the model adjustment, 20 variables out of a total of 22 explanatory variables turn out to be significant and due to the nature of each variable, the parameter associated with each explanatory variable can have a value close to zero without this meaning that the variable does not have an effect. on the response variable. For example, the variable People aged over 60 years (%) has a different nature than Sex ratio (men / women) and these in turn have a very different nature from Investment in health as a percentage of GDP, therefore they are not comparable their effects on each other. I suggest extending the explanation of the equations presented in the subsection \"Beta mixed models\" The proposed beta regression model allows the adjustment of a model that establishes the relationship between the 22 proposed explanatory variables in which the effect of the country on lethality can be controlled and separated from the effect of each explanatory variable. For a more detailed explanation of the model see Verkuilen J, Smithson M. Mixed and Mixture Regression Models for Continuous Bounded Responses Using the Beta Distribution. Journal of Educational and Behavioral Statistics. 2012;37(1):82-113. doi:10.3102/1076998610396895" }, { "c_id": "7107", "date": "21 Sep 2021", "name": "Fred Manrique-Abril", "role": "Author Response", "response": "Given that the response variable (COVID-19 fatality rate) is observed for each country on more than one occasion, it is necessary to control the effect of the country in adjusting a model that explains said response variable. This requires a random intercept model. Most variables have an effect around zero except for three variables (60years, Sex, Health). Based on this result, can someone interpret that just these three variables are worth to be analyzed? I am afraid that this result deserves a deeper discussion. In the model adjustment, 20 variables out of a total of 22 explanatory variables turn out to be significant and due to the nature of each variable, the parameter associated with each explanatory variable can have a value close to zero without this meaning that the variable does not have an effect. on the response variable. For example, the variable People aged over 60 years (%) has a different nature than Sex ratio (men / women) and these in turn have a very different nature from Investment in health as a percentage of GDP, therefore they are not comparable their effects on each other. I suggest extending the explanation of the equations presented in the subsection \"Beta mixed models\" The proposed beta regression model allows the adjustment of a model that establishes the relationship between the 22 proposed explanatory variables in which the effect of the country on lethality can be controlled and separated from the effect of each explanatory variable. For a more detailed explanation of the model see Verkuilen J, Smithson M. Mixed and Mixture Regression Models for Continuous Bounded Responses Using the Beta Distribution. Journal of Educational and Behavioral Statistics. 2012;37(1):82-113. doi:10.3102/1076998610396895" } ] } ]	1	https://f1000research.com/articles/9-1285
https://f1000research.com/articles/9-1284/v1	30 Oct 20	{ "type": "Research Article", "title": "Validation of the wrist blood pressure measuring device Omron RS6 (HEM-6221-E) among obese Sudanese patients according to the European Society of Hypertension International Protocol Revision 2010", "authors": [ "Elrazi A. Ali", "Saeed M. Omar", "Yassin Ibrahim", "Osama Al-Wutayd", "Ishag Adam", "Elrazi A. Ali", "Yassin Ibrahim", "Osama Al-Wutayd", "Ishag Adam" ], "abstract": "Background: Electronic devices for measuring blood pressure (BP) need to go through independent clinical validation as recommended by different authorities, both in general and specific populations. The aim of this study was to assess the validity of the Omron RS6 (HEM-6221-E) wrist oscillometric devices in obese Sudanese patients. Methods: Of 90 obese individuals invited for recruitment, 33 were included in the study, and had their BP at the level of the wrist measured using Omron RS6 and standard mercury sphygmomanometer. Two observations were made and the mean was taken. BP differences between the two methods for the 33 participants were classified into three categories (≤5, ≤10, and ≤15 mmHg), according to the European Society of Hypertension-International Protocol revision 2010 (ESH-IP2) criteria. This was then used to assess the validity of the tested Omron RS6 device. Results: Participants had a mean age of 56.97 years (standard deviation (SD), 8.75; range, 36-79). Average systolic blood pressure (SBP) was 146.21 mmHg (SD, 23.07; range, 107-182), and average diastolic blood pressure (DBP) was 93.82 mmHg (SD, 16.06; range, 67-128). There was a good agreement between the two observations using the OMRON RS6 and the standard sphygmomanometer: −4 to + 3 mmHg for SBP and −4 to +4 mmHg for DBP, with the mean difference of 1.73±1.11 mmHg for SBP and 1.49±1.02 mmHg for DBP. Conclusion: Thus, the Omron RS6 (HEM-6221-E) is a valid and suitable measure of BP according to ESH-IP2.", "keywords": [ "validity", "Omron RS6®", "obesity", "wrist", "blood pressure" ], "content": "Introduction\n\nHypertension is defined as systolic blood pressure (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg1. It is a global public health issue, and is recognized to be the major factor contributing to the burden of heart disease, stroke, kidney failure and premature mortality and disability all over the world2. In spite of this burden, a simple preventive and non-invasive measure such as checking and monitoring blood pressure (BP) is expected to decrease the associated cardiovascular mortality with hypertension dramatically3. Hypertension is mostly symptomless particularly in the early stages unless there are complications or other reasons to reveal it, which is why many people go undiagnosed. Moreover, those who are diagnosed may not have access to treatment and may not be able to successfully control their hypertension long term. In order to ensure that the disease is well-controlled, BP should be monitored regularly.\n\nAccurate BP readings via a reliable device is equally important, since this will influence diagnosis and treatment. Generally, there are electronic, mercury and aneroid devices that are used to measure BP. The World Health Organization recommends the use of affordable and reliable electronic devices that have the option to select manual readings, because mercury is toxic and aneroid devices need calibrations and trained personnel for use1. Therefore, these electronic devices must be of certain accuracy. As a result, many BP measuring devices have been validated to meet international standards for the use by the general population4,5. However, few studies for the validation of electronic devices in specific groups, such as the elderly, infants and obese individuals, have been done.\n\nFor overweight and obese individuals, BP measurement is more challenging. First, obesity itself plays a major role in elevating BP through increasing insulin resistance and creating a cycle that eventually ends to metabolic syndrome6. Secondly, obesity affects individuals in very different ways, and many obese people have an significantly increased arm circumference. Hence, if BP is not measured with an appropriately sized cuff it will lead to false high readings, which may conduce unnecessary treatment7. In order to solve the problem of adiposity, some studies use the forearm instead of the arm to measure BP in obese individuals, which results in overestimated BP by approximately 7-15 mmHg – this has to be corrected with equations8. Recently, a study targeting obese individuals compared the BP readings using two devices, one at the level of the arm (brachial artery) and the other at the wrist level (radial artery) to a standard method. These authors found that the electronic device's readings varied significantly from the readings obtained by the standard sphygmomanometer and did not meet the required criteria for obese adults5. In this study, we aimed to evaluate BP measurements among obese hypertensive Sudanese patients using the radial artery (wrist level) compared to the standard brachial measured using a mercury sphygmomanometer and non-electronic stethoscope to attain more valuable information about the validity of using such a method.\n\n\nMethods\n\nOmron RS6 (HEM-6221-E) is a fully automated device for self/home BP measurement at the wrist level using the oscillometric method. Inﬂation is by automated fuzzy logic–controlled electric pump and deﬂation is by automatic pressure release valve. It measures 87 mm × 64 mm × 14 mm (width × height × depth). It has a single wrist cuff, which can be used for wrist size between 13.5–21.5 cm. The device demonstrates the pulse and BP (SBP and DBP) in a digital liquid crystal screen. The device can detect a BP range from 0 to 299 mmHg and pulse rate from 40 to 180 beats/min. Additionally, it can save the last 90 BP readings and it has a position sensor for the wrist and the ability to detect irregular pulse and body motion.\n\nA cross-sectional study was conducted at Gadarif Hospital outpatient clinic in eastern Sudan. Patients who fulfilled the following inclusion criteria were enrolled in the present study: obese (body mass index (BMI) ≥30 kg/m2), adult (age ≥25 years), male and female hypertensive patients, able to give informed consent. BMI was computed from measured weight and height using standard methods. Individuals with abnormal rhythm or uncertainly DBP were excluded.\n\nThis study was conducted among the obese Sudanese population. A total of 90 participants volunteered to take part in the study and met the inclusion criteria, with at least 30 men and 30 women. Participants were invited to take part in the study during their regular doctor’s appointment.\n\nParticipants were recruited to ensure a uniform distribution of test pressures across the BP range: 90–180 mmHg for SBP and 40–130 mmHg for DBP. The participants were divided into three groups as per their SBP and DBP readings: SBP – low (90–129), medium (130–160), and high (161–180); for DBP – low (40–79), medium (80–100) and high (101–130). Readings outside these ranges were included and were either categorized as low or high. In each of the three SBP and DBP ranges, a minimum of 10 to 12 patient was included according to the European Society of Hypertension-International Protocol revision 2010 (ESH-IP2) guidelines with at least 10 men and 10 women in the sample size (Table 1).\n\n*Had to leave before for personal reason before completing the measurements. SBP, systolic blood pressure; DBP, diastolic blood pressure\n\nProcedure. In the outpatient clinic, BP measurements were taken in a room with a comfortable temperature, and ambient noise kept to minimum to avoid disruption during auscultation. Patients who fulfilled the inclusion criteria were allowed to rest for 10 minutes sitting on a chair in upright position with their legs uncrossed and back supported, with complete exposure of the left arm and forearm (at the time of BP checking) so there were no intervening clothes between the cuff and the arm, which might reduce the blood flow. Participants were encouraged to avoid talking or using mobile phones. The arm circumference was checked, and the suitable cuff applied. All the measurements were obtained from the left arm at the heart level.\n\nBP measurements were obtained first by the standard mercury sphygmomanometer then alternating with the test device, so that we obtained nine consecutive BP measurements from each participant: five measurements from the standard mercury sphygmomanometer and four measurements from the tested device.\n\nBlood pressure measurements. The manufacturer of the Omron RS6 (HEM-6221-E) was asked to provide a standard model, and the automated device was used after familiarization sessions in the outpatient clinic for one week, during which time the team performed 12 test measurements and accustomed themselves to the device and the study. No problems in this familiarization period were encountered.\n\nFor the standard test, two stethoscopes and two mercury sphygmomanometers with different cuff sizes were carefully checked prior to the study.\n\nThe working team composed of three personnel, two observers and a supervisor, well trained in BP measurement using standard mercury sphygmomanometer and stethoscope with well-fitting earpieces. They were of good health, hearing, and sight and able to follow the menisci at eye level from 40 mmHg to 180 mmHg. The two observers measured BP using two mercury sphygmomanometer and these readings were used as a reference. Observers were blinded from each other’s readings and from the device readings. They took BP measurement simultaneously and record them to the nearest 2 mmHg. The supervisor checked the agreement of BP values between the two observers so that any readings that varied by 4 mmHg or more were repeated. The supervisor measured the BP using the test wrist device. Two observations were made.\n\nBefore measuring BP, the observer measured the arm circumference in order to use the appropriate size cuff for the standard mercury sphygmomanometer: small cuff for an arm circumference 17–21.9 cm, a regular cuff for an arm circumference of 22–31.9 cm, a large cuff for an arm circumference of 32–42 cm and for patients with arm circumference more than 42 cm an extra-large cuff is used.\n\nThe ESH-IP29 was used as a guide for data analysis9. Accordingly, data was analyzed, presented and expressed to assess the ability of the device to pass or fail to pass the validation protocol requirements. SPSS (version 22) and Microsoft Excel software were used to perform all data analysis. Measurements obtained by the two observers for the standard device were compared and their average was taken, which was later compared with the test device SBP and DBP. The numbers of differences for the tested device and the observer using mercury sphygmomanometer within 5, 10, and 15mmHg, were calculated for both SBP and DBP then the average differences of the values with the standard deviation between the SBP and DBP of the mercury and the tested device were obtained. Finally, Bland–Altman plots were performed for both SBP and DBP to show the differences of device-observer versus average device and observer values for all the 99 pairs of comparisons.\n\nEthical approval was received from the Ethics Committee at the Faculty of Medicine, Gadarif University, Sudan (reference number: 2017/09). Written informed consent to participate was collected from each participant before taking part in the research.\n\n\nResults\n\nIn total, 90 obese participants were screened for inclusion into this study, 57 were excluded (36 due to range adjustment, 18 due to observers' disagreement and 3 left before completing the sequence of measurements for personal reasons). Therefore, data related to 33 participants (12 men and 21 women) who fulfilled the requirements and the criteria of the ESH-IP protocol were analyzed.\n\nMean (±standard deviation) age of participants was 56.97±8.75 years (range, 36–79 years); mean wrist circumference was 22.58±3.25 (range, 13–28 cm); mean height was 163.52±13.54 cm (range, 116–180; mean weight of 104.45±11.45 kg (range, 88–136); and mean BMI of 38.74±8.26 (range, 31.48–81.75). The mean BP values for SBP was 146.21±23.07 mmHg (range, 107–182 mmHg) and for DBP was 93.82±16.06 mmHg (range, 67–128 mmHg). Respondents' characteristics and BP measurements are summarized in Table 2 and Table 3.\n\nSBP, systolic blood pressure; DBP, diastolic blood pressure\n\nThere was adequate agreement between the tested wrist BP measuring device (Omron RS6 (HEM-6221-E)) and the mercury sphygmomanometer (Table 4). All the dots for both SBP and DBP measurements were inside the ±15 mmHg limits (Figure 1, Table 5).\n\nSBP, systolic blood pressure; DBP, diastolic blood pressure\n\n(A) Systolic blood pressure; (B) diastolic blood pressure.\n\nSBP, systolic blood pressure; DBP, diastolic blood pressure\n\n\nDiscussion\n\nIn this study, the Bland-Altman plot showing the observers differences in SBP and SBP reveal that there is adequate agreement between the tested wrist BP measuring device (Omron RS6 (HEM-6221-E)) and the mercury sphygmomanometer (Figure 1). The SBP and DBP plot reflects the overall distribution of measurements among study subjects. All the dots for both systolic and diastolic blood pressure were inside the ±15 mmHg limits.\n\nA very recent study10 also showed that the OMRON RS7 device fulfils the validation criteria of ESH-IP validation protocol in two independent study centres among subjects, showing inter-centre reproducibility.\n\nIn our study, the screening process to include and exclude participants was time consuming. In addition, there was a difficulty in recruiting participants with high blood pressure. According to the validation protocol of ESH-IP, three studies have already been carried out to confirm the validity of the Omron RS6 (HEM-6221-E) device. Validation among the general population confirmed the general validity of the device11, however another study reported that the device failed to fulfil the ESH-IP revision 2010 requirements among obese subjects5. Deutsch et al. used the Omron RS6 position sensor with PSON or PSOFF, which showed that the position sensor is important for the function of the device at the wrist level and it improves the accuracy of the measurements by decreasing variations in wrist height12.\n\nThis study included only participants meaning that extrapolation of the results should be done with caution to individual who are not obese. Moreover, device accuracy needs to be adjusted to wrist circumference and position during measurement.\n\n\nConclusion\n\nThe tested device, Omron RS6 (HEM-6221-E), achieved all the required standards for self/home measurement of blood pressure at the wrist level set by the ESH-IP, and accordingly would safely be recommended for personal use at home among obese patients provided that the manufacturer’s instructions are followed.\n\n\nData availability\n\nOpen Science Framework: Validation of the wrist blood pressure measuring device Omron RS6 (HEM-6221-E) among obese Sudanese patients compared with a standard mercury sphygmomanometer: a cross-sectional study according to the European Society of Hypertension International Protocol Revision 2010, https://doi.org/10.17605/OSF.IO/7S5D313 (registered on 18th October 2020: osf.io/w7dtn).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors would like to thank all patients who participated in the study\n\n\nReferences\n\nWHO: A global brief on Hypertension WHO 2013. 2013; 18. Reference Source\n\nPoulter NR, Prabhakaran D, Caulfield M: Hypertension. Lancet. Lancet Publishing Group. 2015; 386(9995): 801–812. PubMed Abstract \| Publisher Full Text\n\nFarley TA, Dalal MA, Mostashari F, et al.: Deaths preventable in the U.S. by improvements in use of clinical preventive services. Am J Prev Med. 2010; 38(6): 600–609. PubMed Abstract \| Publisher Full Text\n\nChen W, Zeng ZL, Bing S, et al.: Validation of the Grandway MD2301 digital automatic blood pressure monitor according to the European Society of Hypertension International Protocol. Blood Press Monit. 2016; 21(4): 259–261. PubMed Abstract \| Publisher Full Text\n\nAzaki A, Diab R, Harb A, et al.: Questionable accuracy of home blood pressure measurements in the obese population - Validation of the Microlife WatchBP O3® and Omron RS6® devices according to the European Society of Hypertension-International Protocol. Vasc Health Risk Manag. 2017; 13: 61–69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLandsberg L, Aronne LJ, Beilin LJ, et al.: Obesity-related hypertension: pathogenesis, cardiovascular risk, and treatment: a position paper of The Obesity Society and the American Society of Hypertension. J Clin Hypertens (Greenwich). 2013; 15(1): 14–33. PubMed Abstract \| Publisher Full Text\n\nFonseca-Reyes S, de Alba-García JG, Parra-Carrillo JZ, et al.: Effect of standard cuff on blood pressure readings in patients with obese arms. How frequent are arms of a 'large circumference'? Blood Press Monit. 2003; 8(3): 101–106. PubMed Abstract \| Publisher Full Text\n\nPierin AMG, Alavarce DC, Gusmão JL, et al.: Blood pressure measurement in obese patients: comparison between upper arm and forearm measurements. Blood Press Monit. 2004; 9(3): 101–105. PubMed Abstract \| Publisher Full Text\n\nO’Brien E, Atkins N, Stergiou G, et al.: European Society of Hypertension International Protocol revision 2010 for the validation of blood pressure measuring devices in adults. Blood Press Monit. 2010; 15(1): 23–38. PubMed Abstract \| Publisher Full Text\n\nTasic D, Topouchian J, Dragisic D, et al.: Reproducibility of the European Society of Hypertension - International Protocol for validation of blood pressure measuring devices in obese patients. J Hypertens. 2019; 37(9): 1832–1837. PubMed Abstract \| Publisher Full Text\n\nTakahashi H, Yoshika M, Yokoi T: Validation of Omron RS8, RS6, and RS3 home blood pressure monitoring devices, in accordance with the European Society of Hypertension International Protocol revision 2010. Vasc Health Risk Manag. 2013; 9: 265–272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeutsch C, Krüger R, Saito K, et al.: Comparison of the Omron RS6 wrist blood pressure monitor with the positioning sensor on or off with a standard mercury sphygmomanometer. Blood Press Monit. 2014; 19(5): 306–313. PubMed Abstract \| Publisher Full Text\n\nAdam I, Omar SM, Ibrahim Y, et al.: Validation of the wrist blood pressure measuring device Omron RS6 (HEM-6221-E) among obeseoObese Sudanese patients compared with a standard mercury sphygmomanometer: a cross-sectional study ElrazipPatients according to the European Society of Hypertension International Protocol Revision 2010. 2020. http://www.doi.org/10.17605/OSF.IO/7S5D3" }	[ { "id": "87886", "date": "12 Jul 2021", "name": "James A. Hodgkinson", "expertise": [ "Reviewer Expertise Blood pressure monitoring" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 presents a successful validation of a blood pressure monitor in an obese population. It satisfies the methodological requirements for a validation study and is written clearly and succinctly.\nIt is to be noted (as the authors do) that a validation study of the same monitor in an equivalent population has been previously conducted and found the monitor failed validation. This is perfectly reasonable in itself, but perhaps raises the question of why the authors felt another validation study was warranted, and if they have any insights into the reason for the difference in results. Arguably related to this, some of the discussion of previous studies (at least the positive result for the monitor in the general non-obese population) should be in the background.\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": [] }, { "id": "95237", "date": "08 Oct 2021", "name": "Yoshio Iwashima", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study investigated the validity of the Omron RS6 (HEM-6221-E) wrist oscillometric devices in the blood pressure monitoring for 33 Obese Sudanese patients. This manuscript is well written and obtained results confirming their hypothesis. However, there are several studies that blood pressure levels were different between the level of the arm and that of the wrist. It should be noted that, based on much evidence, blood pressure is recommended to be measured in the upper arms, using an appropriate cuff size for the arm circumference. In the case of home blood pressure monitoring for obese patients, blood pressure should be measured at the level of the arm (brachial artery) using a larger cuff.\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/9-1284
https://f1000research.com/articles/9-1096/v1	04 Sep 20	{ "type": "Method Article", "title": "Using triallelic SNPs for determining parentage in North American yak (Bos grunniens) and estimating cattle (B. taurus) introgression", "authors": [ "Ted Kalbfleisch", "Jessica L. Petersen", "R. G. Tait Jr.", "Jiansheng Qiu", "Veronica Basnayake", "Peter H. Hackett", "Michael P. Heaton", "Jessica L. Petersen", "R. G. Tait Jr.", "Jiansheng Qiu", "Veronica Basnayake", "Peter H. Hackett", "Michael P. Heaton" ], "abstract": "Background: Genetic testing for pedigree accuracy is critical for managing genetic diversity in North American (NA) yak (Bos grunniens), a population expanded mostly from imported zoological park specimens. DNA testing also enhances species conservation by identifying recent B. taurus F1 hybrid ancestors (within three generations). Biallelic single nucleotide polymorphisms (SNPs) can accomplish either task, but increases the marker count and costs necessary to achieve both. Our aim was to identify novel, multifunctional, triallelic yak SNPs (tySNPs), with each having two alleles for yak parentage testing, and a third allele for identifying recent cattle introgression. Methods: Genome sequences were aligned to the cattle UMD3.1 assembly and SNPs were screened for 1) heterozygosity in a NA and a Chinese yak, 2) a third allele at high frequency in cattle, and 3) flanking sequences conserved in both species. Subsequently, tySNPs were filtered for unique alignment to the haplotype-resolved F1 yak assembly. Allele frequencies were estimated in a subset of 87 tySNPs by genotyping 170 NA yak. Results: We identified 610 autosomal tySNPs, distributed in 441 clusters with 5 Mb average genome spacing. The average NA yak minor allele frequency was high (0.296), while average introgressed cattle alleles were low (0.004). In simulations with tySNPs, 28 were sufficient for globally-unique animal identification (PI=5.81x10-12), 87 were able to exclude 19 random bulls from parentage at the 99% level without using the dam’s genotype (PE=5.3x10-4), and 87 were able to detect F1 hybridization events after three generations of yak backcrosses (1/16th B. taurus germplasm). Conclusions: Identifying animals, determining parentage and detecting recent hybridization events was efficient with as few as 87 tySNPs. A similar triallelic approach could be used with other bottlenecked Bos species that hybridize with cattle, such as NA plains bison (B. bison).", "keywords": [ "Yak", "Introgression", "Parentage Testing", "SNP test" ], "content": "Introduction\n\nDomestic yaks (Bos grunniens) are native to the Qinghai–Tibet Plateau of Central Asia and valued around the world for their meat, fiber, milk, fuel, wool, transportation, predator protection, and as pets1. The global yak population is large and diverse with upwards of 14 million domestic yak, and 15,000 wild yak (Bos mutus)2. In contrast, the North American (NA) yak population is small and narrow with only 2,000 to 5,000 yak, all of which are domestic. This herd has mostly arisen from a few dozen animals imported from public and private European zoological parks to NA zoos around the turn of the 20th century. The lack of source diversity may have also compounded the problem. For example, the Smithsonian National Zoo (SNZ) in Washington, DC imported their first yaks in 1898 from the Zoological Society of London, England3. For 23 years the closed SNZ herd was expanded by breeding and surplus animals were sent away until the zoo received a single new yak breeding bull from Parks Canada. This Canadian bull descended from yaks kept at Woburn Abbey in England, approximately 50 miles from the original London Zoo source4. Other than zoological park sources, additional yak germplasm has not been introduced to the NA herds due to strict federal regulation barriers placed on the importation of live animals, embryos, and semen. Thus, highly related surplus yaks from limited introductions are the apparent founders of the current NA yak population. This narrow genetic base of the NA yak population is an ongoing concern for maintaining genetic diversity to protect the health and vigor of these herds.\n\nThe challenge of maintaining genetic diversity within NA yaks is increased by occasional hybridization with cattle (Bos taurus). The issues arising from yak and cattle hybridization predates their export from the Qinghai–Tibet Plateau. The production of yak and cattle hybrids has been practiced for 3000 years, starting with the Yin Dynasty5,6. These ancient hybridization events account for the 2-3% Bos taurus alleles present in domestic yak today7.\n\nGenetic management of ancient B. taurus introgression in domestic yak is not typically a concern for breeders. However, in the small NA yak population there are also documented cattle introgression events in the early 20th century8, as well as undocumented introgression from occasional producer-directed efforts to introduce cattle traits like coat color and carcass yield. In some instances, extant animals may be only a couple of generations removed from a previous F1 cattle-yak hybridization event. Thus, identifying and documenting these recently hybridized animals (within three generations) is essential for preserving authentic Bos grunniens germplasm while producing healthy, genetically diverse animals.\n\nSNP-based tools provide new opportunities for more precise genetic management of a given livestock species. Advances in SNP discovery and testing have made it routine to verify parentage, identify animals, and traceback diseases in cattle, sheep, horse, and swine9–12. Previously, yak parentage testing had used Bos taurus derived microsatellite markers (i.e., short tandem nucleotide repeats)13,14. However, publicly available whole genome sequence (WGS) data from Chinese and NA yak, together with a haplotype-resolved F1 yak-cattle assembly, made it possible to identify informative NA yak SNPs in silico for the potential development of parentage SNP markers15–17. The ultimate utility of a set of parentage SNPs may be measured by their success in accomplishing the most challenging scenario: “one-parent traceback.” For example, using DNA from a lamb carcass to identify its true sire in a multi-sire mating system, but not having the dam’s DNA available to perform the trio’s analysis9. Identifying the offspring’s sire requires the genetic exclusion of all other males exposed to the dams. This approach is based on the principle that the true sire must share an allele with the offspring at every site tested18. Thus, when an offspring and a potential sire are homozygous for different alleles at the same site, the potential sire is excluded from parentage. The ideal SNP set for one-parent parentage testing has markers with a minor allele frequency (MAF) greater than 0.30, is evenly spaced across the genome, and has SNPs with highly conserved flanking DNA sequences for efficient and accurate genotyping9. If available, such yak biallelic parentage SNPs would allow producers to access commercial high-throughput SNP genotyping, use multiple-sire pasture breeding strategies, verify pedigrees, and establish unique animal identification information that could be used for tracing if needed.\n\nMarkers appropriate for biallelic yak parentage SNPs do not typically provide information about B. taurus introgression, which would require an additional set of biallelic SNPs thereby increasing cost while limiting choice of platforms suitable for lower throughput. However, based on our previous interspecies alignments of WGS19, we hypothesized that some biallelic parentage SNP sites in yak may also align with a nearly monomorphic, alternative allele in B. taurus. In this hypothetical triallelic SNP system, the evolutionary source of all three alleles could be inferred by estimating frequencies in samples of Bos species. This idea is consistent with the evolution of the Bos genus as a complex of genetically interconnected species with shared evolutionary trajectories20. For example, an ancestral Bos allele would be common in most of the extant Bos species including yak (Figure 1A, nucleotide “C”). A yak-associated allele used for parentage testing would have arisen since the time of the most recent common ancestor for Bos species (Figure 1A, nucleotide “T”). Likewise, a different B. taurus-associated allele would have arisen in the same time frame (Figure 1A, nucleotide “A”). The evolutionary distance between B. taurus and B. grunniens may be sufficient for each species to have evolved their own distinct alleles at the same genomic sites, allowing identification of novel triallelic yak parentage SNPs (tySNPs) (Figure 1B). If a sufficient number of tySNPs (e.g., one in 26 million) could be identified with distributed genome spacing, high MAF in yak, and high specificity in B. taurus, it would be possible to develop a maximally-informative genetic test with a minimal set of about 100 SNPs. This only requires finding one high-quality tySNP per 26 million genomic positions and This would be ideal for a livestock species whose breeders do not currently have access to higher-density genome-wide SNPs and genotyping technologies.\n\nBos species trees are based on those published by Wu et al. (Wu et al., 2018. Nat Ecol Evol 2, 1139–45). (A) Tree based on whole autosome sequences with the randomized axelerated maximum likelihood (RAxML) method. (B) Tree based on whole autosome sequences with the accurate species tree algorithm (ASTRAL) method with a 100-Kb non-overlapping sliding window. *The tree is rooted at the base of the water buffalo (Bubalus bubalus) branch. (Figure1_BosTrees.TIF)\n\nHere we describe a panel of 610 tySNPs for use in animal identification, parentage testing, and for estimating recent B. taurus introgression events in NA yaks. Markers were sequentially filtered for having: 1) identical heterozygous genotypes in a NA and a Chinese reference yak, 2) identical homozygous genotypes in the other non-taurine Bos species, 3) a third high-frequency allele in B. taurus, 4) conserved flanking sequences in Bos species, and 5) a unique location in the B. grunniens genome. A representative trial set of 87 tySNPs were used to estimate performance in NA yak with a MALDI-TOF genotyping platform. Based on these estimates, we performed computer simulations to predict the power to determine parentage and recent B. taurus introgression in NA yak with increasing numbers of tySNPs. Our ultimate goal was to use a minimal set of tySNPs to accomplish multiple diverse genetic tests.\n\n\nMethods\n\nThis article contains no studies performed with animal subjects. The original sources of archived DNA samples used were either purchased from companies that collected them for artificial insemination and not for research (beef cattle), purchased or donated from individuals that collected them privately for their purposes such as food (bison and yak), purchased or donated from zoological parks in their normal course of caring for animals (gaur [B. gaurus] and banteng [B. javanicus]), or donated to the U.S. Meat Animal Research Center (USMARC) by private individuals that collected the samples privately for their own herd management purposes (beef cattle and yak, see Acknowledgements).\n\nThe NA reference domestic yak female used for WGS and alignment to the B. taurus genome was Queen Allante D171 (QA; Figure 2). QA died of natural causes in January of 2010 at approximately 30 years of age and, at the time of her death, was the oldest known living founder of the NA population. Fresh hide was collected post-mortem by the owner, frozen at -20°C, and shipped to USMARC for DNA extraction and production of genome sequences. Approximately 20-fold coverage of FASTQ files for QA were obtained from BioProject accession number PRJNA325061 (BioSample SAMN05558793, SRX2026482, SRX2026483 and SRX2026485 - SRX2026494). Details of library preparation and sequencing are as described by Heaton et al.21.\n\n(Figure2_QueenAllanteAndCalf600.TIF)\n\nThe Chinese reference domestic yak female used for alignment to the B. taurus genome was QH115. Approximately 60-fold coverage of FASTQ files were obtained from BioProject accession number PRJNA74739 (BioSample SAMN00744358, SRX103173-SRX103192). This animal’s data set was originally used because it was the only other yak data set with sufficient coverage (>10X) for both variant discovery and accurate genotyping. Subsequently, additional yak data were available from BioProject accession number PRJNA285834, including B. grunniens: DYS74, SAMN03766772, SRX1056027; DYS77, SAMN03766773, SRX1056028; DYX12, SAMN03766774, SRX1056029; DYX11, SAMN03766775, SRX1056030; DYY31, SAMN03766776, SRX1056031, and B. mutus: WYX01, SAMN03766777, SRX1056032; WYX02, SAMN03766778, SRX1056033; WYX03, SAMN03766779, SRX1056034; WYX04, SAMN03766780, SRX1056035; and WYX05, SAMN03766782, SRX105603622.\n\nWGS and the haplotype-resolved F1 yak-cattle assembly of “Esperanza,” the calf of a female NA yak and a Highland beef bull17, was also used for evaluating the accuracy of tySNPs (ARS_UNL_BGru_maternal_1.0_p), BioProject accession numbers PRJNA551500 and PRJNA552915 (BioSample SAMN12153487,SRR12094761). In addition, research and commercial genotype data from 170 NA yak samples derived from 36 sources were used to estimate allele frequencies in a subset of 87 tySNPs.\n\nThe beef cattle panel consisted of 96 unrelated individuals from 19 popular U.S. beef breeds (USMARC Beef Diversity Panel version 2.9 [MBCDPv2.9])21. Pedigrees were obtained from leading suppliers of U.S. beef cattle semen and breed associations, and analyzed to identify unrelated individuals for inclusion. On the basis of the number of registered progeny, the breeds were estimated to represent greater than 99% of the germplasm used in the US beef cattle industry, contain more than 187 unshared haploid genomes, and allow a 95% probability of detecting any allele with a frequency greater than 0.01623. The breeds in MBCDPv2.9 were (in descending order of registered progeny circa 2000): Angus (n = 6), Hereford (n = 6), Charolais (n = 6), Simmental (n = 6), Red Angus (n = 6), Limousin (n = 6), Gelbvieh (n = 6), Brangus (n = 5), Beefmaster (n = 5), Salers (n = 5), Shorthorn (n = 5), Maine-Anjou (n = 5), Brahman (n = 6), Chianina (n = 4), Texas Longhorn (n = 4), Santa Gertrudis (n = 4), Braunvieh (n = 4), Tarentaise (n = 4), and Corriente (n = 4). The average genome coverage of FASTQ files for these 96 beef bulls was about 14-fold and is available in the NCBI SRA with links to BioProject accession number PRJNA32482221.\n\nThe other Bos species used consisted of approximately 10 to 14-fold coverage of WGS from gaur (n = 2), banteng (n = 2), and bison (n = 1) and were obtained from BioProject accession number PRJNA325061 including B. gaurus: 199911001, SAMN05558794, SRX2026439-SRX2026446, SRX2026451, SRX2026462, SRX2026473, SRX2026484, SRX2026495 - SRX2026498; 199911002, SAMN05558795, SRX2026447 - SRX2026450, SRX2026452 - SRX2026461, SRX2026463 - SRX2026464; B. javanicus: 200710001, SAMN05558796, SRX2026465 - SRX2026468; 200710002, SAMN05558797, SRX2026469 - SRX2026472; and B. bison: 199912001, SAMN05558798, SRX2026474-SRX202648121.\n\nAll sequence data used in this study were quality trimmed using TrimGalore (version 0.5.0)and mapped to the Bos taurus assembly, UMD3.124, using the Burrows Wheeler Aligner (version 0.6.1)25 module aln. The sam-formatted file was then converted to bam format and sorted with samtools (version 0.1.18)26. Samtools was also used to mark the PCR duplicates with the “markdup” function. The Broad Institute’s Genome Analysis Toolkit27 (GATK version 1.5-32-g2761da9) module “IndelRealigner' was used to ensure that any insertions or deletions were consistently aligned. Variant discovery and genotyping for mapped WGS datasets for yak, gaur, banteng, and bison animals (n = 7) were performed using the GATK module UnifiedGenotyper (version 3.4-46-gbc02625) run with genotyping_mode=DISCOVERY. The VCF file was filtered using a custom program (ParseYakCoarse.java)28 to identify tySNPs where the gaur, banteng and bison animals (n = 5) were homozygous for the same inferred Bos ancestral allele (e.g. Figure 1A, “C/C”), and both reference yaks were heterozygous for the Bos ancestral allele and the yak-associated allele (e.g. Figure 1A, “C/T”). The yak-associated allele was not present in the bovine UMD3.1 assembly or the other three Bos species, and the ancestral allele differed from the allele present in the UMD3.1 assembly\n\nThe VCF file containing these above filtered SNPs was then used as the --alleles argument to the Unified Genotyper with genotyping_mode=GENOTYPE_GIVEN_ALLELES, and the 96 BAM files from the cattle diversity panel as the BAM input files. The “--intervals” option was also used with a .bed file that had a record specifying a locus of 2001 bases centered on each of the SNPs. This limited the scope of the genotyping to only the newly filtered SNPs and produced genotypes for all 96 beef cattle animals across those SNPs. A custom program (ParseYakFine.java)28 was written to filter the VCF record, passing only those records where 183 or more of the 192 possible beef cattle alleles (i.e. >95%) was a cattle-specific, bovine UMD3.1 reference allele (e.g., Figure 1A, “A/A”). More than half of the records in the resulting VCF file had a FILTER value of LowQual since no non-reference allele was found in any animal in those records. In order to use the resulting VCF as input for the next step this FILTER value was edited using the text editor emacs and “LowQual” was replaced with “.”. The edited file was used as the input for genotyping WGS.\n\nA VCF file containing candidate tySNPs was used as the --alleles argument in the Unified Genotyper software with genotyping_mode=GENOTYPE_GIVEN_ALLELES and the bam files created for five wild and four domestic Chinese yak were genotyped. The “--intervals” option was also used with a .bed file that had a record specifying a locus of 2001 bases centered on each of the polymorphisms in order to limit the scope of the genotyping to those loci relevant to this study. A custom program (ParseVCF_OtherYak.java)28 was written to count the number of ancestral, yak-associated and B. taurus-associated alleles present in each yak.\n\nThe B. taurus UMD3.1 chromosome position and alleles for the candidate markers were used with a custom program (ParseYakFinal.java)28 to extract 100 bases of flanking sequence both 3’ and 5’ of the marker position. The bam files for the five wild and four domestic Chinese yak, and the 96 beef cattle diversity panel21, were used to identify neighboring SNPs that could disrupt heteroduplex formation with oligonucleotides used in genotyping assays on a variety of genotyping platforms. The GATK UnifiedGenotyper was run with genotyping_mode=DISCOVERY and the --intervals value was set as a .bed file containing records for all loci specified with 2001 bases centered on each marker. Cattle variants discovered in the 100 bp sequences adjacent to tySNPs on either side were replaced with “N” if their allele frequencies were greater than or equal to 5% (i.e., 9 or more of 192 possible alleles). These variant flags may be used by genotype assay design software to redirect oligonucleotide placement to more conserved flanking sequences. Similarly, yak variants flanking the tySNPs at any frequency (compared to the B. taurus UMD3.1 assembly) were also replaced with N for the same reason.\n\nThe last step in filtering candidate tySNPs was aligning 200 bp of flanking sequence to the haplotype-resolved F1 yak assembly (ARS_UNL_BGru_maternal_1.0_p). This determined the yak chromosome and position for the tySNP. Markers were excluded from the group if they did not map exactly once to the assembly. The remaining tySNPs were manually assigned to marker “bins” based on their chromosome position. Clustered markers (e.g., less than 1 Mb) were typically grouped in the same bin to allow SNP assay design software choices for the most amenable target in the region. Where possible, the goal for spacing between bins was 5 Mb. With perfect 5 Mb spacing in the 2479 Mb yak autosomal genome, there would be about 496 bins plus 29 for the end bins.\n\nPrior to assay development, the cutoffs for call rate (total genotypes obtained/total genotypes possible) and accuracy (correct genotypes/total genotypes obtained) were set at 97% and 99%, respectively. For the purposes of parentage exclusion the cutoff call rate of 97% means that a minimum of 94% of the tySNPs will have a genotype for each of the two animals in a pairwise comparison. Although these cutoffs are relatively high, we consider them to be the “gold standard” in SNP-based parentage testing and are within the capability of today’s DNA testing technology. Sets of parentage SNPs that meet these standards substantially increase the efficiency of testing9. Assay development and genotyping was performed at Neogen Genomics (Lincoln, Nebraska, USA) with the MassARRAY platform and iPLEX GOLD chemistry according to the manufacturer’s instructions (Agena Bioscience, San Diego, California, USA). The multiplex assays were designed with the manufacturer’s assay design software and a preliminary set of 139 bins with 518 tySNPs aligned to the B. taurus UMD3.1 assembly. The software options were set for a maximum of 48 assays per plex and to select one tySNP marker available in any bin. Extension probe concentrations were adjusted empirically to optimize signal across the entire mass spectrum. The three multiplex assays were run on DNA from the NA reference yak (QA) and other NA yak. Specific SNP assays that produced low call rates or high error rates were censored from data sets. For the present report, genotype data was used for any of these 139 binned SNPs that passed the assay criteria above and had a unique mapping position in the haplotype-resolved F1 yak assembly.\n\nPI is an estimate for the probability of a coincidental genotype match between two animals. Assumptions used for analyzing tySNPs included Hardy-Weinberg (HW) distributions of genotypes, a negligible frequency of the B. taurus-associated allele, and that the average MAF for yak parentage alleles in NA yak is representative. Briefly, the PI for locus A with SNP alleles A1 and A2 was the sum of the squares of the three genotype frequencies: PI = (χ11)2 + (χ12)2 + (χ22)2, where χ11, χ12, and χ22 were the relative genotype frequencies of A1A1, A1A2, and A2A2, respectively29. The combined PI for multiple SNP markers was the product of the PI for each individual marker. The underlying assumption was that the marker spacing was sufficient for meiotic recombination to cause alleles to be randomly associated with one another. However, as parentage SNP density increases, the validity of this assumption is decreased. Thus, the combined PI for more than one parentage SNP per chromosome is an underestimate of the probability of a coincidental match between random animals from the population, due to linkage disequilibrium between SNPs on the same chromosome. Also, for the purposes of estimating PI and PE, we assumed that the frequency of the B. taurus-associated allele (A3) was negligible.\n\nPE is the probability that a random animal would be excluded from parentage. PE is also the least complicated method of parentage analysis and estimates the fraction of potential adults excluded from parentage. In this report, all PE estimates stringently used only one parent’s genotype information i.e., the most challenging scenario. Thus, exclusion was based only on the frequency of the opposing homozygous SNP genotypes in the offspring and the purported parent as previously described9. Briefly, the probability of opposing homozygotes (POH) between a random offspring and a random eligible adult at SNP locus A with alleles A1 and A2, was calculated as follows: POH = (χ11offspring)(χ22adult)+(χ22offspring)(χ11adult), where χ11 and χ22 were the relative genotype frequencies of A1A1 and A2A2, respectively for the adults or offspring groups. The frequencies of homozygous SNP genotypes were assumed to be the same within a breed group regardless of age. Thus, for a single biallelic SNP, PE = POH = 2(χ11)(χ22) when one of the parent’s genotypes is unavailable. This represents the fraction of eligible adults that would be excluded from parentage at one locus, averaged over all comparisons between offspring and adults. Without using the other parent’s genotype information, the combined PE for multiple SNPs was as follows: PE(SNPn) = PE(SNP1) + R1PE(SNP2) + R2PE(SNP3) … + Rn-1PE(SNPn), where P represents the fraction of eligible adults excluded by the first SNP and R1 is the remaining fraction of unexcluded adults. R2 to Rn-1 are remaining fractions of unexcluded adults after each round of subsequent testing with n parentage SNPs. Thus, for 29 parentage SNPs (one on each autosome), the combined PE for unrelated parents is given by: PE(29) = PE(1) + R1PE(2) + R2PE(3) …+ R28PE(29). As was the case with combined PI, the combined PE for more than 1 parentage SNP per autosome is an underestimate of the probability that a random alleged parent would be excluded from parentage due to linkage disequilibrium between SNPs on the same chromosome. For related parents, the PE for each SNP was multiplied by a coefficient of relatedness (r), where r = 0.125, 0.250, or 0.500 [34]. Thus, PE(29) for related parents = (rPE(1) + rR1PE(2) + rR2PE(3) … + rR29PE(29)).\n\nTriallelic SNPs can lead to parentage exclusion in ways that biallelic do not, due to the presence of B. taurus-associated alleles in some individuals. Consider the three alleles of a tySNP: the Bos ancestral allele (A1), the yak parentage allele (A2), and the B. taurus-associated allele (A3). An exclusion occurs whenever the calf and the adult do not share an allele. Thus, when evaluating “one-parent” scenarios where the dam’s genotypes are unavailable, the analysis is not limited to only the opposing homozygous genotypes. Heterozygous sites may become informative when the calf or the adult possess a copy of the B. taurus-associated allele. Two common examples of these genotype configurations are when the calf is A1/A1 and the adult is A2/A3 genotype, or when the calf is A1/A3 and the adult is A2/A2. In both examples the adult is excluded from parentage. Similarly, when evaluating “two-parent” scenarios where the dam’s genotype at some sites can be used to determine the sire’s allele in the calf, a heterozygous site in the dam and the calf may become informative if one or the other possesses a copy of the B. taurus-associated allele. For example, when the calf is A1/A2 and the dam is A2/A3, the calf’s sire allele is A1 and will exclude all adults that do not carry the A1 allele. Although parentage exclusions caused by the B. taurus-associated alleles only occur in a few percent of the NA yak genotypes comparisons, it is important to account for them when processing parentage tests in commercial settings.\n\nA triallelic SNP has an increased potential to exclude random adults from parentage compared to a biallelic SNP. However, the exceedingly low frequency of the B. taurus-associated allele in yak results in a negligible contribution to yak parentage exclusion. Consequently, the third allele was ignored for the purposes of these simulations. One million random offspring/adult pairings were simulated with tySNP allele frequencies inferred from genotypes of 170 NA yak with HW assumptions. The offspring/adult pair approximates the one-parent parentage testing scenario since the other parent’s genotypes were not used to phase the offspring’s heterozygous sites. An exclusion from parentage was counted for each tySNP site where the offspring and the adult were homozygous for different alleles. The frequency distribution for exclusions for a given set of tySNPs was determined by summing the exclusive sites for each offspring/adult pairing over all pairings. A second simulation was performed to test the effect of “allelic dropout,” a well-known source of genotyping error, and the systematic error we expect most often with a high-quality sample in this SNP panel. This occurs when other genomic SNPs are present in the binding sites for the three oligonucleotides used in a MALDI-TOF assay. These SNPs may disrupt heteroduplex formation in any of the three required assay primers and cause the linked target allele to be absent from the genotyped alleles. In this simulation, a yak sire was simulated by choosing alleles for each tySNP based on allele frequencies inferred from genotypes of 170 NA Yak with HW assumptions. For each tySNP, one of the sire yak alleles was assigned to the offspring with the second allele chosen at random based on the allele frequencies for the corresponding marker. A fixed genotype error rate was then applied to each genotype. For those genotypes chosen to be in error, one of the two alleles was omitted from the call, and the event was recorded if an artifactual exclusion was introduced.\n\nAn F1 yak/cattle hybridization event is readily detected with tySNPs since the offspring would have a B. taurus-associated allele at every site (Figure 3). However, how many back crossings with yak can occur before the cattle alleles can no longer be reliably detected? The cattle allele transmission probability is 0.5 for every generation past the F1 event. Thus, the offspring would be expected to lose, on average, half of the B. taurus-associated alleles each subsequent generation (Figure 3). This would result in approximately 50%, 25%, and 12.5%, in the first, second, and third generations, respectively, after the F1 event. A simulation was performed where an F1 yak/cattle cross had one cattle allele and either a Bos ancestral or yak-associated allele at each of tySNP positions. From that simulated F1 offspring, a cross was also simulated with a yak that had no cattle alleles at any tySNP. This was accomplished by randomly removing one of the two alleles for the F1 at each site and adding back a second non-cattle allele that would have been contributed by the backcross. Starting with the resulting genotypes from this simulated mating, a new set of genotypes was generated using the same process for an additional five generations. The distribution of the remaining cattle alleles per generation was subsequently plotted.\n\nAbbreviations: P1, parental generation; F1, hybrid generation; B, backcross generations; na, not applicable. (Figure3_CattleIntrogressionChart3.TIF)\n\n\nResults\n\nAligning the QA and QH1 reference yak genome sequences to B. taurus reference genome assembly identified 3133 candidate tySNPs that were: 1) heterozygous in both yak, 2) homozygous in gaur, banteng, and bison, and 3) had a third B. taurus-associated allele. Subsequent filtering of the less frequent cattle alleles (i.e., less than 0.95) reduced the set of tySNPs to 1023. Aligning the flanking sequences of the 1023 candidate tySNPs to the B. grunniens haplotype-resolved yak genome assembly identified 612 tySNPs with unique chromosome coordinate positions. Two additional candidate SNPs were removed for being monomorphic after all tySNPs were genotyped in 170 NA yak in a final round with updated statistics. The remaining 610 tySNPs were grouped into 441 regional clusters (i.e., bins) with an average distance of 5.26 Mb between bins (Table S1)30. Of the 610 tySNPs, 87 overlapped an unpublished set of tySNPs for which we previously developed MALDI-TOF MS genotyping assays (Table S2, see Methods)31. These 87 tySNPs markers predated the availability of the haplotype-resolved yak genome, and thus, their unique alignment and genome positions relative to the yak reference assembly were previously unknown. The 87 tySNPs were placed in the context of all 610 tySNPs on the haplotype-resolved yak genome assembly (Figure 4). Together, all 610 tySNPs, their bins, and sequence information are suitable for input into SNP assay design software for a variety of genotyping platforms.\n\nThe positions of 610 tySNPs are shown aligned to the ARS_UNL_BGru_maternal_1.0_p yak assembly. Yellow dots indicate those 87 tySNPs converted to assays and used in NA yak to estimate allele frequencies and utility. (Figure4_610tySNPs_442bins4.TIF)\n\nGenotypes from MALDI-TOF MS assays for the 87 tySNPs were scored in 170 NA yak from 36 sources to provide an estimate of the allele frequencies of parentage alleles and cattle alleles (Table S5)32. The overall SNP genotyping rate (i.e. “call rate”) was 0.9952 for 87 tySNPs in 170 animals. The Bos ancestral allele was the major allele for most of the tySNPs in NA yak (67%). The average MAF for the yak parentage allele was 0.296 with 53% of them making the 0.30 cutoff (Figure 5A). The overall B. taurus-associated allele frequency was very low (0.0043) with more than half of the NA yaks having zero of 174 possible cattle alleles among the 87 sites tested (Figure 5B). The B. taurus-associated allele frequencies for six of the 87 tySNPs were higher than the rest of the group, although less than 0.1 overall. (Figure 5C).\n\n(A) Yak parentage SNPs MAFs. (B) B. taurus-associated alleles within animals. Panel C, variation in B. taurus-associated allele frequencies among different tySNPs. (Figure5_87_SNPs_MAFCatt4.TIF)\n\nUsing the average MAF for 87 tySNPs (0.296) and HW assumptions, the PI for one SNP was 0.427, meaning that approximately 43% of NA yak would be expected to share identical genotypes at an average tySNP. Extending this to 29 tySNPs distributed equally on 29 autosomes yielded a combined theoretical PI of 1.92 × 10-11. However, selecting the best high-MAF tySNPs gave a slightly better result with only 28 autosomes: PI = 5.81 × 10-12 (Figure 6A, Table S2)31. For determining a calf’s sire without the dam’s genetic information (i.e., one-parent parentage testing), the theoretical PE for one SNP was 0.087 with the same MAF and HW assumptions. Thus, approximately 9% of candidate bulls can be excluded with one tySNP. To exclude a group of 30 candidate bulls at the 99% confidence level would require 87 tySNPs all with the same PE (Figure 6B). There were 19 fewer bulls excluded at the 99% confidence level when using the actual PE calculated from NA genotypes for all 87 tySNPs, likely due to linkage between tySNPs and low MAFs in some markers. By adding the dam’s genotypes, the power of exclusion approximately doubles due to the ability to phase the calf’s heterozygous alleles.\n\n(A) The probability of a coincidental genotype match with tySNPs. (B) The number of tySNPs needed to exclude 99% of the candidate bulls from parentage in the absence of the dam’s genotype information. (Figure6_PI_PE_7.TIF)\n\nThe intrinsic properties and allele distributions of the 610 tySNPs were further evaluated in silico with WGS from yaks, Bos species, beef cattle and an F1 yak-cattle hybrid trio. By design, the two reference yak were heterozygous at all 610 sites, having exactly one Bos ancestral allele and one yak-associated allele (Table 1). Excluding monozygotic twins, these are the only two yaks expected to have all 610 identical heterozygous genotypes, since the tySNP marker screening was targeted to them. Also by design was the Bos ancestral allele frequency being fixed in gaur, banteng, and bison due to selection for this property in the filtering. A notable exception was a single tySNP in bison that was heterozygous for a 4th allele at one site (A/T, ARS1.2-UCD chr12:83562937, Table S2)31. An unexpected genotype result was found in one of the five Chinese domestic yak data sets (DYY31, SAMN03766776), which contained greater than 98% B. taurus-associated alleles. Additional analyses performed on its mapped WGS dataset (Table S6)33 confirmed this to be a B. taurus data set and it was eliminated from subsequent analyses.\n\nThe remaining Chinese yak data sets were analyzed for ancestral allele and cattle allele content. The average ancestral allele frequency was slightly higher in wild yaks (0.624) compared to domestic yaks (0.608), while the B. taurus-associated allele frequencies in these yaks were lower (0.0042 and 0.0076, respectively, Table 1). These B. taurus-associated allele frequencies in Chinese yaks are compared to 0.0043 estimated for 87 tySNPs genotyped in NA yaks. In beef cattle, the Bos ancestral allele frequency was only 0.010 due to the selection of B. taurus-associated alleles in the filtering process. The frequency of B. taurus-associated alleles in the 610 tySNPs was 0.9865 in beef cattle. These B. taurus-associated allele frequencies were consistent with WGS genotypes from the F1 yak-cattle hybrid family trio: 0.9984 for the Highland sire, 0.0082 for the NA yak dam, and 0.5049 for the F1 calf. Thus, the intrinsic properties and allele distributions of the 610 tySNPs genotyped in silico with WGS were consistent with those obtained from multiplexed MALDI-TOF MS assays for 87 tySNPs in the group of 170 NA yaks from 36 sources.\n\nBased on the intrinsic properties of the 87 tySNPs above, it is reasonable to extrapolate their performance in scenarios with larger sets of tySNPs. Efficient parentage exclusion depends on a number of factors including: the MAF, the number of markers, and the genotyping error rate. It also requires the majority of random non-parents to have significantly more exclusions (i.e, opposite homozygous genotypes) than the erroneous exclusions in the true parent due to genotyping errors. With the current set of 87 tySNPs and a 0.296 average MAF, most random calf-adult pairings had 5 to 10 exclusive genotypes (i.e., opposing homozygous genotypes) while most calf-parent pairs have only 0 to 2 false exclusive genotypes at a 1% genotyping error rate (Figure 7A). At a 5% genotyping error rate, approximately 0.05 of the true parents are expected to have three false genotype exclusions and fall into the overlap with 0.03 of the correct exclusions in non-parents. This means that 0.0015 of the calf-adult pairs would be difficult to exclude from parentage due to the overlap between false exclusions in true parents and the correct but few exclusions in non-parents. This overlap can be improved by either increasing the number of similar tySNPs, using markers with higher MAFs, or reducing the genotyping error rate (Figure 7B and 7C). In addition, if the dam’s genotypes are available for the same tySNPs, the power is essentially doubled.\n\nSimulations were run as described in the Methods. Red and pink arrows point to the ambiguous overlap between correct exclusions in non-parents and the false exclusions in the actual parents at 5% and 1% genotyping error rates, respectively. (Figure7_ParentageSim4.TIF)\n\nThe power to detect recent cattle introgression after F1 hybridization and subsequent yak backcrossing was simulated with sets of tySNP. Approximately half of the cattle alleles of an F1 hybrid are lost in subsequent backcross generations and thus fit a binomial distribution model. With 87 tySNPs, a third-generation backcross animal (i.e., 15/16th yak) would be expected to have 10 B. taurus-associated alleles detected (Figure 8A). This is compared to a fullblood NA yak which had, on average, less than 1 B. taurus-associated allele per animal (Figure 5B). Doubling the tySNPs increases the detection level to another backcross generation (Figure 8B and 8C). The modes and shapes of these curves would be complicated and more numerous if there was more than one cattle/yak cross in a yak’s recent pedigree. Regardless, a NA yak with less than 3 of 87 B. taurus-associated alleles would be unlikely to have an F1 hybrid as a parent, grandparent or great grandparent.\n\n(A), (B) and (C) Simulated results with 87, 174, and 350 tySNPs, respectively. Each peak represents one backcross generation removed from the F1 hybridization event. (Figure8_tySNP_BackCrossSim7.TIF)\n\n\nDiscussion\n\nOur aim was to identify a minimal set of novel triallelic SNPs to accomplish multiple diverse genetic tests in NA yaks. We identified a novel set of 610 tySNPs where each marker has two alleles for NA yak parentage testing, and a third allele for identifying recent cattle introgression. In addition to allele frequencies, the markers were stringently selected for reduced negative attributes such as indels, repetitive sequences, and flanking SNPs, to enhance their performance on present and future genotyping technologies. The tySNPs were distributed across the genome in 441 clusters (bins) with an average spacing of 5 Mb, based on the recently completed NA yak genome assembly17. A subset of 87 tySNPs was developed for a MALDI-TOF MS platform and their abilities to: 1) provide each animal with a unique genetic identifier, 2) exclude random adults for parentage determination, and 3) identify animals with F1 hybridization events in their recent ancestry. By selecting the best tySNPs on each available autosome, only 28 tySNPs of the 87 were needed to uniquely identify every NA yak (estimated combined PI = 5.81 × 10-12). Excluding monozygotic twins, this result means the odds of any two random NA yaks having the same genotypes at all 28 sites by chance would be 1 in 172 billion. This is enough power for tracing yaks and their products in the global food chain if needed.\n\nThe power to exclude random adults from parentage with 87 tySNPs was high. Most random adults had between 5 to 10 tySNP sites that excluded them from parentage with a given offspring, i.e., did not share an allele with the offspring at those sites. However, the false exclusions in a true parent due to genotype error was only about 1 in 87 tySNPs with a genotype platform error rate of 1%. More than 99% of random adults can be excluded from parentage even with a genotyping error rate as high as 5% (i.e., four exclusions allowed in a true parent). Thus, most yak calves can be assigned to a single parent without having the other parent’s genotypes available. For multi-sire pasture mating situations this can reduce the cost of parentage testing by nearly 50%. Note that these estimates are for parentage exclusion with unrelated adults. The power to exclude full sibs from parentage is essentially reduced by half and thus may require use of the dam’s genotypes and/or more accuracy in the genotyping system9. When 1000 offspring are involved, one effective strategy is to first assign the high-confidence calf-sire relationships with one-parent testing, and then use the dam’s genotypes to confirm sire exclusions on any remaining ambiguous calf-sire relationships.\n\nThe B. taurus species introgression in 170 NA yaks from diverse sources was low and not distinguishable from that of other yaks. With 87 tySNPs, no significant differences were detected between the average B. taurus-associated allele frequency in 170 NA yak (0.0042) and that of nine domestic and wild chinese yak (0.0057). This suggests the genetic foundation of the NA yak population overall is not significantly influenced with introgressed B. taurus germplasm. A low background of cattle introgression in yaks facilitates the ability of tySNPs to detect F1 hybridization events in the recent ancestry of a yak. With 87 tySNPs, simulations predicted confident detection of F1 hybridization events in yak containing as little as 1/16th B. taurus germplasm (i.e., three backcross generations after the F1 event). This would be sufficient to verify the accuracy of three- or four-generation pedigrees. It should be noted, however, that tySNPs are not informative with regards to which B. taurus animal was involved in the F1 hybridization event, because nearly all B. taurus animals are homozygous for the B. taurus-associated allele. We also noted that six tySNPs had B. taurus-associated allele frequencies between 0.01 and 0.10 in NA yak, while the B. taurus-associated allele frequencies in the other 81 tySNPs were essentially zero. Reasons for this could include: ancient B. taurus introgression and selection and/or the existence of both alleles prior to speciation. A future tySNP filter to consider would be less than 1% prevalence of B. taurus-associated alleles in NA yaks. In spite of these exceptions, overall B. taurus-associated alleles in tySNPs were rare in NA yaks, and thus able to identify animals descended from backcrosses of recent F1 hybrids.\n\nThe primary areas for improving the multiplexes set of 87 tySNPs include: marker abundance, parentage MAF, and genomic distribution. The 87 tySNPs presented here were developed out of necessity, prior to the availability of the NA yak genome assembly, and without prior knowledge of their allele frequencies in NA populations. While only 28 tySNPs provide more than enough power for genetic fingerprinting, additional tySNPs would add significant power for parentage exclusion. Using future WGS from an additional 10–15 NA yak would allow MAF estimates to be confidently assigned for the 610 tySNPs presented here. With this NA yak allele frequency information in hand, choosing tySNPs with the highest MAFs and increasing the number of markers to at least 131 (i.e., 1.5x) would increase the proportion of random adults excluded from parentage, as simulated in Figure 7B. This would also help with excluding highly related animals from parentage, for example, when full-sib bulls are simultaneously exposed to cows. Having a set of 131 tySNPs with high MAF would also somewhat improve the ability to detect F1 hybridizations events. Simulations showed that to gain one more generation in sensitivity for F1 hybridization detection, 175 (i.e., 2x) tySNPs would be needed. As genotyping technologies improve and become more cost-efficient, it may be possible to incorporate additional markers from the 421 bins of 610 tySNPs.\n\nTriallelic SNPs have been systematically identified in other mammals. In Homo sapiens, a set of 1,270 polymorphic tri-allelic SNPs mined from the 1000 Genomes Project was recently used in forensic identification of missing persons34. Interestingly, there are approximately twice as many triallelic sites in humans as expected by chance35. It is conceivable that at least some of these may be due to introgression of archaic species into modern humans36. Regardless of their use in humans, the multipurpose triallelic SNP approach presented here may be useful in other species that can hybridize, including bovids, cervids, odontids, camelids, equids, canids, felids, and ursids. An obvious immediate application is in NA plains bison (B. bison). Plains bison are wild animals native to NA, have experienced a population bottleneck, hybridize with B. taurus, and farmed for their meat and byproducts. Ideally, WGS from a diverse group of 15 unrelated plains bison sampled from the NA herd would be used, together with the banteng, gaur, yak, and cattle to identify suitable triallelic bison SNPs (tbSNPs). By using WGS from 15 bison, the MAF estimates for bison parentage would be known a priori and could be more effectively used in the tbSNPs filtering process. Depending on the aims, WGS from NA wood bison (B. bison athabascae) or European wisent (B. bonasus) could also be included in filtering for MAF estimates and extend the potential utility of a tbSNP panel.\n\n\nConclusion\n\nResults from novel tySNPs presented here demonstrate that one minimal set of markers can be efficiently and accurately used for animal identification, parentage determination, and detecting recent F1 hybridization events to support herd management and breeding decisions of yak producers. The 610 tySNPs, multiplex MALDI-TOF MS assays for the subset of 87 tySNPs, and associated information are available for world-wide use without restriction.\n\n\nData availability\n\nNCBI BioProject: Bos mutus strain:yakQH1 Genome sequencing and assembly. Accession number PRJNA74739.\n\nNCBI BioProject: Bos grunniens Genome sequencing. Accession number PRJNA285834.\n\nNCBI BioProject: Phased trio assembly of yak and cattle genomes. Accession number PRJNA551500.\n\nNCBI BioProject: Bos grunniens x Bos taurus Genome sequencing and assembly. Accession number PRJNA552915.\n\nNCBI BioProject: WGS data from diverse types of U.S. cattle. Accession number PRJNA324822.\n\nNCBI BioProject: WGS data from Cetartiodactyla. Accession number PRJNA325061.\n\nFigshare: Table S1. Genomic locations and sequences features of 610 tySNPs in 441 bins. https://doi.org/10.6084/m9.figshare.12472925.v130.\n\nFigshare: Table S2. Genomic locations and sequence features of a subset of 87 tySNPs. https://doi.org/10.6084/m9.figshare.12473087.v331.\n\nFigshare: Table S3. In silico genotypes derived from WGS for 610 tySNPs in yak, cattle, and other Bos species. m. https://doi.org/10.6084/m9.figshare.12473360.v137.\n\nFigshare: Table S4. Oligonucleotide sequences for MALDI-TOF MS assays of 87 tySNPs. https://doi.org/10.6084/m9.figshare.12473492.v138.\n\nFigshare: Table S5. MALDI-TOF MS genotypes of 87 tySNPs for 170 NA yak. https://doi.org/10.6084/m9.figshare.12473537.v132.\n\nFigshare: Table S6. Analysis of Chinese domestic yak WGS data set DYY31 for B. taurus sequences. https://doi.org/10.6084/m9.figshare.12682331.v133.\n\n\nSoftware availability\n\nAll custom software used to analyze data for this project are available from the GitHub page for this project: https://github.com/kalbflei/YakParentageAndIntrogression.\n\nArchived software at time of publication: https://doi.org/10.5281/zenodo.398845728.\n\nLicense: MIT license.", "appendix": "Acknowledgments\n\nWe thank J. Carnahan and H. Sadd for outstanding technical assistance. This work was conducted in part using the resources of the University of Louisville's Research Computing Group and the Cardinal Research Cluster. TK would like to acknowledge specifically the support of Mr. Harrison Simrall of the University of Louisville Research Computing Group. We would also like to thank the University of Kentucky Center for Computational Sciences and Information Technology Services Research Computing for their support and use of the Lipscomb Compute Cluster and associated research computing resources. TK would like to acknowledge specifically the support of Mr. Vikram Gazula. We thank Dr. E. Bailey thoughtful discussions and improvements to the manuscript; Drs. D. Armstrong and E. Louis from Omaha's Henry Doorly Zoo for providing gaur blood samples, Ms. L. Chemnick and Dr. O. Ryder from the San Diego Zoo's Beckman Center for Conservation Research for providing banteng DNA samples; the late Dianne Latona for historical research in ‘Out of Tibet—Highlights of the Yak’s Journey to the New World’ and her passion for preserving the genetic health and welfare NA yaks, and members from the International Yak Association and USYAKS for donating yak tissues samples. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeaton MP, Chitko-McKnown CG, Grosse WM, et al.: Interleukin-8 haplotype structure from nucleotide sequence variation in commercial populations of U.S. beef cattle. Mamm Genome. 2001; 12(3): 219–26. PubMed Abstract \| Publisher Full Text\n\nZimin AV, Delcher AL, Florea L, et al.: A whole-genome assembly of the domestic cow Bos taurus. Genome Biol. 2009; 10(4); R42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Durbin R: Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010; 26(5): 589–95. 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\nVan der Auwera GA, Carneiro MO, Hartl C, et al.: From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics. 2013; 43: 11.10.1–11.10.33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalbfleisch T: kalbflei/YakParentageAndIntrogression: Initial release (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3988457\n\nHeaton MP, Keen JE, Clawson ML, et al.: Use of bovine single nucleotide polymorphism markers to verify sample tracking in beef processing. J Am Vet Med Assoc. 2005; 226(8): 1311–4. PubMed Abstract \| Publisher Full Text\n\nHeaton M: Table S1. Genomic locations and sequences features of 610 tySNPs in 441 bins. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12472925.v1\n\nHeaton M: Table S2. Genomic locations and sequence features of a subset of 87 tySNPs.figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12473087.v3\n\nHeaton M: Table S5. MALDI-TOF MS genotypes of 87 tySNPs for 170 NA yak. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12473537.v1\n\nHeaton M, Kalbfleisch T: Table S6. Analysis of Chinese domestic yak WGS data set DYY31 for B. taurus sequences. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12682331.v1\n\nPhillips C, Amigo J, Tillmar AO, et al.: A compilation of tri-allelic SNPs from 1000 Genomes and use of the most polymorphic loci for a large-scale human identification panel. Forensic Sci Int Genet. 2020; 46: 102232. PubMed Abstract \| Publisher Full Text\n\nHodgkinson A, Eyre-Walker A: Human triallelic sites: evidence for a new mutational mechanism? Genetics. 2010; 184(1): 233–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen L, Wolf AB, Fu W, et al.: Identifying and interpreting apparent neanderthal ancestry in african individuals. Cell. 2020; 180(4): 677–87.e16. PubMed Abstract \| Publisher Full Text\n\nHeaton M: Table S3. In silico genotypes derived from WGS for 610 tySNPs in yak cattle, and other Bos species. m. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12473360.v1\n\nHeaton M: Table S4. Oligonucleotide sequences for MALDI-TOF MS assays of 87 tySNPs. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12473492.v1" }	[ { "id": "70890", "date": "23 Sep 2020", "name": "Leslie A. Lyons", "expertise": [ "Reviewer Expertise Genetics", "DNA profiling", "development of parentage", "forensic and population DNA panels" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Kalbfleisch et al., explores the identification and use of triallelic SNPs (tySNPs) that are diagnostic for North American yak and cattle. The markers could then be used for population and parentage studies to manage the NA yak populations, attempting to keep inbreeding at a minimum and reducing introgression from cattle breeds. Sequence and assembly data from cattle (UMS3.1) and the F1 yak assembly were used to identify unique candidate tySNPs that had conserved flanking sequence to ensure robust amplification in both species and were dispersed across the genome. Overall, 610 autosomal tySNPs were identified. Allelic frequencies were determined for 87 tySNPs by genotyping in 170 NA yak. By simulations, the power for unique identification, parental exclusions and introgression up to three backcross generations from the F1 were estimated. A unique DNA profile could be obtained with 27 SNPs and the parentage and hybridization determinations required up to 87 SNPs.\nCurrent SNP-based parentage panels for cattle and other species contain over 100 to several hundred SNPs for unique identification and parentage determination. The panels need to be robust and valid for use in a variety of inbred breeds for a given species. Although the global population of domesticated yak is large and diverse, NA yak have a small population derived from founders in zoo populations imported from Europe. The herd has not been diversified using artificial insemination techniques. Ancient hybridization suggests 2 – 3% Bos taurus introgression. Recent, planned and unplanned cattle introgression has occurred in the yak NA population.\nThe manuscript is well written and easy to understand and read. The authors provide extensive background in each section for the reader, however, some of which can be deleted to shorten the manuscript, but likely not a concern for the online publication. The study design is well planned and the bioinformatic approaches are sound. However, in the end, 87 SNPs were selected – but apparently not by the methods presented here. The presentation gets to ~610 candidate SNPs, where these 87 are amongst those candidates, but, the 87 were selected before these methods were conducted. So, how were the 87 SNPs initially selected and where are the other SNPs that likely failed in the development process? The 87 SNP panel also needs to be adjusted. This is a commercial set of SNPs that I fear are being promoted – not considering all the criteria even presented in this publication.\nIntroduction:\nThe background information is very informative, but perhaps the introduction is a bit too long.\n\nWhat is the calculated evolutionary distance (time) between cattle and the yak?\n\nBoth Figure 1A and 1B are not necessary. Perhaps use Figure 2 as Figure 1B?\n\nMethods:\nSample collection – “purchased or donated from zoological parks in their normal course of caring for animals”. Most zoological parks have ACUC protocols and require “study protocol forms” for samples released – even after opportunistic collections during health examinations. Perhaps the protocols should be confirmed?\n\nCan more of the Methods be shortened by using references? Such as details on the identity of the animals used for sequencing?\n\nThe use of the pedigrees to select a strong representation of the beef cattle breeds is a strong aspect of the project.\n\nCorrect spacing: “TrimGalore (version 0.5.0)and”\n\nPerhaps a supplementary table could be used to represent the information in the text regarding the number of each breed and the genome accession numbers, thus, shortening the text.\n\n“the cutoffs for call rate (total geno-types obtained/total genotypes possible) and accuracy (correctgenotypes/total genotypes obtained) were set at 97% and 99%, respectively.” and “For the purposes of parentage exclusion the cutoff call rate of 97% means that a minimum of 94% of the tySNPs will have a genotype for each of the two animals in a pairwise comparison” These are stringent cutoffs for the data and should produce a very robust panel of SNPs.\n\nUsed MALDI-TOF mass spectroscopy for genotyping: a preliminary set of 139 bins with 518 tySNPs aligned to the B. taurus UMD3.1 assembly. 48 assays per plex using one SNP per bin – producing 3 multiplexes.\n\nAlthough very thorough – can the section including the “Estimating the probability of identity (PI) and probability of exclusion (PE) with tySNPs”, and “Identifying exclusions in a tri-allelic SNP system” be shortened by referencing?\n\nOverall, the bioinformatic approaches appear to be sound and robust for this study.\n\nFigure 3 is not really needed.\n\nResults:\nInteresting – “Of the 610 tySNPs, 87 overlapped an unpublished set of tySNPs for which we previously developed MALDI-TOF MS genotyping assays (Table S2, see Methods)31.” Thus, this panel was already developed and in use, but then verified by phased yak genome. Therefore – how were these original 87 tySNPs identified and designed?\n\nIf starting from scratch with 610 tySNP candidates and then designing mass array multiplexes – 441 bins with 48 SNPs per multiplex = 10 assays minimum.\n\nThus – it does not seem like the authors used the presented information to produce the 87 tySNP panel. After the fact – they are validating the panel but not suggesting any improvements from the data produced by the methods performed in the manuscript. For example, at least 5 or so of the 87 tySNPs appear to be very physically close in Figure 4 – and hence should not be in the panel.\n\nThe in silico analysis for the 610 SNP allele frequencies confirmed the allelic frequencies for the 87 SNPs that were genotyped in the 170 yaks. However, could a better set of tySNPs have been selected from this data?\n\nAll the exclusion and introgression calculations are based on the 87 SNPs.\n\nNo animals with documented, known hybridization were genotyped for validation.\n\nDiscussion:\n“Our aim was to identify a minimal set of novel triallelic SNPs to accomplish multiple diverse genetic tests in NA yaks.” The authors do define a robust set of triallelic SNPs – however – the most robust or minimal set has NOT been identified because this paper confirms an a priori set of 87 SNPs that were selected using perhaps a different process as described here. The authors need to confirm the same process was used to select the 87 SNPs – short of using the phased yak assembly for confirmation and positioning, which they indicate.\n\nThis manuscript does indicate the 87 SNPs developed for yak testing are robust and have good exclusion rates, but it does not select the best SNPs from the 610 to produce the panel. No modifications are suggested for the panel – including for SNPs now shown to be in close proximity or the 6 tySNPs with B. taurus – associated allele frequencies. However, the authors do suggest that better SNPs could be selected in more yak WGS data was available. Another way to produce this data would be to make additional multiplexes from the remaining 610 SNPs and genotype the same 170 yaks. If 87 SNPs had already been produced – likely – additional data on tySNPs not selected for the panel is likely available.\n\nIn the end, the NA yak are not significantly influenced with B. taurus introgression, thus, a standard set of SNPs would be likely sufficient, although introgression could not be monitored.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6046", "date": "29 Oct 2020", "name": "Ted Kalbfleisch", "role": "Author Response", "response": "Dear Reviewer 1, Thank you for your detailed comments on our manuscript. Regarding the concern surrounding the 87 markers used, we revised the manuscript to highlight our aim of showing the variety of purposes to which triallelic yak SNPs could be applied, as our intention was not to validate a livestock SNP panel. We regard the identification of the 610 tySNPs and their genomic metadata in Table S1 as a significant outcome of the present report. We can confirm the subset of 87 tySNPs were identified by the same process as the 610 tySNPs, except for the use of the haplotype-resolved Yak reference genome for determining their correct and unique genome positions. Like all the candidate tySNPs, their MAFs were unknown at the time of their original selection. These 87 tySNPs were also identified a second time in the group of 610 as they met all the same selection criteria. None of the 610 tySNPs were excluded for being close to another tySNP (hence binning), since automated assay design software needs options. We have updated the new version of the manuscript as described below to highlight these facts. With respect to the comment “This is a commercial set of SNPs that I fear are being promoted – not considering all the criteria even presented in this publication.” All of the genotype data presented here were produced for research purposes and predate any commercial offering of a yak tySNP test. The data were meant to provide a statistically significant sampling of the intrinsic properties expected in 170 NA yak for the 610 markers identified here. Although a commercial tySNP test is now available and contains the 87 tySNPs, we do not endorse it to the exclusion of other markers or test panels that may be suitable. Page 8, column 1, paragraph 1, line 13 of the first version: “Of the 610 tySNPs, 87 unpublished set of tySNPs for which we previously developed MALDI-TOF MS genotyping assays (Table S2, see Methods)31. These 87 tySNPs were a subset of markers identified by the same process except for alignment to the haplotype resolved yak genome. Their prior selection predated the availability of this novel yak reference assembly. Thus, their unique alignment and yak genome positions were previously unconfirmed. The positions of the 87 tySNPs, together with the other 523 tySNPs, are shown on the haplotype-resolved yak genome assembly (Figure 4). Together, all 610 tySNPs, their bins, and their sequence information are suitable for input into SNP assay design software for a variety of genotyping platforms.” And we have modified the following within the Discussion Page 10, column 2, paragraph 2, line 1 of the first version: Our aim was to identify a set of novel triallelic SNPs. We identified a novel set of 610 tySNPs where each marker has two alleles for NA yak parentage testing, and a third allele for identifying recent cattle introgression. As to the reviewer’s suggestion that there may be a better set of 87 markers available within the 610, we agree. However, the MAF needs to be estimated for each tySNP in NA yak before this can be explored further. That effort is beyond the scope of this report. All 610 tySNPs should be considered viable targets for assay development, with anyone being free to use them in full or to identify a suitable subset within them for their specific purpose. We made the following modification to this sentence in the conclusion: Page 13, column 1, paragraph 2, line 6 of the first version:The 610 tySNPs, their assay design information, multiplex MALDI-TOF MS assays for the subset of 87 tySNPs, and all other associated information are available for world-wide use without restriction. In addition, we have responded point-by-point to your comments below (bold text). With respect to requests to make the manuscript briefer, since length is not a concern in this online format, and the editors had previously requested such information, we have left most of the content unchanged. Introduction: The background information is very informative, but perhaps the introduction is a bit too long. What is the calculated evolutionary distance (time) between cattle and the yak? We modified Page 3 column 2 paragraph 2 to include: In this hypothetical triallelic SNP system, the evolutionary source of all three alleles could be inferred by estimating frequencies in samples of Bos species, a genus that diverged 5 million years ago. Both Figure 1A and 1B are not necessary. Perhaps use Figure 2 as Figure 1B? We believe that the Figure 1 panels show different information. Figure 1A shows how tySNPS would arise, while Figure 1B shows the accurate species distances from the root. Methods: Sample collection – “purchased or donated from zoological parks in their normal course of caring for animals”. Most zoological parks have ACUC protocols and require “study protocol forms” for samples released – even after opportunistic collections during health examinations. Perhaps the protocols should be confirmed? The following information was added to Methods: The banteng dna samples (1 ug each) were transferred under a Research Material Agreement that was executed on February 6, 2007 by the legal directors of the San Diego Zoo and the technology transfer office of the USDA, ARS. The gaur blood samples were collected under IACUC protocols on June 15,1999 at the Omaha Henry Doorly Zoo by the head zoo veterinarian, Dr. Doug Armstrong, during evaluation and preparation for interstate transfer to another zoo. Can more of the Methods be shortened by using references? Such as details on the identity of the animals used for sequencing? This journal requires full method descriptions, efforts to shorten with previous submissions have been declined. The use of the pedigrees to select a strong representation of the beef cattle breeds is a strong aspect of the project. We agree. Correct spacing: “TrimGalore (version 0.5.0)and”. Repaired Page 5 column 2, paragraph 2 Perhaps a supplementary table could be used to represent the information in the text regarding the number of each breed and the genome accession numbers, thus, shortening the text. We have left the text as is. “the cutoffs for call rate (total geno-types obtained/total genotypes possible) and accuracy (correctgenotypes/total genotypes obtained) were set at 97% and 99%, respectively.”and “For the purposes of parentage exclusion the cutoff call rate of 97% means that a minimum of 94% of the tySNPs will have a genotype for each of the two animals in a pairwise comparison”These are stringent cutoffs for the data and should produce a very robust panel of SNPs. We agree. Used MALDI-TOF mass spectroscopy for genotyping: a preliminary set of 139 bins with 518 tySNPs aligned to the B. taurus UMD3.1 assembly. 48 assays per plex using one SNP per bin – producing 3 multiplexes. This is correct. Although very thorough – can the section including the “Estimating the probability of identity (PI) and probability of exclusion (PE) with tySNPs”, and “Identifying exclusions in a tri-allelic SNP system” be shortened by referencing? As described above, F1000Research editors require full method descriptions, efforts to shorten with previous submissions have been declined. Overall, the bioinformatic approaches appear to be sound and robust for this study. We agree. Figure 3 is not really needed. As this work was inspired by input from yak breeders, we wanted to include a figure that shows producers the expected loss of introgressed cattle alleles across generations of yak backcrossing. This figure relates directly to the ability of tySNPs to detect cattle introgression as shown by simulations in Figure 8. Results: Interesting – “Of the 610 tySNPs, 87 overlapped an unpublished set of tySNPs for which we previously developed MALDI-TOF MS genotyping assays (Table S2, see Methods)31.” Thus, this panel was already developed and in use, but then verified by phased yak genome. Therefore – how were these original 87 tySNPs identified and designed? Addressed in the revised manuscript in “Amendments from Version 1”. If starting from scratch with 610 tySNP candidates and then designing mass array multiplexes – 441 bins with 48 SNPs per multiplex = 10 assays minimum. Depending upon the total number of assays required by an interested party, and the assay platform/chemistry, any subset of the proposed bins could be chosen for an assay panel. We have demonstrated with a MALDI-TOF MS platform that 3 multiplexes with 87 total assays will provide enough information to reasonably accomplish animal identification, parentage determination, and detect recent hybridization. Thus – it does not seem like the authors used the presented information to produce the 87 tySNP panel. After the fact – they are validating the panel but not suggesting any improvements from the data produced by the methods performed in the manuscript. For example, at least 5 or so of the 87 tySNPs appear to be very physically close in Figure 4 – and hence should not be in the panel. As addressed in the revised manuscript in “Amendments from Version 1”, our aim was not to validate an elite panel of 87 tySNPs, but to use the population genotypes to estimate their MAF in NA yak. Since all tySNPs were selected based on being heterozygous in only two yaks (1 NA and 1 Chinese), we had initial concerns that the MAF for most of the SNPs would be too low to be useful. Using data from the 87 unmapped tySNPs and 170 yak we were pleasantly surprised there was a useful distribution of MAFs. We document this in Figure 5A and can reasonably infer a similar distribution among all 610 tySNPs. The in silico analysis for the 610 SNP allele frequencies confirmed the allelic frequencies for the 87 SNPs that were genotyped in the 170 yaks. However, could a better set of tySNPs have been selected from this data? Any new panel design starting with these 610 tySNPs would likely end with a different marker composition. However, any randomly selected set of 87 tySNPs distributed among the 441 bins are expected to produce similar results in NA yaks. Moreover, creating specific combinations of markers into multiplex assays (up to 48 at a time) is a tremendous technical challenge. This challenge is increased by having three SNP alleles to assay instead of just two. In addition, flanking polymorphisms near some of the tySNPs reduce the utility of some of the 610 identified tySNPs depending on the genotyping platform and assay requirements. All of these challenges were overcome here as part of a solution to develop as powerful of assay as possible with cost awareness. All the exclusion and introgression calculations are based on the 87 SNPs. Since we had data on 87 SNPs, that number was used as the lower limit. In order to demonstrate the benefit of more markers, we simulated tests with 1.5, 2, and 4-fold increases in marker count. The balance between cost and the benefit of more markers is difficult to strike. However, we wanted to demonstrate what could be expected with these 87 tySNPs since this is the data we had. No animals with documented, known hybridization were genotyped for validation. This assertion is incorrect. We used the F1 cross between a Highland bull and a yak dam for in-silico genotyping of the full panel of 610 markers (Table 1 last row). This hybrid female (“Esperanza”) had a 0.5049 cattle allele fraction. The genotypes are provided in Table S3. Discussion: “Our aim was to identify minimal set of novel triallelic SNPs to accomplish multiple diverse genetic tests in NA yaks.”The authors do define a robust set of triallelic SNPs – however – most robust or minimal set has NOT been identified because this paper confirms an a priori set of 87 SNPs that were selected using perhaps a different process as described here. The authors need to confirm the same process was used to select the 87 SNPs – short of using the phased yak assembly for confirmation and positioning, which they indicate. We confirm. This manuscript does indicate the 87 SNPs developed for yak testing are robust and have good exclusion rates, but it does not select the best SNPs from the 610 to produce the panel. No modifications are suggested for the panel – including for SNPs now shown to be in close proximity or the 6 tySNPs with B. taurus – associated allele frequencies. However, the authors do suggest that better SNPs could be selected in more yak WGS data was available. Another way to produce this data would be to make additional multiplexes from the remaining 610 SNPs and genotype the same 170 yaks. If 87 SNPs had already been produced – likely – additional data on tySNPs not selected for the panel is likely available. The success of SNP assay design is dependent on many factors including chemistry, platform, technology, reagents, sample quality, and operator experience, to name a few. We used all the data from all the tySNPs that met the selection criteria and converted to a reliable assay (>97% call rate and >99% accuracy). Figure 5A shows that some of these 87 tySNPs had low MAFs which is less than ideal for the tasks at hand but were still included. As mentioned above, any new panel design starting with these 610 tySNPs would likely end with a different marker composition. However, any random set of 87 tySNPs distributed among the 441 bins are expected to produce similar results in NA yaks. In the end, the NA yak are not significantly influenced with B. taurus introgression, thus, a standard set of SNPs would be likely sufficient, although introgression could not be monitored. As the reviewer indicates, introgression cannot be monitored with a standard set of bi-allelic SNPs. This was the motivation to identify tySNPs that can detect recent introgression among NA yak without the need for a second, independent set of markers for that task." } ] }, { "id": "70891", "date": "28 Sep 2020", "name": "Oliver A. Ryder", "expertise": [ "Reviewer Expertise population management", "comparative genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 used available genomes from wild and domestic yaks and related bovids to identify SNPs that are informative for partentage analysis in yaks, for which cattle have a nearly fixed alternate allele. Using available information on allele frequencies they estimate the power for parentage, even without data from one parent and introgression detection and conduct simulations to evaluate the number of SNPs required to reach different confidence levels for the two analyses.\nThe paper is well written, highly detailed, and methods are adequately described.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1096
https://f1000research.com/articles/9-657/v1	29 Jun 20	{ "type": "Software Tool Article", "title": "Reproducibly sampling SARS-CoV-2 genomes across time, geography, and viral diversity", "authors": [ "Evan Bolyen", "Matthew R. Dillon", "Nicholas A. Bokulich", "Jason T. Ladner", "Brendan B. Larsen", "Crystal M. Hepp", "Darrin Lemmer", "Jason W. Sahl", "Andrew Sanchez", "Chris Holdgraf", "Chris Sewell", "Aakash G. Choudhury", "John Stachurski", "Matthew McKay", "David M. Engelthaler", "Michael Worobey", "Paul Keim", "J. Gregory Caporaso", "Evan Bolyen", "Matthew R. Dillon", "Nicholas A. Bokulich", "Jason T. Ladner", "Brendan B. Larsen", "Crystal M. Hepp", "Darrin Lemmer", "Jason W. Sahl", "Andrew Sanchez", "Chris Holdgraf", "Chris Sewell", "Aakash G. Choudhury", "John Stachurski", "Matthew McKay", "David M. Engelthaler", "Michael Worobey", "Paul Keim" ], "abstract": "The COVID-19 pandemic has led to a rapid accumulation of SARS-CoV-2 genomes, enabling genomic epidemiology on local and global scales. Collections of genomes from resources such as GISAID must be subsampled to enable computationally feasible phylogenetic and other analyses. We present genome-sampler, a software package that supports sampling collections of viral genomes across multiple axes including time of genome isolation, location of genome isolation, and viral diversity. The software is modular in design so that these or future sampling approaches can be applied independently and combined (or replaced with a random sampling approach) to facilitate custom workflows and benchmarking. genome-sampler is written as a QIIME 2 plugin, ensuring that its application is fully reproducible through QIIME 2’s unique retrospective data provenance tracking system. genome-sampler can be installed in a conda environment on macOS or Linux systems. A complete default pipeline is available through a Snakemake workflow, so subsampling can be achieved using a single command. genome-sampler is open source, free for all to use, and available at https://caporasolab.us/genome-sampler. We hope that this will facilitate SARS-CoV-2 research and support evaluation of viral genome sampling approaches for genomic epidemiology.", "keywords": [ "SARS-CoV-2", "genome-sampler", "QIIME 2", "bioinformatics", "genomics" ], "content": "Introduction\n\nThe intersection of the SARS-CoV-2 outbreak and the genomics revolution has led to the rapid accumulation of viral genomes that are fueling our epidemiological understanding of the global pandemic. However, the rate of genome sequencing is challenging our ability to conduct comprehensive analyses in a timely manner. Local networks of health care professionals, laboratory professionals, and researchers are rapidly generating genome sequences at an unprecedented rate and feeding these data into global community resources, such as GISAID1 and GenBank2. Contextualizing locally-derived genome sequences with those from global resources (e.g., as recently performed by the Arizona COVID-19 Genomics Union3) enables phylogenetic analyses that can provide information about the relative roles of local transmission versus repeated introductions. This can help to evaluate the utility of control measures, such as stay-at-home orders. These sequencing data thus enable a new paradigm in epidemiology, which must be facilitated by computational workflows designed to handle this scale of data.\n\nContextualization of locally derived genome sequences will generally begin with two collections of sequences: those obtained from a global community resource and those obtained locally. The widely used NextStrain4 platform refers to these sequence collections in their documentation as the context sequences and the focal sequences, respectively, and we adopt that terminology here.\n\nTo enable phylogenetic analysis of full-length SARS-CoV-2 genomes, for example with Bayesian methods or maximum likelihood methods with bootstrap support, subsampling the context sequences is essential for computational feasibility. To avoid introducing post-sequencing sampling biases into our analysis, we subsampled the context sequences across three axes: time, space (i.e., geographic dispersion of near neighbors of focal sequences), and viral genome diversity. Sampling across time enables us to reliably infer a molecular clock signal from the data by ensuring that our sample of viral genomes span as much time as possible and include the oldest available genomes. Sampling the context sequences to include near neighbors of the focal sequences that come from different geographic regions enables us to avoid erroneously describing groups of focal sequences as monophyletic. Sampling across viral diversity enables us to represent the known diversity of the virus in our analysis. Each of these steps additionally reduces the chance of over-represented genomes dominating the analysis. When data sets are relatively small, this process can be performed manually, but when numbers of context genomes measure in the thousands, tens of thousands, or even hundreds of thousands (which may be likely as the pandemic progresses), an automated and reproducible subsampling approach is essential to maximize efficiency and to avoid human error.\n\nHere we present genome-sampler5, a QIIME 2 plugin that enables other research teams to apply our context sequence subsampling workflow. Our subsampling workflow is compatible with tools such as NextStrain4, which includes a similar but not identical subsampling process (details provided in the Discussion section). We believe that our workflow can reduce sampling bias in analysis of SARS-CoV-2 genomes, and could be applied for regionally focused analyses, such as ours, or globally focused analyses. QIIME 26 (https://qiime2.org) is a plugin-based bioinformatics software platform developed for microbiome multi-omics analysis. It includes a unique retrospective data provenance tracking system that ensures reproducibility of bioinformatics steps by recording details of all analysis steps (commands called, parameters and input arguments provided, as well as details of the computational environment where the analysis was run, such as versions of underlying software dependencies; see examples at https://view.qiime2.org and in Figure 2 of the QIIME 2 paper6). We built this functionality as a QIIME 2 plugin because, given the pace at which SARS-CoV-2 genomics research is currently being carried out, human error in bioinformatics workflows is likely and the detailed record keeping needed to ensure reproducibility may be inadvertently skipped. QIIME 2 ensures that workflow errors could be detected retroactively and that workflows can be reproduced, even if detailed records are not kept while they are being run.\n\n\nMethods\n\ngenome-sampler5 operates on three input files: a fasta file containing the unaligned context sequences, a fasta file containing the unaligned focal sequences, and a tab-separated text file containing metadata for the context sequences. The context sequences and metadata will typically be obtained by the user from a public repository such as GISAID. The focal sequences will typically be sequences that the team has compiled independently, for example from their locale.\n\ngenome-sampler can be installed in a conda environment on macOS or Linux systems, as described in its installation documentation linked from the project website. The complete workflow can be applied in one step using the included Snakemake7 workflow, or the steps can be applied individually.\n\n\nUse case\n\nHere we describe the series of steps taken by the genome-sampler5 workflow (see Figure 1). In each step, any parameter values that can be overridden by the user are bolded. This description is accompanied by an online tutorial, available from the project website, which illustrates a use case focused on a small set of sequences obtained from GISAID. The tutorial is tested with each release of genome-sampler to ensure that all commands remain up to date.\n\nThis workflow samples context sequences for downstream phylogenetic analysis. Specific steps are represented by boxes: the QIIME 2 plugin name is bolded, and the action name in monospace font. Inputs and outputs are represented by folded-page file icons. The surrounding dashed box represents the Snakemake workflow which automates execution of the contained steps. Given context metadata, context sequences, and focal sequences, the Snakemake workflow will produce a fasta file which is ready for alignment and a summary of the sampling procedure as a QIIME 2 visualization.\n\nThe genome-sampler workflow works as follows:\n\n1. Clean up and filter the context sequences.\n\ni. Filter sequences that contain non-IUPAC characters8 as these characters can be problematic for downstream tools, such as sequence aligners or alignment viewers.\n\nii. Remove any gap (“-” or “.”) characters, as this workflow is intended to work on unaligned sequences. (Aligned reference sequences can be provided as input since they will be unaligned in this step.)\n\niii. Filter sequences that are composed of >10% N characters.\n\n2. Uniformly sample context sequences across time, selecting 7 sequences from each 7-day period between the earliest and latest dates represented in the data set. If there are fewer than 7 sequences in any 7-day period, all sequences from that period are included in the result. These sequences are referred to as the temporally sampled context sequences. The user can optionally supply a start date, in which case any genomes from before that time will be excluded.\n\n3. Search focal sequences against context sequences to identify their 10 closest matches. This is achieved using vsearch’s usearch_global option9 at 99.99 percent identity. The resulting collections of best hits are sampled to select 3 geographically distinct context sequences for inclusion in the subsampled context sequence collection. This sampling procedure is weighted such that each geographic region has an equal probability of selection instead of each genome. This weighting prevents overrepresented regions from dominating the sample. This step ensures that any monophylies of target sequences are not artifacts of our sequence sampling approach. These sequences are referred to as the geographically sampled context sequences. (This step is achieved using sequence metadata, and can be parameterized so that this can be applied over any categorical metadata, not just geography.)\n\n4. Cluster the complete context sequence collection with vsearch’s cluster_fast option at 99.90 percent identity. The resulting cluster centroid sequences represent a divergent collection of the SARS-CoV-2 genomes and are referred to as the diversity sampled context sequences.\n\n5. Combine the temporally, geographically, and diversity sampled context sequences with the focal sequence collection. The resulting collection of sequences will be deduplicated by sequence identifier, so sequences contained in multiple different subsamples are represented only once in the final sequence collection. This final collection of sequences should be used for downstream analysis.\n\n\nDiscussion\n\nThe NextStrain workflow also subsamples context sequences for its phylogenetic tree builds using augur (https://github.com/nextstrain/augur) and scripts in their ncov repository (https://github.com/nextstrain/ncov). Their workflow subsamples the context sequences across two axes: time and geography, prioritizing similarity to focal sequences when selecting sequences from different geographic regions. They sample across time by including a specified number of sequences per month for different regions. When determining the closest matches, percent identity is computed based on a multiple sequence alignment of all sequences, which is computed by aligning each sequence against a reference alignment using mafft10.\n\nStep 2 of our workflow is similar to their time sampling approach, but is independent of other variables such as geography. The workflows diverge more in Step 3, where we begin by identifying near neighbors of all focal sequences using global alignment search with vsearch. We then optionally sample across the geographic source of those sequences such that each geographic region represented in each collection of near neighbors has an equal probability of selection. We follow this with Step 4, where we sample the full genetic diversity of the context sequences by clustering them all against one another and including the resulting cluster centroid sequences in our final sequence collection. As far as we are aware, there is not an analog to our Step 4 in the NextStrain workflow.\n\nOur workflow is modular in design to facilitate benchmarking and optimization of this essential context sequence sampling step. Our three sampling steps can be used individually or in any combination, and can be replaced with a random sampling step (the sample-random action) to allow evaluation of the importance of each step. At this stage, we do not claim that our workflow is better than the one used by NextStrain. We hope the similarity of our interfaces (both of which require the same input and output, are accessible through Snakemake, and use the same terminology to describe data) will allow for independent comparison of these and other approaches. In our next stage of work on this project, we plan to evaluate the impact of each subsampling step and their associated parameters on downstream phylogenetic results.\n\nThe retrospective data provenance tracking system implemented in QIIME 2 differs from other systems such as Snakemake7 or Galaxy11, which we view as providing prospective data provenance tracking. For example, when a Snakemake file is used to run a workflow, that workflow is documented for reproducibility by the Snakemake file. However, if a user were to run the underlying commands independently, they must keep detailed records of their commands to ensure reproducibility of the analysis. This is not necessary with QIIME 2’s retrospective data provenance tracking system, which records steps regardless of whether the workflow is run using a tool like Snakemake or Galaxy, or whether individual components are run independently. Additionally, QIIME 2’s system assigns universally unique identifiers (UUIDs) to all execution steps, inputs, and outputs, so data can be unambiguously linked to workflow descriptions. QIIME 2 is therefore fully compatible with workflow engines such as Snakemake or Galaxy, but provides additional information which further ensures reproducibility.\n\nWe present genome-sampler5, a QIIME 2 plugin that supports subsampling of genomic sequence collections based on time of genome isolation, geography of genome isolation, and genomic diversity, thus facilitating genomic epidemiology based on large numbers of genomes while reducing the possibility of post-sequencing sampling bias impacting conclusions. As the number of available SARS-CoV-2 genomes continues to increase rapidly, approaches such as this will be required to enable phylogenetic and other analyses of genome data.\n\n\nData availability\n\nThe context sequences and metadata used in the genome-sampler Use case were obtained from GISAID. Those genomes were sampled from patients in Arizona, USA, and published to GISAID by the Arizona COVID-19 Genomics Union (ACGU). The focal sequences and metadata used in the genome-sampler Use case were sequenced at a later time than the context sequences, also from patients in Arizona. The focal sequences were generated and assembled by the ACGU and are currently being added to GISAID. These context and focal sequences and associated metadata are all available for download for use in learning genome-sampler (see the project website). For analysis purposes, we recommend obtaining sequences from a public repository, such as GISAID or GenBank, as those sequences will be updated (for example to improve genome assemblies) before our tutorial data is updated.\n\n\nSoftware availability\n\ngenome-sampler source code available at: at https://github.com/caporaso-lab/genome-sampler.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.38918195.\n\nLicense: BSD 3-Clause \"New\" or \"Revised\" License.\n\nDocumentation, written using Myst (https://myst-parser.readthedocs.io/en/latest/) and rendered using Jupyter Book (https://jupyterbook.org/), is available at http://caporasolab.us/genome-sampler/. If you need technical support, please post a question to the QIIME 2 Forum at https://forum.qiime2.org. We are very interested in contributions to genome-sampler from the community - please get in touch via the GitHub issue tracker or the QIIME 2 Forum if you’re interested in contributing.", "appendix": "Acknowledgements\n\nWe are deeply indebted to the patients who provided SARS-CoV-2 samples (via an uncomfortable procedure) while suffering from this new pathogen, to the medical professionals who bravely collected the samples that form the basis for our genomic epidemiology work, and to the laboratory professionals who processed and sequenced genomes from these samples. Furthermore, we would like to acknowledge the GISAID sequence contributors from across the world who have sequenced genomes at an unprecedented rate and made them available for public use.\n\n\nReferences\n\nElbe S, Buckland-Merrett G: Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob Chall. 2017; 1(1): 33–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBenson DA, Cavanaugh M, Clark K, et al.: GenBank. Nucleic Acids Res. 2013; 41(Database issue): D36–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLadner JT, Larsen BB, Bowers JR, et al.: Defining the Pandemic at the State Level: Sequence-Based Epidemiology of the SARS-CoV-2 virus by the Arizona COVID-19 Genomics Union (ACGU). medRxiv. 2020; 2020.05.08.20095935. Publisher Full Text\n\nHadfield J, Megill C, Bell SM, et al.: Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018; 34(23): 4121–4123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCenter for Applied Microbiome Science, Pathogen and Microbiome Institute: genome-sampler: Reproducibly Sampling SARS-CoV-2 Genomes Across Time, Geography, and Viral Diversity (Version 2020.6.0). Zenodo. 2020. http://doi.org/10.5281/zenodo.3891819\n\nBolyen E, Rideout JR, Dillon MR, et al.: Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019; 37(8): 852–857. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKöster J, Rahmann S: Snakemake--a scalable bioinformatics workflow engine. Bioinformatics. 2012; 28(19): 2520–2522. PubMed Abstract \| Publisher Full Text\n\nCornish-Bowden A: Nomenclature for incompletely specified bases in nucleic acid sequences: recommendations 1984. Nucleic Acids Res. 1985; 13(9): 3021–3030. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRognes T, Flouri T, Nichols B, et al.: VSEARCH: a versatile open source tool for metagenomics. PeerJ. 2016; 4: e2584. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfgan E, Baker D, Batut B, et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res. 2018; 46(W1): W537–W544. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "65756", "date": "31 Jul 2020", "name": "James Hadfield", "expertise": [ "Reviewer Expertise Bioinformatics", "phylogenetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a software tool to tackle a pressing, but welcome, problem: the number of publicly shared SARS-CoV-2 sequences (c. 75,000 at the time of this review) are too numerous to be analysed or visualised using currently available methods in a time-frame relevant for understanding local outbreaks. There is a need for researchers to be able to interrogate a particular collection of samples in a wider (often worldwide) context, and the choice of such context will greatly influence the conclusions drawn. The approach presented here samples contextual sequences by considering temporal, geographical and sequence-similarity data.\nAs the paper notes, similar approaches are available and in use by the wider community, however there is value in creating a range of tools for researchers to employ as required and which best suit their needs. This tool is easily installable and provides a ready-to-use solution for a pressing problem. It is purposefully designed in such a way that it is interoperable with other approaches, and will be immediately useful to researchers.\nI agree with the authors that a thorough evaluation and comparison of different subsampling approaches is beyond the scope of this paper, however there are some aspects which were not discussed in the manuscript which should be addressed (i.e. minor revisions required). Please note that this review focuses on the genome-sampler software described here, and is not a commentary on the wider QIIME 2 platform.\nInstallation\nFollowing https://caporasolab.us/genome-sampler/tutorial.html, installation was straightforward and the provided example worked out of the box. The tutorial was well written and didn't require any background knowledge of QIIME 2. The authors should be commended for this.\nPoints to address\nTime required. The example data provided (10 focal, 75 contextual sequences) took c. 40 minutes running on a laptop using a single core. As this tool will commonly be employed using all publicly available data as context (currently c. 75,000 genomes) an overview of the time (as well as memory & parallelizability) required to perform subsampling for various numbers of focal & contextual sequences should be provided.\n\nAligned genomes are not required as input as vsearch is used to compare genomes. My understanding is that vsearch will perform (a relatively fixed number of) pairwise alignments for each focal genome vs. the contextual data set to gauge percent identity. The paper would benefit from a short explanation of why this approach was used rather than aligning all sequences (e.g. to a reference genome).\n\nThere is no ability to subsample focal sequences. As the authors correctly mention in the introduction, the impressive rate of genome sequencing presents a number of challenges. It is already a reality that certain locally-derived (focal) datasets are large enough to require subsampling of their own, and this will become more common over time. The authors should address this by either implementing the ability to sample focal sequences or prescribing that the researcher must define a suitably small focal set.\n\nThe rapid submission of samples to public repositories should to be facilitated as much as possible. It may be commonplace to have focal samples which are also present in the contextual data. Could the authors detail what would happen in this case (e.g. are the contextual \"duplicates\" removed or will they bias steps 3 & 4 as they may preclude the inclusion of other samples?), or does this use-case need to be avoided by the user?\nMinor points\nStep 5 is not annotated on figure 1.\n\n[page 3, paragraph 3] Sampling across time won't necessarily allow the reliable inference of a clock signal, but is rather a prerequisite.\n\n[page 4, step 1(iii)] Are short sequences (i.e. those with lots of gaps) excluded here, or only those with a large proportion of Ns?\n\n[page 3, step 3] Reword to clarify that this step is performed per-sample, i.e. that the (10) closest matches are found for each sample in the focal set.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6047", "date": "28 Oct 2020", "name": "Greg Caporaso", "role": "Author Response", "response": "Thanks for your thoughtful review of our manuscript! We have reviewed your comments and are submitting a revision that addresses them as detailed here. The reviewer comments are presented in italics, and our replies to each follow in-line. InstallationFollowing https://caporasolab.us/genome-sampler/tutorial.html, installation was straightforward and the provided example worked out of the box. The tutorial was well written and didn't require any background knowledge of QIIME 2. The authors should be commended for this.Thank you! Points to addressTime required. The example data provided (10 focal, 75 contextual sequences) took c. 40 minutes running on a laptop using a single core. As this tool will commonly be employed using all publicly available data as context (currently c. 75,000 genomes) an overview of the time (as well as memory & parallelizability) required to perform subsampling for various numbers of focal & contextual sequences should be provided.In response to this request we have added a new figure to this paper presenting a benchmark of CPU time and memory usage for different sized context and focal genome data collections. Our results are presented in Figure 1. Aligned genomes are not required as input as vsearch is used to compare genomes. My understanding is that vsearch will perform (a relatively fixed number of) pairwise alignments for each focal genome vs. the contextual data set to gauge percent identity. The paper would benefit from a short explanation of why this approach was used rather than aligning all sequences (e.g. to a reference genome).In the ideal scenario, for our sample_diversity step, we would align all genomes in a collection against all other genomes in that collection, compute all similarities between genomes, and then use those pairwise similarities to cluster genomes based on their similarity. We could then select one or more sequences from each cluster for downstream analysis, which would ensure that we have represented the diversity of the genome collection. Computing all pairwise alignments is too time consuming for most practical applications however, so we use vsearch’s heuristic approach which tries to achieve the same goal but by reducing the number of pairwise alignments that are computed by trying to prioritize alignments between each sequence and sequences that are suspected to be highly similar to it. This provides higher resolution than the all-against-one approach that the reviewer mentions, because for example, if two query sequences (say ACGTT and AGGTA) are aligned to a single reference sequence (say ACGTA), we know that both are 80% similar to the reference, but we don’t know how similar those sequences are to each other (they could be 60% similar to each or 100% similar to each other in this example). Thus we could represent distance to a reference sequence reasonably well using the all-against-one comparison, but we wouldn’t know if we had sampled the full diversity represented in the genome collection. Since we were interested in using the vsearch approach for the sample_diversity step, it made sense to us to also use this for the other steps in the workflow. There is no ability to subsample focal sequences. As the authors correctly mention in the introduction, the impressive rate of genome sequencing presents a number of challenges. It is already a reality that certain locally-derived (focal) datasets are large enough to require subsampling of their own, and this will become more common over time. The authors should address this by either implementing the ability to sample focal sequences or prescribing that the researcher must define a suitably small focal set.It actually is possible to sample the focal sequences using the same approaches that are applied to the context sequences in our tutorial, though we acknowledge that that was not clear (and undocumented) in our original submission. In the revised documentation included with our updated release we have added a section that specifically discusses this point. See the new section, Sampling focal sequences. The rapid submission of samples to public repositories should to be facilitated as much as possible. It may be commonplace to have focal samples which are also present in the contextual data. Could the authors detail what would happen in this case (e.g. are the contextual \"duplicates\" removed or will they bias steps 3 & 4 as they may preclude the inclusion of other samples?), or does this use-case need to be avoided by the user?This is a great point which we did not address in our initial version of the documentation. This situation would bias the sample-neighbors step in particular, as those duplicated sequences would be very likely to be chosen as the nearest neighbors. We have added a note to the main tutorial, and a new section to the documentation Removing sequences present in both focal and context sequence collections, illustrating how the user can address this. Minor pointsStep 5 is not annotated on figure 1.Thanks for pointing out this omission. We have updated the figure to fix this. [page 3, paragraph 3] Sampling across time won't necessarily allow the reliable inference of a clock signal, but is rather a prerequisite.We modified the text the reviewer is referring to clarify this point. [page 4, step 1(iii)] Are short sequences (i.e. those with lots of gaps) excluded here, or only those with a large proportion of Ns?Since the input sequences for genome-sampler are unaligned, any gap characters (- or .) are stripped from the sequences on import. At this step of the analysis, the user does have the ability to filter sequences based on their length (sequences with length less than a minimum length or greater than a maximum length, both of which can be specified by the user, are optionally filtered). This has been clarified in the text. [page 3, step 3] Reword to clarify that this step is performed per-sample, i.e. that the (10) closest matches are found for each sample in the focal set.We have modified the text to clarify this." } ] }, { "id": "67936", "date": "27 Aug 2020", "name": "C. Titus Brown", "expertise": [ "Reviewer Expertise bioinformatics and software development" ], "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 software package, genome-sampler, that subsamples collections of SARS-CoV-2 genomes with attention to various metadata attributes. The paper is well motivated and well written.\n\nMy review focuses on the tutorial and getting the software running.\n\nI was pleased to see that the authors posted their source code to an archival location (Zenodo).\n\nI would encourage the authors to specify a release (rather than the latest branch) to install in the tutorial. Similarly, the conda install instructions in the tutorial should pin the versions of the software; this could easily be done via an environment yml provided within the genome-sampler github. And (minor nit) specifying '-y' in the copy/paste command line for conda install would be good too!\n\nThe tutorial downloads FASTA data from a Dropbox. This is brittle and should be changed. I suggest using an archive (Zenodo, osf.io, DataDryad) for this data, and making it copy/paste downloadable. They're small enough to put in github and version, too.\n\nThe tutorial downloads spreadsheet data from a Google Docs spreadsheet, and I would encourage the authors to put this data in git as well - even if there's a pedagogical reason to ask users to go through downloading from a spreadsheet, it's brittle to have a content-mutable and unversioned URL be the only location for the tutorial data.\n\nThe approach of downloading the Snakefile is also brittle. I suggest revamping the tutorial so that it does a local install of a git clone; see e.g. this documentation (for an alpha package) that would be a simpler approach, I think: https://github.com/dib-lab/charcoal/tree/51caa9f034f3d301367cb6eea2ee96b5e1ea05bb#quickstart\n\nNote that snakemake supports config files that let you put the overridable configuration parameters in a YAML file. This might be nicer than editing the Snakefile directly. Happy to provide detailed examples on request.\n\nWhile I'm suggesting things, you could also use conda environments in the Snakefile to obviate the qiime etc. install. Then you'd just need to install snakemake and run it with -- use-conda to get it all done.\n\nWhen running the tutorial, I see the following error: ... /qiime2/sdk/util.py\", line 92, in parse_format raise TypeError(\"No format: %s\" % format_str) TypeError: No format: GISAIDDNAFASTAFormat\nI'm not sure where to go from here. I've filed an issue with the full details.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5870", "date": "28 Aug 2020", "name": "C. Titus Brown", "role": "Reviewer Response", "response": "I have managed to run the pipeline successfully, and have the following additional comments -- --- I successfully ran the tutorial, huzzah! It would be good to add a rough estimate of resources required (memory, disk, CPU time) to the tutorial." }, { "c_id": "6048", "date": "28 Oct 2020", "name": "Greg Caporaso", "role": "Author Response", "response": "Thank you for the feedback on our manuscript, and for testing the software and offering suggestions. Below we provide a point-by-point reply. Your comments are presented in italics, and we reply to each in-line. This paper describes a software package, genome-sampler, that subsamples collections of SARS-CoV-2 genomes with attention to various metadata attributes. The paper is well motivated and well written. My review focuses on the tutorial and getting the software running. I was pleased to see that the authors posted their source code to an archival location (Zenodo). I would encourage the authors to specify a release (rather than the latest branch) to install in the tutorial. Similarly, the conda install instructions in the tutorial should pin the versions of the software; this could easily be done via an environment yml provided within the genome-sampler github. And (minor nit) specifying '-y' in the copy/paste command line for conda install would be good too!We agree with the reviewer’s suggestion and have updated our install instructions to install from a specific release. The conda installation now includes specific pinned versions of dependencies. (We prefer to not include the -y option, but rather have the user acknowledge that changes are about to be made to their system, so we have not included that.) The tutorial downloads FASTA data from a Dropbox. This is brittle and should be changed. I suggest using an archive (Zenodo, osf.io, DataDryad) for this data, and making it copy/paste downloadable. They're small enough to put in github and version, too.We also agree with this suggestion. As of our most recent release, the tutorial files are now packaged in the GitHub repository and are included in the updated Zenodo package that we have prepared for the most recent release and paper resubmission. The tutorial downloads spreadsheet data from a Google Docs spreadsheet, and I would encourage the authors to put this data in git as well - even if there's a pedagogical reason to ask users to go through downloading from a spreadsheet, it's brittle to have a content-mutable and unversioned URL be the only location for the tutorial data.As of our most recent release, these files are now packaged in the GitHub repository and are included in the updated Zenodo package. The approach of downloading the Snakefile is also brittle. I suggest revamping the tutorial so that it does a local install of a git clone; see e.g. this documentation (for an alpha package) that would be a simpler approach, I think:https://github.com/dib-lab/charcoal/tree/51caa9f034f3d301367cb6eea2ee96b5e1ea05bb#quickstartWe have updated the tutorial so that this file is downloaded from a release version of genome-sampler, which is less brittle. We prefer to not have users install from a git clone directly, since we are officially supporting conda installation (which tends to be easier for our novice user), and adding a second officially supported installation method would add to our technical support burden. Note that snakemake supports config files that let you put the overridable configuration parameters in a YAML file. This might be nicer than editing the Snakefile directly. Happy to provide detailed examples on request.We thank the reviewer for this suggestion. We now use a Snakemake config file to configure these parameters. While I'm suggesting things, you could also use conda environments in the Snakefile to obviate the qiime etc. install. Then you'd just need to install snakemake and run it with -- use-conda to get it all done.This is an attractive option and we invested time in exploring this approach. We found that our platform-specific environment files complicate this, so we ultimately opted to not take this suggestion. Specifically, users would need to replace the environment path for every rule in the Snakefile (if they were using OS X instead of Linux, for example). We have simplified our installation workflow (both for Snakemake users and non-Snakemake users) and hope that our provided workflow can serve as a template for more sophisticated use-cases, such as rule-specific conda or singularity containers. When running the tutorial, I see the following error:.../qiime2/sdk/util.py\", line 92, in parse_formatraise TypeError(\"No format: %s\" % format_str)TypeError: No format: GISAIDDNAFASTAFormatI'm not sure where to go from here. I've filed an issue with the full details.We haven’t come across this error, but it is likely related to genome-sampler not being installed correctly. Our updated install instructions should simplify this. We’re happy to try to help work out the problem, but we haven’t seen this issue on our issue tracker. We have not had reports of other users running into this (but we have had other support requests, so we know that this is not something that is preventing people from using genome-sampler)." } ] } ]	1	https://f1000research.com/articles/9-657
https://f1000research.com/articles/9-301/v1	28 Apr 20	{ "type": "Research Article", "title": "Where have all the parasites gone? Unusual Plasmodium falciparum monoparasitaemia in a cross-sectional malariometric survey in northern Nigeria", "authors": [ "Usman Nasir Nakakana", "Ben O. Onankpa", "Ismaila Ahmed Mohammed", "Ridwan M. Jega", "Nma Muhammad Jiya", "Ben O. Onankpa", "Ismaila Ahmed Mohammed", "Ridwan M. Jega", "Nma Muhammad Jiya" ], "abstract": "Background: Malaria is caused by one of five currently known Plasmodium parasite species causing disease in humans. While modelling has provided information of the vector, the same is not entirely the case for the parasite. The World Malaria reports of 2014 to 2016 reported 100% of confirmed cases from Nigeria being due to Plasmodium falciparum. Generally, about 98% of cases of uncomplicated malaria in most regions surveyed in Nigeria recently is due to P. falciparum, with the remainder being due to P. malariae. This study aimed to determine the proportions of Plasmodium parasites causing uncomplicated malaria in Wamakko Local Government Area of Sokoto State, north-western Nigeria. Methods: The study was a descriptive, cross-sectional study conducted during the rainy season and dry season in north-western Nigeria. The area has a ‘local steppe’ climate and Sudanian Savannah vegetation. Sampling was via multistage cluster sampling. Selected participants were examined for pallor, palpable splenomegaly and signs of complicated malaria. Blood samples were also taken for rapid diagnosis of malaria and thick and thin films to identify parasitaemia and the parasite species. Participants found to have malaria were treated with Artemether/Lumefantrine and those with complicated malaria were referred to the nearest hospital. Results: We found a parasite prevalence of 34.8% overall, which was higher in the rainy season (49.3%) than in the dry season (20.2%). There was monoparasitaemia of Plasmodium falciparum throughout the study area, irrespective of the clinical status of the participant. Mapping of the parasite was extended throughout the Local Government Area and the State. Conclusions: Despite the intermediate endemicity in the area. P. falciparum monoparasitaemia affirms theories of disappearance of other parasite species, either due to faltering control of P. falciparum or more efficient control of other species.", "keywords": [ "Malaria", "Nigeria", "Plasmodium falciparum", "PfPR2-10" ], "content": "Introduction\n\nMalaria is caused by one of five currently known Plasmodium species causing diseases in humans. These are P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. Scientists have modelled the anopheles mosquito vector extensively based on its characteristics, but not so much the parasite1. An absence of the Duffy antigen on red blood cells of West Africans has long been postulated to be responsible for the absence of P. vivax in these areas, but cases of P. falciparum, P. malariae and P. ovale, in order of decreasing occurrence, have been found. The World Malaria Reports of 20142 and 20153 reported 100% of confirmed cases in Nigeria as being due to P. falciparum. It is generally thought to account for about 98% of all malaria cases, with P. malariae accounting for the rest, often as a co-infection with P. falciparum4. This figure likely over-estimates the proportion of cases as P. falciparum is responsible for most severe cases of malaria; these are the cases most commonly reported alongside confirmed cases of malaria, which are tracked by passive surveillance in Nigeria. It may also be because of limited expertise in identifying other species of Plasmodium.\n\nAvailable data from across Nigeria shows a mixed picture; large areas across northern Nigeria in 1967/68 found an average proportion of 22% of malaria parasitaemia due to P. malariae in a study at a time when most of Nigeria was considered holoendemic for malaria. P. malariae was often seen in coinfection with P. falciparum, particularly among younger age groups. P. ovale was responsible for 5% of malaria infections, being more common in children under five years of age, and P.falciparum ranged between 84.4% and 90.5% across age groups5. The proportion of P. malariae was high, probably because the data was obtained from a survey of both asymptomatic and symptomatic participants.\n\nMore recently, in 2010, prior to the commencement of nationwide LLIN distribution, a study including 4209 individuals in Jos, northern Nigeria, found a P. malariae rate as low as 1.6%, with P. falciparum responsible for 98.7% of infections, sometimes in co-infection with P. malariae4. Children aged less than 10 years and all individuals in every third household were selected for this abridged malaria indicator survey (MIS). Crucially, however, no P. vivax or P. ovale species were seen either in this location or another in south-eastern Nigeria, which was shown to have a higher rate of P. malariae infections (around 30%) and lower rate of P. falciparum infections (68.1%) in a study also conducted in 20106. This study, however, included 2,936 individuals from 1400 clusters in Abia state, spread out across the state. Quality control measures in the identification of parasite species were implemented in the study, including a WHO-certified malaria microscopist, giving credibility to the results obtained6. In south-western Nigeria, a study in Ikorodu in 2012 recruited 1,496 participants of all ages, which included 237 children under the age of five years and 509 children aged 15 years and below. Microscopy and DNA evaluation was used to determine parasite species, which found that 93.6% of participants had P. falciparum infection, with the remainder being P. malariae7. This is in contrast to previous studies in 1976 around the same location in south-western Nigeria, which found that 13% to 16% of parasitaemia was due to P. malariae, with 62 to 76% being due to P. falciparum8. These proportions were the age-group specific parasite prevalence in the study, which included mostly children aged five to 10 years (1,500 participants) in an attempt to compare spleen and parasite rates among individuals with sickle cell trait and those with normal adult haemoglobin. It also found P. ovale in 2% to 3% of participants in 5–10 and 2–4 year age groups, respectively. These findings, although limited in scope, perhaps suggest a changing trend, with the disappearance of P. ovale from Nigeria over time. P. falciparum is an undisputed leader in all the studies performed. Its high proportion perhaps accounts for high rates of malaria-related anaemia in most studies, explained by the ability of the parasite to invade and destroy both young and senescent red blood cells7,9.\n\nWe conducted this study to determine relative proportions of parasites causing clinical malaria in Sokoto, north-western Nigeria.\n\n\nMethods\n\nEthical approval was obtained from the Independent Ethics committee of Usmanu Danfoidyo University Teaching Hospital, Sokoto, Nigeria with ethical approval number UDUTH/HREC/2014/No. 246. Ethical approval was also obtained from the Independent Ethical committee of the Sokoto State Ministry of Health with the approval number SMH/1580/VIV. Permission was also obtained from the district heads of the included communities in the study to visit the communities.\n\nThe study was conducted in Wamakko Local Government Area of Sokoto State, located in the north-western geopolitical zone of Nigeria. It has an area of 732.146km2, with a projected population of 260,860 by 201910. It is located at coordinates 13°2′16″N 5°5′37″E. The geography of the area is predominantly flat plains with Sudan Savannah-type vegetation and it stands at an altitude of 292m above sea level, near to the confluence of the Sokoto and Rima rivers. Its climate is tropical, described as local steppe climate.\n\nThe study was a two–point, cross-sectional prospective descriptive study conducted during the rainy season and dry season. In April and November 2016, we screened and recruited participants simultaneously until we reached the target population.\n\nWe determined the minimum sample size using Cochran’s formula11, assuming a prevalence of 50% based on a previous survey12, and 500 participants gave a power of at least 80% to show reliable results.\n\nAs is the norm for MIS’s, multistage cluster sampling in proportion to size was employed. The primary clusters were four randomly selected wards of the eleven political wards within the Local Government Area (LGA) based on the population within each of the wards, with secondary clusters being eight settlements unevenly selected from the four wards; proportionate to size, making up the total sample size. Based on the assumptions of a 70% response rate and that 80% of households include at least one child less than five years of age, in accordance with a previous Nigeria MIS in 201013, approximately 892 households were required to meet the target of at least 500 participants per season based on the assumptions stated earlier. This was surpassed by the estimated number of households within the eight settlements selected. All children in the secondary clusters who fulfilled the inclusion criteria, and whose parents consented to participate, were included in the study.\n\nParticipants were visited at their homes and while in the household, after identifying the household head. They were provided with information regarding the study and all eligible children within the household invited to participate. Those parents who accepted signed or thumb-printed the informed consent form. All children in the selected settlements who met the age criteria of two to 10 years, with or without symptoms of malaria, were recruited for the study, provided they had been residents of the study area for at least two weeks. They were, however, excluded if they were suspected to have taken any medication with antimalarial properties within the two weeks prior to enrolment36. Recruitment was carried out on consecutive days until the entire village was covered. Each participant was evaluated once except for those who had parasitaemia without symptoms, who were followed up by a field assistant for up to 48 hours for the development of symptoms. The period of recruitment was about a month in each season.\n\nWe conducted the study procedures at a central location in each of the study villages. Field assistants went from house-to-house and recruited the participants and then brought the consenting participants to the central location. A paediatrician screened potential participants for eligibility and the caregivers of eligible participants were required to sign informed consent forms. He performed a physical examination for each participant and graded splenomegaly according to Hackett’s criteria14. The WHO criteria for severe malaria was used15. Using a single use lancet, we collected capillary blood by pricking the index finger of the child’s left hand. A drop of blood was collected each for a thick and thin malaria parasite film for estimation of parasite density and species identification, respectively.\n\nConcomitantly, during the same session, we did rapid diagnosis of malaria using a drop of blood with CareStart® Malaria HRP2 rapid detection tests (RDTs) (Access Bio, Inc., model G0141), which can detect P. falciparum.\n\nEach day, we transported the samples to the paediatric department laboratory of the Usmanu Danfodiyo University Teaching Hospital and fixing of thick films was done with methanol. Thin films were stained immediately and stored in the lab. We analysed the samples in a completely anonymized manner in pairs of thick and thin films; examining the thin film if we found the thick film positive for malaria. The study numbers were the only identifiers for the thick films, which were kept apart from the thin films. A trained malaria microscopist performed the analysis, under the supervision of a medical parasitologist. We examined at least 10 fields before a slide was declared negative for malaria parasites.\n\nThe tail segment of the thin films was viewed to identify the species of malaria parasite, using the typical description of parasite species, having been trained on parasite identification16.\n\nQuality control of the diagnosis of the parasitaemia was provided by a trained medical microbiologist re-examining 10% of the slides selected at random. A discrepancy of 10% or more would have necessitated reanalysis of all the thick films and the thin films subsequently. The discrepancy was 3% (kappa score of 0.71) and as such, this was not necessary.\n\nStudy participants with malaria, determined by the presence of at least one symptom and a positive RDT or thick blood film, were treated by the study paediatrician at home with Artemether-Lumefantrine. Children were dosed according to standard dosing17 but only the first dose was directly observed.\n\nWe analysed the data using SPSS version 22. We determined the prevalence of malaria by parasitaemia and RDT by determining proportions. We used descriptive statistics to determine averages and proportions. Participants with missing data were excluded from the analysis. We carried out sub-group analysis for age, gender and season and used kappa analysis to control the quality of malaria diagnosis.\n\n\nResults\n\nWe screened a total of 1136 participants for inclusion in the study after they consented to participation in the study. We excluded 109 because they had been treated with antimalarials in the two weeks prior to enrolment and excluded 10 from the analysis due to incomplete data. We included 1017 participants in the analysis (Figure 1)18.\n\nThe age-sex distribution showed that all ages were equally represented in the study, as shown in Table 1.\n\nWe found an overall prevalence of malaria for the study of 34.8% using microscopy and 33.8% using RDT as shown in Table 2. There was an agreement between the two diagnostic methods, as shown by the kappa statistic (p <0.001).\n\nKappa agreement κ = 0.764; p <0.001.\n\nWe saw the highest age-specific prevalence among participants aged two years, with the lowest among ten-year olds. Table 3 also shows a significant association between the age of the participants and prevalence of malaria parasitaemia (p= 0.000).\n\nχ2 = 38.453; df = 8; p<0.01.\n\n279 participants were found across the seasons to have clinical malaria and all of them had P. falciparum malaria, irrespective of the season and nature of their clinical presentation. The relative proportions of parasites are depicted in Figure 2. The most common presenting feature among these was fever (92%), followed by vomiting (38%), refusal to feed/poor appetite (32%) and body weakness (25%).\n\nOf the clinical cases of malaria, 9.6% had complicated malaria, as indicated by the WHO criteria for severity19. The number of severe malaria cases was significantly lower in the dry season than the rainy season, as shown in Table 4. Different participants had various combinations of the criteria for severity, although the common criteria were hyperpyrexia, prostration and persistent vomiting.\n\nχ2 = 18.883; df = 1; p = 0.000.\n\nThe prevalence of malaria parasitaemia during the rainy season was significantly higher than the dry season, with prevalence rates of 49.3% and 20.2%, respectively, all due to P. falciparum.\n\nThe mean parasite density was much higher during the rainy season (1006.13) than during the dry season (405.45). The details are shown in Table 5.\n\n\nDiscussion\n\nThe prevalence of malaria in this study, when compared with serial MIS’s performed in 201013 and 201520 shows a progressive reduction; from 48.1% to 37.1% for north-western Nigeria, and a prevalence of 46.6% for Sokoto in 2015 compared with 34.8% for our study. Prevalence in the MIS’s was measured among children aged six to 59 months and is probably higher than for the children included in this study because including the children from 6–10 years is likely to reduce the overall prevalence, as is excluding those aged six months to two years, who generally have a higher prevalence rate21.\n\nAlthough there were studies performed in the past in Sokoto, they are limited in comparison to the present study by virtue of having been conducted in a different age group, or hospital in lieu of community setting and the seasons in which these studies were conducted. The prevalence of 34.8% found here was higher than the 27.9% found by Abdullahi et al.12 in Sokoto; however, samples in the previous study were collected from patients visiting two hospitals within the metropolis and was thus not community-based. Furthermore, all ages from 0 to 65 years were included in the study, which is likely to further dilute the findings and give a falsely low prevalence because the incidence of malaria is generally lower among adolescents and adults, as indicated in the study. The overall picture supports the suggestion of a reduction in the prevalence of malaria, likely owing to better access to malaria prevention and increasing urbanisation; both of which cause a decline in malaria parasite rates generally22.\n\nThe prevalence found in this study is also lower in comparison to the 45.4% prevalence rate found in a study by Jiya et al.23, conducted in Sokoto between 2007 and 2009. Additionally, it was lower than the prevalence of 49.6% found among children under the age of five years in the same study. Considering both age-specific prevalence rates, there is a reduction in prevalence, although being a hospital-based study, the prevalence for the former study is likely to be higher than the current. It is, however slightly, higher than the projected national average of 29% for 2015, with wide inter-regional differences24. The Nigerian MIS of 2015 found a higher prevalence of 46.6% than this study, although the age of included participants ranged from six to 59 months, which will limit the comparability of results from this study due to the different age ranges of participants20.\n\nThe prevalence by age in this study roughly indicated a progressive decline with age. The highest age-specific prevalence was among two-year-olds (50.4%), with a statistically significant difference among the age groups. This finding is in conformity with the steady-state assumption and is similar to findings in previous studies that showed higher prevalence among younger age-groups.\n\nWith respect to the parasite species causing uncomplicated malaria, all parasitaemia in this study was found to be due to P. falciparum. This is in keeping with recent studies in Adamawa25 and Cross River states9 in 2011 and 2013, respectively. Another study from Ihiala, in Anambra state of south-eastern Nigeria, found P. falciparum mono-parasitaemia even though this study considered all types of malaria, both severe and uncomplicated26. This is, however, unlikely to affect the findings, as most cases of severe malaria in this area are due to P. falciparum26. An earlier report from Sokoto between 2005 and 2006, carried out at Usmanu Danfodiyo University Teaching Hospital by Jiya and Sani23,27 likewise did not find any Plasmodium species apart from P. falciparum, although they only considered cases of severe malaria, which are unlikely to be due to a different species of Plasmodium within Nigeria. Meanwhile, only three cases out of 582 (0.01%) were positive for P.malariae in another study by Nwaorgu and Orajaka28 in Awka, south-eastern Nigeria. The finding of P. falciparum mono-parasitaemia supports the fact that P. falciparum is the dominant species of Plasmodium in Sub-Saharan Africa, with the tendency to exclude other forms of parasitaemia, as expounded by Lucas and Gilles29 in 1998 with time and sustained malaria control activities.\n\nOther studies have shown the presence of other forms of parasitaemia, notably with P. malariae either as mono-infection or coinfection with P. falciparum. In Abia and Plateau states (2010), P. malariae accounted for 32.0% and 1.4% of malaria infections, respectively6. In a study in north-central Nigeria, 6.1% of examined participants had P. malariae infection30 and as high as 41% and 4%, respectively, had P. malariae and P. ovale infections in a historical study in Garki, Abuja (1968)31. Outside Nigeria, there has been a shift towards mono-parasitaemia with P. falciparum as well, and this has been documented in the Horn of Africa32 and Benin in West Africa33. Some authors have suggested it be an evidence of failing control measures but this is at variance with data from this study, which shows a reduction in prevalence from previous data, including a reduction in the number of severe cases of malaria, which were very few in this study.\n\nSevere malaria was seen in 26 of the 1017 participants analysed in this study, with an overall prevalence of 2.6%. It was higher during the rainy than the dry season, probably due to higher prevalence of the disease and higher parasitaemia, as earlier discussed. This is lower than expected from other hospital-based studies for which children presenting to the hospital are more likely to be ill than those found in a community-based survey such as this. In one such study in Ilorin by Olanrewaju and Johnson29 found that a third of all children admitted with malaria had a severe form of malaria.\n\n\nData availability\n\nFigshare: complete data.xlsx. https://doi.org/10.6084/m9.figshare.11590542.v118.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWith their permission, we wish to acknowledge Abdurrahman I and Yakubu B for assisting in the laboratory analysis and Jap Van Hellmond for providing guidance with species identification.\n\n\nReferences\n\nMoffett A, Shackelford N, Sarkar S: Malaria in Africa: vector species’ niche models and relative risk maps. PLoS One. 2007; 2(9): e824. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: World malaria report 2014. Nigeria. Geneva; 2014. Reference Source\n\nWorld Health Organization: World Malaria Report 2015. Geneva, Switzerland; 2015. Reference Source\n\nFederal Ministry of Health: Federal republic of Nigeria, National Antimalarial Treatment policy. 2005. Reference Source\n\nVoller A, Bruce-Chwatt LJ: Serological malaria surveys in Nigeria. Bull World Health Organ. 1968; 39(6): 883–97. PubMed Abstract \| Free Full Text\n\nNoland GS, Graves PM, Sallau A, et al.: Malaria prevalence, anemia and baseline intervention coverage prior to mass net distributions in Abia and Plateau States, Nigeria. BMC Infect Dis. BMC Infectious Diseases; 2014; 14(1): 168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAina OO, Agomo CO, Olukosi YA, et al.: Malariometric survey of ibeshe community in ikorodu, lagos state: dry season. Malar Res Treat. 2013; 2013: 487250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoyo AE: Malariometric indices and hemoglobin type. Am J Trop Med Hyg. 1972; 21(6): 863–7. PubMed Abstract \| Publisher Full Text\n\nUdoh EE, Oyo-ita AE, Odey FA, et al.: Malariometric Indices among Nigerian Children in a Rural Setting. Malar Res Treat. 2013; 2013: 716805. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational population commision: Federal Republic of Nigeria Housing Census 2010. 2010; III. . Reference Source\n\nSnedecor G, Cochran W: Statistical Methods. 8TH ed. Iowa State Press; 1989. Reference Source\n\nAbdullahi K, Abubakar U, Adamu T, et al.: Malaria in Sokoto, North Western Nigeria. African J Biotechnol. 2009; 8(24): 7101–5. Reference Source\n\nNPC, NMCP, ICF International: Nigeria Malaria Indicator Survey (MIS) 2010. Abuja, Nigeria.; 2012. Reference Source\n\nHackett LW: Spleen measurement in malaria. NatMalariaSoc. 1944; 3(2): 121–33.\n\nWHO: Treatment of Severe Malaria. In: Guidelines For The Treatment of Malaria. Geneva; 2015; 71–88. Reference Source\n\nWHO: Basic malaria microscopy. 2nd editio. Geneva, Switzerland; 2010; 69–75. Reference Source\n\nMasanja IM, Selemani M, Khatib RA, et al.: Correct dosing of artemether-lumefantrine for management of uncomplicated malaria in rural Tanzania: do facility and patient characteristics matter? Malar J. 2013; 12(1): 446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNakakana U, Mohammed Jiya N, Onankpa BO, et al.: complete data.xlsx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11590542.v1\n\nWHO: Management of Severe malaria, a practical handbook. 3rd ed. Geneva, Switzerland: World Health Organization; 2012; 41–42. Reference Source\n\nNational Malaria Elimination Programmme, National Population Commission, National Bureau of Statistics, ICF International: Nigeria Malaria Indicator Survey 2015: Key Indicators. Abuja, Nigeria and Rockville, Maryland, USA: 2016. Reference Source\n\nGething PW, Patil AP, Smith DL, et al.: A new world malaria map: Plasmodium falciparum endemicity in 2010. Malar J. 2011; 10(1): 378. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTatem AJ, Gething PW, Smith DL, et al.: Urbanization and the global malaria recession. Malar J. 2013; 12(1): 133–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiya N, Sani U, Jiya F, et al.: Prevalence of uncomplicated malaria in a paediatric out patient department of a tertiary health institution in Sokoto, Nigeria. Sahel Med J. 2010; 13(1): 29–34. Publisher Full Text\n\nMalaria Atlas Project: Malaria map of Africa. Maps. [cited 2016 May 10]. 2016; 1. Reference Source\n\nHouben CH, Fleischmann H, Gückel M: Malaria prevalence in north-eastern Nigeria: a cross-sectional study. Asian Pac J Trop Med. 2013; 6(11): 865–8. PubMed Abstract \| Publisher Full Text\n\nAribodor DN, Njoku OO, Eneanya CI, et al.: Studies on prevalence of malaria and management practices of the Azia community, Ihiala L.G.A., Anambra State, south-east Nigeria. Niger J Parasitol. 2003; 24(1): 12–9. Publisher Full Text\n\nJiya N, Sani U: Pattern and Outcome of Severe Malaria among Children in Sokoto, North Western Nigeria. WJBiomed Res. 2016; 3(1): 6–12.\n\nNwaorgu OC, Orajaka BN: Prevalence of Malaria among Children 1 – 10 Years Old in Communities in Awka North Local Government Area, Anambra State South East Nigeria. African Res Rev. 2011; 5(5): 264–81. Publisher Full Text\n\nLucas AO, Gilles HM: A New Short Textbook of Preventive Medicine for the Tropics: Malaria. 2nd ed. Great Britain: ELBS with Edward Arnold; 1998; 188–192.\n\nNmadu PM, Peter E, Alexander P, et al.: The Prevalence of Malaria in Children between the Ages 2–15 Visiting Gwarinpa General Hospital Life-Camp. J Health Sci. 2015; 5(3): 47–51. Reference Source\n\nMolineaux L, Gramiccia G: The Garki project: research on the Epidemiology and Control of Malaria in the Sudan Savanna of West Africa. Geneva, Switzerland; 1980. Reference Source\n\nO’Meara WP, Mangeni JN, Steketee R, et al.: Changes in the burden of malaria in sub-Saharan Africa. Lancet Infect Dis. 2010; 10(8): 545–55. PubMed Abstract \| Publisher Full Text\n\nKleinschmidt I, Omumbo J, Briët O, et al.: An empirical malaria distribution map for West Africa. Trop Med Int Health. 2001; 6(10): 779–86. PubMed Abstract \| Publisher Full Text" }	[ { "id": "69662", "date": "01 Sep 2020", "name": "Manuela Runge", "expertise": [ "Reviewer Expertise malaria epidemiology in school children", "malaria surveillance", "mathematical modelling of malaria" ], "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 presents findings from a cross-sectional study conducted in one LGA (Wamakko) in Sokoto State in Nigeria that aimed to determine the proportions of plasmodium parasites causing uncomplicated malaria. The study included data collection in both, rainy as well as the dry season. The diagnosis of malaria in study participants was done via rapid tests and dried blood spots, which in the study closely aligned with each other (3% difference). The key finding is monoparasitaemia in the study population with all of the malaria infections caused by plasmodium falciparum. However, it is not exactly clear why this study was performed. The authors did not make a clear statement of the gap in the literature or how their study complements the literature. The studies that they cited suggests that the monoparasitemia as it relates to malaria in Nigeria is a known fact. Is their goal to confirm this for a different geographical location? A more clearly written aims and study motivation will convince readers of the importance of their study\nMajor comments\nPlease elaborate on your methods.\nIf cluster sampling proportionate to size was used, list all the wards and settlement in a table with their population sizes. This can be included in the supplement. How was cluster selection done? Did you stratify by ward and settlement sizes before random sampling? Be explicit about what was done and how it was done.\n\nPlease use the discussion section to discuss the implications of your study for treatment and control and the wider scientific community. What are the study limitations? The bulk of this section should not be spent comparing your findings with other studies. We would refer the authors to this link on how to write a discussion section. There are other resources online as well.\n\nIn the discussion, studies are cited that report monoparasitaemia, which seems circular/contradictory to the title of ‘unusual’ monoparasitaemia, as the discussion indicates that this could have been expected? The authors may consider rephrasing the title or to add a stronger argumentation in the introduction and discussion (see major comment 1).\n\nThe conclusions are missing in the main text, but included in the abstract.\n\nThe potential reasons for the disappearance “either due to faltering control of P. falciparum or more efficient control of other species” (last part of the abstract conclusion) could be discussed more in the discussion as one would expect from the title.\n\nSevere versus clinical malaria. The study aim states a focus on uncomplicated malaria, however, Table 4 only shows severe malaria, which should also show uncomplicated malaria (or instead). The WHO criteria for severe malaria were used, does that include criteria for uncomplicated malaria if not which criteria were used? (Paragraph on Procedures, line 9). How were severe cases treated in the study did they also receive ALu at home or were referred to the next hospital or health facility? Moreover, it would be relevant to show uncomplicated malaria per age in addition to parasitaemia in Table 3.\n\nMinor comments\nPrevious studies reported the highest parasitaemia among school-aged children 1 and prevalence curves that peak among school children while incidence curves peak in children under the age of five 2. The article here found that parasitaemia is highest at age two and then decreases by age. A sentence for clarification on this would be helpful that explain the decrease over age?\n\nThe authors found 65% to be non-malaria fevers, this is interesting and the authors may consider highlighting it as a finding and adding it to the discussion (Fig 2).\n\n“Scientists have modelled the anopheles mosquito vector extensively based on its characteristics, but not so much the parasite” the reference to this study and to niche models of vectors is confusing, as the author’s study is not doing niche modelling or geographic mapping of parasites. It would seem more appropriate to have a description of the geographical occurrence of parasites leading over to the next sentence.\n\nMissing reference for the Duffy antigen sentence line 6 to 9 in the first paragraph (“An absence of the Duffy antigen on red blood cells of West Africans has long been postulated to be responsible for the absence of P. vivax in these areas, but cases of P. falciparum, P. malariae and P. ovale, in order of decreasing occurrence, have been found”).\n\n“Participants were visited at their homes and while in the household” Perhaps should be “while being at home”? Also, were participants visited after identifying the head, or happened both at the same time, visiting the household and then identifying the household head?\n\nConfusing use of household head – parent - caregivers, are these refer all to the same (i.e. are household heads always parents?)\n\nThe authors cite the Nigerian national treatment policies for parasite co-infections, which however is not the main source which remains unclear, is there an actual study that could be cited?\n\n“five to 10 years” write out numbers below twenty, or use numbers consistently.\n\nPossible gender bias in writing ‘he’ for the pediatrician “He performed a physical examination for each participant and graded splenomegaly according to Hackett’s criteria”.\n\n“Concomitantly, during the same session…” consider removing duplication?\n\n“The study numbers” is not explained before; do the authors mean participant identifiers or study participant numbers?\n\nUnclear sentence: “The tail segment of the thin films was viewed to identify the species of malaria parasite, using the typical description of parasite species, having been trained on parasite identification”, who has been trained?\n\nUnclear sentence: “We determined the prevalence of malaria by parasitaemia and RDT by determining proportions.” Maybe the authors could add formula pos mRDT/tested by mRDt; pos BS/ tested by BS to be clearer?\n\nConsider reducing the black background in figures and tables and not using 3D plots, see some general rules on scientific figures in 3.\n\nBe consistent with ‘malaria’ or ‘clinical malaria’ to distinguish malaria infection from clinical malaria, i.e. Fig 2 caption and title, and text description.\n\nConfusing Fig 2, as it reads as if ‘no parasite can also cause malaria’? Would a barchart be more appropriate? Or consider rephrasing the figure title.\n\nReference number 36 is missing or not needed (sample procedures line 11), reference list goes only up to 33.\n\n‘The finding of P. falciparum mono-parasitaemia supports the fact that P. falciparum is the dominant species of Plasmodium in Sub- Saharan Africa’ Consider rephrasing like ‘confirms/ support the other findings of mono-parasitaemia’ to avoid the term ‘fact’.\n\nThe discussion structure might need some revision for better understanding, for example, the last paragraph on severe malaria in the discussion might fit better after discussing uncomplicated malaria and before discussing monoparasitaemia in other studies.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5947", "date": "27 Oct 2020", "name": "Usman Nasir Nakakana", "role": "Author Response", "response": "Please elaborate on your methods. If cluster sampling proportionate to size was used, list all the wards and settlement in a table with their population sizes. This can be included in the supplement. How was cluster selection done? Did you stratify by ward and settlement sizes before random sampling? Be explicit about what was done and how it was done This has been done, there were two strata, selected independently. The first stratum was proportional to size, i.e at the ward level. Please use the discussion section to discuss the implications of your study for treatment and control and the wider scientific community. What are the study limitations? The bulk of this section should not be spent comparing your findings with other studies. We would refer the authors to this link on how to write a discussion section. There are other resources online as well.I have added sections and modified the discussion as per your recommendations. Implications to the wider scientific community are included. In the discussion, studies are cited that report monoparasitaemia, which seems circular/contradictory to the title of ‘unusual’ monoparasitaemia, as the discussion indicates that this could have been expected? The authors may consider rephrasing the title or to add a stronger argumentation in the introduction and discussion (see major comment 1).It has been explained why the results are unusual, given the population studied i.e community-based. this is reflected in the introduction and conclusions as suggested The conclusions are missing in the main text, but included in the abstract.They have now been included in the main text. The potential reasons for the disappearance “either due to faltering control of P. falciparum or more efficient control of other species” (last part of the abstract conclusion) could be discussed more in the discussion as one would expect from the title.This has been included in the discussion. Severe versus clinical malaria. The study aim states a focus on uncomplicated malaria, however, Table 4 only shows severe malaria, which should also show uncomplicated malaria (or instead). The WHO criteria for severe malaria were used, does that include criteria for uncomplicated malaria if not which criteria were used? (Paragraph on Procedures, line 9). How were severe cases treated in the study did they also receive ALu at home or were referred to the next hospital or health facility? Moreover, it would be relevant to show uncomplicated malaria per age in addition to parasitaemia in Table 3.Table 3 has been modified to include other parameters such as the age-specific prevalence of uncomplicated and severe, complicated malaria. Figure 2 has also been modified for more clarity Minor comments Previous studies reported the highest parasitaemia among school-aged children 1 and prevalence curves that peak among school children while incidence curves peak in children under the age of five 2. The article here found that parasitaemia is highest at age two and then decreases by age. A sentence for clarification on this would be helpful that explain the decrease over age?This has been explained as the steady-state assumption. The authors found 65% to be non-malaria fevers, this is interesting and the authors may consider highlighting it as a finding and adding it to the discussion (Fig 2).The figure has been changed for clarity. What is reported was that 34.8% of all children had malaria parasitaemia and the nature of parasitaemia i.e symptomatic and asymptomatic has been clarified. “Scientists have modelled the anopheles mosquito vector extensively based on its characteristics, but not so much the parasite” the reference to this study and to niche models of vectors is confusing, as the author’s study is not doing niche modelling or geographic mapping of parasites. It would seem more appropriate to have a description of the geographical occurrence of parasites leading over to the next sentence.The sentence has been rephrased. Missing reference for the Duffy antigen sentence line 6 to 9 in the first paragraph (“An absence of the Duffy antigen on red blood cells of West Africans has long been postulated to be responsible for the absence of P. vivax in these areas, but cases of P. falciparum, P. malariae and P. ovale, in order of decreasing occurrence, have been found”).Reference provided. “Participants were visited at their homes and while in the household” Perhaps should be “while being at home”? Also, were participants visited after identifying the head, or happened both at the same time, visiting the household and then identifying the household head?Clarified that it happened in the house. Confusing use of household head – parent - caregivers, are these refer all to the same (i.e. are household heads always parents?)Clarified, the household head gives permission and it could be a parent or other person. The authors cite the Nigerian national treatment policies for parasite co-infections, which however is not the main source which remains unclear, is there an actual study that could be cited?The treatment guidelines are the ones cited. “five to 10 years” write out numbers below twenty, or use numbers consistently.I thought this was one to nine, it has been edited. Possible gender bias in writing ‘he’ for the pediatrician “He performed a physical examination for each participant and graded splenomegaly according to Hackett’s criteria”.Clarified, the lead investigator is the corresponding author and is male. “Concomitantly, during the same session…” consider removing duplication?Done. “The study numbers” is not explained before; do the authors mean participant identifiers or study participant numbers?Study number is explained. Unclear sentence: “The tail segment of the thin films was viewed to identify the species of malaria parasite, using the typical description of parasite species, having been trained on parasite identification”, who has been trained?Clarified. Unclear sentence: “We determined the prevalence of malaria by parasitaemia and RDT by determining proportions.” Maybe the authors could add formula pos mRDT/tested by mRDt; pos BS/ tested by BS to be clearer?Formula provided for each instance. Consider reducing the black background in figures and tables and not using 3D plots, see some general rules on scientific figures in 3.Figure changed. Be consistent with ‘malaria’ or ‘clinical malaria’ to distinguish malaria infection from clinical malaria, i.e. Fig 2 caption and title, and text description.Figure 2 has been modified. Confusing Fig 2, as it reads as if ‘no parasite can also cause malaria’? Would a barchart be more appropriate? Or consider rephrasing the figure title.Figure 2 changed. Reference number 36 is missing or not needed (sample procedures line 11), reference list goes only up to 33.References updated. ‘The finding of P. falciparum mono-parasitaemia supports the fact that P. falciparum is the dominant species of Plasmodium in Sub- Saharan Africa’ Consider rephrasing like ‘confirms/ support the other findings of mono-parasitaemia’ to avoid the term ‘fact’.Fact has been deleted The discussion structure might need some revision for better understanding, for example, the last paragraph on severe malaria in the discussion might fit better after discussing uncomplicated malaria and before discussing monoparasitaemia in other studies Some modification has been done" } ] }, { "id": "69658", "date": "15 Sep 2020", "name": "Richard Mwaiswelo", "expertise": [ "Reviewer Expertise Malaria" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article presents findings from a cross-sectional study conducted in Wamakko Local Government Authority, in Sokoto State in Nigeria with a broad objective of determining the prevalence of Plasmodium parasite species causing uncomplicated malaria in human in the area. The diagnosis of malaria was performed using mRDTs (for the detection of the presence of infection) and thick (for assessing parasite density) and thin (for species identification) smears. The key finding is Plasmodium falciparum monoinfection in the study area.\n\nMajor Comment The authors have failed to answer the following questions so as to convince readers, and thus should be addressed: What is the aim of this study? Why do you want to understand the prevalence of different malaria parasite species present in that part of Nigeria? How does the understanding of the prevalence of the parasite species contribute to the management, control and prevention of malaria in general? Try to explain this so as to lay down the significance of this study.\n\nAbstract Background\nThe first sentence…………'Malaria is caused by one of five currently known Plasmodium parasite species’…the sentence is not clear. Where do the authors refer to where malaria is caused by that one of the five species?\n\nWhat information on vector does the modeling provide? The sentence is redundant; I suggest to be omitted.\n\nThe sentence………’This study aimed to determine proportions of Plasmodium parasites’………is not in line with the true aim of the study. It would sound clear if it was stated that the aim was to determine the proportions of Plasmodium species infecting humans in the study area.\n\nMethods\n‘Selected participants were examined for ………. signs of complicated malaria'. However, there is nowhere in the abstract were it has been stated that this was either a hospital or community based study, and whether only symptomatic or both symptomatic and asymptomatic individuals for malaria infection were included in the study. How were the study participants selected, randomly or conveniently? What was the age limit of the study participants?\n\n‘…………and thick and thin films to identify parasitemia and parasite species’. Which one of the tests was used to identify parasitemia, and which one was used to identify species? Be specific.\n\n‘………. with complicated malaria were referred to the nearest hospital’………but you are silent about those individuals with uncomplicated malaria, were they treated?\n\nResults\n‘There was monoparasitemia of Plasmodium falciparum’…………. I suggest to rewrite the sentence and read……………. There was Plasmodium falciparum monoinfection throughout…….\n\nOn the other hand, detection of parasite species was based on microscopy thin smears, where there any efforts to detect the parasite species using more sensitive methods such as PCR?\n\n‘Mapping of the parasite…….’This sentence is not part of the results but rather of the methods, move it to the methods section.\n\nConclusion\nOmit the sentence……………...’Despite the intermediate endemicity in the area’…………. otherwise, how does the endemicity determine the presence of a certain malaria parasite specie?\n\nIntroduction\nThe first sentence is confusing, same as in the abstract. See the above comment.\n\nThe third sentence………...’scientists have modelled the Anopheles………...but not so much on the parasite’. This sentence is misleading, and which characteristics are you referring to? The sentence itself is redundant and I suggest to be omitted.\n\nThis figure likely overestimated the proportion of cases………...tracked by passive surveillance in Nigeria’. The sentence is not clear, rephrase it to increase clarity.\n\nThese proportions were the age-group specific parasite prevalence……………...sickle cell trait and those with normal adult hemoglobin. Rephrase this sentence to increase clarity.\n\n‘Plasmodium falciparum is an undisputed leader’……………. I don’t think the word leader suits well in this sentence, I suggest the use of the word ‘predominant/prevalent’.\n\nThere is no aim of the study in the whole introductory section.\nMethods Study settings\nHow many rainfall seasons are there in the study area? When does the rainfall season start and end? What are the major economic activities in the area? What are the major vectors? What are the major malaria control tools employed in the area?\n\nStudy design\nWhere the participants selected randomly or conveniently?\n\nWas this a community-based or health facility-based study?\n\nSample population\nRewrite sentences 1 and 2 to increase clarity……...’after identifying the household head. They were provided with information regarding the study’………. To who was the information provided, the household head, or everyone in the household? Who gave the consent?\n\n‘Residents of the study area for at least two weeks’…………...with this very short time of being a residency in the study area, how sure are you that the malaria parasite species found in the assessed individuals have not originated out of your study area (where the assessed individual was living before moving into the study community)?\n\nExcept for those who had parasitemia without symptoms……...followed up………. for up to 48 hours for the development of symptoms. Is it ethical to not treat an infected individual and only wait for symptoms to occur?\n\nProcedures\nDoes this mean that the field assistant recruited the study participants prior to obtaining consent from parents/guardians? Is this ethical?\n\n‘We examined at least 10 fields before a slide was declared negative’……….what is the standard number of fields that are required to be read before declaring a slide negative?\n\nStatistical analysis\n‘We determined the prevalence of malaria by parasitemia and RDT by determining proportions'. The sentence is not clear, rewrite it.\n\nResults\nCombine tables 1 and 3\n\nCombine tables 4 and 5\n\nFigure 2………..the prevalence of other parasite species is 0%, I suggest you find another method for presenting this, otherwise the figure is not convincing.\n\nThe following sub-sections can be combined: Prevalence of malaria, age-specific and sex-specific prevalence of malaria, the prevalence of malaria by seasons, and parasite density by seasons. All these can be under one sub-section ‘Malaria prevalence’.\n\nYou should have at least 3 sub-sections to include: Included participants, Prevalence of malaria, and Plasmodium parasite species.\n\nParasite density across the season\n‘…………density was much higher during rainy…………., but the p-value is not given and the authors states that details are shown in Table 5, this is not proper.\n\nDiscussion\nEvaluation of the prevalence of malaria in the study area was not the main objective of the study, but rather the evaluation of the prevalence of Plasmodium species infecting humans in the study area was the main objective. Why then the first paragraph concentrate on the prevalence of malaria rather than of the infecting parasite species?\n\nThe second sentence in the first paragraph is not clear, and what are you trying to suggest by ……...’as is excluding those aged 6 months to 2 years? How is malaria prevalence stratified by age, I mean what is the standard?\n\nThe discussion has concentrated much on malaria prevalence rather than on the main objective of the study, which is the prevalence of Plasmodium species infecting humans in the study area.\n\nThe conclusion is missing.\n\nReferences\nReference 2. World Health Organization…………. Nigeria. Geneva, 2014. Remove the name Nigeria.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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? No", "responses": [] }, { "id": "69663", "date": "18 Sep 2020", "name": "Linda Eva Amoah", "expertise": [ "Reviewer Expertise Recombinant DNA technology and Malaria transmission", "Plasmodium species identification", "gametocytes and serology as well as host genetics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract/Introduction:\nWhat is monoparasitaemia? Do you mean mono species parasitaemia?\n\nThe first sentence of the abstract/introduction should be revised as malaria in other animals is caused by other Plasmodium species.\n\nThe reference WHO 2014 and 2016 are not current, what is the current data?\n\nWhat is meant by ‘parasite mapping’ and why is this important to the reader?\n\nCheck the text and remove spaces before full stops as well include a space before the falciparum in P. falciparum.\n\n‘not so much the parasite’? I do not understand what this means. There is no modeling in this study so the second sentence in the introduction is not relevant, especially as it is very confusing.\n\nWhat is the definition of ‘confirmed cases?’\n\nWhat does ‘crucially’ mean?\n\nMethods:\nIs April the peak of the malaria season? The description of malaria indices in the study site is not sufficient.\n\nThe blood draw procedure can be rewritten to enhance clarity. Overall, how many drops of blood were collected? And what processes/procedures were performed?\n\nTables:\nTables 1, 2, 3, and 5 can easily be merged. It would reduce the repetition.\n\nTable 2 can be stratified by age as the other tables.\n\nTable 4 should be age stratified and other cases separated into asymptomatic and uncomplicated cases.\n\nFigure 2:\n\nThe title is wrong as it includes ‘no parasites’. No parasites do not cause malaria.\n\nThere are items in the legend that are not on the graph, it would be best to remove and just state in the foot notes that none of those other species were identified.\n\nResults:\nWas the parasite density at the two time points significantly different?\n\nDiscussion:\nIt would be very easy to compare the results from the current study with the data in the MIS by comparing similarly aged children. The statement can be revised if the analysis is redone for children aged 5 years and below.\n\nI think the last paragraph should be a summary of the major findings and provide information related to the main aim or relevance to the title but it is presently not so.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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? Partly", "responses": [ { "c_id": "5960", "date": "27 Oct 2020", "name": "Usman Nasir Nakakana", "role": "Author Response", "response": "Abstract/Introduction: What is monoparasitaemia? Do you mean mono species parasitaemia?Yes, this is clarified in the text The first sentence of the abstract/introduction should be revised as malaria in other animals is caused by other Plasmodium species.It has been rephrased for clarity The reference WHO 2014 and 2016 are not current, what is the current data? The data was collected in 2016 and written up in 2017, the data is referencing the time around which the data was collected to ensure comparability, considering the constantly changing data What is meant by ‘parasite mapping’ and why is this important to the reader? Parasite mapping refers to the description of geographical spread of the prasite species and how it can be extended to other parts of the state, based on climatic and geographic information. Check the text and remove spaces before full stops as well include a space before the falciparum in P. falciparum.Checked and corrected ‘not so much the parasite’? I do not understand what this means. There is no modeling in this study so the second sentence in the introduction is not relevant, especially as it is very confusing.Reference to modelling is deleted, the statement has been rephrased What is the definition of ‘confirmed cases?’ confirmed cases is defined as those with a positive malaria parasite test and clinical symptoms.This has been elaborated What does ‘crucially’ mean?Changed to 'notably' Methods: Is April the peak of the malaria season? The description of malaria indices in the study site is not sufficient.Some information has been provided The blood draw procedure can be rewritten to enhance clarity. Overall, how many drops of blood were collected? And what processes/procedures were performed?It has been re-written to increase clarity Tables: Tables 1, 2, 3, and 5 can easily be merged. It would reduce the repetition. Table 3 contains more data including data on the age distribution Table 2 can be stratified by age as the other tables.The data is provided in table 3 Table 4 should be age stratified and other cases separated into asymptomatic and uncomplicated cases.The data is provided in table 3 Figure 2: The title is wrong as it includes ‘no parasites’. No parasites do not cause malaria. There are items in the legend that are not on the graph, it would be best to remove and just state in the foot notes that none of those other species were identified.The figure has been changed entirely in version 2 Results: Was the parasite density at the two time points significantly different? There was no test of significance performed but it is much higher in the rainy than the dry season. Discussion: It would be very easy to compare the results from the current study with the data in the MIS by comparing similarly aged children. The statement can be revised if the analysis is redone for children aged 5 years and below.The prevalence of malaria is described as it relates to the parasite species distribution and the impact of malaria endemicity. The metric for endemicity is the prevalence of falciparum malaria among children 2 to 10 years of age, which is the age group we studied. We also did not collect any data in children 6 months to 2 years and so even if we consider children under 5 years, we still would not be able to compare the data directly with MIS data. I think the last paragraph should be a summary of the major findings and provide information related to the main aim or relevance to the title but it is presently not so.It has been revised" } ] } ]	1	https://f1000research.com/articles/9-301
https://f1000research.com/articles/9-1193/v1	02 Oct 20	{ "type": "Research Article", "title": "The worldwide clinical trial research response to the COVID-19 pandemic - the first 100 days", "authors": [ "Perrine Janiaud", "Cathrine Axfors", "Janneke van't Hooft", "Ramon Saccilotto", "Arnav Agarwal", "Christian Appenzeller-Herzog", "Despina G. Contopoulos-Ioannidis", "Valentin Danchev", "Ulrich Dirnagl", "Hannah Ewald", "Gerald Gartlehner", "Steven N. Goodman", "Noah A. Haber", "Angeliki Diotima Ioannidis", "John P. A. Ioannidis", "Mark P. Lythgoe", "Wenyan Ma", "Malcolm Macleod", "Mario Malički", "Joerg J. Meerpohl", "Yan Min", "David Moher", "Blin Nagavci", "Florian Naudet", "Christiane Pauli-Magnus", "Jack W. O'Sullivan", "Nico Riedel", "Jan A. Roth", "Mandy Sauermann", "Stefan Schandelmaier", "Andreas M. Schmitt", "Benjamin Speich", "Paula R. Williamson", "Lars G. Hemkens", "Perrine Janiaud", "Cathrine Axfors", "Janneke van't Hooft", "Ramon Saccilotto", "Arnav Agarwal", "Christian Appenzeller-Herzog", "Despina G. Contopoulos-Ioannidis", "Valentin Danchev", "Ulrich Dirnagl", "Hannah Ewald", "Gerald Gartlehner", "Steven N. Goodman", "Noah A. Haber", "Angeliki Diotima Ioannidis", "John P. A. Ioannidis", "Mark P. Lythgoe", "Wenyan Ma", "Malcolm Macleod", "Mario Malički", "Joerg J. Meerpohl", "Yan Min", "David Moher", "Blin Nagavci", "Florian Naudet", "Christiane Pauli-Magnus", "Jack W. O'Sullivan", "Nico Riedel", "Jan A. Roth", "Mandy Sauermann", "Stefan Schandelmaier", "Andreas M. Schmitt", "Benjamin Speich", "Paula R. Williamson" ], "abstract": "Background: Never before have clinical trials drawn as much public attention as those testing interventions for COVID-19. We aimed to describe the worldwide COVID-19 clinical research response and its evolution over the first 100 days of the pandemic. Methods: Descriptive analysis of planned, ongoing or completed trials by April 9, 2020 testing any intervention to treat or prevent COVID-19, systematically identified in trial registries, preprint servers, and literature databases. A survey was conducted of all trials to assess their recruitment status up to July 6, 2020. Results: Most of the 689 trials (overall target sample size 396,366) were small (median sample size 120; interquartile range [IQR] 60-300) but randomized (75.8%; n=522) and were often conducted in China (51.1%; n=352) or the USA (11%; n=76). 525 trials (76.2%) planned to include 155,571 hospitalized patients, and 25 (3.6%) planned to include 96,821 health-care workers. Treatments were evaluated in 607 trials (88.1%), frequently antivirals (n=144) or antimalarials (n=112); 78 trials (11.3%) focused on prevention, including 14 vaccine trials. No trial investigated social distancing. Interventions tested in 11 trials with >5,000 participants were also tested in 169 smaller trials (median sample size 273; IQR 90-700). Hydroxychloroquine alone was investigated in 110 trials. While 414 trials (60.0%) expected completion in 2020, only 35 trials (4.1%; 3,071 participants) were completed by July 6. Of 112 trials with detailed recruitment information, 55 had recruited <20% of the targeted sample; 27 between 20-50%; and 30 over 50% (median 14.8% [IQR 2.0-62.0%]). Conclusions: The size and speed of the COVID-19 clinical trials agenda is unprecedented. However, most trials were small investigating a small fraction of treatment options. The feasibility of this research agenda is questionable, and many trials may end in futility, wasting research resources. Much better coordination is needed to respond to global health threats.", "keywords": [ "COVID-19", "clinical research agenda", "hydroxychloroquine" ], "content": "Introduction\n\nOn December 31, 2019, the World Health Organization (WHO) China Country Office was informed of pneumonia cases of unknown etiology1; on January 30, 2020, the WHO declared coronavirus disease 2019 (COVID-19)2 a public health emergency and on March 11 a pandemic3. Radical public health measures, including quarantine, social distancing, school and workplace closures, and others have been implemented worldwide, affecting the lives of billions of people. The pandemic resulted in rapid generation and dissemination of studies and their results4. However, information on trials that are planned, ongoing, finished, or published are spread across trial registries, preprint servers, publication databases and other repositories. In June, 2020, the number of ongoing trials outweighed by far completed trials; however, no overview of COVID-19 trials has followed up on actual enrolment in ongoing trials5–8.\n\nWe established the COVID-evidence platform (www.covid-evidence.org) to collect this information in a central database of COVID-19 trials testing any interventions for treatment or prevention. We used COVID-evidence to describe the worldwide clinical research response to COVID-19, its evolution over the first 100 days since the first cases were officially reported, and the expected feasibility and risk of waste of resources. We describe the trials’ characteristics, their place in the research landscape, and how they changed over time.\n\n\nMethods\n\nCOVID-evidence includes trials from international registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform [ICTRP]), preprint servers (medRxiv, bioRxiv), PubMed, the WHO COVID-19 literature database, and a listing of all trials with ethical approval in Switzerland9.\n\nOur protocol and details on the search strategies and specific definitions used for COVID-evidence are available on the Open Science Framework (OSF)10. We searched the relevant data sources using peer-reviewed search strategies developed by information specialists. Results from literature databases and preprint servers were pre-screened by a single reviewer who excluded unsuitable publications (e.g. opinion papers or observational, non-interventional studies). Cases with unclear eligibility were discussed by at least three reviewers until consensus was reached.\n\nWe included any planned, ongoing or completed trial that tested any intervention to treat or prevent COVID-19 in humans that was registered or published within the first 100 days of the COVID-19 outbreak, i.e. after the first cases reported to the WHO to 100 days later (January 1 to April 9, 2020). We considered as a trial any study prospectively assigning an intervention11. This included randomized and non-randomized, controlled or non-controlled trials regardless of language, geographical region, or setting. Epidemiological studies or studies of diagnostic test accuracy (without any health-related outcome) were excluded.\n\nFor each trial, we extracted dates of registration and publication, design characteristics and details of the population, intervention, comparison, outcomes, geographic region, funding and setting. We categorized drugs and biologicals according to major pharmacological classes and main clinical indications.\n\nA team of 19 reviewers (including clinicians, clinical researchers, clinical pharmacologists, meta-researchers and systematic reviewers) either manually extracted information or verified information that was obtained with automatic data scraping methods. All details on scraping, variable definitions and extraction/verification procedures are available on OSF10,12. For all trials registered up to April 9, 2020, we extracted data through April 30. The status for each trial was updated on July 6, 2020 (using ClinicalTrials.gov where possible; if not, we used the ICTRP; for trials originally registered in the Chinese Clinical Trial Registry available through ICTRP we used the former if it was more up to date).\n\nWhen a trial had entries in different data sources, we gave first priority to publications, second to preprints, and third to registries (here, ClinicalTrials.gov was preferred).\n\nFrom May 12 to July 3, 2020, we emailed the corresponding investigators of all trials, except discontinued ones, inquiring about their enrolment accrual. Replies were collected up to July 6, 2020.\n\nAll analyses were descriptive and reported as percentages, medians (interquartile range, IQR) or means. We used Ninox (Ninox Software GmbH, Berlin, Germany; version 2.6), and R (version 3.6).\n\n\nResults\n\nWe identified 689 trials registered or published over the pandemic’s first 100 days, testing interventions to treat or prevent COVID-19 (see Underlying data)12 with a total planned sample size of 396,366 participants. As of July 6, 2020, 19 trials (including 4,378 participants) had been completed and had published results, and 16 were completed without available results (5,173 participants). Thirty (4.4%) were active but no longer recruiting (59,259 participants), 384 (55.7%) started recruiting (217,357 participants), 174 (25.3%) had not yet started (97,406 participants), 50 (7.3%) were discontinued (12,048 participants), and 4 (0.6%) were terminated (577 participants). The status was unknown for 12 (1.7%; 168 participants).\n\nThe 689 trials’ median target sample size was 120 (IQR 60 to 300; Table 1); 40.7% (n=280) planned to enroll fewer than 100 participants, 8.3% (n=57) over 1,000, and 1.6% (n=11) over 5,000 (see Underlying data)12. 75.8% (n=522) trials were randomized and 59.2% (n=408) did not use blinding (Table 1). Randomized trials were on average three times larger than non-randomized trials (median sample size 150 vs. 50).\n\nAdditional categories such as other countries can be found in the extended data\n\na 4 trials assessed interventions for both treatment and intervention\n\nb For 6 trials the randomization was unclear\n\nc The sample size is missing for 17 trials\n\nd Some trials compared different types of interventions: 3 trials biologicals and drugs; 6 trials drugs and traditional medicine; 5 trials other interventions and drugs; 1 trial other interventions and traditional medicine; 2 trials procedures and drugs; 1 trial vaccine and device\n\ne Included for example use trials that used a standard of care arm and an active control arm\n\nf a public/not-for-profit sponsor was reported but the absence of an industrial sponsor does not exclude an industrial funder\n\nAlthough few trials focused on health-care workers (3.6% [n=25]), they were larger: 96,821 planned health-care workers (median 700 [IQR 400 to 2,486]) versus 155,571 planned patients (median 100 [IQR 50 to 240]) for the inpatient trials (76.2% [n=525]) (Table 1). Overall, 46.4% of the trials intended to use mortality as a primary (n=98) or secondary outcome (n=369; Table 1). Out of the 525 inpatient trials, 55.6% (n=292) planned on reporting mortality as an outcome.\n\nOut of the 689 trials, 607 (88.1%) assessed treatment interventions (187,209 planned patients); drugs were more frequent (349 trials [57.5%]), encompassing a vast range of substances. The two most common pharmacological classes were antiviral drugs (assessed in 144 trials; e.g. lopinavir/ritonavir [n=45]) and antimalarial drugs (112 trials; e.g. hydroxychloroquine [n=83]). There were 106 trials investigating traditional medicine and 70 exploring highly diverse pharmaceuticals of various classes, e.g. bismuth potassium citrate, ebastine, pirfenidone, dipyridamole and hydrogen peroxidase (Figure 1 and see Extended data)12. The comparators were predominantly standard of care or no intervention (47.1% [n=286]), placebo (17% [n=103]) or other interventions (18%; [n=109]) (Table 1).\n\nInterventions for treatment assessed in more than 25 trial: antiviral drugs were assessed in 144 trials; (e.g. lopinavir/ritonavir [n=45]), antimalarial drugs in 112 trials; (e.g. hydroxychloroquine [n=83]), monoclonal antibodies in 52 trials (e.g. tocilizumab [n=26]), traditional medicine in 106 trials, other drug intervention in 70 trials, nonspecific anti-inflammatory/immunosuppressive drugs in 42 trials (e.g. colchicine [n=4]), antibiotic/anti-parasitic drugs in 34 trials (e.g. azithromycin [n=28]), biologicals in 80 trials (e.g. convalescent plasma [n=27]), procedures in 28 trials (e.g. renal replacement therapy [n=4]), other non-drug interventions in 27 trials (e.g. physical activity). More information can be found in the Extended data12.\n\nThe first column represents the proportion of all trials that were of the specified type. The second column represents the proportion of all trials registered that week that were of the specified type (i.e. within week, between trial types). The third column represents the distribution of when trials of this type were registered (i.e. within trial type, between week), and can be interpreted as either a percentage or count (not specified). A trial might assess more than one intervention category and detail for prevention and treatment trials are given in the extended data12.\n\nOverall, 78 trials (11.3%) focused on prevention (205,841 planned participants), mainly prophylactic drug use (n=41), vaccines (n=14; 9 already started recruitment; see Extended data)12 and non-pharmaceutical interventions (n=10) (e.g. masks or the use of media and influencers in people’s compliance to hygienic practices). Four trials (0.6%) assessed interventions both for prevention and for treatment. No trial planned to assess benefits or harms of implementing or de-implementing any social distancing or lockdown measures.\n\nThe number of trials increased rapidly; on average 0.5 trials per day were registered in January, 8.1 in February, 8.4 in March, and 17.6 in April 2020.\n\nTrials were conducted in 42 countries and through international collaborations (Table 1; see Extended data)12. Half were from China (51.1% [n=352]), which dominated initially (Figure 2); starting March 2020, more trials came from other countries. Trial characteristics were similar across the five most frequent geographical locations (China, USA, France, Spain and international) contributing to 73.3% (n=505) of the global trial research (Table 1). Traditional medicine was assessed in 30.4% of trials from China (n=107) but rarely in other countries.\n\nCumulative number of registered trials over time (a) by continent, and (b) for countries with at least 10 registrations (excluding China). Four trials not shown were registered in 2019 or earlier, with a study design subsequently adapted to address COVID-19 (EUCTR2015-002340-14-NL; NCT03680274; NCT03331445; and NCT03808922). For 18 trials the registration date was unknown.\n\nLarger trials were initiated later. In February, fewer than 8% of trials included more than 500 participants in contrast to 29.6% of trials in March (Figure 3). Later trials more often used blinding, placebo and mortality as primary outcome (Figure 3). Participations of healthcare workers and healthy people also started later. When the proportion of trials from China decreased, so did trials assessing traditional medicine (from 46.9% to 0.9%) while the proportion of trials assessing drugs rose (from 38.1% to 77.2%). Antivirals came under investigation earlier than antimalarials (Figure 1).\n\nThe first column represents the proportion of all trials that were of the specified type. The second column represents the proportion of all trials registered that week that were of the specified type (i.e. within week, between trial types). The third column represents the distribution of when trials of this type were registered (i.e. within trial type, between week), and can be interpreted as either a percentage or count (not specified).\n\nOut of the 689 trials, 6.7% (n=46) planned to enroll 1,000 to 5,000 participants. Most were randomized (89.1% [n=41]), assessed drugs (80.4%; n=37), and many were not blinded (52.2% [n=24]). Five were cluster-randomized. The top three regions were the United States (21.7%; n=10), France (13% [n=6]) and international collaborations (10.9% [n=5]) (see Extended data)12.\n\nEleven (1.6%) trials, registered between February and April 2020, planned to enroll over 5,000 participants (see Extended data)12. There were 10 randomized (one cluster RCT), eight not blinded and five conducted in multiple countries. These trials tested drugs (n=9), masks (n=1) and traditional medicine (n=1). Three trials are described as platform trials (i.e. WHO Solidarity trial13, RECOVERY trial14 and CROWN-CORONATION trial15) and use an adaptive design.\n\nSix drug interventions tested in these 11 larger trials (seven for treatment and four for prevention) were simultaneously investigated in at least 10 smaller trials (see Extended data)12. Overall, 169 trials (143 for treatment, 24 for prevention and two for treatment and prevention) with fewer than 5,000 participants assessed at least one intervention that was also assessed in a larger trial (median sample size 273 [IQR 90 to 700]; 134 had fewer than 1000 participants). For 107 of those (63.5%) the larger trial was registered before. For example, 106 trials with fewer than 5,000 participants tested hydroxychloroquine and 88 of them (83%) were registered after the first large trial testing this drug and 83 (77.6%) assessed hydroxychloroquine as treatment (Figure 4; see Extended data)12. These 106 trials had a median sample size of 334, but cumulatively, they planned to enroll as many patients as the four larger trials testing hydroxychloroquine (76,617 vs 77,000).\n\nThe dashed lines represent the registration of the four trials planning to enroll over 5,000 participants; two were registered on April 2, 2020.\n\nOut of the four trials planning on enrolling over 5,000 participants, two assessed hydroxychloroquine for treatment and two for prevention. Out of the 106 smaller trials, 83 assessed hydroxychloroquine for treatment, 22 for prevention and one for both treatment and prevention\n\nBy the end of 2020, 414 trials (60.1%) with a total of 160,107 planned participants were expected to be completed (i.e. last patient, last visit), including 240 drug trials (97,846 participants) and 22 over-1,000 participants trials and five over-5,000 participants trials. For vaccines, five trials expected completion in 2020, five in 2021 and another four between 2022 and 2024 (see Extended data)12. However, of the 270 trials that planned to start recruiting by the end of February, 190 started (70.4%), but 80 had not (as of July 6, 2020) (see Extended data)12.\n\nBy July 6, 2020, we received enrolment information for 112 out of the 604 trials listed as planned or ongoing (18.7%). Of the 112 trials, 16 had not started recruiting although their start dates were overdue; one was discontinued. Among the 112 trials, 55 had recruited fewer than 20% of the target sample size, 27 between 20-50%, and 30 more than 50% (median recruitment 14.8% [IQR 2.0 to 62.0%]; median duration of recruitment 72 days [IQR 53.5 to 83 days])). Median recruitment was similar in treatment and prevention trials (15.9% (IQR 2 to 61.1%] vs 14.8% [IQR 4.3 to 62.5%]). For 19 trials, investigators mentioned difficulties in recruitment due to a fortunate decrease in the number of COVID-19 cases.\n\n\nDiscussion\n\nThe global clinical research community has mounted a massive, unprecedented volume of research in response to the COVID-19 pandemic. Almost 700 trials within 100 days planned to include almost 400,000 participants globally. Many treatments were planned for investigation, mostly drugs, often antivirals, and sometimes substances that may seem rather unexpected for an infectious disease (e.g. colchicine or dipyridamole) reflecting the huge heterogeneity of disease manifestations and therapeutic targets16. Few trials focused on prevention, but some were very large and focused on healthcare workers (i.e. 24.4% of planned trial participants are healthcare workers). Trials from China dominated the research agenda before research activities followed the spread of COVID-19 throughout the world. Most trials were planned as randomized, clearly demonstrating that such designs are possible within a pandemic17 and within a very short time, unlike the 2014–2015 Ebola outbreak where only a few therapeutic trials had a randomized design, and none started within 100 days17.\n\nThe emergence of 689 trials in a 100-day period is unparalleled. Between 250 to 342 HIV/AIDS trials are registered per year on ClinicalTrials.gov18 and only three were registered for Middle East respiratory syndrome (MERS) coronavirus during 2007–201718. While efforts being put into clinical trials were initially welcome, the vast majority of COVID-19 trials are at risk of being abandoned if they cannot recruit enough patients or if other trials on the same treatment provide conclusive results (favorable or unfavorable).\n\nThomas Chalmers highlighted in 1977 the need to ‘Randomize the first patient’19, and a reassuring 75% of trials are indeed randomized. However, we identified areas of concern. Most trials are not blinded, and even if placebos may be not available in such short time, blinding of outcome collection would be preferable. Blinding may not be required for mortality outcomes; however, it was rarely a primary outcome. Half of the trials include fewer than 120 patients and many small trials were initiated after public registration of very large trials addressing similar questions. They may have some heterogeneity in design that might be desirable or focus on specific situations, for example early Phase 1 vaccine trials, but it seems unlikely that such small trials would add meaningfully to the overall evidence. The extensive worldwide discussions about limited evidence from small trials reflect the substantial uncertainty patients and decision-makers face about the merits of popular interventions, such as hydroxychloroquine20.\n\nFor hydroxychloroquine, over 100 smaller studies with over 76,000 patients were planned in the first 100 days to investigate this single therapeutic option out of many potential options. This case, possibly fueled by media attention relayed by decision-makers and politicians, highlights the urgent need for early evidence-based research and priority setting. Such proliferation may reflect best intentions of clinical researchers to actively contribute to evidence generation and inform timely treatments locally instead of awaiting published evidence or using experimental treatments outside of clinical trials. It may also indicate a lack of research structures allowing them to contribute to larger, synergistic trials. With the emergence of results, the entire agenda may shift. Many hydroxychloroquine and chloroquine trials’ enrolment was temporally halted due to harmful effects in an observational study21, the publication of which was subsequently retracted22. However, the release of the randomized RECOVERY trial results showing no benefit (in fact, a trend for increased mortality)23 with hydroxychloroquine and another “negative” trial on hydroxychloroquine-prophylaxis24 created uncertainties about the feasibility (i.e. inability to recruit planned sample sizes) or futility (i.e. inability to demonstrate treatment effects) of all the ongoing and planned hydroxychloroquine trials.\n\nThere are excellent examples of how efficient structures allow for rapid response to evidence needs, such as the UK RECOVERY trial25. Strongly endorsed and prioritized by authorities and medical representatives26, it is running as streamlined pragmatic platform trial in over 176 hospitals, randomizing over 12,000 patients in just over four months14. It has already provided evidence on the lack of benefit for hydroxychloroquine23 and lopinavir/ritonavir27, and a reduction of mortality with dexamethasone28 (still awaiting results for azithromycin, tocilizumab and convalescent plasma). Such key trials have had major impact on decision-makers such as the FDA revoking the Emergency Use Authorization of hydroxychloroquine29 on June 15.\n\nConversely, we found other large trials with major recruitment difficulties. The DisCoVeRy trial, for example, was designed as an adaptive trial of 3200 patients, running in 35 countries. However, while DisCoVeRy recruited 758 patients in France, only one was recruited in the rest of Europe30, as of June 17, 2020.\n\nThe lack of coordination in the research response created substantial research waste, exposed many patients to unnecessary risks, and harms medical progress by creating competition among trials investigating similarly promising therapeutic alternatives31–33. However, in absence of such desirable research synergies, all these scattered activities can and should be bundled to contribute to rapid evidence generation in living meta-analyses. The COVID-evidence database provides a unique opportunity to surveil the planned, ongoing and completed trials that can then be synthesized – it would only need systematic sharing of trial data.\n\nAs many countries are facing restrictions of movement and lifestyle at various severity levels, affecting the physical and mental health of billions of people, it is remarkable that not a single trial was initially planned to evaluate these measures. While the lack of controlled experiments evaluating their implementation may not be unexpected (given the initial urgency, ethical considerations, and organizational challenges), it would now be highly desirable that the de-implementation or re-implementation be subject to systematic evaluation in high-quality trials. The diverse options to ease or reinforce lockdown would be amenable to randomization, such as alternative time points or extents of re-opening schools or kindergartens, of ways to protect elderly in nursing homes, of home office programs, or of contact restrictions. Such evidence would be critical to inform future pandemics or the management of possible second waves of COVID-19, yet it was not on the initial agenda.\n\nSeveral limitations merit attention. First, unclear reporting in registries might have introduced inaccurate results. Some ambiguously reported items required discussion among several reviewers but were resolved to the best of our ability. Second, we rarely identified protocols or manuscripts, precluding more detailed analyses of trial designs. Third, some control groups receiving “standard of care” interventions were not clearly described, likely some of these included interventions that were or are still under investigation in other trials. Fourth, we may have missed a few cases of duplicate entries across registries or of multiple national parts of an international trial, thus slightly overestimating the number of trials but not affecting the overall interpretation. Fifth, we arbitrarily selected a period of the first 100 days, which is traditionally used to benchmark early outcomes of policies or presidencies. Finally, we do not assess the actual research output from all these early trials. This unprecedentedly fast-moving research body is scattered across data sources and registries without uniform updates. More definitive answers will require more time, but our results allow for the diagnosis of “system cracks”34 that may become symptomatic in this pandemic, such as infrastructure limitations, and also identify best practices.\n\n\nConclusion\n\nThe incredible volume and speed of trial research observed in the first 100 days of the COVID-19 pandemic should not hide the fact that in its early days the global clinical trial research agenda lacked clear coordination, efficiency and exploitation of synergies. There are excellent examples of very large trials implemented with impressive efficiency, likely providing the clearest evidence. However, early coordination and a unified approach are needed - otherwise futility and waste of resources may be prominent features of such an ambitious research agenda.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: COVID-evidence: a living database of trials on interventions for COVID-19 / The worldwide clinical trial research response to the COVID-19 pandemic - the first 100 days; https://doi.org/10.17605/OSF.IO/PJEM312.\n\n- 2020-07-06-Dataset_manuscript.xlsx. (Raw trial metadata.)\n\n- 2020-06-03_COVe_Procedures_Variables_manuscript.pdf. (Procedures for screening and extracting data.)\n\n- 2020-09-08-Extended_data_Manuscript.docx. (Extended data Figures 1–5 and Tables 1–5.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank Constantin Sluka (University of Basel) for his help in setting up the COVID-evidence database, Andreas Widmer (University Hospital Basel) for administrative support and Ninox Software GmbH, Berlin, Germany, for freely providing the database. We would also like to thank all the investigators we reached out to and took the time to respond to our inquiries.\n\n\nReferences\n\nWHO: Pneumonia of unknown cause – China. WHO. Accessed May 26, 2020. Reference Source\n\nZhu R, Gao R, Robert SH, et al.: Systematic Review of the Registered Clinical Trials of Coronavirus Diseases 2019 (COVID-19). medRxiv. 2020. Publisher Full Text\n\nWang C, Horby PW, Hayden FG, et al.: A novel coronavirus outbreak of global health concern. Lancet. 2020; 395(10223): 470–473. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYan W: Coronavirus Tests Science’s Need for Speed Limits. The New York Times. Accessed May 26, 2020. Reference Source\n\nNasrallah AA, Farran SH, Nasrallah ZA, et al.: A large number of COVID-19 interventional clinical trials were registered soon after the pandemic onset: a descriptive analysis. J Clin Epidemiol. 2020; 125: 170–178.. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiovani D, Pansieri C, Peyrin-Biroulet L, et al.: A snapshot of the ongoing clinical research on COVID-19 [version 1; peer review: 2 approved]. F1000Res. 2020; 9: 373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEjaz K, Kauser T, Siddiqa A: Comprehensive overview of COVID-19 clinical trials. J Pak Med Assoc. 2020; 70(Suppl 3)(5): S158–S161. PubMed Abstract \| Publisher Full Text\n\nFajgenbaum DC, Khor JS, Gorzewski A, et al.: Treatments Administered to the First 9152 Reported Cases of COVID-19: A Systematic Review. Infect Dis Ther. 2020; 9(3): 435–449. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwiss Association of Research Ethics Committees: Approved and submitted projects. swissethics. Accessed May 27, 2020. Reference Source\n\nJaniaud P, Axfors C, Saccilotto R, et al.: COVID-evidence: a living database of trials on interventions for COVID-19. 2020. Publisher Full Text\n\nICMJE, International Committee of Medical Journal Editors: Clinical Trials Registration. Accessed May 17, 2020. Reference Source\n\nJaniaud P, Saccilotto R, Axfors C, et al.: The worldwide clinical trial research response to the COVID-19 pandemic - the first 100 days. 2020. http://www.doi.org/10.17605/OSF.IO/PJEM3\n\nSolidarity trials: An international randomised trial of additional treatments for COVID-19 in hospitalised patients who are all receiving the local standard of care (EUCTR2020-001307-16-ES). Accessed May 27, 2020. Reference Source\n\nRECOVERY Trial: Welcome — RECOVERY Trial. Accessed May 17, 2020. Reference Source\n\nCROWN CORONATION: Chloroquine RepurpOsing to healthWorkers for Novel CORONAvirus mitigaTION - Full Text View - ClinicalTrials.gov. Accessed May 27, 2020. Reference Source\n\nBauchner H, Fontanarosa PB: Randomized Clinical Trials and COVID-19: Managing Expectations. JAMA. 2020; 323(22): 2262–226. PubMed Abstract \| Publisher Full Text\n\nNational Academies of Sciences E: Integrating Clinical Research into Epidemic Response: The Ebola Experience. 2017. Publisher Full Text\n\nJaffe IS, Chiswell K, Tsalik EL: A Decade On: Systematic Review of ClinicalTrials.gov Infectious Disease Trials, 2007-2017. Open Forum Infect Dis. 2019; 6(6): ofz189. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChalmers TC: Randomize the first patient! N Engl J Med. 1977; 296(2): 107. PubMed Abstract \| Publisher Full Text\n\nLenzer J, Brownlee S: Pandemic Science Out of Control Issues in Science and Technology. Accessed May 27, 2020. Reference Source\n\nMehra MR, Desai SS, Ruschitzka F, et al.: RETRACTED: Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis. Lancet. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehra MR, Desai SS, Ruschitzka F, et al.: RETRACTED: Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis. Lancet. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNo clinical benefit from use of hydroxychloroquine in hospitalised patients with COVID-19 — RECOVERY Trial. Accessed June 9, 2020. Reference Source\n\nBoulware DR, Pullen MF, Bangdiwala AS, et al.: A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19. N Engl J Med. 2020; 383(6): 517–525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHow to set up a trial in nine days — University of Oxford, Medical Sciences Division. Accessed May 27, 2020. Reference Source\n\nThe importance of covid 19 clinical trials.pdf. Accessed May 27, 2020. Reference Source\n\nLopinavir-Ritonavir results — RECOVERY Trial. Accessed July 3, 2020. Reference Source\n\nHorby P, Lim WS, Emberson J, et al.: Effect of Dexamethasone in Hospitalized Patients with COVID-19: Preliminary Report. medRxiv. 2020. Publisher Full Text\n\nFood and Drug Administration: Coronavirus (COVID-19) Update: FDA Revokes Emergency Use Authorization for Chloroquine and Hydroxychloroquine. FDA. Accessed July 3, 2020. Reference Source\n\nCovid-19 : Essai Discovery, les raisons d’un flop français. LExpress.fr. Accessed July 3, 2020. Reference Source\n\nHalpern SD, Karlawish JHT, Berlin JA: The continuing unethical conduct of underpowered clinical trials. JAMA. 2002; 288(3): 358–362. PubMed Abstract \| Publisher Full Text\n\nGelinas L, Lynch HF, Bierer BE, et al.: When clinical trials compete: prioritising study recruitment. 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PubMed Abstract" }	[ { "id": "72379", "date": "05 Oct 2020", "name": "Margaret McCartney", "expertise": [ "Reviewer Expertise I am a general practitioner with an interest in evidence based medicine." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI am very pleased to see this paper and congratulate the authors. It answers an important question on what research has been done and critically appraises them. This team already have my respect and admiration for their online COVID trials tracker.\n\nThese aren't criticisms:\n\nI had difficulty trying to find what the small amount of trials using non drug/device interventions tested. I appreciated the appendixes with a list of all the trials, broken down into types of trials and interventions. It would perhaps help to draw out the non-drug interventions in full in the text especially as the authors list non-drug interventions that could be planned for testing.\n\nIn terms of further work, there should be data available about the trials that were planned in the next pandemic (some of this was in systems for epidemiological work across centres). I think we have missed a chance to plan non-drug intervention trials and appreciate the authors call to do this. It might be helpful to know how many planned trials actually happened to help model non drug trial options.\n\nIs there any way to graphically explain types of funders in relation to types of trial? It might be a way to hold funders to account - I suspect this will show that it is better to fund fewer bigger trials but I don't know - it might be helpful for funders reading this to appreciate where to put their dollar.\n\nThank you for writing this, I think it does a great job of holding the research communities to account.\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?\nI cannot comment. A qualified statistician is required.\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": [ { "c_id": "6031", "date": "26 Oct 2020", "name": "Perrine Janiaud", "role": "Author Response", "response": "Dear Margaret McCartney,Thank you very much for your kind review and feedbacks. Some elements of answers to your comments below and we have submitted a revision: We have added a few examples in the Results section “Intervention to treat COVID 19 […] Regarding non-drug interventions, 28 investigated procedures (e.g. renal replacement therapy), 8 devices (e.g. various respiratory devices), and 27 trials investigated other non-drug interventions (e.g. physical activity and pulmonary rehabilitation).” We agree, there’s clearly a need to learn from the challenges and successes of the COVID-19 research agenda. Such knowledge would help for future health challenges but on a more immediate level it could definitely inform what would be the best design for non-drug trials. Even more so important, as many countries are currently taking actions to reinforce restrictions. As the funding type was unclear for 41.8% of the assessed trials (i.e. a public/not-for-profit sponsor was reported but the absence of an industrial sponsor does not exclude an industrial funder), we believe that a graph may not be useful to reflect the reality." } ] }, { "id": "72375", "date": "06 Oct 2020", "name": "Atle Fretheim", "expertise": [ "Reviewer Expertise Research methods", "health systems- and policy research", "systematic review." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addresses an important topic, and was an interesting and relatively easy read. Thanks for that!\n\nI have only managed to find one issue of some substance to comment on, and one very minor thing.\nIn the Discussion section you raise the argument that lack of blinding is a problem, especially since many trials don’t have mortality as a main outcome. This is in line with the general understanding that “subjective” outcomes, i.e. outcomes based on some degree of judgement, are more prone to bias due to lack of blinding than “objective” outcomes (with regards to validity of outcome assessment)1. However, I miss a mention of what outcomes the trials actually did include. All I find in the text about types of outcomes concerns whether or not mortality was included - nothing about what other types of outcomes were in use. This information is of some importance for assessing how crucial blinding is in these trials. I did manage to find the column on outcomes in the Extended Data file, but I think a sentence or two, or three, summarizing the general picture on outcome-types would be good to include in the main text.\nOne minor details, on language, which I hesitate to mention being a non-native English speaker:\nIs there a word missing in this sentence (an “a” before “streamlined”, perhaps)? (Discussion, 5th paragraph):\n“Strongly endorsed and prioritized by authorities and medical representatives, it is running as streamlined pragmatic platform trial in over 176 hospitals, randomizing over 12,000 patients in just over four months”.\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?\nNot applicable\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": [ { "c_id": "6032", "date": "26 Oct 2020", "name": "Perrine Janiaud", "role": "Author Response", "response": "Dear Atle Fretheim,Thank you very much for your kind review and feedbacks. Some elements of answers to your comments below and we have submitted a revision: We have not assessed the outcomes in detail, mainly due to the heterogeneity of the reporting for the different outcomes. We unfortunately do not have additional data to present. However, we have added clarifications in the Discussion and Limits: Discussion paragraph 3: \"Blinding may not be required for mortality outcomes; however, mortality was rarely a primary outcome. Other objective outcomes commonly used according to the COVID-19 core outcome sets, such as hospitalization and mechanical ventilation, may still be impacted by subjective decisions and the awareness of the randomly allocated intervention and thus may benefit from a blinded assessment.”Fourth limit: “Fourth, we did not assess in detail all the different outcomes being used, and the blinding of the outcome collection was not systematically reported preventing us from fully apprehending the impact of the lack of blinding” Thank you for spotting the missing “a”" } ] } ]	1	https://f1000research.com/articles/9-1193
https://f1000research.com/articles/8-1994/v1	26 Nov 19	{ "type": "Research Article", "title": "Relationship and career challenges faced by people infected with HIV in Malaysia", "authors": [ "Tuan Norbalkish Tuan Abdullah", "Ruhani Mat Min", "Mosharaf Hossain", "Siti Salina Abdullah", "Tuan Norbalkish Tuan Abdullah", "Mosharaf Hossain", "Siti Salina Abdullah" ], "abstract": "Background: This study was completed at two general hospitals in Malaysia that provide treatment for HIV patients. The aim of the study was to explore the experiences of people infected with HIV (PIWH) and how they cope with HIV. Methods: This qualitative study was based on a social constructivist and grounded theory approach. A total of 12 PIWH were selected by purposive sampling, all of whom participated in semi-structured and audio-recorded interviews, which were supported with non-participant observations and diary entries on three occasions for each participant. The interviews and diaries were transcribed and analysed using the grounded theory approach, which was assisted by utilizing NVIVO-8 to identify the themes related to the experiences of the participants. Results: PIWH experienced challenges related to their career and relationships with family and others. These challenges led to difficulties in gaining employment and career development, as well as feelings of denial, being uncomfortable, rejection, and labelling. They found that their lives were totally and dramatically changed after being tested positive for HIV. Conclusions: Among PIWH, HIV impacted relationships with significant others and career development. The absence of support and acceptance from significant others affected the ability of PIWH to cope with their daily challenges. The results of this study have implications for policymakers in terms of gaining sufficient knowledge and awareness to provide prevention programmes for HIV/AIDS.", "keywords": [ "people affected with HIV", "challenges", "qualitative research", "social constructivist", "grounded theory" ], "content": "Introduction\n\nThe global number of new human immunodeficiency virus (HIV) infections continues to decline. New infections across all ages, as demonstrated by modelled estimates, declined from 3.4 million in 1996 to 1.8 million in 2017; however, progress towards the 2020 milestone of less than 500,000 new infections is far slower1. An estimated 47% of new infections occurred among key populations: men who have sex with men (MSM); people who inject drugs (PWID); people in prisons and other closed settings; sex workers and their clients; transgender people (TG); and the partners of people within these populations. In 2017, globally, there were roughly 21.7 million people living with HIV and receiving antiretroviral therapy (ART)2. In Malaysia, the first reported cases of HIV/AIDS were in 1986, where PWID were largely driving the country’s epidemic. However, the Ministry of Health Malaysia (2018) indicated that the ratio of Malaysian people infected by HIV has declined from 4 in 2000 to 0.2 in 20153. The same report also described the populations most affected by the epidemic, with infection rates exceeding 5% among PWID, female sex workers (FSW), TG, and MSM populations. Among the FSW, TG and MSM populations in Malaysia, in 2017, the HIV prevalence was highest (25%) for MSM3.\n\nAlthough UNAIDS reported that HIV infection has no cure4, PIWH and those at substantial risk can enjoy healthy, long, and productive lives through the use of effective antiretroviral (ARV) drugs to control the virus and prevent transmission2. HIV is also recognized as a highly stigmatized disease5. It challenges an individual physically, socially, and psychologically. Moreover, it can also threaten one’s sense of meaning, purpose, and significance in life6. PIWH may find it difficult to face the reality that they are infected with HIV7, which can contribute to self-denial8 and changes in behaviour9.\n\nHIV/AIDS can affect the physical, social, economic, and psychological condition of the patient. In relation to this, PIWH may encounter numerous problems, such as discrimination, losing social status and role, changes in the patterns of intimate relationships, losing jobs and financial resources, and problems with acquiring the necessary medication10. Many of these problems are common among patients with other chronic diseases, but the stress associated with social and family problems arising from the disease, such as social stigma and exclusion, especially from family and friends, is intensely and uniquely threatening to people with HIV/AIDS11.\n\nThe issue of poor relationships can be related to Adler’s concept of social interest as it refers to the awareness of individuals of being part of the human community and the attitude to dealing with the social world. This concept involves the capacity to cooperate and contribute to something bigger than oneself12. The problems faced by PIWH, such as being stigmatized and excluded by others, may contribute to their denial of the infection with HIV, as well as impact their engagement with society12. In relation to this, PIWH may face criticism, stigma, social discrimination, and ruptures in relationships and life projects13. These negative responses can cause social mortality14, as well as have an impact on their physical and mental health15.\n\nA previous study informed that members of both the community and social network may fear being infected with HIV and be frightened of taking care of HIV/AIDS patients16. The stigma or negative views from society can lead to the isolation and low self-esteem among PIWH until they feel depressed, which can contribute to feelings of inferiority17. In contrast, support and positive relationships with others can be sources of motivation to strive for skills, success, and the completion of their goals, which, ultimately, might convert the feeling of inferiority to that of feeling positive12. Failure to notice and concentrate on patients’ problems may lead to lower levels of accountability and an increase in the pessimism of the infected persons towards society, and thus, lead to the further spread of the virus10.\n\nHowever, as there has been a lack of research concerning the experience of challenges faced by PIWH based on the mode of HIV transmission, this study examines different experiences and challenges among PIWH participants. The main purpose of this study is to understand the experiences of PIWH living with this disease from their own perspectives. This research will explore the experiences of PIWH, with a focus on their lives after being infected with HIV. Qualitative inquiry will be used to capture the challenges and experiences, as well as to allow for any new themes or ideas to emerge. To the researcher’s knowledge, this is the first study to explore these experiences among PIWH based on the mode of HIV transmission.\n\n\nMethods\n\nThis qualitative study using a social constructivist approach was conducted between March 2017 and February 2018 at two selected public hospitals in Malaysia (Hospital Sultanah Nur Zahirah and Hospital Sungai Buloh). Ethical approval to conduct the study was obtained from the Medical Research and Ethics Committee of the Ministry of Health Malaysia KKM/NIH/P17-93, dated 28th February 2017 (12). The participants gave their written informed consent to voluntarily participate in this research, and their participation was based on their choice, free from the elements of assault, threat, injustice or manipulation18; consent was not given to share their data18.\n\nThe reasons underlying the selection of the two public hospitals were firstly, that these hospitals provide HIV treatment and counselling to the patients and secondly, because the HIV patients from these hospitals are continuously engaging with the treatment. Moreover, these hospitals reported high numbers of HIV infected patients based on the yearly report of HIV statistics20. In order to obtain information about the rich experiences of the participants, purposive sampling was used based on the HIV patients registered at the hospitals from February 2015 to February 2017. In March 2017, the medical staff at both selected hospitals helped by providing a list of names of PIWH. The selection of participants was based on inclusion and exclusion criteria. The inclusion criteria were being over 18 years old, having been infected with HIV for at least two years, and being literate. The exclusion criteria were being under 18 years old, having been infected with HIV for less than two years, and being illiterate.\n\nBased on the inclusion and exclusion criteria, a total of 25 PIWH were identified as potential participants. An initial meeting was conducted with the potential participants to explain the purpose, ethical principles, and the duration of the study. The explanation of the research, which was conducted face-to-face and not part of the HIV treatment, was given by the researcher (TN) with the purpose of obtaining voluntary participation. The briefing session was conducted by the researcher and attended by the medical staff of each of the selected hospitals and the potential participants. Although the briefing session would disclose the HIV status of the participants to the researcher, who was not affiliated with the hospitals, the researcher was fully aware of the confidentiality required throughout this research21.\n\nAll 25 PIWH were willing to participate in this research. The first stage of the interview sessions was conducted with four voluntary PIWH who were selected based on their mode of HIV transmission; PWID, heterosexual, vertical transmission and MSM. Throughout the study, a total of 12 PIWH participated in this research based on the data saturation22, which involved different types of HIV transmission as PWID, heterosexual, MSM or vertical transmission. The selection of 12 participants is consistent with grounded theory research, in which data gathering is carried out on datasets collected from 8–20 participants22,23.\n\nThis study used the grounded theory approach for data gathering. This approach identifies theories from data via logical assumption or inductive processes based on the observation and exploration of a phenomenon24. It begins with the researcher identifying a research question that is broad, open ended, and action oriented22,24. In this study, it began with ‘How do PIWH experience life after being affected with HIV?’\n\nThen, a group of people or settings that exemplify different facets of this question was chosen22.\n\nSemi-structured interviews, non-participant observations, and diary writing were involved in this study, with the main data provided by the semi-structured interviews. Semi-structured interviews involved one-to-one interaction between the researcher (TN) and the participants based on proposed protocols. Each of the interview sessions was conducted face-to-face between the participants and the researcher; a female and registered counsellor. The relationship between the researcher and the research participants started at the initial meeting, which was prior to the interview session. The participants were aware of the purpose of the research and the goals of the interview, which was to gather experiences as PIWH going through a counselling session. The researcher engaged in the interviews with the participants with prior knowledge of HIV and an interest in understanding the experiences of PIWH going about their lives. All the interview sessions were conducted in Bahasa Malaysia, which is the spoken language of the participants22. All the interviews were audio recorded for the purpose of data gathering. Each of the interviews was conducted within one and half hours, and each of the participants was allowed time to respond to the open-ended questions to ensure they were covered in adequate depth21. It was expected that the interviews would capture the key issues described by the research participants. Gathering this type of information requires flexibility and the use of semi-structured interviews is beneficial as it allows the participants to share their experiences using their own words and ways25. The use of open-ended questions with the grounded theory approach as a guideline for the interviews was done to avoid assumptions23,18,21.\n\nEach of the participants had the chance to decide the date and time for the interview, which was decided according to their choice and was also free from the element of threat19. All the interviews were conducted at the HIV clinic in the selected hospitals. Each participant participated in three interviews. The details of the interview questions for each of the three interviews are provided in Table 1. The interview questions were prepared prior to the actual data gathering, through preliminary interviews conducted with non-research participants before the actual research.\n\nThis study also collected data by the observational method to describe the setting, activities, and people who participated in the study26. Non-participant observations were performed in this study, each with a duration of one and half hours, and with each participant being observed at three different times after the interview sessions. The researcher (TN) visited the HIV clinic in the selected hospitals and conducted the observations without being involved in the counselling sessions between the medical staff and the participants21.\n\nDiary entries by the participants were also required as part of the data in this study as they represent a record of thoughts, feelings, opinions, or actions. The participants had the opportunity to share their experiences, actions, and personal information about themselves through the diaries27. The diary entries allowed the researcher to compare the information shared by the participants during the interview and self-reported experiences written in the diary19. Three diary entries were required for each participant during the study period. The participants were required to record their experiences in this study by completing pre-prepared statements (Table 2), which were open-ended as this gives freedom to the participants to select their own words and style of writing27. These diaries were completed by the participants at home.\n\nThe study was conducted in Bahasa Malaysia, and the researcher (TN) transcribed all the interviews for the process of data analysis into English. The grounded theory approach was used in analysing the data, allowing categories, themes, and patterns to emerge23. The stages of coding and the identification of the emergent themes was through the use of NVivo 8. The first stage of data analysis started immediately after the first data gathering process. Then, through a process of constant comparison, the three main sources of data – interviews, non-participant observation and diary writing – were compared and contrasted with each other. Discussions among all the researchers at all stages of data analysis were conducted. Triangulation of the three main data and discussions among the researchers was to ensure the trustworthiness and credibility of the data analysis. Throughout all the stages of data collection and analysis, the researcher kept memos and drew diagrams. Details of the data collections and analysis can be seen in Figure 1.\n\n\nResults\n\nA total of 12 participants contributed to the study, of which seven (58.33%) were male and five (41.66%) were female. Of these, three (25%) were PWID who contracted HIV via intravenous drug use, two (16.66%) contracted HIV via vertical transmission, four (33.33%) were heterosexual and contracted HIV via sexual transmission, and three (25%) were MSM who contracted HIV via sexual transmission. Overall, two (17%), three (25%) and seven (58%) of the participants were in the age groups <30 years old, 30–40 years old, and >40 years old, respectively. Regarding occupation, seven (58.33%) were employed, three (25%) were unemployed, and two (16.66%) were businessman. The summary of the background of the PIWH in the study is summarised in Table 3.\n\nMYR, Malaysian Ringgit; HIV, human immunodeficiency virus; PWID, people who inject drugs.\n\nDifficulties in coping with life and facing struggles to survive are among the challenges that cause negative feelings in affected people28,29. The PIWH in this study shared their experiences of facing life challenges in relation to their career as well as their relationship with family and others. The participants indicated that they had experienced career challenges in that it was difficult to get a job or advance their career. Besides that, the participants also shared the experience of having problems in their relationships with family and others. These challenges were found to be very much related to the mode of HIV transmission (intravenous drug use in PWID, vertical transmission, sexual transmission in MSM or heterosexual sexual transmission). The experiences shared by the research participants are outlined below.\n\nCareer challenges underpinned the experiences among those infected with HIV via intravenous drug use, vertical transmission and heterosexual sexual transmission. Those infected via vertical transmission indicated that they experienced some difficulties in getting a job, while those infected via intravenous drug use and heterosexual sexual transmission experienced disruption to their career development.\n\nDifficult to get a job. One of the research participants infected via vertical transmission indicated that they faced difficulties in getting a job. According to Case 4 (aged 21, female), as HIV positive, her time was spent on regular treatment appointments, which made it impossible to think about getting a job. The participant was also tied by the way of life as an HIV positive individual:\n\n“After I finished my study, I started to plan for my future. I had a dream about a good job like others. But it remained a dream because I know that getting a job is not easy for HIV affected. I need to go to the hospital and have treatment regularly. Also, I have to be more careful about certain things like having healthy food and I need to stay alert to the changes happening in my body.”\n\nAnother participant, Case 5 (aged 19, female), who was infected via vertical transmission also said that getting a job is impossible for PIWH. She had experienced being rejected as soon as the employer knew her status:\n\n“I am totally sure that getting a job is impossible for HIV affected. Before this, I had experience in applying for a job. I told the employer about my background and after he knew it, he said that I am not qualified to have the job.”\n\nThe participants indicated that as HIV positive, they have no chance of getting a job because of their status.\n\nCareer development disrupted. Being HIV positive can also disrupt career development, as experienced by those infected via intravenous drug use and heterosexual sexual transmission. According to Case 3 (aged 40, female), after being diagnosed with HIV, the participant started to lose motivation when working and missed opportunities to develop her career because of difficulties after being infected with HIV.\n\n“In working, I started to feel meaningless. I am good as a worker but after being diagnosed with HIV I cannot focus properly on my work. I need to go to the hospital to get treatment, and I think I spend more time in hospital than the workplace. Frequently, I was not feeling well because of HIV. I also started to miss the opportunities to get a bonus from the company.”\n\nAnother participant who was infected via intravenous drug use, Case 2 (aged 51, male), mentioned in his diary that HIV had raised some barriers in his working life. The barriers were related to expanding his business and making more profit.\n\n“It is really difficult to live with HIV. It gives me more stress, especially in working. After the diagnosis, I started to have some struggles in expanding my work. I did not always feel well, and am totally dependent on medicines because of my CD4 getting lower. Now, I am unable to work overtime and cannot focus on my work.”\n\nIn addition, during the non-participant observation, Case 2 verbally indicated to the medical staff that he was worried about his business situation because of the amount of time he spent in the hospital for treatment.\n\nAnother participant who was infected via heterosexual sexual transmission, Case 7 (aged 42, female), said that as HIV affected, she started to have some problems in her working life. The participant said that her work was disrupted when she had some emotional struggles in facing her HIV infection. The difficulties in handling these emotions affected her career development.\n\n“Only God knows how I felt during the HIV diagnosis. I was totally out of myself and I planned to quit my job. At the same time, I was offered a salary increment. I just did not know what was the best thing to do. I was at a loss.”\n\nAnother participant who was infected via heterosexual sexual transmission, Case 6 (aged 48, female), also stated in her diary that HIV infection made her life worse as her business was almost ruined due to her situation. The participant needed to focus on her business, while, at the same time, she was struggling to adhere to the treatment regularly and sometimes she got depressed about continuing the treatment.\n\n“Every day I kept thinking about my business. This business should be continued even if I am not feeling well. I had to be strong to make my business run smoothly. Sometimes I was really afraid that my condition would not allow me to focus on it.”\n\nThe participants indicated that HIV infection is a barrier to them developing their career. This was related to challenges in dealing with HIV infection as the participants had to deal with regular treatment and the daily activities as HIV infected.\n\nThe participants also reported that HIV affected their relationship with others. This was experienced by those infected via heterosexual sexual transmission and MSM who were infected via sexual transmission. The participants started to feel uncomfortable about being with others and chose to isolate themselves from others.\n\nFeeling uncomfortable. The participants infected via heterosexual sexual transmission indicated that they felt uncomfortable being with others as they wanted to ensure that their status would not be disclosed. Case 7 shared the uncomfortable feeling of being with others and stated that the feeling was related to the changes in her physical appearance after being infected with HIV.\n\n“When I knew the result of my HIV status, I noticed that I started to feel awkward being with people, especially my friends. I did not want them to know my HIV status. I changed my routine and I just spent more time with my children at home. Besides that, I felt uncomfortable about the physical changes as I became thinner and my skin looked so dull.”\n\nIn addition, in her diary, Case 7 mentioned that she was not really comfortable about attending the treatment sessions in hospital if there were other patients in the same session.\n\n“During the treatment, I was not comfortable if I had to be with a lot of patients in the same session. I was worried that someone would notice me attending HIV treatment in the hospital.”\n\nAnother participant who was infected via sexual transmission, Case 6, also shared the feeling of being uncomfortable since being affected with HIV. It changed her a lot, especially her physical appearance, and she worried about facing people when her physical appearance was different.\n\n“With HIV, I looked so dull. I was so particular in my appearance as a businesswoman. I have to face and meet people in my career. The treatment affected me a lot. I was so not comfortable with the medicine I was taking. The medicine had some effect, especially in terms of my physical appearance.”\n\nThe participants indicated that the feeling of being uncomfortable affected their life regarding their relationships with others. In addition, the feeling was also related to the changes in their physical appearance as the participants faced people in their daily life.\n\nDenial. The participants also reported being in denial about being HIV positive. This was experienced by those infected via heterosexual sexual transmission and MSM who were infected via sexual transmission. Case 10 (aged 36, male) said that it was difficult to accept being infected with HIV. This was because the participant realized that his wife could not accept the situation.\n\n“When I received the HIV result, the first thing that came into my mind was my wife. I couldn’t imagine how she would react towards this matter. I just could not accept this.”\n\nAnother MSM participant, Case 11 (aged 44, male), also indicated about being in denial. The participant said that it was very hard to accept the reality of being affected with HIV.\n\n“It was so unbelievable. I was a very particular person in any matter related to myself, especially my health. I felt like it was not me. I tried to think positive, but I knew this was the reality. It was so hard for me.”\n\nIn addition, Case 10 mentioned in his diary that he would never accept that he was HIV positive.\n\n“Even for thousand years upward, I would never accept HIV is inside my body.”\n\nAnother MSM participant, Case 12 (aged 45, male), also said that it was very hard to accept the reality of being infected with HIV.\n\n“I prayed that it was not me. I hoped the result was not mine. I repeated the test about three times just to make sure that the result was true. It hurts me.”\n\nThe participants indicated experiencing a feeling of being uncomfortable with others and denial in terms of accepting the fact that they are HIV positive. These experiences also contributed to the struggles faced in their daily life.\n\nThe participants also indicated having challenges in their relationships with their families. They had experienced rejection and labelling from the family members.\n\nRejection. Being HIV affected, Case 10 said that he could never face his family since he experienced rejection from his mother and siblings.\n\n“I never informed my family about my HIV status. One day, my sister came into my room and she found the documents of treatment on my bed. Then, the news was spread. The situation was so tense. Then I knew that no one in my family would accept this.”\n\nCase 10 also mentioned in his diary that he would never put on his family the burden of living with a HIV affected person.\n\n“I can’t even manage my feelings when I see my mother crying. All I can say is that this burden (HIV affected) should never be laid on them (family).”\n\nLabelling. Participants infected via heterosexual transmission and MSM who were infected by sexual transmission reported that they were also labelled by their family members as HIV positive. Case 7 stated that she had experienced being labelled by her siblings.\n\n“I have experienced this, my brother came to me and said it to my face that I have HIV. So, he said that the family need to be more careful in sharing the things in the house with me.”\n\nIn addition, Case 11 also said that his family labelled him as being disgusting, dirty, and irritating in the eyes of others, including his siblings.\n\n“When they asked about the plate I had used, or when they asked which plate I had used, it clearly showed that they were not really happy about it.”\n\nIn summary, their experiences in facing life challenges caused changes in the reality of life for PIWH. Being HIV positive, they need some space or area to be accepted and heard. Their sharing of these experiences showed that these challenges were experienced when they were engaging with others or being in public.\n\n\nDiscussion\n\nThis study provides knowledge of the challenges experienced by PIWH. These challenges have been found to influence PIWH’s relationships with family and others and affect their careers. HIV could have a severe impact on the career opportunities30 and the stability of relationships31. This can cause social isolation and conflictual social interactions that may increase stress, resulting in poorer overall social functioning32. In this study, it was found that the challenges experienced by PIWH were different based on the mode of HIV transmission among PWID who were infected via intravenous drug use, those infected via vertical transmission, those infected via heterosexual sexual transmission, and those infected by homosexual sexual transmission.\n\nThe participants reported that career development was among the challenges faced by them. This was experienced by PWID and those infected via vertical transmission and heterosexual sexual transmission. They struggled to get a job and found it was very difficult to continue their career once they started to notice changes after being affected with HIV. The reasons for these experiences were related to them having to concentrate on regular treatment, needing an adjustment in terms of arranging the schedule for treatments and struggling to concentrate on their work. These findings are related to the view33 that PWID infected with HIV are concerned about being different due to the need to take medication throughout the day and the decision of whether or not to confide in their supervisors and co-workers about the reason why they are frequently taking this medication. Besides that, these feelings may lead PIWH to decrease their achievements, classify their lives as not important, and resist recognizing their long-term goals34. For example, Case 2 and Case 3 reported that it was very difficult to perform well in their career after changes to their routine since being HIV positive, as they needed to focus on the treatment provided. Therefore, the findings of this study can be related to Adler’s concept of social interest, as the problems of the individual are related to the feeling of being unaccepted in their social community12. Hence, the participants need to have some support system that can help them to adapt to a new routine of life after being infected with HIV.\n\nThis study also indicated that those who were infected via heterosexual sexual transmission, and MSM who were infected by sexual transmission struggled to maintain their relationships with others. For example, Case 7 was not comfortable being with others. The participant became worried about not being accepted if their HIV status was disclosed to other people. This can be seen from the Adlerian perspective as human behaviour is determined by the capacity to interpret the events according to the social interest concept12. In addition, Adler stated that people express social interest through shared activity, cooperation, participation in the common good, and mutual respect. With regard to Case 7, the uncomfortable feeling felt when being with others was based on the thinking that no one would accept the reality of her HIV status. Besides that, the participants also reported denial of the situation. For example, Case 9 stated that it was very hard to accept the fact that they were infected with HIV. According to the participant, this feeling was related to feelings of worry about his spouse if his HIV status was known. This is because the disease not only affects the patient, but also their family members, especially their spouse10. In other words, HIV status creates challenges not only with others but also a personal dilemma among those who are HIV affected.\n\nThe participants who had been infected via heterosexual sexual transmission and MSM who were infected by sexual transmission also reported challenges in facing their family. Under the category of relationships with family, the most important issues raised by participants were rejection and labelling from family members. In relation to this, appropriate support from families enables HIV infected MSM to have appropriate responses to the stress caused by illness, and, therefore, they would have fewer mental health issues10. For example, Case 8 stated that he chose not to see his family when he noticed their rejection and showed that he was stressed from the experience of rejection from the family. This situation can be seen through the Adlerian perspective; an individual seeks a space in the family and society to fulfil their need for security, acceptance, and worthiness35. Moreover, Case 7 also experienced being labelled by family members, which contributed to their feelings of isolation. In relation to this, when families and society treat patients negatively, so that they are isolated and excluded when found to be HIV positive, the disease will spread in a clandestine mode of transmission and a major part of the infected population will remain hidden36.\n\n\nConclusions\n\nThe experiences that PIWH reported related to their career challenges and uncomfortable relationships with other people, which can lead to physical and mental health issues, such as physical pain and emotional struggles. Their experiences of these challenges can affect their daily lives as human beings. Even though the rates of HIV infection have already declined, the findings showed that PIWH still needed help to handle the challenges of living with HIV. Therefore, knowledge and awareness about HIV prevention among the community, non-governmental organisations and HIV treatment providers should be highlighted by the policymakers. Policies concerning the prevention of HIV need to be improved and can be emphasized in collaboration with government agencies to ensure the success of these prevention programmes.\n\nThese findings also have important implications for counselling services for HIV/AIDS prevention in Malaysia as well as in similar settings in other low- and middle-income countries. There is a need for further similar research to be conducted, particularly counselling services for HIV infected people. There is a limitation to the study. The findings cannot be generalized to broader populations of PIWH with the same degree of certainty. This is because the findings of this study were not tested to determine whether they were statistically important or due to chance.\n\n\nData availability\n\nThe underlying data for this study cannot be openly shared since the consent to participate obtained from the HIV patients explicitly stated that their data would remain confidential and only be reported in an aggregated manner. Anyone wishing to access the underlying data should first contact the corresponding author (ruhani@umt.edu.my), who will facilitate contact with the ethical review board that approved the study. Access will be granted upon approval from the ethical review board.", "appendix": "Acknowledgments\n\nThe authors express their sincere thanks to the Ministry of Health Malaysia and University Malaysia Terengganu for allowing and assisting the researcher to collect the data smoothly.\n\n\nReferences\n\nUNAIDS Global Report: Joint United Nations Programme on HIV/AID. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmitz MF, Crystal S: Social relations, coping, and psychological distress among persons with HIV/AIDS. J Appl Soc Psychol. 2000; 30(4): 665–683. Publisher Full Text\n\nHunt B, Jaques J, Niles SG, et al.: Career concerns for people living with HIV/AIDS. Journal of Counseling & Development. 2003; 81(1): 55–60. Publisher Full Text\n\nKose S, Mandiracioglu A, Mermut G, et al.: The social and health problems of people living with HIV/AIDS in Izmir, Turkey. Eurasian J Med. 2012; 44(1): 32–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorey G: Theory and practice of counseling and psychotherapy. Nelson Education. 2015. Reference Source\n\nAshraf M, Sitwat A: Personality dimensions, positive emotions and coping strategies in the caregivers of people living with HIV in Lahore, Pakistan. Nurs Pract. 2016; 22(4): 364–374. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66794", "date": "10 Aug 2020", "name": "Syed Mohamed Aljunid", "expertise": [ "Reviewer Expertise Public Health Medicine and Health Policy and Management" ], "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 excellent paper reporting on the outcome of qualitative research carried out on HIV positive patients to understand the challenges they face in life. The authors have reported the details of their encounter with 12 respondents using a semi-structured questionnaire in a face-to-face interview, non-participant observations, and respondents' diaries. There are a number of issues as follows that the authors need to address in order to strengthen the paper for the final submission:\nMethods: On page 4, right column para1, the authors stated that they used the open-ended questionnaire with grounded theories to avoid assumptions. Please elaborate on the kind of assumptions that they want to avoid in this study\n\nMethods: On page 4, right column para 2: it was mentioned that the date and time of interview were selected by respondents to avoid \"threat\". Please elaborate what kind of threat are they concerned about since the interviews were carried out in the clinics.\n\nMethods; Regarding Diaries entries, what are the authors expectation on the respondents entrance of the diaries? What kind of guidances were given to the respondents on what to record in the diary.\n\nMethods: Explain how items in Table 2 on page 5 were used in this study to guide the respondents?\n\nResults: What do you mean by vertical transmission here? Are you sure this information is accurate? Who gave this information? Was it from the respondents or the doctors who treat the patients? How do you verify this information\n\nDiscussion: Page 9, first line on right column: Clarify what is the meaning of \"feeling felt\"?\n\nDiscussion: Page 9, right column, 2nd paragraph: Please explain in more detail how Adlerian perspective is relevant to the findings of this research.\n\nConclusion: Please revise the first sentence of the Conclusion. It is not clear what the authors trying to say here.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5887", "date": "07 Sep 2020", "name": "Tuan Norbalkish Tuan Abdullah", "role": "Author Response", "response": "CommentsRespond to the reviewMethods: On page 4, right column para1, the authors stated that they used the open-ended questionnaire with grounded theories to avoid assumptions. Please elaborate on the kind of assumptions that they want to avoid in this study This study conducted open-ended semi-structured interviews.Methods: On page 4, right column para 2: it was mentioned that the date and time of interview were selected by respondents to avoid \"threat\". Please elaborate what kind of threat are they concerned about since the interviews were carried out in the clinics.Open-ended semi structured interviewsMethods: Regarding Diaries entries, what are the authors expectation on the respondents entrance of the diaries? What kind of guidances were given to the respondents on what to record in the diary.see documentExplain how items in Table 2 on page 5 were used in this study to guide the respondents?See documentResults: What do you mean by vertical transmission here? Are you sure this information is accurate? Who gave this information? Was it from the respondents or the doctors who treat the patients? How do you verify this informationSee documentDiscussion: Page 9, first line on right column: Clarify what is the meaning of \"feeling felt\"?See documentDiscussion: Page 9, right column, 2nd paragraph: Please explain in more detail how Adlerian perspective is relevant to the findings of this research.See documentConclusion: Please revise the first sentence of the Conclusion. It is not clear what the authors trying to say here.See document" } ] }, { "id": "68394", "date": "17 Aug 2020", "name": "Kristina Lindvall", "expertise": [ "Reviewer Expertise Public Health", "Nutrition", "Qualitative methodology" ], "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 want to thank the authors and the Editor of F1000 Research for the opportunity to review this interesting manuscript.\n\nBelow are my comments alternatively suggestions for revision divided into the sections in which they appear.\n\nAbstract:\nThe background of the abstract does not include any motivation for the need of this study. Instead, it describes the setting and the aim of the study. If the word limit allows I would suggest adding one sentence motivating the need of the study. If there is a need to save words I would suggest deleting the first sentence in the abstract alternatively some of the details in the methods. For example, to end the sentence starting with “The interviews and diaries were transcribed… after the word “approach”.\nIntroduction (Aim):\nI would suggest using the same aim in the abstract as in the main manuscript. Now they are slightly different (for example in the abstract the authors mention “coping”). I would also suggest either using the word “aim” or “purpose” in both the abstract and the main text for consistency.\n\nMethods:\nFirst paragraph: Would it be possible to add where in the country the two hospitals are situated, alternatively how large areas of the country that these hospitals cover?\n\nParagraph labelled “Participants”: The authors mention that the final number of participants was decided based on saturation. Would it be possible to add some more information on how you determined that you had reached saturation (either in this paragraph or in “data analysis”)?\n\n“Procedure”, fifth paragraph: The authors describe, “This study also collected data by the observational method to describe the setting, activities, and people who participated in the study”. Where can the reader see the results of these observations?\n\nData analysis: The authors describe, “The first stage of data analysis started immediately after the first data gathering process”. Which “firs data gathering process” is referred to here? For example, does it mean that data analysis started after the first one or two interviews, after all interviews had been conducted or something different?\n\nData analysis: Did the authors use initial coding and focused coding during the analytical process? If yes, I suggest adding this to the “data analysis” paragraph. I would also suggest using the same terminology in the methods and in the results. I.e if categories and themes and/or sub-categories are mentioned in the results then I believe it would be helpful if these are also referred to in the analytical process described in the paragraph “data analysis”.\n\nData analysis: Did the authors start to analyze one source of material (for example the interviews) and then continued with the next or were all three sources (interviews, observations, diary entries) analyzed simultaneously.\n\nData analysis (Trustworthiness): Were there any other criteria used to ensure trustworthiness than credibility (for example, transferability, dependability confirmability)? If, yes. It would be helpful if the authors could add some more information in relation to this.\n\nTable 1: One alternative table heading could be “Interview questions used in three rounds of interviews”.\n\nTable 2: One alternative table heading could be “Pre-prepared statements used for diary entries”.\n\nFigure 1. Could the authors please add some more detail to the figure text? For example, where can the reader see the data collection in the figure? What do the arrows, lines and squares in the figures represent? If some squares represent participants and others, for example represent the results. Could these differences be indicated by squares being different in the frame, shape or shading?\n\nResults:\nFirst paragraph: I wonder if it perhaps is enough with one decimal for the percentages presented?\n\nFirst paragraph: If possible, it would be interesting to also the age range of participants.\n\nDid the authors collect any information on if the participants lived in a more urban or rural setting alternatively in a smaller or larger city? If this is available, perhaps this could be added to table 3.\n\nJust after the first paragraph of the results and before the paragraph with the heading “Challenges of PIWH” it would be helpful if the authors could add an introduction of the results. I.e how many themes or categories that emerged and how many sub-categories (depending on how the authors prefer to label these) that emerged and which these were. In this way, the reader is also helped to see what the headings and the sub-headings of the results represent.\nDiscussion:\nFirst paragraph: Here the authors state that “In this study, it was found that the challenges experienced by PIWH were different based on the mode of HIV transmission among PWID who were infected via intravenous drug use, those infected via vertical transmission, those infected via heterosexual sexual transmission, and those infected by homosexual transmission”. Here it would be helpful if the authors could add one or two sentences summarizing these differences.\n\nSecond paragraph. Do the authors see any limitations with the current study being based on a qualitative study design? To me, the limitation currently raised more to the study not having a quantitative study design.\nConclusions:\nIt would be helpful if the authors could highlight what they find as their main findings in the first two-three sentences of the conclusion.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5889", "date": "07 Sep 2020", "name": "Tuan Norbalkish Tuan Abdullah", "role": "Author Response", "response": "Abstract: The background of the abstract does not include any motivation for the need of this study. Instead, it describes the setting and the aim of the study. If the word limit allows I would suggest adding one sentence motivating the need of the study. If there is a need to save words I would suggest deleting the first sentence in the abstract alternatively some of the details in the methods. For example, to end the sentence starting with “The interviews and diaries were transcribed… after the word “approach”.Answer: Agreed with your comments and already revisedIntroduction: I would suggest using the same aim in the abstract as in the main manuscript. Now they are slightly different (for example in the abstract the authors mention “coping”). I would also suggest either using the word “aim” or “purpose” in both the abstract and the main text for consistencyAnswer: Thank you for suggestionsMethods: 1. First paragraph: Would it be possible to add where in the country the two hospitals are situated, alternatively how large areas of the country that these hospitals cover?Answer: This is related to the ethical issue, which in Malaysia, there are only general hospitals from each state that manage HIV cases.2. Paragraph labelled “Participants”: The authors mention that the final number of participants was decided based on saturation. Would it be possible to add some more information on how you determined that you had reached saturation (either in this paragraph or in “data analysis”)?Answer: Agreed with your comments and already revised3. “Procedure”, fifth paragraph: The authors describe, “This study also collected data by the observational method to describe the setting, activities, and people who participated in the study”. Where can the reader see the results of these observations?Answer: Agreed with your comments and already revised4. Data analysis: The authors describe, “The first stage of data analysis started immediately after the first data gathering process”. Which “firs data gathering process” is referred to here? For example, does it mean that data analysis started after the first one or two interviews, after all interviews had been conducted or something different? Answer: Agreed with your comments and already revised5. Data analysis: Did the authors use initial coding and focused coding during the analytical process? If yes, I suggest adding this to the “data analysis” paragraph. I would also suggest using the same terminology in the methods and in the results. I.e if categories and themes and/or sub-categories are mentioned in the results then I believe it would be helpful if these are also referred to in the analytical process described in the paragraph “data analysis”.Answer: Agreed with your comments and already revised6. Data analysis: Did the authors start to analyze one source of material (for example the interviews) and then continued with the next or were all three sources (interviews, observations, diary entries) analyzed simultaneously.Answer: Agreed with your comments and already revised 7. Data analysis (Trustworthiness): Were there any other criteria used to ensure trustworthiness than credibility (for example, transferability, dependability confirmability)? If, yes. It would be helpful if the authors could add some more information in relation to this. Answer: Triangulation8. Table 1: One alternative table heading could be “Interview questions used in three rounds of interviews”. Answer: Agreed with your comments and already revised9. Table 2: One alternative table heading could be “Pre-prepared statements used for diary entries”. Answer: Agreed with your comments and already revised10. Figure 1. Could the authors please add some more detail to the figure text? For example, where can the reader see the data collection in the figure? What do the arrows, lines and squares in the figures represent? If some squares represent participants and others, for example represent the results. Could these differences be indicated by squares being different in the frame, shape or shading?Answer: Agreed with your comments and already revisedResults: 1. First paragraph: I wonder if it perhaps is enough with one decimal for the percentages presented?Answer: Agreed with your comments and already revised2. First paragraph: If possible, it would be interesting to also the age range of participantsAnswer: It is already stated in the table the range of participants3. Did the authors collect any information on if the participants lived in a more urban or rural setting alternatively in a smaller or larger city? If this is available, perhaps this could be added to table 3. Answer: The information of the participants was not involved/considered of urban and rural setting.4. Just after the first paragraph of the results and before the paragraph with the heading “Challenges of PIWH” it would be helpful if the authors could add an introduction of the results. I.e how many themes or categories that emerged and how many sub-categories (depending on how the authors prefer to label these) that emerged and which these were. In this way, the reader is also helped to see what the headings and the sub-headings of the results represent.Answer: Agreed with your comments and already revisedDiscussion: 1. First paragraph: Here the authors state that “In this study, it was found that the challenges experienced by PIWH were different based on the mode of HIV transmission among PWID who were infected via intravenous drug use, those infected via vertical transmission, those infected via heterosexual sexual transmission, and those infected by homosexual transmission”. Here it would be helpful if the authors could add one or two sentences summarizing these differences.Answer: Agreed with your comments and already revised 2. Second paragraph. Do the authors see any limitations with the current study being based on a qualitative study design? To me, the limitation currently raised more to the study not having a quantitative study design.Answer: Agreed with your comments and already revised (Conclusion Para 2)Conclusion: It would be helpful if the authors could highlight what they find as their main findings in the first two-three sentences of the conclusion.Answer: Agreed with your comments and already revised" } ] } ]	1	https://f1000research.com/articles/8-1994
https://f1000research.com/articles/9-1269/v1	23 Oct 20	{ "type": "Research Article", "title": "A first draft genome of the Sugarcane borer, Diatraea saccharalis.", "authors": [ "Lucas Borges dos Santos", "João Paulo Gomes Viana", "Fabricio José Biasotto Francischini", "Sofia Victoria Fogliata", "Andrea L. Joyce", "Anete Pereira de Souza", "María Gabriela Murúa", "Steven J. Clough", "Maria Imaculada Zucchi", "Lucas Borges dos Santos", "João Paulo Gomes Viana", "Fabricio José Biasotto Francischini", "Sofia Victoria Fogliata", "Andrea L. Joyce", "Anete Pereira de Souza", "María Gabriela Murúa", "Steven J. Clough" ], "abstract": "Background: The sugarcane borer (Diatraea saccharalis), a widely distributed moth throughout the Americas, is a pest that affects economically important crops such as sugarcane, sorghum, wheat, maize and rice. Given its significant impact on yield reduction, whole-genome information of the species is needed. Here, we report the first draft assembly of the D. saccharalis genome. Methods: The genomic sequences were obtained using the Illumina HiSeq 2500 whole-genome sequencing of a single adult male specimen. We assembled the short-reads using the SPAdes software and predicted protein-coding genes using MAKER. Genome assembly completeness was assessed through BUSCO and the repetitive content by RepeatMasker. Results: The 453 Mb assembled sequences contain 1,445 BUSCO gene orthologs and 1,161 predicted gene models identified based on homology evidence to the domestic silk moth, Bombyx mori. The repeat content composes 41.18% of the genomic sequences which is in the range of other lepidopteran species. Conclusions: Functional annotation reveals that predicted gene models are involved in important cellular mechanisms such as metabolic pathways and protein synthesis. Thus, the data generated in this study expands our knowledge on the genomic characteristics of this devastating pest and provides essential resources for future genetic studies of the species.", "keywords": [ "Diatraea saccharalis", "sugarcane borer", "draft genome", "assembly", "Lepidoptera" ], "content": "Introduction\n\nDiatraea saccharalis (Fabricius), commonly known as the sugarcane borer, is an important moth pest of the family Crambidae (Lepidoptera; Crambidae) that is distributed throughout the Americas, including South America, the Caribbean, Central America, and the southeastern United States (Box, 1931; CAB International, 1989; Dyar & Heinrich, 1927). D. saccharalis host plants include important crops such as sugarcane (Saccharum spp.), sorghum (Sorghum bicolor), wheat (Triticum spp.), maize (Zea mays L.), and rice (Oryza sativa L.) (Myers, 1932; Rodríguez-del-Bosque et al., 1988; Roe, 1981). The damage caused by the D. saccharalis larvae in young plants produce a characteristic damage, that is the appearance in series of holes across the leaves that are still rolled up, and posteriorly begin to feed off the apical meristem that is killed, resulting in a condition known as \"dead heart\". In the developed plants, the larvae, after hatching, start to feed on the leaves and migrate to the sheath region where they are protected, and feed on their sheath and start to scrape the stalk. As the borer develops, generally third instar and older, it feeds almost exclusively within tunnels in stalks (Flynn et al., 1984). As a result of the borer behavior, the physical strength of the mature plant stalk is reduced, decreasing the plant biomass and sugar content in more developed crops, and have an increased susceptibility to plant pathogens due to the holes made by the larvae (Cruz, 2007; Wilson et al., 2017). In maize fields, a new behavior of the D. saccharalis was observed, where the larva may burrow into corn ears (Rodriguez-del-Bosque et al., 1990).\n\nAlthough the sugarcane borer has a wide distribution across the Western Hemisphere (Dyar & Heinrich, 1927), it is treated as a single species and its identification relies on morphological characteristics despite the high variability shown in these traits between populations (Dyar & Heinrich, 1927; Francischini et al., 2017; Pashley et al., 1990). Several studies have developed molecular markers to provide a solid differentiation from other species of the same genus (Bravo et al., 2008; Francischini et al., 2017; Joyce et al., 2016; Pavinato et al., 2013; Pavinato et al., 2017) and to investigate the genetic variation among populations of D. saccharalis (Joyce et al., 2014; Joyce et al., 2016; Silva-Brandão et al., 2015). Geographical comparisons using individuals from the southeastern United States and Argentina have also suggested that the species might be treated as a cryptic species complex due to the significant genetic divergence among lineages (Fogliata et al., 2019; Joyce et al., 2014). The high genetic variation and a wide plant host range have also supported the success of this pest in colonizing regions with different environmental conditions, and human migration may likely be a major factor that contributed to its spread throughout the Americas (Francischini et al., 2019). Furthermore, the great increase of genetic diversity of the sugarcane borer in Brazil coincides with the expansion of agricultural production of sugarcane and maize crops in the Brazilian landscape (Pavinato et al., 2018). This makes D. saccharalis an interesting organism to study a wide range of questions including ecological adaptation and evolutionary dynamics. However, such studies require a significant number of molecular resources which are currently missing. A reference mitochondrial genome (Li et al., 2011) and a transcriptome assembly have been described (Merlin & Cônsoli, 2019); however, a well-assembled reference genome of D. saccharalis is not yet available.\n\nAcross the approximate 170,000 lepidopteran species, there are about 80 published genomes to date (Triant et al., 2018), however only a few of them have scaffolds assigned to the chromosome level [Bombyx mori (Kawamoto et al., 2019), Heliconius melpomene (Heliconius Genome Consortium, 2012), Plutella xylostella (You et al., 2013), Melitaea cinxia (Ahola et al., 2014), Trichoplusia ni (Chen et al., 2019), and Cydia pomonella (Wan et al., 2019)]. Recently, a few de novo genome assemblies of moth species have been released, such as Spodoptera frugiperda (Gouin et al., 2017; Kakumani et al., 2014), Thaumetopoea pityocampa (Gschloessl et al., 2018b) and Achroia grisella (Koseva et al., 2019). Due to the complexity of insect genomes, the draft assemblies have poor continuity and contain many gaps, which may result in a loss of a large number of genomic regions which relate to biological features of the species (Li et al., 2019). The lack of genetic information of this clade makes it difficult to understand some evolutionary processes such as those underlying the adaptation to plant defense systems under different environments (Davey et al., 2016). The rise of next generation sequencing technologies and the improvement in assembly algorithms have been providing more resources to assemble large and more complex genomes of non-model organisms at a lower cost (Wachi et al., 2018). Nevertheless, many challenges still remain when selecting the best assembly software and setting the best parameters in order to get the most accurate genome assembly.\n\nIn this study, we report the first genome sequence of D. saccharalis which was assembled using paired-end (PE) short reads generated by the Illumina HiSeq platform with high sequencing depth coverage. This draft genome assembly reveals important aspects of the genetics for this species such as genome size and heterozygosity levels. In addition, we identified hundreds of protein-coding gene models using a homology-based approach using B. mori protein information and investigated the functional annotation of these proteins. However, the difficulties in assembling long tandem-repeat regions using a short-read technology also resulted in fragmented sequences within the assembly. These data provide a vastly improved genome that will serve as a valuable resource for further genomic studies of the species in order to develop more effective approaches for the management of this pest.\n\n\nMethods\n\nD. saccharalis moths used for the DNA extraction were from an inbred colony which originated in Houma, Louisiana, and was reared on an artificial diet (Southland Products, Lake Village Arkansas). The insects were raised for about 70 generations, with new insects occasionally added to the colony. The adult moths were placed in 100% ethanol and shipped to the lab for DNA extraction. Genomic DNA was isolated from thorax and leg tissues derived from a single adult male specimen. The tissues were ground up to become a fine powder in a 10 mM Tris-HCl (Sigma-Aldrich, USA), 400 mM NaCl (Fisher Scientific, USA) and 2 mM EDTA (Fisher Scientific, USA) solution with the addition of RNase A (New England BioLabs, USA), and the high molecular weight DNA was precipitated using 120 µl 5M NaCl (Fisher Scientific, USA) (Miller et al., 1988). Prior to sequencing, we assessed the DNA concentration (620 ng) using a NanoDrop ND-1000 spectrometer (v3.8.1, Thermo Scientific, USA) and fragment size (18.2 kb) in a 0.7% agarose gel (Fisher Scientific, USA). A PCR-free shotgun strategy was used to prepare the genomic library with insert sizes ranging from 200 to 500 bp. Raw sequence data from paired-end libraries with read lengths of 2x250 were generated by an Illumina HiSeq 2500 sequencer at the University of Illinois at Urbana-Champaign.\n\nPrior to the assembly, the quality of raw read data from sequencing was assessed using FastQC v.0.11.8 (Andrews, 2010) and preprocessed to remove adapter sequences, base-calling duplicates, as well as the elimination of low-quality reads (Phred Score < 30) using the Trimmomatic tool v.0.39 (Bolger et al., 2014). Based on assembly statistics, an optimal setup was found using ~40 million reads randomly selected which corresponds to a coverage close to 60X.\n\nGenome size and heterozygosity rate estimations were made using a k-mer frequency distribution approach (Li & Waterman, 2003). Initially, the occurrence of unique 21-mers (where k=21) was counted and a distribution histogram generated using Jellyfish software v.2.0 (Marçais & Kingsford, 2011). The results were plotted using the Genome Scope tool v.0.1 (Vurture et al., 2017) with recommended parameters.\n\nThe genome assembly was constructed using SPAdes v. 3.11.1 (Bankevich et al., 2012). Briefly, the assembly process starts with the selection of multiple k-mer sizes (k = 21,33,55,77,99, and 127), followed by the construction of individual De Bruijn graphs for each size of k. Afterward, graphs were combined to calculate the distances between the k-mers and to map the edges of the final assembly graph. A set of contiguous DNA sequences, defined as contigs, were generated as output from SPAdes. Scaffolds were produced by combining contigs using read pair information and gap filling.\n\nFollowing the genome assembly, we used an established MAKER v.2 pipeline (Holt & Yandell, 2011) to predict gene structure within the D. saccharalis scaffolds longer than 50 kb. First, repeat sequences were masked using RepeatMasker v.4.1.0 (Smit et al., 2013–2015), based on known repeat elements in RepBase (Bao et al., 2015). A total of 22,510 protein sequences from the lepidopteran silkmoth B. mori reference assembly (Duan et al., 2010) were used for the homology-based prediction approach. A Python script (Santos, 2020) was made to filter out gene models having low-similarity scores (Aligned identity < 50%) when compared to the reference protein.\n\nThe predicted protein dataset was imported into Blast2GO Basic software v.5.2.5 (Götz et al., 2008) for functional annotation analysis. Blast alignments were made using the NR database (NCBI, 1988) with Taxonomy Filter parameter set to “moths” and E-value threshold ≥ 1.0e-03. GO annotations were made in accordance with the Blast2GO protocols.\n\nTo access the fraction of the genomic sequences from D. saccharalis that corresponded to repetitive elements we first identified repeated families using RepeatModeler v.1.0.11 (Smit & Hubley, 2010) with the parameter “-engine ncbi” set. The custom library generated in this step was then used to mask the genome on RepeatMasker v.4.1.0 (Smit et al., 2013) with default parameters. The same procedures were also applied to the B. mori reference genome (Kawamoto et al., 2019) to compare the proportion of repeat content between these two species and verify the accuracy of this approach.\n\nTo assess the accuracy of the genomic sequences, we mapped the total set of Illumina raw paired-end reads to the assembled scaffolds using the BBMap software v.38.36 (Bushnell, 2014) to analyze the accordance rate between the primary dataset and the final assembly. Subsequently, we searched for the presence and completeness of highly conserved single copy orthologs from the “Insecta” database using BUSCO v.3.0 (Simão et al., 2015). The results were then compared to B. mori reference assembly results.\n\n\nResults\n\nThe genome of D. saccharalis was sequenced using the Illumina HiSeq 2500 system with paired-end 2x250 nt reads. We obtained over 306 Megabase pairs (Mb) of paired-end sequence data, representing 212X average genome coverage.\n\nThe haploid genome size estimate was made using the abundance of unique k-mers (k = 21), which corresponds to the second peak, highlighted by the dotted line in the plot (Figure 1). GenomeScope estimated a haploid genome size of 359 Mb. Additionally, this analysis revealed 0.69% of variation across the genomic sequences, indicating that the D. saccharalis genome of the selected individual had low heterozygosity properties, most likely due to the inbred nature of the colony used in this experiment.\n\nThe first peak corresponds to the heterozygous and the second, the homozygous peak. Estimate of the heterozygous portion is 0.69%. Values evidenced in the subtitle correspond to the inferred total genome length (len), genome unique length percent (uniq), overall heterozygosity rate (het), mean k-mer coverage for heterozygous bases (kcov), read error rate (err), average rate of read duplications (dup) and k-mer size (k).\n\nAn optimal coverage size of input data was defined as 60X, corresponding to 46 million randomly selected reads obtained by a Python script (Santos, 2020). The assembly of the reduced data set using SPAdes resulted in 50,460 scaffolds larger than 1 kb having a cumulative length of 453 Mb, and a scaffold N50, an indication of assembly contiguity, of 16.3 kb. Additionally, the genomic GC content corresponds to 33.75% of the bases. Summary statistics of the assembly are described in Table 1.\n\nThe alignment of raw input short-read data resulted in a total of 96.40% of the reads mapped to the assembly, of which 89.64% were properly paired to the scaffolds. To access the completeness of our D. saccharalis genome assembly, we used BUSCO (Simão et al., 2015) and a set of 1,658 conserved, single-copy Insecta genes. We found that our assembly was highly complete, with 87.1% (1,445 out of 1,658) of these BUSCO genes being present in the genomic sequences (86.5% single copy, 0.6% duplicated genes). The B. mori reference genome contains 98.5% complete BUSCO genes (Figure 2).\n\nBars show the proportions of conserved single-copy genes found in each assembly as a proportion of the total gene set (n).\n\nOur analysis revealed that approximately 186 Mb of the D. saccharalis genome were identified to be various nucleotide repeat elements of which represent over 41% of the assembled sequences (Table 2). Although the majority of these elements were unclassified (35.44%), the repeats are composed by 14.8 Mb of long interspersed nuclear elements (LINEs), 4.5 Mb of simple repeats, 3.9 Mb of DNA transposons, as well as retrotransposons and low complexity elements. The consistency of our findings to the sugarcane borer assembly was assessed by analyzing the repeat content present in B. mori chromosomes through the same pipeline. B. mori assembly has shown a similar pattern of high repeat genomic content, having 46.32% of its genome composed of repeated elements. Moreover, the number of unclassified elements also compose a significant part of the repeats (112.6 Mb), followed by LINEs (70 Mb), DNA transposons (11.2 Mb), retrotransposons (4.8 Mb) and simple repeats (6.1 Mb).\n\n1LINE, long interspersed nuclear element; LTR, long terminal repeat retrotransposon.\n\nA total of 548 D. saccharalis genomic sequences longer than 50 kb were used in the MAKER homology-based gene prediction pipeline along with a library of repeated elements generated using RepeatModeler v.1.0.11 and the B. mori protein dataset as reference for building the models. The analysis resulted in a total of 1,394 predicted gene models among the D. saccharalis scaffolds, of which contain a certain level of similarity to a given B. mori protein sequence (Additional table S1, extended data (Santos, 2020)). Subsequently, we selected 1,161 gene models presenting high similarity to its respective reference protein (Identity ≥ 50%). In total, 1,094 proteins are related to the selected gene models and a small fraction of these proteins (4.6%) are the product of multiple gene models. The functional annotation pipeline was further applied using the 1,094 protein sequences, which were further assigned into different categories of their respective Gene Ontology (GO) terms using the Blast2GO software (Götz et al., 2008) (Additional table S2, extended data (Santos, 2020)). The NCBI “moth” protein database identified 1,002 proteins (91.5%) with information, and 826 (75.5%) having a gene ontology assignment. The most abundant GO categories of these proteins are shown in Figure 3.\n\nThe results are categorized in three main categories: cellular component (orange), molecular function (green), and biological process (purple). The y-axis indicates the number of hits identified for each category.\n\n\nDiscussion\n\nThe rise of low cost third generation sequencing technologies has been motivating the development of innovative genomic studies involving non-model species (Ellegren, 2014). Consequently, we notice an emergent increase in the demand for genome assemblies of more organisms. Although initial “draft” assemblies might not be contiguous enough to elucidate more complex events such as whole genome duplications, these projects have been demonstrated to be essential in the investigation of important genomic characteristics of poorly studied organisms, such as repetitive regions, genome size estimation, occurrence of variation, synteny with other species, gene size evolution and protein sequence evolution (da Fonseca et al., 2016; Fournier et al., 2017; Liyanage et al., 2019).\n\nThe key objective of this study was to provide the first genome representation of the sugarcane borer, D. saccharalis, using a high throughput sequencing approach to produce de novo genomic sequences. Our analyses indicate that the species has an estimated genome size of 359 Mb and produced a total length of 453 Mb of assembled genomic sequences representing the D. saccharalis genome. The genomic GC content was 33.74%, which is in the range of other lepidopteran species such as Ostrinia scapulalis (37.4%) (Gschloessl et al., 2018a), S. frugiperda (32.9%) (Kakumani et al., 2014), and B. mori (38.8%) (Kawamoto et al., 2019).\n\nRepetitive element estimates from the de novo assembled sequences revealed that the D. saccharalis genome is over 40% composed of repetitive sequences. This observation, in addition to the discrepancy between the estimated genome size and the total assembly length, suggests that the assembly of repetitive-rich regions is more difficult when using short-reads, leading to the formation of an accurate but more fragmented assembly with fairly small contigs (Peona et al., 2018; Treangen & Salzberg, 2012).\n\nThe consistency of our pipeline was confirmed by analyzing the repetitiveness present in B. mori chromosomes. The percentage of repetitive elements found in this species (46.32%) was significantly similar to the findings from Kawamoto et al., 2019 (46.45%). Additionally, the high proportion of repetitiveness also appears in other lepidopteran members such as H. vespertilio (50.3%) (Pippel et al., 2020), T. pityocampa (45.3%) (Gschloessl et al., 2018b) and S. frugiperda (29%) (Gouin et al., 2017).\n\nIn terms of completeness of genic regions, our D. saccharalis assembly showed the presence of a significant number of conserved insect single-copy genes. Out of 1,658 Insecta BUSCO gene orthologs, only 3.9% were missing in the assembly. When compared to a well-studied species, B. mori, which has its genome assembled at a chromosome level, the number of missing genes is 1%, suggesting that some of these genes are likely absent from the genomes of some lepidopteran species.\n\nGenetic similarities between the two species were also evidenced through functional annotation of predicted genes using a homology-based approach. The analyses identified over 1,000 gene models on D. saccharalis genome using protein information from B. mori. Further gene ontology annotation revealed that proteins with high identity levels between the two species are related to important cellular components such as the nucleus, plasma membranes and cytoplasm, as well as being involved in molecular mechanisms essential for cell maintenance such as the transcription process, protein synthesis and metabolic pathways. However, future additional transcriptome evidence would improve the annotation and validate these gene models, providing more solid information about the genes present in the D. saccharalis genome.\n\nTo this end, the combination of a de novo assembly algorithm and several predictive models has successfully identified important features of the D. saccharalis genome for the first time, and generated sequence data that can be immediately used at several levels: the discovery of additional genomic features, the identification of SNP markers (Boutet et al., 2016) and improvements on the genome annotation by the combination with transcriptomic data (Holt & Yandell, 2011). Furthermore, this will provide valuable resource for the investigation of potential novel techniques to control this pest on crops (Kirk et al., 2013).\n\n\nData availability\n\nWhole-genome sequencing project of Diatraea saccharalis. BioProject, Accession number: PRJNA647758. https://www.ebi.ac.uk/ena/browser/view/PRJNA647758.\n\nRaw gDNA Illumina reads of Diatraea saccharalis. Biosample, Accession number: SAMN15594284. https://identifiers.org/biosample:SAMN15594284\n\nThe complete draft genome assembly of Diatraea saccharalis. GenBank, Accession number: JACGTY000000000. https://www.ncbi.nlm.nih.gov/nuccore/JACGTY000000000.\n\nZenodo: bslucas98/Dsaccharalis_genomeassembly: A first draft genome of the sugarcane borer, Diatraea saccharalis https://doi.org/10.5281/zenodo.4067084 (Santos, 2020).\n\nThis project contains the following extended data:\n\n• Select_subset_reads.py (script used to select a subset of raw reads)\n\n• SupplementaryTables_DsacGenome.xlsx (Additional Table 1 and 2)\n\n• protein_identity.py (Script used to select protein gene models having >=50% similarity to the B. mori model)\n\nThis data is licensed under the terms of the Creative Commons Attribution 0 1.0 Universal (CC0).", "appendix": "References\n\nAhola V, Lehtonen R, Somervuo P, et al.: The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera. Nat Commun. 2014; 5(1): 4737–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrews S: FASTQC. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "78017", "date": "28 Jan 2021", "name": "Jay Ghurye", "expertise": [ "Reviewer Expertise Genome assembly", "scaffolding", "metagenomics" ], "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 enjoyed the manuscript describing the assembly of the first ever D. sachharalis genome. Overall, the text is easy to follow.\n\nHere are the suggestions:\n\nPlease specify exact parameters and commands for all the tools used in the manuscript. That would help with the reproducibility of the results.\n\nThe description of de novo genome assembly is quite convoluted. Just saying the genome was assembled and scaffolded using SPAdes assembler would be sufficient.\n\nGenome assembly evaluation can be made more thorough. Please consider reporting metrics reported by paired end reads based evaluation tools such as ALE (https://academic.oup.com/bioinformatics/article/29/4/435/1992221).\n\nPlease report the base level accuracy of the genome assembly (Assembly QV). That would help evaluate the how good assembly is at a single base pair resolution.\n\nCan you use closely related species of D. sachharalis to scaffold your draft assembly to chromosome level? There are tools to do that if you can use such a genome.\n\nIn table 1, the total scaffold length is much smaller than the assembly size. Why is that the case?\n\nIn table 1, please consider formatting the number with commas.\n\nIn both the tables, please make numbers right formatted.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "85607", "date": "25 May 2021", "name": "Steven M Van Belleghem", "expertise": [ "Reviewer Expertise Population genomics" ], "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\nLucas Borgos dos Santos and colleagues describe the first draft genome assembly of the sugarcane borer D. sachharalis, a common pest species throughout the Americas. They used high-coverage short-read sequencing (Illumina 2x250 bp) to assemble a ~453 Mb genome fragmented over 50,460 scaffolds with an N50 of 16.3 kb and 87% completeness based on BUSCO analysis.\nI believe the manuscript is well-written and provides a helpful guide into understanding this genomics resource, its quality and its usability.\nThese are some remarks I hope can improve the manuscript:\nAbstract:\nResults: Please provide estimated completeness (in %) based on BUSCO analysis. N50 to get a sense of the fragmentation would also be interesting here.\n\nConclusion: “Functional annotation reveals that predicted gene models are involved in important cellular mechanisms such as metabolic pathways and protein synthesis.” I personally see very little point in reporting functional annotation results if it is not for making comparisons. Metabolic pathways and protein synthesis are common to all organisms.\n\nIntroduction:\nSuggested edit: \"… that is the appearance OF A series of holes across the leaves that are still rolled up…\".\n\nResults:\n“We obtained over 306 Megabase pairs (Mb)” I assume you mean 306 million paired reads, for a total of 2x250x306,000,000 = 153 Gb?\n\n“…this analysis revealed 0.69% of variation across the genomic sequences…”. Please explain by giving comparative examples to demonstrate that this is on the low side.\n\nPlease comment on why the cumulative length of the assembly (453 Mb) is longer than the genome size estimation (359 Mb). Could it also include haplotypes that are split up as two scaffolds due to regions of high heterozygosity? Using ‘haplomerger’ could improve this.\n\n“The alignment of raw input short-read data resulted in a total of 96.40% of the reads mapped to the assembly, of which 89.64% were properly paired to the scaffolds.” Please comment on what this means about the assembly. Does this mean scaffolds are 89.64% accurate?\n\nBUSCO genes: How many were completely identified and how many were identified as fragments?\n\nTable 1: Please use commas to separate 1,000 everywhere.\n\n“…of which contain a certain level of similarity to a given B. mori protein sequence…” Please specify “certain level”.\n\n1,394 predicted gene models seem rather low. Is this due to the fragmentation of the genome? The stringency levels?\n\nDiscussion:\n“The consistency of our pipeline was confirmed by analyzing the repetitiveness present in B. mori chromosomes” It was unclear to me that you performed this analysis yourself.\n\n“Further gene ontology annotation revealed that proteins with high identity levels between the two species are related to important cellular components such as the nucleus, plasma membranes and cytoplasm, as well as being involved in molecular mechanisms essential for cell maintenance such as the transcription process, protein synthesis and metabolic pathways.” I am not convinced this analysis provides a biologically meaningful result, given the low number of genes identified and there not being an alternative dataset to compare the GOs to. I think this type of analysis makes more sense in RNA-seq and differential gene expression studies.\n\nI find the discussion slightly repetitive with the results and would suggest merging the results and discussion and having a short conclusion paragraph.\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/9-1269
https://f1000research.com/articles/9-1267/v1	22 Oct 20	{ "type": "Data Note", "title": "Dataset: percent of population covered by local government mask orders in the US", "authors": [ "Philip Jacobs", "Arvi Ohinmaa", "Arvi Ohinmaa" ], "abstract": "We present a dataset covering the extent of local mask orders between April and August 2020, in states which did not have statewide orders (and hence 100% coverage). We obtained data from national and regional newspaper and broadcaster web-based articles, and city and county web pages. The information that we abstracted included: city or county of ordinance, date that the ordinance took effect, and the population of the city or county. In 14 states, city or county governments issued mask-wearing orders, and from our dataset it can been seen that the median population covered in the states was 37.5%; the coverage ranged from 1.6% (New Hampshire) to 77.1% (Arizona). The dataset can be accessed from: https://doi.org/10.7939/DVN/A9C1UU", "keywords": [ "COVID-19", "Mask orders", "Local government", "Cities", "Counties" ], "content": "Introduction\n\nBy August 9, 2020, governors in 35 US states had issued statewide mandates for persons to wear COVID-19 protective face masks1. These mandates ensured that the entire state populations were covered, although population adherences to the mandates were not complete1. In 15 of the remaining 16 states, until September 9, some local governments (county or municipal) had also issued mandates for persons to wear masks. Mask wearing in public has become a bulwark against COVID-19, and it is desirable to determine the population proportions in the states that are only covered by local ordinances. We present a dataset that provides this information.\n\n\nMethods\n\nStarting with the 14 states with only local mandates (see Figure 1), we searched for lists of counties and towns or cities that introduced mask orders effective September 9 or earlier. We conducted a Google search for each state with the combined terms “State name” (e.g., Florida), “COVID-19”, “mask” or “face mask”, and “county order” or “city order,” Usually, we found at least one article with a mention of counties or cities with face masks. In some states the list was large; in those states we searched for internet articles with complete lists; we found lists for Arizonaa, Floridab, South Carolinac, Tennesseed, and Wisconsine. We then searched Google for newspaper or broadcast sites that covered the mask order for each county or city using “State name” (e.g., South Carolina), “city name” (e.g.,” Columbia”), or “county name” (e.g., “Richland County”), “COVID-19”, and “mask order”. We also searched for the ordinances on the city, town, or county government web pages.\n\nThe number of mandates ordered by separate city or county governments in each of 14 states which did not have a central mandate. Blue indicates cities ordering a mandate, brown indicates counties and black indicates a tribal council.\n\nWe collected the following data: the state and city or county name; the date each order became effective; the 2019 population of each relevant city or town2 and county3. We used these populations as estimates of the number of people in each area who were under mask orders. If both city or town and county had issued mask orders, we used only the county population as our measure of the number of people covered.\n\n\nDataset description\n\nIn the accompanying Excel file (Underlying data), in the column ‘NAME’ we list the counties, cities and towns with mask orders. We identify cities or towns with an “M” (municipality) and counties with a “C.” We also show the date that each order came into effect. We embed the internet address of the related newspaper or broadcast article in the “Date in effect” column. We also record the population of each city, county and state. If both a city and its county had a mask order, we used the county population as our indicator of coverage. We recorded the city population in a separate column.\n\nData on the number of ordinances for cities and counties in each relevant state is shown in Figure 2. Counties took the initiative in Wyoming, Nebraska, Utah, and Tennessee. In states with more orders, including Arizona, South Carolina and Florida, cities took the initiative. In Arizona there was also an order from a Tribal Council. In Figure 1, we show the country map, identifying states with statewide orders (blue), local government only orders (brown), and no orders (light red). In this figure, we also present the percent of each state’s population that was covered by mask orders. For the states with statewide orders, coverage is complete (100%). One state, North Dakota, did not have any orders up until our cutoff date of September 9 (in South Dakota, one city, Brookings, enacted an order on September 9). In Figure 3, we show the ranking of states by percent population covered, for states with only local mandates.\n\nThe figure shows the percent of each state’s population that was under a local or statewide mask order by September 9, 2020.\n\nThe average coverage for all 14 states, which have only local coverage, is 37.5%.\n\n\nSummary\n\nOur dataset shows the population coverage for mask mandates in states where local governments took policy initiatives. Coverage in these states varied widely and is an important component of any analysis of COVID-19 prevention policies.\n\nThere is little nationwide information available on the degree of coverage in states with local mandates. There is no central body that collects and organizes this data and makes it publicly available. This dataset addresses that deficiency. However, there are limitations in collecting this information. Firstly, mask order enactment dates keep changing and local governments keep adding or terminating enactments as the local COVID-19 situation changes. Secondly, news bureaus do not always provide the current situation. Finally, data on county and city orders are not always kept in a central place for public information.\n\n\nData availability\n\nUniversity of Alberta Library Dataverse: Mask Orders: Local Government, https://doi.org/10.7939/DVN/A9C1UU4.\n\nDatabase contains detailed collected data for 15 states with local orders and more general data for 34 states with statewide orders:\n\nPart 1. Detailed data\n\nA. State\n\nB. Location\n\nC. Location’s designation: Municipality or County\n\nD. Date order became in effect + source data (embedded)\n\nE. Population that is contributed to the state population measure\n\nF. Actual population (some double counting)\n\nG. Blank\n\nPart 2 State-level data\n\nH. State\n\nI. State population\n\nJ. Population under mask orders in state\n\nK. Per cent of population under mask orders in state\n\nL. Number of municipalities with orders\n\nM. Number of counties with orders.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nNotes\n\na Source: https://floridapolitics.com/archives/342364-beyond-the-veil-what-face-mask-requirements-are-in-place-in-florida\n\nb Source: https://www.tennessean.com/story/news/2020/07/06/does-your-tennessee-county-require-face-masks-worn-public/5387850002/\n\nc Sources: https://ktar.com/story/3298944/heres-where-arizona-cities-stand-on-requiring-face-masks/; https://www.fox6now.com/news/list-wisconsin-cities-with-mask-mandates\n\nd Source: https://www.thestate.com/news/coronavirus/article244628692.html\n\ne Source: https://www.wistv.com/2020/07/08/full-list-face-mask-ordinances-place-across-sc/", "appendix": "References\n\nJacobs P, Ohinmaa AP: The enforcement of statewide mask wearing mandates to prevent COVID-19 in the US: an overview [version 1; peer review: awaiting peer review]. F1000Res. 2020; 9: 1100. Publisher Full Text\n\nUnited States Census Bureau: City and Town Population Totals: 2010-2019. Reference Source\n\nUnited States Census Bureau: County Population Totals: 2010-2019. Reference Source\n\nJacobs P: Mask Orders: Local Government. UAL Dataverse, V1, UNF: 6:EcojjgiWVb/t8xWj7xVF7A== [fileUNF]. 2020. http://www.doi.org/10.7939/DVN/A9C1UU" }	[ { "id": "73597", "date": "04 Jan 2021", "name": "Mayvis Rebeira", "expertise": [ "Reviewer Expertise Health Economics", "Econometrics", "Disease Risk Factor", "Health Policy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 dataset provides information on the proportion of the population covered by mask mandates in states where local governments had taken policy initiatives. The period of the data collection is between April and August 2020. Data was obtained from national and regional newspaper and broadcaster web-based articles, and city and county web pages.\nThe data includes two parts: Part 1 contains municipal/county data and date the mask order was in effect and the population. The source data does not seem easily accessible here. Part 2 contains data on states where there was state-wide enactment of masks and the number of counties/municipality with local ordinances. Based on these data, the authors found that in 14 states, city or county governments issued mask-wearing orders, and from the dataset, the authors conclude that the median population covered by mask enactments in the states was 37.5% and the coverage ranged widely from 1.6% in New Hampshire to 77.1% in Arizona. The method used to derive the dataset from public sources is explained including the search terms.\nThe authors noted several important limitations to the dataset including the fact that mask order enactment dates keep changing and local governments keep adding or terminating enactments. Further, the authors rely on publicly available sources for the information as not all news bureaus may have provided such information. As such there may be data that is missing and further validation and update of the dataset may need to be conducted at a later stage when used to analyze COVID-19 policies. Since the data is publicly available, the verification of data can be conducted and further additional data can be added as more publicly information on mask ordinances become available publicly.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "77749", "date": "08 Feb 2021", "name": "Jonathan Harris", "expertise": [ "Reviewer Expertise County government and related areas" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, the authors created a new, useful dataset on mask mandates across the country and the share of the population these mandates cover in each state. Since no centralized database is available for these policies, the researchers used sound methodology to create the dataset through targeted Google searched.\nThe authors covered the primary limitations of their methodology, including (1) the difficulty it will be to keep this dataset updated with ever-shifting policies, (2) having to rely on news outlets for information and (3) the lack of a centralized database for this information. On the other hand, this dataset will be easy to check, since it is all publicly available.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1267
https://f1000research.com/articles/9-1266/v1	22 Oct 20	{ "type": "Method Article", "title": "The evaluation of feeding, mortality and oviposition of poultry red mite (Dermanyssus gallinae) on aging hens using a high welfare on-hen feeding device", "authors": [ "F. G. Nunn", "K. Bartley", "Javier Palarea-Albaladejo", "Alasdair J. Nisbet", "K. Bartley", "Javier Palarea-Albaladejo", "Alasdair J. Nisbet" ], "abstract": "A study was performed to examine any effect of hen age on the feeding ability and mortality of different life-stages of Dermanyssus gallinae [Poultry Red Mite (PRM)] when fed using a high welfare, on-hen mite feeding device. Mite feeding assays were carried out every two weeks on a cohort of five Lohman Brown hens with devices containing adult and deutonymph PRM or adult and protonymph PRM. Feeding rates and mortality of each PRM life stage and oviposition of adult female PRM were evaluated over an 18-week period. There was a significant reduction in oviposition rates of female PRM as they fed on hens of increasing age. However, no clear trend was detected between the feeding rates of all three haematophagous life stages and hen age. The same conclusion was reached regarding mite mortality post-feeding in both deutonymph and adult female PRMs, although a weak positive association was apparent between hen age and protonymph PRM mortality. This study shows that the on-hen feeding device can be used both for short term studies to assess novel anti-PRM products (new acaricides, vaccines etc.) and longer, longitudinal studies to determine longevity of the effects of such novel anti-PRM products. It also demonstrates that blood feeding by mites on older hens is less able to sustain PRM populations than feeding on younger hens. This on-hen mite feeding device directly impacts upon reduction and refinement by greatly reducing the numbers of birds required per experimental group compared to traditional PRM challenge infestation models and by eliminating the need for birds to be exposed to large numbers of mites for extended periods of time that can cause welfare concerns. This paper describes the methodology for these studies and how to assemble pouches and handle mites both before and after feeding assays.", "keywords": [ "poultry red mite", "feeding device", "high welfare" ], "content": "\n\nEnables small-scale longitudinal trials to test novel mite control compounds and interventions.\n\nThe on-hen feeding device can be used on hens of any age up to 36 weeks without affecting feeding rates or baseline mortality of all blood feeding life stages of Dermanyssus gallinae.\n\nAllows for more reliable testing of the efficacy of novel mite control compounds.\n\nReduction: Only requires small experimental groups of hens. This greatly reduces the number of field trials needed, which typically require several hundred hens per group in randomised block design trials.\n\nRefinement: In vitro testing demands invasive sampling of hens and unreliable in vitro tests require high numbers of replicates, requiring even more invasive sampling. The on-hen feeding device requires no invasive sampling.\n\nRefinement: The use of these devices allows the birds to remain free from the parasites for the vast majority of the experimental period, with parasites only accessing the birds for short (three hour) periods every 2-3 weeks instead of the continual exposure encountered in field trials.\n\nHens can be housed at a low stocking density in high quality and environmentally-enriched floor pens, which is supportive of natural behaviours, whereas field trials necessitate the usage of commercial style cage systems where hens are kept at high stocking density in minimally enriched environments.\n\nMaterials are not specialised or expensive and all are readily commercially available.\n\nEnables small scale, robust testing of novel control compounds before the expense and complexity of a field trial.\n\nDoes not require housing for large numbers of hens.\n\nMite feeding assays are easily carried out within a working day.\n\nFor use in testing of vaccines for efficacy in short and in longitudinal studies against poultry red mite.\n\nFor use in testing novel systemic acaricides, feed additives or water additives for efficacy in short and in longitudinal studies against poultry red mite.\n\nUse in other blood feeding parasite-host models.\n\nCould be employed in disease transmission studies.\n\n\nIntroduction\n\nPoultry red mite (Dermanyssus gallinae De Geer, 1778) costs the hen egg-laying industry in excess of €231 million per year in the European Union (Flochlay et al., 2017) and is a major welfare problem in the egg laying industry where the birds are kept in situ for long periods of time (~1 year) allowing them to form large populations in the hens’ accommodation. This blood-feeding ectoparasite has a five-stage life cycle (Figure 1) and the protonymph, deutonymph and adult stages are all haematophagous, feeding on the hens at night time. Even moderate infestations of mites (approximately 50,000 parasites per hen), have a negative impact on hen welfare, leading to behaviours such as restlessness at night, feather pecking and cannibalism. Severe infestations (500,000 mites per hen) can induce substantial welfare issues including anaemia, death and losses in production (Sparagano et al., 2014; Van Emous, 2005). Not only do the infestations have direct effects on hen welfare and production, D. gallinae is also recognised as a vector for several key avian and zoonotic diseases. These include Salmonella Enteritidis (Sparagano et al., 2014) and avian influenza virus (Sommer et al., 2016) and PRM has been demonstrated to act as a reservoir for fowl typhoid between sequential hen flocks (Pugliese et al., 2019).\n\nEggs hatch into six-legged larvae which moult into octopod protonymphs. Deutonymphs are markedly smaller than the adult females. Both protonymphs and deutonymphs must feed in order to moult to the next stage. In optimal conditions, it can take just seven days to complete its reproductive cycle. Image K. Bartley, used with permission.\n\nMany of the current chemotherapeutic control methods are ineffective, leading to unsuppressed infestations with the associated severe welfare issues and commercial losses. This had led to a recent surge in scientific research activity for novel control and treatment methods (Flochlay et al., 2017; Sparagano et al., 2014).\n\nTesting potential new mite treatments generally uses a small numbers of mites in in vitro efficacy assays. Positive results are followed by field testing, which by necessity involves large numbers of parasites and hens in order to account for the many variables involved in infestation of hens within a henhouse (e.g. Bartley et al., 2015; Bartley et al., 2017; Thomas et al., 2018; Wright et al., 2016). The in vitro method of testing novel systemic control compounds (e.g. novel vaccines, systemic acaricides in feed and water), while convenient, has in the past suffered from a high background mite mortality, device failure and variable feeding rates (Bartley et al., 2015; Wright et al., 2016). These shortcomings mean that a high replicate number is required, thus leading to increased invasive sampling of hens. Bartley et al. (2017) demonstrated that results obtained using the in vitro device for testing vaccine efficacy do not always lead to mite population reduction in a field trial situation.\n\nThe experimental design of previous field trials incorporated a large number of hens (n = 768) arranged in a cage system (e.g. Bartley et al., 2017) with substantial replicate blocking of the test and control groups to account for potential spatial variation in mite population growth that can occur due environmental differences across the poultry accommodation (Moro et al., 2009). These trials therefore involve large numbers of birds continually exposed to parasites for prolonged periods. In addition to the impacts of poultry red mites on hen health and welfare described for prolonged mite exposure (Van Emous et al., 2005; Sparagano et al., 2014), a rise in corticosterone and adrenalin levels in continually-infested hens is indicative of moderate to severe levels of stress (Kowalski & Sokół, 2009).\n\nTo address these issues, we have developed a novel on-hen, in vivo feeding device for mites and we previously described the optimisation and development of this methodology for all hematophagous life stages of the parasite (Nunn et al., 2019). This method offers an alternative to the in vitro tests, to allow much more accurate and reliable assessment of vaccines and other mite control methods on small numbers of hens before progressing to field studies. This strategy addresses the \"Reduction\" aspect of the 3Rs principles by greatly reducing the number of hens used, as it would accurately discriminate between effective and poorly-performing vaccines or other control methods (e.g. new pesticides) before they were progressed to field trials. We propose that this on-hen system may also be used to test the effectiveness of mite control methods across prolonged periods on small numbers of hens (<10 per treatment group, as opposed to several hundred per treatment group previously used in field trials (Bartley et al., 2017; George et al., 2010) without continually exposing birds to the parasites, and the ability to use the device in such circumstances is the subject of this current study. This would address a second 3Rs principle - \"Refinement\" as it would allow the birds to remain free from the parasites for the vast majority of the experimental period, with parasites only accessing the birds for short (three hour) periods every 2–3 weeks instead of the continual exposure encountered in field trials.\n\nThere is substantial potential for using such a system in the development of novel interventions for PRM: Specifically, for vaccine testing, there are currently at least four active academic teams worldwide involved in early-stage vaccine development. Once tested in the laboratory, these vaccines will require field testing over prolonged periods to test duration of immunity and correlation with protection. If each group produces three different vaccines and, if the optimised on-hen in vivo feeding device was able to detect poor duration of vaccine efficacy in two of the methods for each group, that would lead to an overall reduction in hen use of ~3,000 hens in field trials. One of these groups has recently started an antigen-identification programme (Makert et al., 2016) and has identified a group of 10 proteins of interest as vaccine antigens through a combined immunoproteomics/in vitro feeding device method. One of the proteins that this group has identified is identical to one on the list of 22 that we previously published (Wright et al., 2016) but the other nine are unique. If each of these proteins were to be used in field trials, this would use ~7000 birds (based on previous field trial design parameters) whereas pre-screening each of the nine candidates with an optimised on-hen in vivo feeding device would use ~100 birds and could therefore substantially reduce the numbers of hens entering subsequent field trials.\n\nBeyond vaccine development, several academic groups worldwide are involved in the development of novel acaricides for PRM, using in-feed and in-water applications as well as sprays. These include the use of essential oils, combinations of essential oils and entomopathogenic fungi and novel botanicals as well as groups investigating novel uses for existing acaricides including macrocyclic lactones, amitraz, carbamates and synthetic pyrethroids. In addition to these University and Research institute-based academic teams, multiple Contract Research Organisations (CROs) are involved in testing new compounds against PRM. Each of these academic and industry applications could exploit the technology being developed in the work described herein to refine and select the most appropriate candidates for field trials before performing large-scale trials with products which may not behave in the same way on-hen as they do in vitro.\n\nThe purpose of the study described here is therefore to evaluate feeding and mortality of all blood-feeding life stages, and oviposition rates of adult female PRM on aging hens to assess the device’s utility for longer-term longitudinal trials.\n\nIt is envisaged that benefits arising from this research will be cross-disciplinary, benefiting researchers working on methods of controlling ectoparasites of livestock and humans as well as those working on vector-borne disease. The successful development of a method for assessing novel interventions for parasites and disease vectors before large-scale field trials are performed will promote the uptake of the 3Rs principles by greatly reducing the number of birds required per study, and is a refinement compared to the traditional infestation model that requires birds to be exposed to large numbers of mites for extended periods of time. It will have the added economic benefit of substantial reduction in the costs of product development.\n\n\nMethods\n\nEvery effort was made to emeliorate harm to the hens used in this study: the staff involved with the feeding assays were already experienced having optimised the device previously (Nunn et al, 2019). Plucking of feathers was kept to a minimum, just enough to allow the pouches to be applied and plucking is not required once the hens reach ~22 weeks of age and adult feathers are present. Bandaging and tape was veterinary grade and had previously been shown to cause no harm and both handlers were practised in its usage. Hens were kept together at all times to prevent stress.\n\nAll experimental procedures described here were approved by the Moredun Research Institute Animal Welfare Ethical Review Body (approval E20/18) and were conducted under the legislation of UK Home Office Project License (reference P46F495BD) in accordance with the Animals (Scientific Procedures) Act of 1986. The manuscript was written in adherence with the ARRIVE guidelines.\n\nFive Lohman brown pullets (18-weeks-old at the start of the experiment), purchased from a local pullet breeding facility, were housed in an enclosed, loose litter floor pen of 2.5 m × 2 m with temperature (18 °C) and lighting (16 h light/8 h dark) controlled to mimic commercial hen laying conditions. Hens were in good condition with no evidence of previous exposure to D. gallinae. Hens had ad libitum access to layer’s pellets and water, perches and nest boxes were provided.\n\nFor provision of parasite material, mixed stage and sex D. gallinae were collected every two weeks into vented 75 cm2 canted tissue culture flasks (Corning, New York, USA) from a commercial egg laying unit. The optimum quantity of collected mites/detritus placed into a tissue culture flask was ~ 2-3 cm deep when the flasks are upright. Less than this quantity and mites desiccated over the subsequent “conditioning period”; more than this and mites were overcrowded and died. Any overcrowded culture flask (becomes wet and dirty inside) had the contents transferred into fresh flask(s) as many times as required during the seven days after collection, when the flasks were stored at room temperature (RT). Adult females and deutonymphs were isolated (see Figure 1) from this ‘mite pool’ for feeding assays following conditioning.\n\nProtonymphs were provided by hatching mite eggs and allowing the emerging larvae to moult, as follows: Mite eggs were harvested by collecting freshly fed mites from the caps of 75 cm2 tissue culture flasks and transferring ~1 cm3 fed mites to individual 30 ml polystyrene Universal tubes (Thermo Scientific, Mass. USA), sealed with a double layer of AeraSeal™ film (Sigma, MO, USA) and incubated overnight at 25°C and 85% relative humidity (RH). The tubes were unsealed and placed onto glass Petri dishes in a containment moat of Flash™ multipurpose cleaning fluid (Procter and Gamble, Harrogate, UK) in a 250 ml plastic weighing boat (Scientific Laboratory Supplies, Nottingham UK). Mite eggs were then able to be picked out of the polystyrene Universal tube using a fine paintbrush and collected in 5 ml polystyrene bijoux tubes (Thermo Scientific, UK), sealed with a double layer of AeraSeal™ film (Sigma) and incubated at 25°C and 85% RH for a three-day period to allow eggs to hatch into hexapod larvae and these larvae to then moult into octopod protonymphs. The protonymphs were stored in the bijoux tubes and stored at 4°C until use. This protonymph hatching protocol made counting and handling of the small protonymphs for subsequent use easier than attempting to pick them from the mites and detritus from ‘mite pool’ field collections.\n\nFor mite conditioning prior to feeding assays, deutonymphs and adults were stored in detritus in vented 75 cm2 canted tissue culture flasks (Corning) at RT for seven days after field collection to allow for complete digestion of the blood meal. All stages of mites were then stored at 4°C for two weeks (deutonymphs) or four weeks (protonymphs and adult females) before being used in feeding assays. These optimal conditioning periods had previously been empirically determined to be necessary for maximum feeding of each stage (Nunn et al., 2019).\n\nAssays were carried out, on the same hens as they aged, at two-week intervals over an 18-week period. At each assay point hens either wore feeding devices containing 50 adult female mites and 50 protonymph mites or 50 adult female mites and 50 deutonymph mites. Hens were randomly-allocated feeding devices (“haphazard allocation”) and assays were carried out on Tuesday and Thursday mornings in each week in which assays were performed. At the end of the feeding period, hens were selected randomly (“haphazard selection”) for removal of the feeding devices. Blinding of hen identification numbers to the mite counter was considered to be unnecessary during experimental procedures and analysis as the single experimental group had no specific treatments to be compared with another group. Preliminary work carried out during the optimisation of the feeding device, had demonstrated that the nymph stages of D. gallinae feed better when in the presence of adult mites (“co-operative” feeding; Table 1).\n\nDevices were left on the hen for three hours in all experiments. Table 1a shows data from two experiments with feeding devices in which no adults were included and Table 1b shows data when adults were included in the feeding devices containing nymphs. These data were taken from studies during optimisation of the on-hen feeding device. As feeding the different lifestages simultaneously is more convenient as well as improving feeding data, this was done in all subsequent trials.\n\nDevices were left on the hen for three hours in all experiments. Table 1a shows data from two experiments with feeding devices in which no adults were included and Table 1b shows data when adults were included in the feeding devices containing nymphs. These data were taken from studies during optimisation of the on-hen feeding device. As feeding the different lifestages simultaneously is more convenient as well as improving feeding data, this was done in all subsequent trials.\n\nPower calculations were based on simulated mite feeding data using a binomial generalised linear mixed model (GLMM) to establish the numbers of hens required to detect a change from expected scenario of 40% to worst case scenario of 15% in mean feeding rate, with baseline values for variability in feeding rate within bird over time (SD = 0.25) and between birds (SD = 0.6) estimated from two pilot previous assays using birds of different ages (Table 2). The estimated time SD was multiplied by two to consider the case of feeding rates doubling in variability over an extended trial. The results indicated that four birds is the minimum to reach the standard 80% statistical power threshold at a 5% significance level under those conditions, however we used five to cover for unexpected deaths in this long study.\n\nThree groups of four hens had the on-hen feeding device in place for three hours. Each device contained around 50 adult female mites and Assay 1 was carried out when hens were aged 18 weeks and Assay 2 when hens were aged 22 weeks. Groups 1 and 2 refer to “Control” groups of hens in vaccine trials.\n\n*Feeding device detached;-no feeding.\n\nTo compare mite mortality and feeding rates over time, GLMMs were fitted by the maximum likelihood method, using the Laplace approximation and a logit link function, to the data including lifestage (protonymph, deutonymph or adult), time (weeks after the start of the experiment) and the interaction between them as fixed effects and hen identity as a random effect. No criteria were set a priori for inclusion/exclusion of animals or data points although one data point was excluded as considered an extreme outlier in mite mortality being 21 times the upper limit of the interquartile range for the protonymph mortality data collected n that week (mortality 57.14%, time death observed 48h, week 10). For mite mortality, time at which death post-feeding was observed was included as an additional random effect. The effect of hen aging on oviposition was formally tested using a Poisson GLMM fitted by maximum likelihood, based on the Laplace approximation and a logarithmic link function, including an offset (in logarithmic scale) to account for the number of fed mites, with time as a fixed effect and hen identity as a random effect. Statistical testing from these model fits was conducted using type-II Wald chi-square statistics. Pairwise tests of differences in mean between lifestages and timepoints were conducted based on the predicted marginal means from the GLMM estimates. The corresponding p-values were adjusted for multiplicity using the Benjamini and Hochberg’s method (Benjamini & Hochberg, 1995). Significance tests were assessed at the usual 5% significance level. All analyses were performed using R Core Team, 2018.\n\nStep 1: Manufacture of feeding devices. Feeding devices were made in advance of each feeding event, as shown in Video 1, described in the text and shown in Figure 2.\n\nThe tape overhangs assist with handling and the blue ‘brace’ acts both as a refuge for mites and in keeping apart the mesh sides.\n\nTo make the pouch (Figure 2), a strip of polyester mesh [aperture width of 105 μm, pore depth 63 μm (Plastok Ltd, Birkenhead, UK)] was cut to 6cm wide by 15 cm long. Using 1.25 cm diameter Leukoplast® tape (BSN Medical, Germany), a seal was made on the long edge to make a tube, ensuring that the Leucoplast tape was adhered to the inside of the mesh tube. A strip of AeraSeal™ (Sigma-Aldrich Co Ltd, Dorset, UK) tape was attached to the outside of the mesh tube, to cover the Leukoplast® tape join. The tube was then cut into 3 cm segments and one of the open ends sealed with c.a. 3 cm strip of 1.25 cm diameter Leukoplast® tape, leaving an overhang at each end of the pouch. To make the plastic insert, the shaft of a blue plastic bacterial spreader loop (Quadloop Sphere, Sterilin) was gently heated in a Bunsen flame and placed onto a hard surface then bent into a U shape. The ends of the loop were trimmed and filed to remove sharp edges. The plastic inserts were then placed in pouches (one per pouch) ready to fill with mites (Figure 2).\n\nTechnical tips:\n\n1. Use fabric shears or a paper guillotine to cut the mesh to prevent the mesh from fraying.\n\n2. Keep all edges straight to minimise the amount of sticky Leukoplast® tape adhesive that the mites come into contact with.\n\n3. Covering the outside of the mesh tube long join with AeraSeal™ tape, covers the Leukoplast glue and makes mite recovery easier after the feeding assay.\n\n4. Making the bottom and top seals longer than the pouch width (overhangs) enables handling of the pouches without squashing mites-it also helps in pouch removal from the hen.\n\nStep 2: Filling the devices with mites. To fill the feeding devices (pouches) with mites, flasks containing conditioned mites were removed from storage at 4°C, and then kept at RT for 20–30 minutes to allow the mites to become mobile. Two centimetres of wet ice were placed into two large (280ml) chemical weigh boats (Fisherbrand). The cap of the flask was unscrewed and tapped out onto a glass Petri dish and the mites allowed to disperse for a few seconds before placing Petri dish on the ice in the weigh boat (Figure 3a). Mites were sorted using a fine paint brush and transferred to the second glass Petri dish on ice until sufficient numbers for one feeding device were obtained. The mites are then gently gathered into a ball using the brush and placed into the feeding device which was then quickly sealed with a piece of Leukoplast® tape (BSN Medical, Germany) (Figure 3b). Filled feeding devices were kept overnight in a loosely sealed plastic container placed in polystyrene box containing wet ice. The plastic container was raised above the wet ice on a metal support, so that it was not in direct contact with the ice. This kept the temperature sufficiently low (around 10°C) and the relative humidity level high, so that the mites did not desiccate.\n\nTechnical tips.\n\n1. When transferring mites from the flask cap to Petri dish, do not use a cold Petri dish or put Petri dish on ice first as the mites will ‘ball up’ and make it very difficult to pick individuals.\n\n2. Work quickly to limit the amount of condensation on Petri dishes - once a dish gets very wet it makes picking mites up more difficult and they won’t be in the best condition going into the feeding device.\n\n3. When transferring the selected mites from the Petri dish to the device, place the device close to the mites on the cold Petri dish and try to transfer them in one movement. Work quickly! The blue plastic brace is a convenient place to quickly wipe the mites off the paint brush.\n\n4. If it is convenient to fill the devices the day before application to the hens, store the devices overnight in a loosely sealed plastic container placed in an insulated box containing wet ice. Raise the container above the wet ice on a support, so that it isn’t in direct contact with the ice. This keeps the temperature sufficiently low (around 10°C) and the RH level high, so that the mites do not desiccate.\n\nStep 3. Application of feeding devices to hens. The pouch application kit is shown in Figure 3c. A video clip demonstrating pouch application can be seen in Video 1.\n\nPrior to the first feeding assay, feathers were plucked from the outer thigh areas of each hen and thereafter whenever needed. Before laying the hen on its side, the pouch was prepared for application by cutting four strips of 1.25cm wide Leukoplast® tape. Two of those strips were cut slightly shorter for the shorter sides of the pouch. Holding the pouch with the central join facing upwards, a strip of tape was placed on each of the four sides of the pouch. The size of the pouch with the tape strips added was not larger than the plucked area of the hen’s leg (Figure 4, panels A and B), thus preventing the tape from causing irritation to the hen while it is attached and stops the tape pulling on feathers when it is removed. The device was further secured with cohesive bandage (Central Medical Supplies Ltd., Pontefract, UK) wrapped over the feeding device which not only secures the device onto the hen but also provides dark conditions to encourage mite feeding. Feeding devices were left in place for three hours at 9am in the morning, to allow for mite processing in the afternoon. During this time hens were allowed to carry out normal foraging and nesting behaviours. Handfuls of mixed corn were scattered in the litter to encourage foraging behaviour while the pouches were attached. Following the completion of feeding (three hours after the application of the device), the devices were removed and fully engorged and partially fed mites were recovered from the devices as described below.\n\nThe stages of pouch application with Panel A showing hen restraint and plucked thigh. Hens are placed gently on their side with the handler using one hand to restrain the hen and the other to carefully straighten the leg being used. Panel B shows how the four strips of Leukoplast® tape are used to loosely adhere the pouch to the skin (described in Step 3) before the final bandaging using the cohesive bandage (Panel C). The cohesive bandage should be firm enough to be secure but not so tight as to cause any discomfort.\n\nTechnical tips\n\n1. Practise bandaging on yourself or a colleague first –too tight will be uncomfortable for the hen, too loose and mites might not be able to attach for feeding.\n\n2. A piece of leucoplast tape can be used to secure the end of the cohesive bandage if necessary.\n\n3. If required, cohesive bandage can be cut in half (i.e. from 5 cm width to 2.5 cm width). This can be done while bandage is still on the roll, using a Stanley knife or scalpel blade.\n\nStep 4. Mite recovery from the feeding device. A video demonstrating how to dissect the pouch can be found in Video 1. The bandage can be unwound and removed completely. Slowly and carefully remove the device from the hen’s skin, peeling it off in the same direction of feather growth as this prevents damage to the hen’s skin.\n\nPouches can then be dissected as shown in Figure 5a and 5b.\n\na) A pouch post feeding assay; b) partially dissected pouch; c) tissue culture plates containing fed mites for monitoring.\n\nWhen taking the feeding device apart, the tape that was used to stick the pouch to the hen was removed first. Then, one of the tape seals was removed by holding one end tab on one side of the device and gently pulling the tab on the opposite side as shown (see Figure 5a and 5b). Once the seal on one side of the device had been peeled off, the blue plastic brace was then removed using forceps. The blue plastic brace and the semi-dismantled device was placed onto a glass Petri dish on ice to prevent mites escaping. Then fed/unfed mites were recovered from it. Mites generally crawled out from the feeding device during this time and were recovered from the surface of the device. The feeding device was then carefully dissected with scalpel and forceps to allow collection of and remaining mites.\n\nFed mites, identified by the presence of internalised hen blood, were counted, any dead mites (judged by curled legs and lack of movement) counted and then placed individually into wells (Figure 5c) of 96-well tissue culture plates (Costar, Corning, NY, USA), sealed with AeraSeal™ tape (Sigma-Aldrich Co Ltd, Dorset, UK) and incubated at 25°C with 85% relative humidity and monitored for mortality and fecundity, 48-hour intervals for six days. Egg counting per fed mite was used as the measure for fecundity.\n\nTechnical tips:\n\n1. If visualisation of the mites under light microscopy is required following feeding, isolating them in a 96-well tissue culture plates (Costar, Corning, NY, USA) allows clear microscopical visualisation of the mites through the underside of the plate. Moulting, oviposition and mortality can be scored using this system.\n\n2. We have found AeraSeal™ to be the best tape to use as mites are less likely to stick to than to other porous tapes.\n\n\nResults\n\nThere were statistically significant overall effects of mite lifestage (when comparing protonymph, deutonymph or adult) and of the interaction between this and time (i.e. hen aging) on the proportions of mites feeding on hens when applied to the hens in the feeding devices (p < 0.0001 in each case) (Nunn et al., 2020a). The differences in predicted feeding rate (proportion of all mites in each feeding device expected to feed successfully) by mite lifestage are shown in Figure 6 (and the raw data supporting this in Table 3).\n\nEach data point represents the predicted feeding rate of mites, aggregated across all timepoints in the trial (± 95% CI) based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint.\n\nFeeding rates of each lifestage of Poultry Red Mites (PRMs) in on-hen feeding devices placed on hens across an 18-week period. Each data point “rate” represents the predicted feeding rate of mites, aggregated across all timepoints in the trial (± 95% CI) based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nFeeding rates averaged over all 10 timepoints across the 18-week period were approximately 1.5 times higher for adult mites than for either deutonymphs or protonymphs (p < 0.001 in both cases) but there were no statistically significant differences between the feeding rates of the two nymphal stages (p = 0.2437). When examined between timepoints in the experiment (i.e. as the hens aged) there was notable variation in feeding rates at different timepoints for deutonymphs and protonymphs (Figure 7, panels B and C with supporting data in Table 4). For example, at week 6 deutonymphs had much higher feeding rates than at every other timepoint (p < 0.0001 in each case), though no overall trend of feeding rates across time was evident (Figure 7B). There was substantially less variation in adult feeding across timepoints (Figure 7A), though feeding rates at week 16, for example, were significantly lower than on all other dates (p < 0.05). Again, no overall trend of feeding rates across time was obvious for adult mites.\n\nPanel A shows data for adult females, Panel B for deutonymphs and Panel C for protonymphs. Each data point represents the predicted feeding rate of mites (± 95% CI), based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint.\n\nFeeding of each lifestage of Poultry Red Mite (PRM) on hens across an 18-week period. Table 4a shows data for adult females, Table 4b for deutonymphs and Table 4c for protonymphs. Each “rate” represents the predicted feeding rate of mites (± 95% CI), based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nFeeding of each lifestage of Poultry Red Mite (PRM) on hens across an 18-week period. Table 4a shows data for adult females, Table 4b for deutonymphs and Table 4c for protonymphs. Each “rate” represents the predicted feeding rate of mites (± 95% CI), based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nFeeding of each lifestage of Poultry Red Mite (PRM) on hens across an 18-week period. Table 4a shows data for adult females, Table 4b for deutonymphs and Table 4c for protonymphs. Each “rate” represents the predicted feeding rate of mites (± 95% CI), based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nThere was no statistically significant overall effect of mite lifestage (when comparing protonymph, deutonymph or adult) on mite mortality post-feeding (p = 0.2907). However, there was a significant effect of the interaction between lifestage and time (i.e. hen aging) (p = 0.0006). This was examined further by investigating post-feeding mortality rates by time point for each lifestage (Figure 8 and Table 5).\n\nMortality of each lifestage of Poultry Red Mite (PRM) which had fed on aging hens across an 18-week period with Panel A showing data for adult females, Panel B for deutonymphs, and Panel C for protonymphs. Each data point represents the predicted mortality rate of mites (± 95% CI), where a value of 1 would represent 100% mortality, based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint\n\nMortality of each lifestage of Poultry Red Mite (PRM) which had fed on aging hens across an 18-week period with Table 5a showing data for adult females, Table 5b for deutonymphs, and Table 5c for protonymphs. Each “rate” represents the predicted mortality rate of mites (± 95% CI), where a value of 1 would represent 100% mortality, based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nMortality of each lifestage of Poultry Red Mite (PRM) which had fed on aging hens across an 18-week period with Table 5a showing data for adult females, Table 5b for deutonymphs, and Table 5c for protonymphs. Each “rate” represents the predicted mortality rate of mites (± 95% CI), where a value of 1 would represent 100% mortality, based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nMortality of each lifestage of Poultry Red Mite (PRM) which had fed on aging hens across an 18-week period with Table 5a showing data for adult females, Table 5b for deutonymphs, and Table 5c for protonymphs. Each “rate” represents the predicted mortality rate of mites (± 95% CI), where a value of 1 would represent 100% mortality, based on recorded percentages of 100 adult mites or 50 protonymph or deutonymph mites which had fed on each of five replicate hens at each timepoint. “LCL” and “UCL” are the lower and upper limits of the 95% confidence interval, respectively.\n\nMortality rates were variable for adults and deutonymphs across the time period of the experiment. For adults (Figure 8A), mortality at week 4 was significantly lower than at weeks 0, 6, 8 and 18 (p < 0.05) and, in addition, mortality rates for adults at week 18 was significantly higher than at weeks 4 and 16 but no clear pattern over time could be discerned across the whole experiment. For deutonymphs (Figure 8B), mortality at week 12 was significantly higher than at weeks 0, 4, 10, 14 and 16 (p < 0.05) and mortality at week 18 was significantly higher than at week 16 (p < 0.05). Again, no discernible trend in mortality over time could be determined for deutonymphs. In contrast, for protonymphs, there appeared to be a weak positive trend in mortality as the hens aged (Figure 8C), with significant increases in mortality of protonymphs between week 0 and weeks 6, 10, 14, 16 and 18 (p < 0.05). Overall, levels of mortality were very low in each lifestage albeit with variability in mortality rates observed for all lifestages as indicated by the wide confidence intervals.\n\nAnalysis of the oviposition of adult mites (total eggs per surviving, fed female mite monitored over the six-day period for each time point) over time demonstrated that there was a statistically significant overall effect of hen age on the capacity of adult mites to lay eggs after feeding on these hens (p < 0.0001; Figure 9 and Table 6), with a trend of oviposition dropping as the hens aged.\n\nData shows the total number of eggs laid per fed, surviving female mite as monitored over a six-day period following each assay week.\n\nOviposition of Poultry Red Mites (PRMs) which had fed on aging hens across an 18-week period (boxplots and actual values of egg counts in logarithmic scale). Data shows the total number of eggs laid per fed, surviving female mite as monitored over a six-day period following each assay week. “Q1” and “Q3” are the first and third quartiles of the data distribution, respectively.\n\nThus, average mite oviposition at week 0 was statistically significantly higher than that at all subsequent weeks (p < 0.0001) and average oviposition at week 18 was significantly lower than that at all previous timepoints (p ˂ 0.0012).\n\n\nDiscussion\n\nThis study demonstrated that, while feeding rates of adult females were significantly higher than those of nymphal stages, there was no evidence of a decreasing trend in feeding rates of any life stage associated with hen age, with only punctual significant deviations. Mite mortality was generally very low using this in vivo feeding device, certainly substantially lower than recorded when using in vitro feeding devices (e.g. 27% in Wright et al., 2009). There were no significant overall differences in fed mite mortality rates between life stages and no significant increased mortality was observed in deutonymph or adult life stages with increased hen age, although a weak positive association was apparent in protonymphs. A significant decrease in egg-laying by fed adult females was demonstrated with increased hen age.\n\nThis effect of hen age on the egg-laying capacity of D. gallinae following feeding on the hens may be responsible for the observed limits in expansion of mite numbers in prolonged field trials (e.g. see Bartley et al., 2017; Mul et al., 2017) but the cause of the observed effect is not clear. The historical dogma is that no effective, protective, natural immune response develops in hens against PRM in natural infestations and the limited exposure (in terms of time and mite numbers) of hens to the parasite in our experiment would almost certainly preclude an effective humoral immune response. The effect may therefore come from a change in the physiology of the hens during that period making feeding less nutritionally rewarding to the mite as the hen ages.\n\nThe feeding behaviour of D. gallinae is poorly understood, but from our previous work it seems that nymph feeding is greatly increased when nymphs and adults are allowed to feed simultaneously (Table 2). Further work is required to definitively assess any co-operation in adult and nymph feeding as well as between nymph stages. Feeding rates across the time course described here were variable for all life stages, although less variable for adults than for either of the nymph stages. This variability may at least in part be attributed to mite quality, which may be inconsistent as mites are collected from field populations separately for each individual feed. This could be addressed by having a continual lab supply of mites from infested hens under controlled conditions (e.g. see Wang et al., 2018) or, preferably, from a continuous in vitro culture system. We have previously described optimisation of mite conditioning (Nunn et al., 2019) for the on-hen feeding assay and this helps in addressing some of the issues around variability in feeding rates. While a certain amount of conditioning is crucial to allow the collected mites time to digest previous blood meals, a degree of flexibility may be required in this conditioning period to maximise feeding rates (Lima-Barbero et al., 2019) and the use of real time controls for comparison is imperative in order to account for background mortality rates.\n\nMite population dynamics in field infestations are not well characterised but differences in ratios of life stages have been noted previously in collections from hen houses (George et al., 2010), and have been observed in collections by our own group. The reasons for this are unclear but may relate to the variable effects of hen aging on the post-feeding survival of different life stages noted here (Figure 3). If required for short term in vivo experiments to determine the effects of novel mite interventions (e.g. novel acaricides) the increase in protonymph mortality and decrease in oviposition associated with hen age, seen in this study, can be avoided in by choosing younger hens for use in mite feeding assays. Equally, for longer term, longitudinal studies, the expected effects of hen aging on the mites should be incorporated into any formal sample size calculations if the anticipated effects of the novel intervention are likely to impact protonymph mortality or adult mite fecundity.\n\nThe results of this study can therefore be used in designing shorter trials to ascertain whether a vaccine or some other systemic acaricide has any effect on fed mites. Short-term trials such as that described in Price et al. (2019) can be relatively easy to carry out with young hens and mites used from the same collection point to give a more consistent feeding rate. Price et al. (2019) demonstrated that although feeding rates were variable for three assays using the same batch of conditioned mites, there was no significant difference in feeding rates between the different treatment groups. For longer trials, designed to look at the longevity of an IgY response to novel vaccine antigens, adequate controls must be in place to assess background mortality and oviposition of the batch of the mites in use.\n\nThe exploitation of the device being developed in this proposal is not limited to the PRM - academic beneficiaries working on all ectoparasites and arthropod disease vectors including fleas, biting and chewing lice, cimicids (e.g. bedbugs) and ticks could easily adapt the device to produce a refinement of their research involving animals which would minimise parasite exposure of the test animals during blood-feeding in experiments where novel interventions (both insecticide/acaricide effects and anti-disease vector effects) are being tested and reduce numbers of animals by weeding out poorly performing interventions prior to field testing.\n\n\nData availability\n\nFigshare: The evaluation of feeding, mortality and oviposition of poultry red mite (Dermanyssus gallinae) on aging hens using a high welfare on-hen feeding device_DATA.xlsx. https://doi.org/10.6084/m9.figshare.12848432.v1 (Nunn et al., 2020a).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank the Bioservices Group at Moredun Research Institute for their ongoing help and expertise.\n\n\nReferences\n\nBartley K, Wright HW, Huntley JF, et al.: Identification and evaluation of vaccine candidate antigens from the poultry red mite (Dermanyssus gallinae). 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PubMed Abstract \| Publisher Full Text\n\nSparagano OAE, George DR, Harrington DWJ, et al.: Significance and control of the poultry red mite, Dermanyssus gallinae. Annu Rev Entomol. 2014; 59: 447–466. PubMed Abstract \| Publisher Full Text\n\nThomas E, Zoller H, Liebisch G, et al.: In vitro activity of fluralaner and commonly used acaricides against Dermanyssus gallinae isolates from Europe and Brazil. Parasit Vectors. 2018; 11(1): 361. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Ma Y, Huang Y, et al.: An efficient rearing system rapidly producing large quantities of poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae), under laboratory conditions. Vet Parasitol. 2018; 258: 38–45. PubMed Abstract \| Publisher Full Text\n\nWright HW, Bartley K, Huntley JF, et al.: Characterisation of tropomyosin and paramyosin as vaccine candidate molecules for the poultry red mite, Dermanyssus gallinae. Parasites and Vectors. 2016; 9(1): 544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWright HW, Bartley K, Nisbet AJ, et al.: The testing of antibodies raised against poultry red mite antigens in an in vitro feeding assay; preliminary screen for vaccine candidates. Exp Appl Acarol. 2009; 48(1–2): 81–91. PubMed Abstract \| Publisher Full Text\n\nVan Emous R: Wage war against the red mite! Poultry Int. 2005; 44: 26–33." }	[ { "id": "73574", "date": "10 Nov 2020", "name": "Cristian Magdas", "expertise": [ "Reviewer Expertise Veterinary Parasitology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very good manuscript which provide the results of a study what brings a new method for feeding of poultry red mite, very useful in testing the effectiveness of new systemic control compounds against this mite, feeding method which could be used also for other arthropods. The manuscript brings also important data regarding the decrease in egg-laying by adult females fed on increased hen age, with the recommendation of younger hens for use in mite feeding assays. The methods are very well described, really appreciated the technical tips. Anyhow, I have some suggestions/questions for the authors:\nMethods-ethical statement-first line – “ameliorate”.\n\nPlease check on the page 5 and replace “bijoux” with “bijou”. I think that the correct version is “bijou containers” and please check if not 7 ml instead of 5 ml? Also, “30 ml polystyrene Universal containers” I think is better, just a suggestion.\n\n1,25 cm isn’t the width of the Leukoplast, but not the diameter?\n\nIn the legends of figure 6, 7 and corresponding tables is mentioned “100 adult mites”. Why recorded percentages refers to 100 adult mites, but not to 50 adult mites?\n\nExperimental design and statistical analysis: in the week in which the assays were performed, the feeding devices were applied 2 times on the hens?\n\nMaybe it would be good if the authors explained why the feeding devices were left fixed for only 3 hours?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "73832", "date": "23 Nov 2020", "name": "Antonio Camarda", "expertise": [ "Reviewer Expertise Dermanyssus gallinae vectorial role", "Acaricidal evaluation", "Dermanyssus gallinae population dynamics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 focuses on the application of an innovative on-hen in vivo technique for feeding mites, improving previous studies about the same topic. As explained by the authors, this device could represent an important alternative for testing new acaricides and vaccines, as well as for studying the vectorial role of Dermanyssus gallinae. The technique is well described and rich in details, in particular the section concerning the device construction. Furthermore, the presence of figures, videos and technical tips, which could be very helpful for both understanding and reproducing the experiment, is remarkable. Moreover, the obtained results are supported by a strong and effective statistical analysis. Anyway, I have few observations for the authors:\nIntroduction: “If each group produces three different vaccines and, if the optimised on-hen in vivo feeding device was able to detect poor duration of vaccine efficacy in two of the methods for each group, that would lead to an overall reduction in hen use of ~3,000 hens in field trials.” The authors should explain how these numbers are estimated.\n\nMethods – Ethical statement: it is not clear the reason why plucking feathers is not necessary in hens older than 22 weeks of age. Please, clarify the concept.\n\nMethods – Parasite material: the paragraph sounds a bit confusing. It could be useful to adopt a short step-by-step scheme. Methods – Parasite material: how did you distinguish sexes and stages of mites?\n\nMethods – Parasite material: taking into account that the life cycle of Dermanyssus gallinae requires 2-3 days for eggs to hatch and 1-2 days for larvae to molt into protonymphs, can a three-day period of incubation be long enough to obtain an adequate number of protonymphs?\n\nMethods - Protocol for each mite feeding event – Step 1: According the other reviewer’s suggestion, 1,25 is the tape’s width and not the diameter.\n\nMethods – Protocol for each mite feeding event – Step 3: is there a specific reason why the device has been left in place for a three-hour period?\n\nMethods – Protocol for each mite feeding event – Step 4: “The feeding device was then carefully dissected with scalpel and forceps to allow collection of and remaining mites”. Did you intend any remaining mites?\n\nDiscussion: “The reasons for this are unclear but may relate to the variable effects of hen aging on the post-feeding survival of different life stages noted here”. Could the differences in ratios of life stages observed in field collected mite populations be related, not only to the effects of hen aging, but also to the adopted mites collection technique? It could be useful to reconsider the paragraph taking into account this concept.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1266
https://f1000research.com/articles/9-789/v1	29 Jul 20	{ "type": "Case Report", "title": "Case Report: An extremely rare occurrence of recurrent inguinal low-grade fibromyxoid sarcoma involving the scrotum", "authors": [ "Samy Chitayat", "Rodrigo Barros", "José Genilson Ribeiro", "Heleno Augusto Moreira Silva", "Flávio Rondinelli Sá", "Bruno de Souza Bianch Reis", "Angelo Maurilio Fosse Junior", "Samy Chitayat", "José Genilson Ribeiro", "Heleno Augusto Moreira Silva", "Flávio Rondinelli Sá", "Bruno de Souza Bianch Reis", "Angelo Maurilio Fosse Junior" ], "abstract": "Low-grade fibromyxoid sarcoma (LGFMS) is a rare sarcoma subtype. The most common tumor locations are the deep soft tissue of extremities or trunks. We report a rare case of recurrent LGFMS in the inguinal region involving the scrotum and both testicles. A 38-year-old male patient reported a history of multiple nodular lesions in the left inguinal region accompanied by local inflammation. The patient was submitted for local resection of the lesion at our institution, with histopathological diagnosis of LGFMS. He missed his follow-up, returning with a large bulge in the left inguinal region involving the scrotum with signs of tissue necrosis and local purulent discharge. Surgical exploration was performed and the patient underwent tumor resection in the left inguinal region and the entire scrotum, with bilateral orchiectomy, with the margins enlarged to the right inguinal region and proximal surface of the penis. Local reconstruction was performed with a left fascia lata tensor muscle flap and ipsilateral thigh coverage using partial skin graft. On microscopic examination, the tumor showed spindle cells arranged in bundles, with abundant collagen and myxoid stroma with interspersed prominent vessels. The immunohistochemical study carried out showed immunoreactivity with Ki67 (<5%), immunonegativity with desmin and S100, confirming the diagnosis of LGFMS. Postoperative recovery was good and no recurrence was seen after two years. The patient is in good health, realizing multidisciplinary outpatient follow-up and performing continuous testosterone replacement. Surgical resection with negative margins for localized disease remains the standard treatment for LGFMS.", "keywords": [ "Low-grade fibromyxoid sarcoma", "sarcoma", "scrotum sarcoma" ], "content": "Introduction\n\nLow-grade fibromyxoid sarcoma (LGFMS) is a rare sarcoma subtype, first described by Evans in 19871. The most common tumor locations are the deep soft tissue of extremities or trunks2. The etiology still unknown and the incidence is 0.18 per million, representing 0.6% of all soft tissue sarcomas3.\n\nMicroscopy reveals bland spindle cell tumors with angulated nuclei, scant cytoplasm arranged in a whorled pattern with cells that are frequently immunoreactive to mucin 42. Despite its deceptively indolent clinical behavior and benign histological appearance, LGFMS has a high tendency for local recurrence and late distant metastasis4. The current treatment includes surgical excision with clear margins for localized disease with or without radiotherapy, while conventional systemic therapy has limited efficacy in advanced LGFMS5.\n\nHere, we report a rare case of recurrent LGFMS in the inguinal region involving the scrotum and both testicles. To the best of our knowledge, there is no case described with this rare presentation.\n\n\nCase presentation\n\nA 38-year-old male patient reported a history of multiple nodular lesions in the left inguinal region accompanied by a local inflammatory process since the age of 13. Since then, he had undergone multiple surgical procedures performed by different health services, with clinical and histopathological diagnosis of complicated hidradenitis. The patient was submitted to local resection of the lesion at our institution, with histopathological diagnosis of LGFMS. He missed his follow-up in 2009, only returning in 2017 with a large bulge in the left inguinal region involving the scrotum with signs of tissue necrosis and local purulent discharge (Figure 1).\n\nMagnetic resonance imaging (MRI) showed a mass of lobulated contours and partially defined limits, located in the left scrotum and extending to the perineal region and the medial aspect of the thigh, with invasion of the ipsilateral adductor muscles, not separable from the left testicle (Figure 2A and 2B).\n\nA) Cross section revealing inseparable inguinal mass with structures of the scrotum. B) Sagittal section showing the relationship between the tumor mass and the adductor musculature of the left thigh.\n\nGiven this case of a large recurrent LGFMS, the patient was scheduled for surgical intervention. Under general anesthesia, the patient was placed in supine position and the intraoperative findings were compatible with the MRI results, additionally revealing the involvement of the right testicle. The patient underwent tumor resection in the left inguinal region and the entire scrotum, with bilateral orchiectomy, with the margins enlarged to the right inguinal region and proximal surface of the penis, this stage of the surgery being performed by the urology team (Figure 3). Local reconstruction was performed by the plastic surgery team, with a left fascia lata tensor muscle flap and ipsilateral thigh coverage using partial skin graft (Figure 4).\n\nOn microscopic examination, the tumor showed an admixture of hypocellular zone and more cellular, spindle cell nodule. Arcades of small vessels with perivascular sclerosis were seen (Figure 5). The immunohistochemical study carried out showed immunoreactivity with Ki67 (<5%), immunonegativity with desmin and S100, confirming the diagnosis of grade 2 LGFMS according to American College of Pathology staging.\n\nPostoperative recovery was good and no recurrence was seen after two years. The patient is in good health, realizing multidisciplinary outpatient follow-up at least every six months in the departments of oncology, urology, plastic surgery and endocrinology, where interviews, physical examination, image exams and testosterone replacement therapy control are carried out. MRI of the abdomen and pelvis, in addition to chest CT are performed every six months. Testosterone replacement is being performed continuously with intramuscular injections of 1,000 mg of testosterone undecanoate once every 12 weeks, keeping testosterone levels in the reference range without side effects.\n\n\nDiscussion\n\nLGFMS is a recently recognized soft tissue tumor that was first reported in 1987 by Evans as a metastasizing tumor with a deceptively benign histological appearance, affecting predominantly adults during the fourth decade of life1. Patients are often misdiagnosed with fibromatosis, neurofibroma or other benign conditions instead of LGFMS6. In our case, the patient underwent multiple procedures without success due to misdiagnosis of hidradenitis. LGFMS generally occurs in the lower proximal extremities and trunk, but is also infrequently described as arising from the inguinal region and the chest wall7. Despite its relatively low-grade histology, local postsurgical recurrence and metastases to lungs and bone are frequently seen and can appear several years after primary surgery8. We present a case in which a primary tumor of the inguinal region relapsed after multiple surgical treatments and aggressively invaded adjacent structures, including both testicles.\n\nRadiological examination has an important role in the diagnosis of LGFMS. However, it is difficult to distinguish LGFMS from other mesenchymal tumors due to the rarity. CT scan and MRI can identify the lesions and help evaluate relationships with adjacent structures. In imageological examination, LGFMS is generally seen as a solitary and well circumscribed lesion, although it can be present as multiple infiltrating masses upon recurrence9. MRI techniques can be more helpful than CT to detect the fibrous and myxoid components of the tumor according to the T1/T2 signal intensity10. In our case, MRI was essential for surgical programming, presenting findings similar to those during surgery.\n\nThe definite diagnosis depends on histopathological examination. Histologically, the tumor has a deceptively benign appearance, making diagnosis a challenge, although immunohistochemistry can exclude entities in differential diagnosis. Therefore, it is essential for the diagnosis to be confirmed by an expert soft-tissue pathologist11. The patient in our study had undergone multiple surgical resections without success, due to mistaken histopathological diagnosis of hidradenitis, before being examined at our institution.\n\nSurgical resection with negative margins for localized disease remains the standard treatment for LGFMS. However, treatments for advanced disease are limited. Radiotherapy has questionable efficacy, being reserved for cases of positive margins, recurrence or metastasis. Chemotherapy is usually reserved for patients with metastatic disease. However, there are no data to support the use of any systemic or locoregional treatments3. Chamberlain et al. recently described their experience with non-surgical therapies to treat LGFMS. According to the authors, systemic therapy has limited efficacy in advanced LGFMS5. Despite tumor recurrence in our case, the patient did not present metastasis after aggressive surgical treatment and it was not necessary to perform adjuvant treatment.\n\nTo the best of our knowledge, there is no case described with recurrent LGFMS in the inguinal region involving the scrotum and both testicles. The patient was properly treated through tumor resection and local reconstruction. However, this study has limitations due to the short follow-up period.\n\n\nConclusion\n\nLGFMS is a rare sarcoma subtype but one which should be considered in nodular lesions in the inguinal region. Histologically, the tumor has a deceptively benign appearance, making diagnosis a challenge. If missed, adjacent structures such as the scrotum can be aggressively involved. Surgical resection with negative margins for localized disease remains the standard treatment.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "References\n\nEvans HL: Low-grade fibromyxoid sarcoma. A report of two metastasizing neoplasms having a deceptively benign appearance. Am J Clin Pathol. 1987; 88(5): 615–9. PubMed Abstract \| Publisher Full Text\n\nMohamed M, Fisher C, Thway K: Low-grade fibromyxoid sarcoma: Clinical, morphologic and genetic features. Ann Diagn Pathol. 2017; 28: 60–67. PubMed Abstract \| Publisher Full Text\n\nMaretty-Nielsen K, Baerentzen S, Keller J, et al.: Low-Grade Fibromyxoid Sarcoma: Incidence, Treatment Strategy of Metastases, and Clinical Significance of the FUS Gene. Sarcoma. 2013; 2013: 256280. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurisaki-Arakawa A, Suehara Y, Arakawa A, et al.: Deeply located low-grade fibromyxoid sarcoma with FUS-CREB3L2 gene fusion in a 5-year-old boy with review of literature. Diagn Pathol. 2014; 9: 163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChamberlain F, Engelmann B, Al-Muderis O, et al.: Low-grade Fibromyxoid Sarcoma: Treatment Outcomes and Efficacy of Chemotherapy. In Vivo. 2020; 34(1): 239–245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDomanski HA, Mertens F, Panagopoulos I, et al.: Low-grade fibromyxoid sarcoma is difficult to diagnose by fine needle aspiration cytology: a cytomorphological study of eight cases. Cytopathology. 2009; 20(5): 304–14. PubMed Abstract \| Publisher Full Text\n\nCowan ML, Thompson LD, Leon ME, et al.: Low-grade fibromyxoid sarcoma of the head and neck: A clinicopathologic series and review of the literature. Head Neck Pathol. 2016; 10(2): 161–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEvans HL: Low-grade fibromyxoid sarcoma. A report of 12 cases. Am J Surg Pathol. 1993; 17(6): 595–600. PubMed Abstract \| Publisher Full Text\n\nHwang S, Kelliher E, Hameed M: Imaging features of low-grade fibromyxoid sarcoma (Evans tumor). Skelet Radiol. 2012; 41(10): 1263–72. PubMed Abstract \| Publisher Full Text\n\nYue Y, Liu Y, Song L, et al.: MRI findings of low-grade fibromyxoid sarcoma: a case report and literature review. BMC Musculoskelet Disord. 2018; 19(1): 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFolpe AL: Low-grade fibromyxoid sarcoma: A review and update. Pathol Case Rev. 2002; 7(4): 139–45. Publisher Full Text" }	[ { "id": "68357", "date": "19 Aug 2020", "name": "Felipe Lott", "expertise": [ "Reviewer Expertise I am an expert in urology oncology at Brazilain National Cancer Institute." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 is well described, with good quality photos and image exam. A well documented pathology analysis was made. It is a rare pathology and this article brings more substance for future diagnosis alert. The treatment is correctly described and clear. All case history is acceptable. I think there is a reason for the article to be indexed without necessity for revision.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "72099", "date": "12 Oct 2020", "name": "Nasir Ud Din", "expertise": [ "Reviewer Expertise I am a histopathologist with expertise in soft tissue and bone sarcomas." ], "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 reported a case of recurrent scrotal LFMS. The initial and recurrent tumor size needs to be reported. Was recurrent tumor bigger than primary tumor?\n\nThe authors claim their case to be first case in this location. However, a previous publication (Unlü et al., 20151) has reported two paratesticular LGFMS. Please read that article and cite.\n\nApart from morphology, the diagnostic marker for LGFMS is MUC4. Was this marker not available in the lab to perform on this case?\n\nIn the pathology section, a differential diagnoses should be first described followed by mentioning appropriate IHC.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-789
https://f1000research.com/articles/9-1265/v1	22 Oct 20	{ "type": "Opinion Article", "title": "The case for an inclusive scholarly communication infrastructure for social sciences and humanities", "authors": [ "Maciej Maryl", "Marta Błaszczyńska", "Agnieszka Szulińska", "Paweł Rams", "Agnieszka Szulińska", "Paweł Rams" ], "abstract": "This article presents a vision for a scholarly communication research infrastructure for social sciences and humanities (SSH). The COVID-19 pandemic has highlighted the pressing need to access research outputs without the traditional economic and temporal barriers. This article explores the current scholarly communication landscape, assessing the reasons for the slower uptake of open access in SSH research. The authors discuss such frontiers as commercial interests, sources of academic prestige and discipline-specific genres. This article defines and discusses the key areas in which a research infrastructure can play a vital role in making open scholarly communication a reality in SSH: (1) providing a federated and easy access to scattered SSH outputs; (2) supporting publication and dissemination of discipline-specific genres (e.g. monographs, critical editions); (3) providing help with evaluation and quality assurance practices in SSH; (4) enabling scholarly work in national languages, which is significant for local communities; (5) being governed by researchers and for researchers as a crucial factor for productive, useful and accessible services; (6) lastly, considering the needs of other stakeholders involved in scholarly communication, such as publishers, libraries, media, non-profit organisations, and companies. They conclude that a scholarly-driven, inclusive, dedicated infrastructure for the European Research Area is needed in order to advance open science in SSH and to address the issues tackled by SSH researchers at a structural and systemic level.", "keywords": [ "scholarly communication", "SSH", "open access", "open science", "research infrastructure" ], "content": "Introduction\n\n“I call on all countries, companies and research institutions to support open data, open science, and open collaboration so that all people can enjoy the benefits of science and research” (Ghebreyesus, 2020). Although we have heard such statements many times from different actors in many countries, this one was profoundly different thanks to the unique context: it was delivered by the director-general of World Health Organisation, Tedros Adhanom Ghebreyesus, at a media briefing on the COVID-19 pandemic. Furthermore, it was followed by a proposal to create “a pool of rights to tests, medicines, and vaccines, with free access, or licensing on reasonable and affordable terms, for all countries” (Ghebreyesus, 2020). It put access to knowledge in the right perspective, as a basic human right to access the outputs of scientific research.\n\nThe COVID-19 pandemic has deeply influenced our thinking about scholarly communication and the future of open access. It will take time to realise how deep this change is, but one thing is certain: this emergency has forced academics to look differently at the practices they had believed to be indispensable, both in the connections within the scholarly community, and between scholars and the public. The sudden rise in remote meetings and conferences is the best example here. However, this change also affects access to scientific outputs. Big commercial content providers like Elsevier or EBSCO, opened some of their resources to address the issue of students and scholars being stranded at home without library access, as well as to “accelerate the fight against coronavirus,” which proves, indirectly, that paywalls and closed access actually slow down research. This sudden eruption of open access to scientific content proved how indispensable it is for international and interdisciplinary collaboration in addressing the great challenges of humankind. On the other hand, this situation gave additional visibility to open access publishers and repositories, which didn’t have to make their resources public for a limited timeframe as they had already been freely available; and so they concentrated on creating dedicated collections1 and guidelines2 about the open content.\n\nIt is against this background that we sketch the scientific case for the future infrastructure for open scholarly communication. Every discussion of the future should be deeply rooted in the past in order to avoid the fallacy of the status quo, i.e., treating the current situation as a given, and every novelty as unprecedented. This is very true for the scholarly communication that evolved throughout the centuries together with technological means. We will thus follow the recent EC report Future of Scholarly Publishing and Scholarly Communication, which proposes to define scholarly communication broadly as “any form of exchange used by scholars and researchers to participate in the elaboration of knowledge through critical discussions and conversations with fellow humans. This encompasses all the procedures, from the purely informal conversation to the highly formalised stage of ‘publishing’” (Expert Group to the European Commission, 2019, 14). In this approach scholarly publishing is a formalised sub-set of scholarly communication. This perspective allows us to look beyond technology to the source of current communication practices: platonic dialogues, debates, treaties, and letters. Similarly, we need to perceive modern research infrastructures as new versions of the traditional ones, like libraries, archives, and museums, which have been serving the main purpose of facilitating research and knowledge exchange by using available technological means.\n\nThe digital transformation brought about new opportunities for creating, sharing, discovering, and accessing scholarly resources. Although we have seen a gradual shift towards open practices, some disciplines—notably social sciences and humanities—seem to lag behind (Ferwerda et al., 2017, 19). It appears that further progress, as well as acceleration, requires a dedicated, inclusive infrastructure that can streamline the fragmented initiatives, address the specific challenges faced by these disciplines, and attune the services to their particularities. In other words, we need to “critically appraise what we need from a scholarly communications infrastructure and to simultaneously build pragmatic and non-damaging transition strategies to harness the full power of open, digital dissemination” (Eve, 2015, 15). This is a pertinent issue in the light of the European Commission’s strategy to create a European Open Science Cloud, a service enabling European researchers to “store, share, and re-use data across nations and scientific disciplines through the open science cloud and without leaving their desk” (Burgelman et al., 2019). This calls for a coordinated effort on the part of social sciences and humanities (SSH) researchers, and for infrastructures to achieve a proper representation of disciplinary needs in this new landscape. A research (and researcher-driven) infrastructure for scholarly communication in SSH should respond to these challenges and opportunities, and enable scholarly communication that would take full advantage of digital affordances, but would not forget its core values.\n\n\n1. Scholarly communication: from theory to infrastructure\n\nDefinition of scholarly communication. The term scholarly communication, in the narrow sense, is usually employed to describe traditional and institutionalised publishing practices as “the system through which research and other scholarly writings are created, evaluated for quality, disseminated to the scholarly community, and preserved for future use” (ACRL Scholarly Communications Committee, 2006). Yet, as the definition in the introduction has highlighted, formalised publishing practices are just a subset of a larger pool of various communication practices, which would entail, for instance, all sorts of formal and informal communication through various channels like emails, social media, blogs, press, etc., both between scholars and between scholars and the public. Thus, apart from researchers, the group of potential actors involved in scholarly communication includes “students, educators, policy makers, public administrators, funders, librarians, journalists, practitioners, publishers, public and private organisations, and interested citizens” (Kraker et al., 2016, 2).\n\nMoreover, communication, conceived broadly, “includes both the dissemination and access to scholarship and research in a variety of formats and states of completion, such as published books or journal articles, research results and data sets, and drafts of papers” (Husain & Nazim, 2013, 405). While it is true that the relationship between communicating and publishing becomes “increasingly muddy” (Edmond, 2020, 4), traditional publishing practices still remain the established sources of academic prestige and suppress the engagement with innovative solutions. We aim to show how artificial these distinctions are by discussing the role of scholarly communication in actual research practices.\n\nThe research lifecycle. In an often-referenced paper that contributed to the modern understanding of humanities research, John Unsworth (2000) proposed a tentative set of scholarly primitives, i.e., “self-understood” terms describing scholarly activities: discovering, annotating, comparing, referring, sampling, illustrating, and representing. Although this list was not meant to be exhaustive, one omission is striking, namely, the exclusion of communicating. It is even more visible once one realises that all the examples Unsworth provides in the paper for comparison (Babble), linking (Blake Archive), and sampling (VRML visualisation of Dante’s Inferno), have an indispensable communication component attached to them. Perhaps communication is even more self-understood, and thus becomes transparent and eventually omitted. To use Unsworth’s nomenclature, communication takes advantage of the additive characteristics of scholarly primitives and enters into combinations with all other scholarly primitives, i.e., it proves to be essential across all stages of research workflow. This means that dissemination should no longer be perceived as the final stage of a research process, running somewhat separately from the actual research, but should be treated as an integral part of all scholarly activities (cf. Giglia, 2019; Nielsen, 2013).\n\nThe process of scholarly activities is usually presented as a lifecycle, comprising such activities as discovering/collecting, organising, annotating, analysing, and disseminating (see: Dallas et al., 2017, 4). If we take a closer look, we see how deeply communication practices are embedded in those processes: literature review, data discovery, communication to peers, peer review, editing, dissemination, marketing, and quality assurance (see: Mounier, 2018, 301). Communication is thus an integral part of all stages of a researcher’s workflow, and each of these stages has “specific communication needs that can be addressed by proper services” (Mounier, 2018, 301). Different stages of the workflow are linked to different operations and software, as evidenced in the Innovations in Scholarly Communication Survey conducted by Utrecht University Library. As Edmond and Romary point out, “[a]t certain stages of the research process, it is often not as important to produce an in-depth scholarly summation so much as to provide short snapshots of an experiment’s current developments (as in the hard sciences), or an analysis of a source (in the humanities)” (Edmond & Romary, 2020, 65). Each phase may entail the use of different formats to communicate early results, observations, data, annotations, etc.\n\nResearchers assume different roles at different steps of this process. They are not only authors, but also information-seekers, annotators, bibliographers, editors, reviewers, etc. These roles are very important for research but do not carry the same level of prestige in academia as the role of author. This has a negative impact on introducing and sustaining changes in scholarly communication (Edmond, 2020, 13). However, such roles, and the scope of responsibilities attached to them, are constantly evolving because of the imprint that emergent technologies and their applications leave on scholarly communication. For instance, Jason Priem predicts, “the reviewer will metamorphose from gatekeeper to interlocutor and collaborator” (2013, 438). This is also true for institutional actors in scholarly communication, who start to enact new roles, such as repositories that already serve some of the functions traditionally reserved for publishers.\n\nCommunication and the community. Communication is thus not only an important factor, but the actual enabler of scholarship. The development of the postal network during the sixteenth century allowed for unprecedented knowledge exchange between scholars within (and often beyond) Europe, forging the idealistic notion of the republic of letters: a common, intellectual space (Hotson & Wallnig, 2019, 7–11). Within the scientific dimension, these letters allowed for the dissemination of important intellectual breakthroughs, becoming prototypes for the first learned journals in the seventeenth century (ibid., 8). This example illustrates Nielsen’s point that the science remains tentative until communicated: “the meaning of scientific knowledge is not only established by its internal qualities or the method by which it has been produced, it also depends on what other scientists make of it, that is, how scientific knowledge is being communicated” (2013, 2071). Consequently, communication is what constitutes science.\n\nCommunication presupposes the existence of a community, where knowledge is exchanged and negotiated leading to a “common narrative and meaning” (Neylon, 2017a, 3; see also: Nielsen, 2013, 2068). Such communities, whether we call them “thought collectives” (Fleck, 1979), “epistemic cultures” (Knorr-Cetina, 1999), or “interpretive communities” (Fish, 1980), share certain beliefs, concepts, and practices for evaluating new knowledge. We can observe these at different levels of organisation, from small research teams through to scholarly associations, disciplines, or groups of disciplines (e.g. SSH). However, such communities have one thing in common: scholarly communication is essential for fostering their group identity (Hartley et al., 2018, 6).\n\nNielsen likens communication to “knowledge travel,” a movement of knowledge between communities, as “[s]cience fundamentally is a shared form of knowledge, and conventional ways of communicating among peers and across epistemic boundaries are central to the collective and collaborative character of science” (Nielsen, 2013, 2070). Hence, we are able to observe scholarly communication both within a community and between communities, which Kulczycki defines as “external communication science” (the “process of explaining and popularising academic research” to non-scientists), and “internal communication science” (“communication of scientists with scientists”) (2013, 5–6). Fleck suggested a spherical understanding of scientific communities, with “specialised experts” at the core, surrounded by “general experts,” who, in turn, border onto “educated amateurs,” with all encircled by the general public (Fleck, 1979, 111–12). Elsewhere he notes that, paradoxically, the more widespread the knowledge is the more exclusive it becomes due to its complexity and specialisation. The accessibility of knowledge calls for its constant translation by scholar after scholar, adapting it and interpreting within their own research and thus making it more understandable for the general public or specialists from other areas (Fleck, 1979, 109). This process of “knowledge travel” and “translation” between different epistemic cultures is especially important for inter- and transdisciplinary endeavours, serving as a bridge that allows for the building of shared understandings and joint research agendas.\n\nSo, effective communication would ensure a constant, bi-directional flow of ideas and expertise between those circles and communities. The lack of such communication impedes the innovative and inclusive potential of those collectives (Kraker et al., 2016; Okune et al., 2018). This could lead to the creation of disciplinary siloes, “knowledge clubs” that operate on the basis of the “joint production and consumption of scholarly output by the scholarly community,” whereas “knowledge is most intensively produced at group-boundaries, in interaction with other, competing groups” (Hartley et al., 2018, 5; cf. Neylon, 2017a, 5–6). Scholarly communication without the actual community of peers exchanging ideas is reduced to the outward signs of academic prestige, but devoid of its communitarian substance that only a vibrant space for exchange of knowledge can create. This is where “predatory” or “deceptive” publishing flourishes, encompassing a range of problems of academic publishing, from poor content quality to deceptive journals (Eriksson & Helgesson, 2018).\n\n“An old tradition and a new technology have converged to make possible an unprecedented public good;” thus begins the Budapest Open Access Initiative (BOAI), signed in February 2002, and hinting at the recreation of the old tradition of the republic of letters in the digital environment. Now, almost two decades later, the situation has somewhat improved, but the appetite of open access proponents has also increased, crystallizing in a movement for open science.\n\nRecent initiatives by various institutions reflect the urge to speed-up this transition. The European Commission’s working document that lays the foundations for the next framework programme, Horizon Europe, has identified “rather limited progress at the EU level in the transition towards open science, including on open access to research output” (European Commission, 2018, 104). The EC adopted a “holistic policy to Open Science,” engaging stakeholders in key aspects ranging from open publications to research data and interoperable services (Burgelman et al., 2019). For instance, Horizon Europe is to “fully embrace and support Open Science as the new research modus operandi,” including support for open data, FAIR principles, financial incentives, and lack of funding for hybrid journals (European Commission, 2018, 105–6). On the same note, Plan S, proposed by a coalition of funders, explicitly states that starting from 2021 all outputs of grants provided them must be available in open access without embargo. On a global scale, UNESCO has launched The Global Consultation on Open Science, to ensure “a better distribution and production of science in the world”. These actions are aimed at stimulating the communities in its transition to open access.\n\nBOAI had famously advocated “open-access to peer-reviewed journal literature,” either through self-archiving or “a new generation of open-access journals,” which are now commonly known as green, gold, and diamond open access (BOAI). Open science, with regard to scholarly communication, currently has a much wider scope as it applies to all stages of the research process, “from designing the question and methods, to collecting and analysing data, through to the communication and dissemination of findings” (Hillyer et al., 2017, 18; cf. Krlev, 2019). So, in other words, all elements of scholarly communication discussed above should be freely accessible. Simply (and broadly) put, open access “refers to the removal of price and permission barriers to scholarly research” (Eve, 2014, 3), with the underlying assumption that everyone should have unrestricted access to knowledge (Tennant et al., 2016, 15).\n\nThere are many arguments in support of open science, which Ulrich Herb (2010) helpfully categorised into groups: science–related arguments (improving scientific communication), financial arguments (response to rising subscription prices), social arguments (reducing the digital divide, fostering societal impact), democracy–related arguments (enabling participation), and socio–political (or moral) arguments (levelling disparities). But still, despite these arguments, recent reports note there are obstacles and slow progress affecting the open access (OA) movement. A recent review of the academic, economic, and societal impacts of OA has concluded that although “Open Access supersedes all potential alternative modes of access to the scholarly literature through enabling unrestricted re-use, and long-term stability independent of financial constraints,” it is still endangered by competition in the unregulated scholarly publishing market (Tennant et al., 2016, 1–2). The uptake of OA policies in Europe varies from country to country, which will be discussed briefly using a handful of examples3.\n\nIn Poland, the 2018 report on the implementation of OA policies reveals that the fragmentation of open science efforts—such as the rise of many small repositories that are not linked to each other (Szafrański, 2019, 11)—calls for a stronger central coordination (MNiSW, 2018). The number of institutional OA mandates is small but growing, and reports suggest that the infrastructure is fragmented and in need for more coordination (MNiSW, 2018; Sójkowska & Gruenpeter, 2019; Święćkowska, 2013). The studies show that the most successful repositories belong only to those institutions where self-archiving is compulsory, but these are still in the minority (Święćkowska, 2013, 8). Similarly, the Ministry of Science and Higher Education in Poland has focused on giving general recommendations rather than on setting up compulsory, nationwide requirements (Gowin, 2017; MNiSW, 2015).\n\nIn the same way, there is still not enough awareness of the benefits associated with the transition to open access in Greece, and the engagement of key stakeholders has been relatively low (Picarra et al., 2015). Even in cases where open access platforms (such as Greece’s EKT ePublishing platform, discussed later in this paper) have been adopted, the full transition to online editorial processes has not necessarily followed (Sachini et al., 2009). Despite the OA initiatives, the majority of scholarly communication efforts in Greece are still market-driven.\n\nConversely, France remains one of the leaders in terms of national coordination and support for open science initiatives (such as HAL repository), which perhaps stems from its centralised tradition, with the National Centre for Scientific Research (CNRS) as the focal point for scholarly activities. OpenEdition is its signature national infrastructure for SSH (see: MESRI, 2018). In 2019, the National Open Science Fund was created to support research infrastructures, digital platforms, and initiatives concerning open journals and books. One may still note the tension between the private and the public publishing sectors however, with the former opposing OA and the latter embracing it (Ferwerda et al., 2017, 74–78).\n\nIn Croatia, the Ministry of Science and Education is currently adopting the open science agenda and although there are other strategic documents addressing OA, funders and policy-makers still haven’t developed key top-down policies. Numerous OA and OS initiatives have emerged in Croatia and have sought to alleviate this lack of national policies, and to provide the infrastructure necessary to support openness, such as the Croatian Scientific Bibliography (CROSBI), and HRČAK, a common platform for OA journals. Over half of the Croatian OA journals are from SSH disciplines, mostly following the Diamond OA route without article processing charges, grounding their business models on government subsidies. Research and higher education institutions can establish their own institutional and disciplinary, or thematic repositories free of charge via the national infrastructure for digital repositories, DABAR.\n\nIn recent years open science has been strongly supported in Switzerland. Since October 2017, the Swiss National Science Foundation (SNSF) has required researchers to include a data management plan (DMP) in their funding application to most of the funding schemes. As of 2020, all results have to be made available in open access. In April 2020, the State Secretariat for Education, Research and Innovation (SERI) agreed with various stakeholders to introduce a national strategy for open research data in 20214. In this landscape the social sciences in Switzerland have been supported by the Swiss Centre of Expertise in the Social Sciences (FORS) infrastructure, whereas humanities are under the lead of the Data Service Center for the Humanities (DaSCH); both institutions are supervised by the Swiss National Science Foundation (SNSF).\n\nAlthough the whole open access landscape has not yet been thoroughly researched and analysed in Luxembourg, it is worth mentioning some significant top-down initiatives, such as the Policy on Open Access adapted by FNR (the National Research Fund), as well as its Open Access Fund programme (focused on promoting OA in projects receiving FNR funding). Moreover, the Scientific Monographs programme for printing books (RESCOM) covers the OA-related costs.\n\nThese examples suggest that, despite the long history of the movement, the uptake of open science is still in need of a major boost. Moreover, progress in various European countries is uneven, which calls for more coordination at the EU level. However, there is a key obstacle impeding this transformation, which Martin Paul Eve calls “digital economics,” i.e., “the economics of scholarly publishing in the two interlinked senses of an ‘economy’ of academic prestige and of finance” (Eve, 2014, 16, 43–85). As for the financial side, academic publishing became a profitable business, functioning in an oligopoly of big commercial companies offering access to scholarly content at very high prices. This concerns the cost of both individual access and library subscription (see: Eve et al., 2017, 122). A good example indicating that something is not right is that access to a single scholarly article tends to cost more than a monthly subscription to a streaming service with blockbusters and popular TV shows. For instance, in order to access some recent work on scholarly communication (e.g. Chisita & Chiparausha, 2019; Sotudeh et al., 2019; Wang et al., 2019; Young & Brandes, 2020) without an institutional subscription, authors would have to pay USD 41.95 per PDF (at the time of writing this article). This is more than the price of many printed monographs, which poses an actual barrier, not only for independent scholars and the non-academic public, but also for institutions that cannot afford costly subscriptions.\n\nThe recent report Untangling Academic Publishing (Fyfe et al., 2017) reconstructs the history of scholarly communication, which has become increasingly commercialised since World War 2 in response to the professionalisation of academia and internationalisation of research: “The older model of academic publishing practised by learned societies and university presses had prioritised the wide circulation of high-quality scholarship, with little or no expectation of making money. The new commercial model demonstrated that, in the new world order, it was possible not merely to break even but to make profit” (Fyfe et al., 2017, 10). This commercial strategy entailed setting up new journals, selling their content to institutions (as they can be charged more) and focusing on the international market to address a larger audience (Fyfe et al., 2017, 10). Digital distribution only strengthens this model, reducing the actual service cost and allowing archival issues to be charged for on the basis of constantly renewed subscriptions, rather than one-time payments for physical copies. Moreover, it also allows the most popular journals to be bundled together with lesser-known ones in order to increase the breadth (and price) of institutional subscriptions. Thus, the profits of large commercial scholarly publishers are based on the unpaid labour of authors and reviewers, as well as the revenue from public funding either in the form of subscriptions or article-processing charges.\n\nThis model has been criticised due to the immense subscription costs that make access to resources uneven, limiting the transfer of knowledge to the community (Kraker et al., 2016). This is also a concern in developing countries, which struggle to foot the bill for access prices (Arunachalam, 2017; Tennant et al., 2016). Another issue is that the money spent on access to paywalled content could be used instead for the benefit of scholars: “it means less support is going back into our library communities and that we have less agency in the future development of scholarly communication infrastructure” (Wipperman et al., 2018, 244).\n\nThese economic barriers are even more puzzling if we consider the cost structure of digital dissemination. Eve points out that OA entails “nonrivalrous commodity exchange,” in which “the ‘use’ of a commodity does not entail somebody else’s inability to use it,” so there is no cost attached to reproducing the object (Eve, 2014, 16). In other words, there is no particular cost associated with a single article download. And acquiring a digital copy of an article surely does not impede somebody else’s ability to do the same, which makes the high prices even less justified. There are many successful OA business models for scholarly publishers that allow for a scholarly-led spending of public resources (for an overview see: Milloy et al., 2012; Speicher et al., 2018; Withey et al., 2011), but the dominant position of the big commercial players keeps them fragile and difficult to sustain (e.g. Björk, 2017), and slows down wider change. Moreover, even if the OA practices are adopted by big commercial providers, there is still a risk “that a small number of companies will own most of the critical data assets, analytics, and platforms used by the scientific community” (Aspesi & Brand, 2020, 576), thus maintaining the dominant position.\n\nA second barrier, closely linked with the financial aspects, is that of academic prestige. Ulrich Herb, who analysed the uptake of OA through the lens of Bourdieu’s concept of scientific capital, concludes that although “the moral vibrancy [of OA] is overwhelming,” scholars are concentrated on accumulating such capital, which is usually achieved through publishing in journals with a high impact factor (2010). Similar conclusions could be drawn from the analysis, by Fyfe et al. (2017), of the history of the relationships between commercial actors, academic prestige, and scientific communications. They conclude that prestige in academia is currently linked with “traditional forms of academic publishing, many of which are controlled by commercially-motivated firms” (Fyfe et al., 2017, 18). The sad outcome is that there are no “credible, prestige-generating alternatives” and that the players in the online publishing business tend to see “online publishing as a valuable income stream, rather than seeking ways to use the potential of the Internet to carry out their traditional ideals of promoting the circulation of knowledge” (Fyfe et al., 2017, 18).\n\nIt should be noted that economic observations and trajectories stem from the analyses of the ‘global West’. A slightly different case could be made for national communities where the scholarly communication is conducted in local languages and with a strong tradition of state-funded publishing, such as Poland and other countries of the former Eastern bloc. Apart from the issues discussed earlier, like high subscription rates, we also observe a diminishing position of the local journals. For instance, the Polish evaluation system for journals is based on the Scopus database which has an uneven, to say the least, representation of scholarship in local languages and discipline-specific outputs, including monographs. If local journals and monographs are not indexed, the impact of a publication could be measured solely on the basis of the international reception, what gives a false impression of a lower impact especially in the case of humanities (e.g. Polish studies). Thus, the prestige of a publication became closely linked with a business product with opaque inclusion policies beyond the control of the scholarly community. This is a dangerous situation as it creates a clear conflict of interests for a company which both publishes journals and ranks their impact. Hence, the pressure for internationalisation, aptly described by Kulczycki & colleagues, (2019b), has a clear business dimension with global corporations benefiting from state policies, which are at the same time suppressing the local high-quality journals.\n\nSo, although there seems to be a consensus that OA is generally good for research, the broader adoption of open practices will not happen without a substantial transformation of the practices of prestige allocation in academia. Eve describes the systemic aspects of this mechanism: since the venue’s prestige is used as a proxy measure for quality, it encourages publication in acclaimed outlets and has a cooling effect on innovation, while conservative evaluation mechanisms petrify this system (Eve, 2014, 50). And, according to Edmond, it is a “generational fallacy” to believe that the new generation of scholars will bring change to academia, as the proxies for prestige are also used for funding allocations and job searches, so the system reinforces itself (Edmond, 2020, 8). Instead, we need systemic action that can address the system of scholarly communication economics in its entirety, addressing both business models, and prestige.\n\nTo sum up, the European Commission tries to address these challenges through its ‘holistic approach’, a top-down measure, which needs to be met by a bottom-up movement towards open science. Robert-Jan Smits, the former European Commission’s Special Envoy for Open Access observed that “[a]lthough researchers all say that they are supporting open access, their dream is still to publish in the most prestigious journals with the highest impact factor, which are often subscription journals. And the universities are obsessed by the traditional rankings using mainly one metric – number of publications in high impact journals” (Smits, 2018). That is why a comprehensive approach towards open science should address the issues of perceived academic prestige, which can differ across communities of practice and thus require different remedies. A proper, sustainable, and far-reaching response to these challenges requires a synergy of initiatives conducted by various actors at different levels, which could be provided by a research infrastructure. The recent actions undertaken by the European Commission, which have led towards the creation of the European Open Science Cloud (EOSC), are meant to put those ideas into practice. The EOSC Declaration, widely adopted by numerous European institutions, research infrastructures (RIs), and societies5, sketches out a vision of a pan-European meta-infrastructure “federating existing resources across national data centres, European e-infrastructures and research infrastructures” (European Commission, 2017, 3). The key to the EOSC is its unique governance model, which intertwines community-driven and multi-governmental movements (Budroni et al., 2019, 130–31). Thus, it is essential that the needs of scholarly communication are properly addressed by the relevant communities, allowing for the proliferation of open science. The EOSC seems to be the ultimate embodiment of the ‘holistic approach’, as it recognises that OS can be achieved only through “considerable cultural change,” in which “no disciplines, institutions, or countries must be left behind” (European Commission, 2017, 1).\n\nThe European Commission defines research infrastructures broadly as\n\nfacilities, resources and related services that are used by the scientific community to conduct top-level research in their respective fields and covers major scientific equipment or sets of instruments; knowledge-based resources such as collections, archives or structures for scientific information; enabling Information and Communications Technology-based infrastructures such as Grid, computing, software and communication, or any other entity of a unique nature essential to achieve excellence in research. (European Commission, 2010, 10)\n\nThe authors of the European Science Foundation (ESF) Report on research infrastructures in digital humanities insist that “an RI cannot be defined in an abstract, absolute and immutable way; rather, it is a term that is adapted for and by different disciplines” (Moulin et al., 2011, 5). The humanities, for instance, have a long tradition of physical infrastructures: “Archives, museums, galleries and libraries have always housed collections of physical objects such as archaeological fragments; paintings or sculptures; inscriptions or manuscripts; books and journals” (Moulin et al., 2011, 4). All of them have been through the digital transformation and adapted to modern scholarly needs, and adjusted to particular research communities. Thus, the crucial difference between e-infrastructure and research infrastructure is the latter’s situatedness in a particular research community, responding to disciplinary research needs (see: Duşa et al., 2014, 21–39), whereas the former addresses more generic needs; as in the case of OpenAIRE, which provides services for institutional repositories of all disciplines (Artini et al., 2015).\n\nIn order to perform its role for the community, a research infrastructure should be inclusive, i.e., facilitate the work of diverse kinds of actors. Okune et al. propose the term “inclusive knowledge infrastructures” to define “tools, platforms, networks and other socio-technical mechanisms that deliberately allow for multiple forms of participation amongst a diverse set of actors” (Okune et al., 2019, 2). This inclusivity is crucial for reconfiguring power relations and involves diverse communities in knowledge-creation processes (Okune et al., 2019, 7). In the case of scholarly communication, that would mean a broader pool of actors and all types of scholarly outputs, beyond the refereed ones (Ren, 2013, 745).\n\nIn the following section we will look at the existing scholarship and will try to reconstruct the actual needs of SSH scholars with regard to a scholarly communication infrastructure.\n\n\n2. What SSH researchers need\n\nParadoxically, the vocal proponents of open science emerged from the humanities; but the uptake of OA in SSH is still lower than in science, technology, engineering, and mathematics (STEM) disciplines (Ferwerda et al. 2017, 19). Moreover, different models of funding between STEM and SSH influence their opportunities to engage in particular open access practices, with SSH researchers usually having less chances to receive funding for article processing charges (APCs) (Rowley et al., 2017, 1204). This discrepancy in the humanities, Eve observes, is often explained as stemming either from different communication practices of those disciplines, or the lack of a critique of their own disciplinary communication practices (Eve, 2014, 24). Suber (2005) listed several differences, arguing that, apart from funding difficulties and less need for immediate access to results, SSH differs in terms of its major communication format, which is the monograph.\n\nMonographs occupy a special place in humanities scholarship. They manifest a crucial specificity of the humanities communication paradigm as a more discursive format, serving different functions to journal communication, allowing for a thick description and complex narrative (Crossick, 2015, 13–14). As Geoffrey Crossick contends in his study on open access monographs, “[t]he process of constructing and writing a book is often a core way to shape the ideas, structure the argument, and work out the relationship between these and the evidence that has emerged from the research process” (Crossick, 2015, 3). Moreover, and paradoxically, this complexity may contribute to SSH scholars’ reluctance to share publications in OA, which Fitzpatrick connects with a fear of the wider, non-specialised audience: “[t]he world at times fails to understand what we do and, because our subject matter seems as though it ought to be universally comprehensible (You’re just writing about books, or movies, or art, after all!), readers often are not inclined to wrestle with the difficulties that our work presents” (Fitzpatrick, 2012, 353). Another crucial issue is that, unlike STEM, SSH tends to be deeply rooted in local contexts and languages. As Mounier puts it, “the diversity of publication venues reflects the epistemic diversity of SSH communities that need their own ways of communicating knowledge to diverse audiences” (2018, 302).\n\nHowever, it would be a mistake to treat SSH and STEM as completely different, as separated academic worlds that compete with each other for the legitimacy of performing the true and “right” kind of research. As Edmond, Bagalkot, and O’Connor suggest, instead of seeing polarities, we need to perceive these disciplines on “a sliding scale of ‘epistemic cultures,’ which, like human cultures, blend with and branch from each other in a wide variety of modes at a number of border regions” (2016, 2–3). The ongoing project on interdisciplinary practices in Europe (SHAPE-ID) tries to answer the question of why SSH is poorly integrated in inter- and transdisciplinary (ID/TD) endeavours. Early results have identified the root of this problem “in a lack of understanding by researchers, policy makers and funders, about what the [arts, humanities and social sciences] are and what these disciplines can contribute to solving societal problems” (Vienni Baptista et al., 2020, 14). The authors also observed that these disciplines are invisible in certain contexts and fields (Vienni Baptista et al., 2020, 19), and have analysed the systemic and institutional obstacles that hinder such cooperation. Bibliometric analyses performed in this study have clearly indicated a certain self-referentiality in the discourse on ID/TD efforts within arts and humanities, and to a lesser extent, in social sciences (Vienni Baptista et al., 2020, 63), leading to disciplinary siloses that hinder cooperation.\n\nRecognising the potential of SSH would contribute greatly to the European Union’s Grand-Challenges’ approach, which entails defining strategic missions that should be addressed by long term, transdisciplinary research programmes (Mazzucato, 2018). A timely example of such a contribution, though on a smaller scale, is the OPERAS’ response to the COVID-19 crisis. While STEM disciplines collate data and papers relevant to fighting the disease and finding a cure, the Beyond COVID-19 project aims to create an annotated bibliography of SSH outputs relevant to the pandemic so the actors involved in policy measures have easy access to scholarly outputs that could help them understand the societal and cultural impacts of their decisions. A dedicated infrastructure for SSH could have a positive effect on bridging various disciplines through scholarly communication, and maintaining the visibility and accessibility of SSH outputs that should become an integral part of such missions to achieve better impact. We will now look at the actual needs of SSH scholars with regard to scholarly communication in more detail.\n\nThere is something distinctive about the research process in the humanities. On the basis of interviews with scholars, Edmond et al. noted the “nomadic nature of the humanistic knowledge creation process, with its constant refreshing of sources and inspirations” (2016, 6). They pointed out the uniqueness and individuality of scholarly apparatuses (Edmond et al., 2016, 10–11), which are currently met by digital tools and methods, that remediate all stages of scholarly workflow, from discovery to publication.\n\nThis transformation is particularly visible in the development of digital humanities (DH), i.e., an approach being taken up across all disciplines of the humanities and social sciences, which incorporates digital tools in research workflows6. DH focuses on both the production and analysis of digital-born or digitised data, as well as on the implementation of digital information and communication technologies during the different stages of the research process, as evidenced in a Taxonomy of Digital Research Activities (TaDiRAH)7 and the methods ontology proposed by the Network for Digital Methods in the Arts and Humanities (NeDiMAH)8. These remediated operations include the capturing of sources (discovery), creation (e.g. writing, programming), data enrichment by annotating or editing, storage (archiving), and various forms of dissemination, that is, from collaboration, to commenting (reviewing), crowdsourcing, publishing, and sharing.\n\nThe uptake of these methods is versatile as evidenced in numerous quantitative and qualitative studies that have analysed the nature of digital methods and tools in the humanities and social sciences (e.g. Antonijevic, 2015; Edmond et al., 2016; Hughes et al., 2015). The digital turn stimulated the rapid changes in these areas across all disciplines in a twofold manner: as cutting-edge digital-humanities work, pushing the boundaries of the state-of-the-art; and digitally-enabled research, characterised by the selective use of particular tools by a largely non-digital community to answer a specific, disciplinary research problem9. For instance, the massive use of electronic communication and social media by research communities facilitated collaborative annotation, writing, and the evaluation of scientific argumentation by a community that connects authors, readers, and reviewers, thus creating a productive environment for the emergent publishing initiatives (Ren, 2013, 745). Scholarly blogs became creative catalysts, offering an opportunity to reach multiple audiences and to receive quick feedback on early findings (Kjellberg, 2010). Such open publishing initiatives that allow for open commentary early in the publication process have an immediate benefit for the quality of research through tapping into a larger number of reviewers and the broadest scope of expertise. This “open system,” as Xiang Ren calls it, does not discredit the principle of peer review but instead makes an attempt “to reorganize and democratize” it through “expanding the scale of ‘peers’ and making it more transparent” (Ren, 2013, 747). In this sense, open access can be seen as a chance “to systematize direct scientific communication,” which does not need formal venues, and instead takes place within specific infrastructures such as archival depots, platforms, personal websites, etc.” (Mayeur, 2017, 75–76).\n\nNovel means of writing, co-authoring, team-collaboration, and versioning of scientific content have sparked the creation of new genres of scientific communication that allow for a better connection between the thought and its expression. These open and innovative communication practices give an insight into researchers’ thought processes, methodology, and spontaneous discussions instead of just the formal results of their work (Ren, 2013, 745). The use of the web for disseminating one’s research results also allows authors to experiment with different forms of scholarly communication (often a tweet, an infographic, a blog post, or an extended comment might be more appropriate for conveying certain messages): “What the journal did for a single, formal product (the article), the Web is doing for the entire breadth of scholarly output” (Priem, 2013, 437).\n\nOpen access to research publications, strengthened by robust discovery tools, enables access to vast resources across disciplines, fields, and national/linguistic boundaries, stimulating knowledge exchange and advancement, all of which has clear benefits for the scholarly community. The discoverability of open access resources is crucial, since one of the main goals within the field of scientific communication is to “enable research to be carried out effectively and efficiently” (King, 2019, 2). Furthermore, the proponents and signatories of the recent Helsinki Initiative on Multilingualism in Scholarly Communication highlight the role of access to multilingual resources, which allows for more voices to be heard; it also ensures a wider scholarly and societal impact, which will be discussed in greater detail later in this paper. So, what are the actual needs for a scholarly communication infrastructure in the humanities?\n\nUser-centred research, providing information about users’ actual needs, is key in designing new products or services, as it “can reduce the potential for poorly designed or misused products” (Lofthouse & Lilley, 2006, 741). Otherwise, there is no guarantee that the infrastructure will address the existing needs of a scholarly community, or that scholars are going to use it (see: van Zundert, 2012; Warwick et al., 2008). The user-centric approach should also be applied to designing a future scholarly communication infrastructure, and researchers as its users with unique habits, needs and expectations (Kemman et al., 2014, 3). Such needs pertain to all stages of the research lifecycle and users expect infrastructures to support the whole research process (Boukhelifa et al., 2018; Dallas et al., 2017).\n\nThese issues are captured by research on digital practices in the humanities (meta-research), which does not focus solely on the advanced proponents of digital methods, but also analyses digital needs and competences of the SSH community, allowing for the transfer of skills and knowledge between researchers (Maryl et al., 2020; Thoden et al., 2017). Frequently, digital tools are applied to speed up the existing (traditional) research methods rather than for methodological innovation (Gibbs & Owens, 2012, 9).\n\nThe need to link the development of infrastructure to thorough user research is visible in recent studies. For instance, the Survey and Analysis of Basic Social Science and Humanities Research at the Science Academies and Related Research Organisations of Europe (SASSH) showed the reality of multilingualism across European researchers, thus proving the need for infrastructure that accommodates different languages (Leathem & Adrian, 2015, 132). There are a number of recent and ongoing projects in which user research is conducted in order to build and enhance research infrastructures such as the Social Sciences & Humanities Open Cloud (SSHOC) (Barbot et al., 2019, 15–22), and DARIAH ERIC Sustainability Refined (DESIR) (Tasovac et al., 2018, 9).\n\nIn the field of scholarly communication, two current projects that are affiliated with OPERAS are conducting user-research. The Open Scholarly Communication in the European Research Area for Social Sciences and Humanities – Preparation (OPERAS-P), an EU-funded project, has launched a survey on SSH scholarly communication and the challenges of openness, together with focus studies aimed at gaining a more in-depth understanding of communication practices. WP 3, Co-design and user research, within the current OPERAS project Transforming Research through Innovative Practices for Linked Interdisciplinary Exploration (TRIPLE) conducted another survey on sources discovery as well as series of interviews with researchers and other stakeholders. They aimed to capture current scholarly discovery practices, and the users’ needs that would be incorporated into building a new platform for finding, accessing, using, and re-using research materials. Indeed, users will continue to be involved and consulted at all phases of the project.\n\nThe picture that emerges from these studies shows a rapidly changing communication landscape to which scholars are trying to adapt. Giglia points out that the changing idea of the “scholarly record,” which now also encompasses materials generated in the process, results in the emergence of a more liquid output and blurs the roles of the different actors in scholarly communication (Giglia, 2019, 142). Currently, sharing practices involve not only concluded research and published outputs but also the byproducts and beta-results of the research process. This, in turn, results in a wide range of objects that could be communicated, like texts, data, methods, and software (Bardi, 2019, 4).\n\nThe DARIAH survey of scholarly practices and digital needs in the arts and humanities showed that open dissemination channels such as OA publications, repositories, blogs, websites, and scholarly social networks (e.g. Academia.edu and Researchgate) are used frequently by 10–15% respondents and regularly by 35–45% (Dallas et al., 2017, 4). Yet, even more interesting is the fact that 10% regularly use generic online content services like Slideshare, Flickr, and Youtube for this purpose (ibid.). It is an indicator of the increasing need for new and open avenues of distributing research outputs. Researchers promote their work on social media and engage in less formal discussions about their output using such outlets as Twitter (Estelle, 2017; Kulczycki, 2013). Online communication tools like WhatsApp are employed to foster communication within the research team (Estelle, 2017, 7).\n\nThe main obstacle to this productive proliferation of new communication practices lies in the fact that these informal communication channels remain invisible to the research assessment system and, consequently, to academic prestige (Tóth-Czifra, 2019). For instance, research on academic blogging discussed by Brown and Woolston shows that despite general approval for this form of communication, especially among early career-researchers, some doubts remain as to whether it would be considered serious enough by peers, especially when applying for an academic position (Brown & Woolston, 2018, 137). Therefore, it is crucial for researchers to see new means of communication as being high quality and effective, because otherwise they might not want to use them. The researchers would support a communication model that is “high impact, rigorously refereed, and of good reputation” (Rowley et al., 2017, 1210). In the next section, we will try to address these issues, sketching out the key areas for the scholarly communication infrastructure in SSH.\n\n\n3. Key areas for the future scholarly communication infrastructure in SSH\n\nShortly before his untimely death, Jon Tennant, a vocal proponent of open science, sketched out its future priorities and challenges (2020), which now constitutes his legacy. What is interesting about his relatively short proposal, published as a blog post, is its scale. Tennant looked not only at the immediate, direct measures for achieving progress, but rather at a complex reconfiguration of scholarly communication institutions that should take place if progress is to be achieved. These priorities entail research evaluation reform, rethinking the role of scholarly publishers, increasing global participation, community building, and creating alternatives to the commercial platforms (Tennant, 2020). This latter point also consisted of a risk analysis as to “whether or not the scholarly community is truly ready to take on the burden and bureaucracy associated with controlling a global scholarly communication infrastructure” (Tennant, 2020).\n\nA recent report by the EC Expert Group, dealing with the future of publishing, puts researchers “at the heart of the scholarly communication of the future,” advocating inclusivity in terms of participants, purposes, and methodologies (Expert Group to the European Commission, 2019, 25). The authors defined the following principles, which articulated their vision of future scholarly communication: maximising accessibility and usability; supporting and expanding the range of contributions; a distributed, open infrastructure; equity, diversity, and inclusivity; community building; promoting high-quality research; facilitating evaluation; promoting flexibility and innovation; and cost-effectiveness (Expert Group to the European Commission, 2019, 25–29). In the following section we discuss the main areas where such future infrastructure is needed with regard to SSH, and how it can tackle these challenges.\n\nThe discussion will be based on the core values of scholarly communication as defined in the Vienna Principles, which were prepared by Open Access Network Austria (OANA). We have taken a similar approach, not treating openness as a goal in itself but rather as a means to achieve broader principles, which are implicitly present in the discussions on open science (Kraker et al., 2016, 2). Kraker and colleagues have identified the following deficits in the current scholarly communication system: restricted access to scholarly materials and inhibited collaboration opportunities between the various actors due to closed communication; inefficient production, evaluation, and dissemination processes, which do not fully embrace and exploit the possibilities of digital technologies; a lack of transparency in evaluation, and a lack of access to data and contextual material on research that hinders the reproducibility of results; technical and legal restrictions; and a constraining reward structure (Kraker et al., 2016, 4–5).\n\nAddressing these multiple challenges in a meaningful and effective way requires a coordinated effort by multiple stakeholders; this could be provided by a research infrastructure. As discussed above, the specificity of SSH scholarly communication, its outputs, and practices, calls for dedicated research infrastructures. There are several European Research Infrastructure Consortia attuned to SSH, like DARIAH, focused on arts and humanities (A&H) research data; CLARIN, working with linguistic resources; CESSDA and ESS with social science data; and E-RIHS,10 dedicated to cultural heritage objects. Although scholarly communication remains embedded in the operations of these RIs, none of them address this issue comprehensively. On the other hand, in all disciplines we observe that “[a] growing number of digital publishing initiatives are approaching scholarly communication in new ways and incorporating dynamics of openness, networking, and collaboration into their most basic functions” (Ren, 2013, 745), which calls for coordinated action to streamline these movements and take advantage of their momentum so that scholarly communication is aligned with researchers needs.\n\nThe discussions presented in the sections above have led us to define the following key areas in which an infrastructure can play a vital role in making open scholarly communication a reality in SSH: open access to outputs, discipline-specific genres, evaluation and quality assurance, impact on local societies through multilingualism, scholarly guidance, and the inclusion of various stakeholders. The remainder of this paper addresses the role a research infrastructure could play in these areas in order to assist a systemic change in SSH scholarly communication.\n\nThe first issue is access. Although its crucial component is mere accessibility, i.e., the free, unrestricted, and sustainable dissemination of knowledge within a community; access should be understood more broadly as allowing for the discoverability and reusability of resources (Kraker et al., 2016, 7). This strengthens both the efficient and effective identification of resources and scientific dialogue within and between scholarly communities (ibid.).\n\nThese principles could easily be illustrated by everyday research practices in SSH. While conducting several systematic literature reviews we repeatedly stumbled over the same telling obstacle. While it is relatively easy to search through the vast collections of commercial databases like Scopus or Web of Science with an elaborated, tailored search string that employs a proximity search for various keywords in selected groups of journals, the situation is dramatically different for the open-access content. Although a very valuable contribution to scholarly content discovery has been delivered by the aggregators for both open publishing (e.g. DOAJ, DOAB) and repository content (e.g. OpenAire, CORE), they provide different search options, rarely allow for full-text search, and do not cover the open content scattered throughout smaller, national or regional infrastructures. In consequence, a comprehensive literature review covering open access papers would have to be conducted through dozens of smaller outlets. As Mounier observes, “[g]iven the multiplicity of dissemination platforms that currently exist, researchers have to browse through too many websites if they want to cover all publications in their field or, alternatively, use Google” (Mounier, 2018, 304).\n\nMoreover, large commercial databases tend to underrepresent SSH outputs (Kulczycki et al., 2018); hence, scholars need to resort to web browsing and popular search engines to retrieve interesting content (Dallas et al., 2017), with all the biases that come with search and personalisation algorithms. The variety of resources encompassing “electronic publications, digital libraries, repositories of full-text papers, algorithms, datasets of scientific data, terminological knowledge bases,” thus requires dedicating “greater efforts to discovering, examining, comparing, and integrating these resources” (Marcondes, 2012, 73). So, instead of struggling with scholarly content overflow on the web, we need to follow Marcondes suggestion and harness the potential of the digital environment for better content discovery.\n\nThis process should run in both directions and also enable researchers to “publish intermediate and relevant products of the research process, i.e. raw data, secondary data, and publications, in a way that they are discoverable, meaningfully interlinked, and re-usable by others” (Castelli et al., 2013, 155). Modern technology should allow their work to be sustainably stored, discoverable, and easy to reuse. Hence, the scholarly research infrastructure needs to make these scattered resources available for discovery. In addition, gathering and interlinking such materials and metadata strengthens the transparency of research by providing contextual information that helps in the assessment of the source’s credibility (Kraker et al., 2016, 8).\n\nThere is a clear “need for a single discovery platform dedicated to SSH, indexing all types of content (primary sources, publications, grey literature) in different languages and across different countries” (Mounier, 2018, 304). One step towards this goal was recently made by the Open Access Publishing in European Networks (OAPEN) Library, which moved to a new platform with better browsing options, and improved metadata and API. Another platform is currently being developed in the TRIPLE project, as one of the OPERAS services’ aims is to address these shortcomings by simplifying access to OA materials for researchers and other stakeholders. This platform will be based on the CNRS’s Isidore search engine and will provide the user with a single access point for OA resources, including publications, projects, researchers’ profiles, and data. European diversity is at the heart of the TRIPLE project so it will ensure that SSH research is more discoverable across different cultures and languages: apart from the three languages already managed by ISIDORE (English, French, Spanish), six additional ones will be used: Croatian, German, Greek, Italian, Polish, and Portuguese. Importantly, the initiative is well aligned with the broader EU strategy and will be integrated into EOSC, thus serving as the gateway for SSH open-science outputs in this pan-European endeavour.\n\nA scholarly communication research infrastructure should not restrict, but rather enable, the successful transfer of knowledge in all genres and formats used in a given discipline. This resonates with the European Commission’s ‘holistic approach’ to EOSC, as described earlier, whereby no discipline must be left behind. Hence, we shall embrace disciplinary specificities to successfully attune infrastructures to the actual needs. The results of SSH work are still often mainly published using traditional channels such as monographs and articles (Bulger et al., 2011), and the infrastructure should cater for these and provide tools for their successful dissemination. There are some differences between how these particular genres are supported by current infrastructures given the stress on journal communication in other disciplines. Monographs are often neglected in terms of OA funding and mandates (Deville et al., 2019). Also, the SSH publishing landscape should be taken into consideration, as it consists of scattered, smaller publishing houses (Ferwerda et al., 2016; Tanner, 2016). There are numerous business models and initiatives to support open publication in SSH (Speicher et al., 2018); hence, the role of infrastructures in this respect encompasses both streamlining the initiatives and also advocating for policy measures that are relevant to diverse outputs.\n\nA good example of the role that infrastructures can play in such endeavours is OpenEdition, which works with a freemium model and allows open access to the full texts of books and articles while delivering premium services, such as other file formats, to libraries, thus providing revenue for smaller publishers who want to disseminate their content through digital means. Another example is Language Science Press, which is supported by a network of cooperating institutions, and provides a platform and technical support for a community of linguist authors, engaging volunteer researchers in the process (Nordhoff & Kopecky, 2018).\n\nHowever, an equally important task for research infrastructures is to support scholars in embracing the innovative potential of new technologies. “These potentials include real-time exchange and dissemination, ubiquitous and simultaneous availability of resources, zero marginal cost for dissemination, new workflows, improved reusability of data and results, the ability to process huge volumes of data and new forms of presenting” (Kraker et al., 2016). This applies to the linking of various outputs together, as already discussed in section 3.2 above, but also to allowing innovative and less formalised genres of communication.\n\nAlthough digital publishing has been around for more than two decades, we still think of communication in Gutenbergian terms. The publishing process is slow and focused on the final output, and is thus reluctant to accept versioning. Yet, open, digital communication allows for the rapid exchange of outputs, also in formats “that would have been considered unpublishable by traditional publishers” (Ren, 2013, 74). While an article in a journal freezes the research at a certain point, “the Web opens the workshop windows to disseminate scholarship as it happens, erasing the artificial distinction between process and product” (Priem, 2013, 437). Instead of freezing the outputs, the infrastructure may support the paradigm of the “continual improvement in scholarly publishing” proposed by Juhas et al. (2018), whereby a digitally-enabled service would allow for the text to be enriched, and commented on at any point. Instead of thinking about a scientific paper in static terms we can understand it “as a dynamic document evolving in time, which can have different versions and releases, published online, enabling incremental and continual improvement in analogy to software” (Juhas et al., 2018, 245).\n\nA good example of an innovative genre can be drawn from Open Notebook Science (ONS), which entails providing up-to-date information on research progress by putting the lab notebook online. The audience has access to the raw descriptions of methods, results and research data, and code, which makes the entire research process transparent. The Open Digital Archaeology Textbook is an example of such collaborative work; it is integrated with live open code notebooks that can be reused, altered, or extended. A similar genre is living books (for examples, see the series Living Books About Life), an open access publication that is “open to ongoing collaborative processes of writing, editing, updating, remixing, and comment by readers.”\n\nResearch infrastructures for SSH should support quality assurance and evaluation through transparent peer review practices of the scholarly outputs and metrics used for assessing their impact (Kraker et al., 2016, 9). Historically speaking, “since the 1960s and 1970s, control of the measures of academic prestige—starting with the management of peer review, and extending to the development of metrics—has been silently transferred from communities of academic scholars to publishing organisations” (Fyfe et al., 2017, 13). Nowadays, many institutions and countries base performance indicators on those metrics, disregarding biases (e.g. underrepresentation of SSH works, monographs, and critical editions) and differences in citation practices (e.g. in the humanities the impact of research is achieved more slowly and could be measured differently). There is also a risk of bias, as “article-level metrics may also be skewed by the advantages available to big publishers (such as inclusion in key bibliographical databases, more effective marketing and publicity, or the direct ownership of key analytical tools)\" (ibid.).\n\nSSH should thus evaluate their own products without delegating—as is common practice in STEM—the selection of metrics and indicators to commercial databases. Research infrastructure can gather data for metrics tailored to SSH, providing guidance, support, and services. The European Commission’s Working Group on Rewards under Open Science argues for using such multi-dimensional criteria in evaluation, as researcher’s “merits, achievements, usefulness are a complex set of different variables, impossible to be summarised by a single figure” (Working Group on Rewards under Open Science, 2017, 7). The findings of the working group led to the proposing of the Open Science Career Assessment Matrix (OS-CAM), which represents a range of evaluation criteria for assessing open science activities, i.e., publishing datasets according to FAIR principles, adopting quality standards, contributing to public engagement, sharing results through non-academic outlets, and translating research into other languages (ibid. 4–5). Such an evaluation should also incorporate altmetrics in the evaluation process to assess “the wider societal impact of research articles,” which, “in conjunction with citation-based metrics can lead to a clearer picture of societal impact of scientific research” (Tennant et al., 2016, 7–10). These details could be harnessed by infrastructures, as some repositories already provide altmetrics data for the digital objects they store. Yet, the challenge is to translate them into an instrument for evaluation. Some initiatives, like ImpactStory, are already collecting various data to trace their actual impact (Priem, 2013, 439). An RI could play a role here by collecting scattered data that is adjusted for evaluation purposes in SSH.\n\nApart from evaluation and impact measures, RIs can also support the assessment of scholarly quality. Innovative peer review practices, on the other hand, are meant to make the process more transparent, for example, through revealing the names of reviewers in the open-identity review (Kulczycki et al., 2019a, 3), or to foster the exchange of ideas by making the reviews openly available during the process of the open peer review.\n\nThe openness of the review process is ensured by publishing reports alongside articles and by strongly urging, but not necessarily mandating the disclosure of the identity of reviewers.... The review process is turned into a collaborative effort either through the communication among editors and authors, or through initiating discussion within research communities. (Schmidt & Görögh, 2017, 66)\n\nIt could also replace the pre-publication review with the post-publication open peer review, aligned with the abovementioned “continual improvement process,” in the course of which “any researcher can write a peer review of a version of an already published paper or comment the paper, give the paper a rating etc.” (Juhas et al., 245).\n\nWorks published at F1000Research (e.g. Tennant et al., 2016) serve as a good example of such a peer review practice, in which consecutive versions of the paper are reviewed and the comments are published together with the paper. Although this may sound revolutionary, these proposals are actually recreating a scholarly dialogue as is the case of scholarly debates around controversial texts. For instance, Critical Inquiry opened a forum discussion around Nan Z. Da’s (2019) article, which stirred the digital humanities community and resulted in many responses that critically assessed the work. Scholars can follow and contribute to a discussion that not only focuses on the given work but also expands its scope, proving that access to actual reviews may be genuinely beneficial for the community. The online version of Debates in Digital Humanities (Gold, 2012) serves a somewhat similar purpose by allowing readers to annotate and discuss the content of articles, thus maintaining the debate not only by presenting different perspectives but also allowing the involvement of readership. Similarly, some book projects, like Kathleen Fitzpatrick’s Generous Thinking, or Exploring Big Historical Data: The Historian’s Macroscope by Shawn Graham, Ian Milligan and Scott Weingart, use online tools to publish open drafts of their work and solicit feedback from the community that could improve the final output.\n\nOne of the roles of infrastructure in respect to this lies not only in providing the right procedures and tools to carry out the process, but also in advocating for policy and institutional changes that could recognise and apply the results of these practices. Tennant & Ross-Hellauer (2020) sketched a roadmap for research on peer-review that may identify its shortcomings and biases, leading to the design of shared-data services in this field. Schmidt & Görögh (2017) provided an extensive review of emergent peer review services with such functionalities, for example, Pubpeer.com, a platform for post-publication peer review, where “collaboration between authors, editors and reviewers is strongly encouraged in order to improve the paper and the overall review experience” (Schmidt & Görögh, 2017, 66). Publons.com, on the other hand, records the peer review contributions of authors and adds it to their Open Researcher and Contributor ID (ORCID) (ibid., 67). There are also tools for open annotation that allow additional commentary layers to be appended on top of content, like PaperHive (repository-based), or hypothes-is (web-based) (ibid., 68-69). The latter tool was recently adapted, as an output of the High Integration of Research Monographs in the European Open Science (HIRMEOS) project (discussed later), so it could annotate digital monographs. However, for such approaches and tools to succeed in transforming our practices, we need to ensure their respectability among the researchers who will use them. Such actions should be integrated with reward practices to make sure that they become a source of prestige in a given discipline.\n\nAnother potential role for scholarly communication RI is in supporting publications in local languages, which is crucial in SSH as they are often addressed to local communities. The danger of the monopoly of English is also well recognised in open access publishing. For example, according to Shen, who analysed the impact of Chinese publications, only 24% of journals included in the Directory of Open Access Journals (DOAJ) are published in other languages (Shen, 2017, 2). Academics are under pressure, often amplified by national evaluation policies, not to publish in local languages, which in turn puts non-English-language journals in “danger of losing high-quality academic papers authored by domestic researchers, which will lead to a decline in journal impact and poses a challenge to the survival of such journals” (ibid., 1–2). Such pressures downplay the need for local impact and sustaining knowledge exchange among domestic researchers (ibid., 13). Hence, what is at stake is the future of multilingual scholarly practices and thus the sustainability of local languages as equally valid media for scientific and cultural communication.\n\nSignatories to the Helsinki Initiative on Multilingualism in Scholarly Communication addressed this problem by preparing a set of recommendations for policy-makers, institutions, funders, libraries, and researchers that aimed to “support dissemination of research results for the full benefit of the society,” and “promote language diversity in research assessment, evaluation, and funding systems” (Federation Of Finnish Learned Societies et al., 2019). These ideas resonate with the postulate of “bibliodiversity,” i.e., “cultural diversity applied to the world of books,” proposed by the International Alliance of Independent Publishers (2018), highlighting “the need to encompass a diversity of languages, scientific areas, publication formats, and actors” (Leão & Balula, 2019, 1). Jussieu Call for Open Science and Bibliodiversity, an initiative of scientific publishing stakeholders, views this concept as challenging the power relations in scientific communication by “putting an end to the dominance of a small number among us imposing their terms to scientific communities.” In a follow-up call for action, Shearer et al. enumerated the barriers to bibliodiversity, such as the already discussed dominance of English, the concentration of infrastructures and services, the limited funding models, and the narrow focus on journal-based policy measures (2020, 5–10).\n\nBibliodiversity has, therefore, a clear infrastructural dimension: the accessibility, described earlier as the fundament of open-access communication, should embrace access to multilingual content and allow the diversity in the system to be reconstituted (see: Mounier, 2018, 304; Leão & Balulam, 2019, 4; Shearer et al., 2020, 10–11). The Helsinki Initiative explicitly calls for the protection of national infrastructures that publish and disseminate research results of local relevance. Signatories call for the provision of sufficient resources for these initiatives and support for them in maintaining “high standards of quality control and research integrity” (Federation Of Finnish Learned Societies et al., 2019). A good example of this mission being fulfilled by an infrastructure is provided by SciELO, an electronic library of Brazilian research articles created in response to the underrepresentation of content in Portuguese and Spanish in international databases, and thus in global knowledge exchange. After two decades of operation, it “provides visibility to the journals it hosts in international indexes such as Scopus, WoS, Latindex, and others, resulting in more than 1.5 million COUNTER-certified daily downloads from all over the world in 2017 for the whole platform” (Mounier, 2018, 301). Redalyc.org, a similar project for Latin America, the Caribbean, Spain, and Portugal, currently hosts over 600 thousand articles from 1.3 thousand journals. Both initiatives signify the need for such infrastructures and exemplify a possible solution.\n\nIf scholarly communication is to serve the research community, it has to be led by researchers. The Vienna Principles endorse the idea of the validating progress of scholarly research: “A system of scholarly communication should identify research gaps and highlight fields that need engagement and contribution ... Therefore, [it]should also promote the reproduction and continual validation of existing knowledge” (Kraker et al., 2016, 10). Thus, communication should be attuned to provide the best possible services that can support the advancement of knowledge. Only a scholarly-led, transparent, and researcher-oriented infrastructure will truly address the existing and emerging needs of scholars of the digital age, by basing its activities on the actual, empirically-evidenced needs of the community, not on the pursuit of commercial revenue.\n\nAs Fyfe and colleagues showed, the control over scholarly communication had gradually been handed over to commercial companies and “academics as authors are not yet free to act entirely in the interests of the most efficient system of research communication\" (2017, 18). The logic of commercial revenue is often at odds with scientific needs, especially in terms of open access publishing (Hartley et al., 2018). To be clear, reclaiming scientific communication does not mean excluding commercial players, but rather providing a healthy balance between the commercial interests of publishers, providers, and researchers, which would protect the interests of scholars and smaller players. This would require close cooperation between all stakeholders, such as “governments, funders, universities, learned societies and publishers” (Fyfe et al., 2017, 19). A good example of such collaboration is the 2.5% Commitment, an initiative that encourages academic libraries to commit this percentage of their budgets to supporting the development of open scholarly content and infrastructures (Lewis, 2017). Although Neylon (2018) partially agrees with this proposal, he also points out its weaknesses, which sheds more light on the question of cooperation. The first issue is that of coordination mechanisms, which are crucial for the success of the infrastructure and should be community-driven, as “communities that understand and can work with knowledge products are better placed to support them than either the market, or the state” (2018). There needs to be an understanding of the shared cause, as “infrastructures need to be seen as both sustaining and being sustained by the communities that they serve” (Neylon, 2017b, 8). Second, what emerges from this argument is that fees should be treated as investments, not costs, because they provide “direct benefits to contributors that arise as a side effect of contributing to the collective resource” (Neylon, 2018). The emerging Invest in Open Infrastructure initiative aims to address these issues by building a recommendation system for funders based on a regular census of infrastructural projects to ensure coordination in the infrastructural response to the scholarly needs of various communities.\n\nAs to the benefits for the scholarly community, the HIRMEOS project is an example of an action that could be adopted by a scholarly research infrastructure in the interests of researchers and for the benefit of “small-scale independent partners with limited resources,” allowing them “to cooperate and gain economies of scale by sharing the costs and resource for technical development in order to implement services that are normally accessible only to larger companies who have much greater financial resources and expertise to draw on” (Mounier, 2018, 304). The project developed a set of services dedicated to identifiers certification, annotation, named-entity recognition, and metrics, that could streamline communication between the resources held by smaller institutions.\n\nFinally, scholarly communication is efficient only if it encompasses both communication within and beyond the research community. Kraker et al. define it as a principle of understandability, entailing adapting the communication for “different stakeholder groups inside and outside of academia, by taking into account specific requirements in order to make it more meaningful and allowing for further involvement and participation\" (2016, 8). It is also linked with the principle of collaboration, which “leads to a better understanding of research among stakeholders, and stakeholders can point out research questions that are important to them\" (ibid., 8–9). It is even more crucial in the case of SSH, where knowledge legitimation “demands not only scientific peers but also society” (Kulczycki et al., 2019b, 10).\n\nThere are many types of stakeholders at various levels of scholarly research infrastructure, starting from content creators, and progressing to providers and consumers. Such stakeholders as researchers, publishers, libraries, media, non-profit organisations, and companies can participate in all stages of this process. In order to maximise the usefulness, and thus the impact, of the infrastructure it should be inclusive and flexible enough to accommodate the needs of different groups.\n\nIn this respect some lessons could be learned from The Open and Collaborative Science in Development Network (OCSDNet), established in 2015 in order to foster the contribution of open science to achieve developmental goals. The first goal is to build a common language, i.e., enabling a reflective process “around shared principles and goals, to ensure that everyone is striving towards a common objective” (Hillyer et al., 2017, 29). Second, the authors stress the importance of adjusting goals to suit different stakeholders, as “there is no one-size-fits-all approach to open science, but it is instead a flexible concept that should be adapted to reflect local norms and realities” (ibid., 30). Finally, stakeholders should be empowered by deciding which data should be open to the public (ibid.).\n\nEKT ePublishing, a project developed by the National Documentation Centre in Greece, provides a useful example of how multiple stakeholders can be accommodated by a research infrastructure. The project provided an infrastructure for scholarly eJournals, eBooks, and eProceedings that could be used by non-profit institutional publishers to disseminate their publications (Nafpliotis et al., 2014). Creating a vast community of stakeholders resulted in increasing researchers’ awareness of modern scholarly communication tools and created a demand from scholarly communities (ibid.,114). Another example is TOME (Toward an Open Monograph Ecosystem), a project that brings together different stakeholders in the United States (i.e., researchers, universities, and libraries) to create a sustainable open monograph ecosystem and open SSH scholarship to a wider readership. TOME recognises the deficiencies in the funding model for monographs and aims to subsidise these outputs through institutionally funded faculty book subsidies. The cooperation, under an ongoing five-year pilot project, is based on the universities providing baseline grants for publications and the publishers committing to producing open-access editions of TOME books. The programme also encourages innovative book formats, enabling the incorporation of multimedia, annotation, and commenting tools.\n\n\nConclusions\n\nWe began this article on an optimistic note, recognising the recent change in attitudes towards open access, which could lead to a durable reconfiguration of the scholarly-communication landscape. Let us conclude by addressing some of the threats.\n\nIn a recent blog post that addressed open-access policies during the COVID-19 pandemic, Samuel Moore expressed some scepticism as to whether the impact of the current situation will last, as “paywalls have been lifted temporarily, unilaterally and unsystematically, purely in response to a global pandemic crisis. Once this crisis has passed, or at least when publishers deem it to have passed, there is no suggestion that anything other than business as usual will return” (Moore, 2020). This prediction has already been proven correct, for example through Elsevier’s subsequent announcement that the free access to ‘Coronavirus Research Hub’ will end after 28 October 2020. Moreover, Moore predicts that the crisis may contribute to a further petrification of the communication oligopoly, as smaller publishers may be hit badly by the economic aftermath of the pandemic, which will cripple the budgets in higher education. The situation is becoming even more complicated, because big publishers, as Aspesi and Brand recently observed, may wish to substitute the diminishing subscription revenue with the income from “combined offerings that condition open access to journals upon purchase of other services,” like data analytics or hosting (2020, 574).\n\nThis is a wider problem, too. Mariana Mazzucato has recently called for a rethink of public-private partnerships in the wake of the upcoming economic crisis, as “[t]oo often, these arrangements are less symbiotic than [they are] parasitic” (Mazzucato, 2020). She expressed worries that the global scholarly effort to develop a COVID-19 vaccine may become “yet another one-way relationship in which corporations reap massive profits by selling back to the public a product that was born of taxpayer-funded research” (ibid.). The situation in scholarly communication seems to be awfully similar.\n\nBut there is a way out. Moore posits that we should be emancipated “from the idea that knowledge and education can only ever be understood as a commodity and disseminated in a market;” instead, he suggests we need to recognise that “there should be no financial qualification to either accessing or producing such knowledge, and that both could be supported through non-market and economically just means” (2020). And this is precisely where he sees the role of infrastructural projects: to create “commons-based alternatives that point to a better future,” by reinstalling the academic oversight over the scholarly communication (ibid.). As Aspesi and Brand put it, “[t]he time for the academic community to act in coordination is now” (2020, 577).\n\nHowever, the economy is only one of the complex factors in the scholarly-communication system we have tried to disentangle in this paper. A successful change to, and implementation of, open-science principles in SSH will require a fundamental reconfiguration of the entire landscape that addresses all stakeholders. Kathleen Fitzpatrick provides a tentative list of practices that will have to change: business models; editorial practices; text structures; copyright ownership; archival and preservation practices; relationships between university libraries, presses, technology centers, and academic units; funding models; and the relationship between academia and the surrounding culture (Fitzpatrick, 2011, 13). And the stakes are high, as she concludes: “As new systems of networked knowledge production become increasingly prevalent and influential online, the university and the scholars who comprise it need to find ways to adapt those systems to our needs, or we will run the risk of becoming increasingly irrelevant to the ways that contemporary culture produces and communicates authority\" (ibid.).\n\nAs we have argued in this paper, only a scholarly-driven, inclusive research infrastructure for scholarly communication could be up to the task of addressing these aspects, as this comprehensively addresses both structural and systemic frontiers. The numerous papers, projects, and initiatives discussed here prove that scholars have many ideas about how to improve scholarly communication, along with the specific needs that have to be addressed. A dedicated research infrastructure may eventually make this vision a reality.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgments\n\nWe would like to thank the following colleagues for their suggestions for improving this article as well as for the insights on the OA situation in their respective countries: Marina Angelaki (The National Documentation Centre, EKT); Claire Clivaz (DH+, SIB Swiss Institute of Bioinformatics), Suzanne Dumouchel (TGIR Huma-Num, CNRS); Mateusz Franczak (Institute of the Literary Research of the Polish Academy of Sciences); Elena Giglia (University of Turin); Delfim Leão (University of Coimbra); Pierre Mounier (OpenEdition, EHESS); Aleš Pogačnik (The Research Centre of the Slovenian Academy of Sciences and Arts); Valérie Schafer (C²DH, University of Luxembourg); Judith Schulte (Max Weber Stiftung); Jadranka Stojanovski (University of Zadar); Graham Stone (Jisc); Piotr Wciślik (Institute of the Literary Research of the Polish Academy of Sciences); Lars Wieneke (C²DH, University of Luxembourg).\n\n\nFootnotes\n\n1 E.g. OpenAire Zenodo Community and COVID Gateway, Fairsharing collection, and the European Commission's COVID-19 Data Portal.\n\n2 E.g. OpenBookPublishers.\n\n3 Many thanks to Marina Angelaki (EKT), Pierre Mounier (OpenEdition), Valérie Schafer (C2DH), and Lars Wieneke (C2DH) for their help in grasping the specificities of OA solutions and the scholarly communication landscape in different European countries.\n\n4 See more: http://www.snf.ch/en/researchinFocus/newsroom/Pages/news-200409-national-strategy-on-open-data.aspx.\n\n5 See: https://ec.europa.eu/research/openscience/index.cfm?pg=open-science-cloud\n\n6 For a general introduction to DH see: (Gold, 2012; Schreibman et al., 2016)\n\n7 For discussion see: (Borek et al., 2016).\n\n8 For discussion see: (Pertsas & Constantopoulos, 2017)\n\n9 For further elaboration of this duplicity using the example of contemporary historians, see: (Maryl et al., 2019, chap. 5)\n\n10 Digital Research Infrastructure for the Arts and Humanities (DARIAH); European Research Infrastructure for Language Resources and Technology (CLARIN); Consortium of European Social Science Data Archives (CESSDA); European Social Survey (ESS); European Research Infrastructure for Heritage Science (E-RIHS).\n\n\nReferences\n\nACRL Scholarly Communications Committee: Principles and Strategies for the Reform of Scholarly Communication. 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Reference Source" }	[ { "id": "73616", "date": "09 Nov 2020", "name": "Ioana Galleron", "expertise": [ "Reviewer Expertise Digital text analysis", "French Literature", "Research Evaluation in the SSH" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStarting from an observation of the recent changes in the scholarly communication system (multiplication of forms of engagement due to the multiplication of on-line communication tools and to the digitisation of resources), this paper advocates in a convincing way for the development of an integrated research infrastructure dedicated to the SSH. Such a platform would facilitate the discovery and the reuse of scientific publications and data, would contribute to preserving linguistic, methodological and epistemic diversity, especially in the humanities, and would stimulate the creation and/ or the expansion of interdisciplinary communities. The needs of SSH scholars with regards to scholarly communication are attentively scrutinized and well understood, both through personal engagement of the authors in user-research within the OPERAS project, and thanks to the perusal of an extensive bibliography. However, the paper seems to me, for the moment, insufficiently focused. The first part (p. 3 to 11) appears to propose a rather theoretical discussion about the gaps impeding a streamlined, multi-lingual, multi-purposed scholarly communication, but the second part (p. 12 and subsequent), abandons the “looking at the future” perspective and sounds much more as a kind of monitoring report on the advances towards an integrated platform for scholarly communication. This second part is, indeed, much more focussed on existing projects, products or initiatives (the TRIPLE project, the OpenEdition endeavour p. 12, the HIRMEOS annotation tool p. 13 and 15, EKT ePublishing p. 15, etc.), the larger part of them being under development within OPERAS realm. This oscillation between “should” and “has been done” is somewhat confusing for the reader, and could be avoided through putting forward the second focus from the very beginning, as an opinion paper allows to do. I also recommend to shorten the background part of the paper, that can be deemed in places as breaking open doors (such as the role of communication as enabler of science, or the negative effects of scholarly publishing industry), to the detriment of elaborating more on the solutions provided by OPERAS and some other providers. As an example, the difference between the TRIPLE project and the OAPEN Library should be explored in more depth, so as to avoid giving the impression of competing products that tackle the same issue, or to justify the need for such a competition. Also, when the authors discuss the post-publication peer-review and the commentary layers appended on already published content (p. 13-14), it is unclear what the contribution of the proposed platform would be. Solutions seem to be already in place, where is the problem and what’s the innovative idea we are talking about here? More generally, the discussion about the challenges related to the streamlining of fragmented initiatives for new forms of scholarly communication could be more developed. It may also be useful to revise the discussion about the proposed platform contribution to research evaluation. Too many perspectives and ideas are conflated here, as shown by the following sentence that artificially separates evaluation (probably reduced to “reading of metrics”) and quality: “apart from evaluation and impact measures, RI can also support the assessment of scholarly quality”. The proposed (or existing but incomplete) platform is presented as answering a need (give visibility to alternative communication forms), but also as a lobbying tool that may ultimately result in imposing unwanted obligations to the scholarly community (“blog, tweet or perish”?). In advocating for taking into account all the forms of scholarly communication in career evaluation, the paper underestimates the difficulty for the scholarly communities to think about acceptable standards and rewards for such activities if they are not to become just a form of “posh” or “negatively oriented modern research”.1 Finally, some compelling affirmations are embraced without further discussion. Undoubtedly, the idea of a publication as a “dynamic document evolving in time” is stimulating. Still, it may be worthwhile to look at what happened in the field of scholarly editing, where the digital format and the TEI freed the scholars 30 years ago from the “frozen document” paradigm. As Elena Pierazzo puts it, this led to the creation of a culture of “perpetual prototype(s)”. Nowadays, platforms and consortia struggle with a form of publishing procrastination, with many resources existing in a digital format, but unavailable for the large public, because deemed insufficiently “scholarly” for publication. Alternatively, the “fluid” model may stimulate “premature” publications, whose improvement is promised but never realised for lack of time or other (good) reasons. Therefore, “formal” publication model still has some clear advantages, and I invite the authors of the paper to reflect more on the place it may take in their comprehensive communication eco-system, and on the links, the complementary or the tensions it may have with the other forms of communication.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "77402", "date": "19 Jan 2021", "name": "Amy Brand", "expertise": [ "Reviewer Expertise Scholarly communications", "academic publishing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 applaud Maryl, Błaszczyńska, Szulińska, and Rams for their extremely thorough literature review on scholarly communication infrastructure across the sciences, social sciences and humanities. I learned a tremendous amount from reading the article and approve it for indexing in F1000Research. That said, I believe the article could be made more concise and more forceful than it is currently. As it stands, the article is a description of what’s broken and a statement of need for change, stopping short of providing actionable proposals or a theory of how transformation will proceed.\n\nTo the extent that this article does sketch a vision of the target state of scholarly communications infrastructure, that vision is one based mostly on coordinated planning and centralized management of some sort, even while scholar-led. I remain skeptical about the likelihood of success of monolithic solutions, given what we know about the important role that entrepreneurship plays in innovation, and about how universities and other parts of the research ecosystem function. (I am aware that US research universities are more operationally decentralized than European universities, and that definitely colors my own views.)\n\nMost of this long, ambitious, and at times meandering article is spent reviewing the current state of scholarly communications infrastructure on an international scale, and making the case for an alternative model, one that supports the values of diversity, inclusivity, and multilingualism, and is researcher governed as opposed to market driven. All to the good thus far. However, the reader is then left wanting suggestions for actionable next steps. What form will real progress take, and where will needed innovations come from? Do the values underpinning open knowledge and tools necessarily run counter to competition-based innovation and distributed models of change?\nIf the F1000’s editors and article authors are inclined to go further, I propose that the authors summarize the literature review here more concisely, and devote more space to spelling out how to make progress, however gradual, towards a researcher-driven, inclusive infrastructure for open science in the social sciences and humanities (SSH). From the authors’ perspectives, this may be another article entirely — and that’s fine — but I’ll lay out my own thoughts and questions here in order to help guide potential revisions or future work.\nFirst, I take issue with the implicit premise here that, because the natural sciences have advanced further and faster than SSH fields towards open access and enriched knowledge infrastructures, models in the sciences are the right ones to follow for SSH. If models of effective scholarly communication in SSH are inherently more diverse and inclusive, as the authors substantiate in their review, then it may be that SSH communities can lead the “harder” sciences in defining at least some new models and infrastructures. For example, why would gold open access be an aspiration for SSH when we’ve seen the problems it has perpetuated in the sciences? And perhaps the more qualitative ways in which we evaluate excellence and prestige in SSH knowledge production have advantages over quantitative citation-based methods that natural science communities can learn from.\nNext, the paper could benefit from a clear definition of scholarly communications infrastructure early on. The authors devote lots of space in this paper to defining scholarly communications within the context of formal and informal communication systems broadly defined, but wait until section 1.3 (page 8) to supply the European Commission’s definition of research infrastructures. So too, that definition stops short of a full depiction of well established scholarly communications infrastructures. In addition to physical and computational edifices and publishing platforms writ large, these include persistent identifier systems such as Crossref and ORCID, metadata frameworks and other technical standards to enable interoperability among systems, content sharing norms such as Creative Commons licenses, and even business models themselves.\nWhen you embrace a broader definition of knowledge infrastructures, it expands your levers of change. If you describe the challenge in an entirely top-down way — that, for example, in order to effect desired transformation in scholarly communication you must convince the powers that be to tear down old, expensive edifices and cooperate in designing and funding the construction of new, expensive edifices — you may be setting yourself up for many years of frustration.\nYes, fixing scholarly communications is a large and complex challenge, with multiple stakeholders, and change will take time. But I would urge the authors to investigate the question of to what extent we can let’s accelerate the process by directly giving researchers, societies and mission driven publishers tools to help drive it from the ground up, as it were. What do I mean by that? Standards like the NISO CRediT taxonomy (http://credit.niso.org/) for representing contributor roles in multi-authored works enable scholars to describe their contributions to scholarship in enriched ways. If we empower scholars with new impact narratives, aren’t we enabling change in academic evaluation that wouldn’t be possible otherwise? I think we are, and believe that seemingly small advances like growing the use of alternative metrics, expanding which scholarly outputs are assigned DOIs, what contributions and works are tracked by ORCID, etc. amount to a powerful enabler of desired change. It’s all infrastructure. Similarly, creating publisher-independent signals of quality and rigor, as proposed in our Peer Review Transparency work https://www.prtstandards.org/report, helps lay the foundation for academic evaluation processes that are less dependent on publisher brands and traditional publication genres to signal contribution and prestige.\nAnother grassroots way to increase the variety of “signals” that inform our assessment of scholarly contribution and excellence is via launching new open access publications. There are several examples of SSH outputs and genres in the article. But I am thinking here of new publications like Reviews in Digital Humanities, “a pilot of a peer-reviewed journal that facilitates scholarly evaluation of digital humanities work and its outputs. This may include, but is not limited to: digital archives, multimedia or multimodal scholarship, digital exhibits, visualizations, digital games, digital tools, and digital projects… The journal responds to the challenge of the growth of the number and scale of digital projects outpacing review opportunities in existing journals. As such, it intervenes by bridging a gap in the evaluation of digital projects by arranging for peer review of digital scholarship.” (See https://reviewsindh.pubpub.org/about).\nAnother very promising “bottom up” approach to change is through the systems that universities use to track and report on faculty activities. Expanding the catalog of works in such systems to include pre-prints, non-traditional publication outputs and genres, or informal communications such as blogs is an indirect but powerful way of deconstructing the institutional norms that hold back change in scholarly communications. Such faculty activity reporting systems can also be directly integrated with institutional repositories (Hanrath, 20161).\n\nInfrastructure is a big, weighty concept and tends to be talked about in scholarly communication circles, including in this excellent paper, in terms of top-down change and centralized organization and funding. I believe distributed innovation and distributed institutional investment are key to the sustainability of new scholar-led infrastructures. I’ve tried above to spark awareness of some complementary bottom-up approaches to these challenges, colored by admittedly deeper knowledge of US than European academia. I hope these reflections prove helpful to the authors and the readers of this article.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1265
https://f1000research.com/articles/9-1061/v1	28 Aug 20	{ "type": "Research Article", "title": "Determinants of newborn care utilization in Pakistan: Findings from the Demographic and Health Surveys", "authors": [ "Sathirakorn Pongpanich", "Abdul Ghaffar", "Najma Ghaffar", "Hafiz Abdul Majid", "Sathirakorn Pongpanich", "Najma Ghaffar", "Hafiz Abdul Majid" ], "abstract": "Background: Information on determinants of postpartum care is essential for public health action, yet this information is scarce in Pakistan. Hence, the current study aimed to determine the factors of newborn postpartum care utilization from the Pakistan Demographic and Health Surveys conducted from 2006–2018. Methods: We analyzed data from three rounds of cross-sectional, nationally representative Pakistan Demographic and Health Surveys (PDHS) 2006–07, 2012–13, and 2017–18. Multivariable logistic regression models were applied to explore factors associated with utilization of newborn postpartum care within two months. Results: This study included 5724 women from the 2006–07 PDHS, 7461 from the 2012–13 survey, and 8287 from the 2017–18 survey. The proportion of women receiving newborn postnatal care within the first two months of delivery increased from 13% in 2006–07 to 43% in 2012–13 but dropped to 27% in 2017–18. Respondent’s occupation and prenatal care utilization of maternal health services were common factors that significantly influenced newborn postnatal care utilization within two months. The utilization of postnatal care was greater among women having educated husbands and where the first child was a male in PDHS 2007 round. Higher wealth index and educated respondent had higher postnatal care utilization odds in DHS 2012 and DHS 2018. However, the odds of using postnatal care decreased with the number of household members and total number of children ever born in DHS 2012 and 2018 rounds. Conclusions: There was a general increase in the proportion of women who utilized postnatal care for their newborns during 2006–2013 but a decrease in 2018. The decreased utilization in 2018 warrants further investigation. Improving women’s economic status, education, employment, and antenatal care attendance and reducing parity may increase newborn postnatal care utilization.", "keywords": [ "determinants", "newborn", "postnatal care", "utilization", "Pakistan demographic health survey" ], "content": "Introduction\n\nThe postnatal period – defined as the first six weeks after birth – is the most critical phase in the lives of mothers and their newborns. Approximately 50% of all maternal and neonatal deaths occur within 24 hours after birth, approximately 60% occur during the first week of life, and the rest occur within six weeks after birth1,2.\n\nIn low-income countries, problems such as preterm birth, birth asphyxia, and infections are the leading causes of neonatal deaths2. A striking 99% of the global maternal and neonatal deaths occur in developing countries including Pakistan. Only ten countries, mostly from Asia, account for two-thirds of neonatal deaths. Pakistan reports 7% of global neonatal deaths2 and an estimated 298,000 deaths annually at a mortality rate of 42 per 1000 live births3.\n\nNowadays, we can save the lives of many newborns through interventions that require only simple technology4. These interventions can be delivered effectively by a skilled birth attendant at home from the first 24 hours of life to 6 weeks4.\n\nVarious policies and health programs5 have been introduced in Pakistan since 1990 to reduce maternal and infant mortality. These include the National Health Policy, National Maternal Newborn and Child Health Program, Pakistan initiative for mothers and newborns, People’s primary healthcare initiative, and Lady Health Worker programs5. However, the effectiveness of a program in improving health indicators depends on the utilization and quality of the services provided. Moreover, reproductive status, family influence, community context, and social and cultural beliefs were found to be significant determinants of postnatal care (PNC)6.\n\nStudies have mentioned that Pakistan does not have a national policy on newborn health, and programs aiming on newborn care are partial in coverage7. Most of the previous studies have focused on assessing the utilization of antenatal or antepartum care services, but only a few have tried to look at postnatal care delivery and utilization. We sought to explore the determinants of newborn PNC utilization over the period from 1991 to 2018 in Pakistan.\n\n\nMethods\n\nThis is a secondary analysis of data from three rounds of Pakistan Demographic and Health Survey (PDHS): PDHS 2006/07, PDHS 2012/13, and PDHS 2017/18. The PDHS 1990–91 did not collect data on postnatal care and hence was excluded from this analysis. The PDHS are nationally representative cross-sectional surveys conducted by the Pakistan Bureau of Statistics with technical support from Opinion Research Corporation (ORC) Macro and funding from US Agency for International Development (USAID). The surveys used a multistage cluster sampling design to collect data on reproductive health, fertility, mortality, family planning, nutrition, and health care utilization. Details about the design of PDHS can be found in published reports3.\n\nThis paper is based on previously published data and did not require ethical approval. Permission to use the PDHS datasets was obtained from the DHS Program.\n\nThe study population comprised women of reproductive age (15–49 years) who gave birth during the last five years preceding the surveys. This included 5725 women from the PDHS 2007/08, 7461 from PDHS 2012/13, and 8287 from the PDHS 2017/18.\n\nA conceptual framework proposed by the World Health Organization to explore the social determinants of maternal health was used to consider the various sociodemographic factors that might affect postnatal care utilization. We considered only the variables that were common across the rounds of PDHS.\n\nAs there are no nationwide separate newborn health interventions, newborn services are provided with different health programs also considering low postnatal care utilization in Pakistan; the outcome variable constructed was postnatal care of the newborn within two months8. Independent variables were categorized as shown in Box 1.\n\n\n\n1. Place of residence (urban, rural)\n\n2. Wealth index quintile (poorest, poorer, middle, richer, richest)\n\n3. Number of household members (1–10, 11–20, >20)\n\n4. Number of children younger than five years in household (None, 1–2, 3–4, >4)\n\n5. Births in the last five years (1, 2, >2)\n\n6. Total children ever born (1–2, 3–4, >4)\n\n7. Age of the respondent mother (15–24 years, 25–34 years, 35 years and above)\n\n8. Education status of the respondent mother (No education, primary, secondary, higher)\n\n9. Occupation of the respondent mother (unemployed, employed)\n\n10. Husband’s age (15–24 years, 25–34 years, 35 years and above)\n\n11. Husband’s education (no education, primary, secondary, higher)\n\n12. Husband’s occupation (unemployed, employed)\n\n13. Number of antenatal care (ANC) visits received (no ANC received, <4 visits, ≥4 visits)\n\nDescriptive statistics were used to summarize participants’ characteristics. Because PDHS collected information on postnatal care during the past 5 years, we used the information on the date of birth of the child and receipt of PNC to calculate the coverage of newborn PNC by year for each round of DHS. We used logistic regression models to determine unadjusted and adjusted odds ratios with 95% confidence intervals for the association between the independent variables and newborn PNC. Variables with p <0.1 in the unadjusted (univariable) analysis were included in adjusted (multivariable) analysis. Data were analyzed using SPSS 22.0 software, and P-values < 0.05 were considered to indicate statistical significance.\n\n\nResults\n\nThe mean age of the mothers and her husband was 29.59 and 34 years, respectively, across the three rounds with a little variation as shown in Table 1. The median number of household members was 9, 8, and 8 respectively over the three rounds. However, the number of children ever born was the same in the three rounds. This indicates that the age and family characteristics were nearly the same for Pakistan over the period of 2007–18 as depicted in Table 1.\n\nTable 2 shows the results of univariable analysis of factors associated with newborn PNC utilization. The following factors are significantly associated in positive relation in all three surveys: rural residence (p=<0.001), wealth index (p=<0.001), education status of the respondent (p=<0.001), no prenatal care (p=<0.001), and number of antenatal care visits received (p=<0.001). While the number of household members (p=<0.001), number of children aged 5 years or below in the household (p=<0.001), total children ever born (p=<0.001), and births in the last 5 years (p=0.004, p=0.001) were negatively associated with newborn PNC in the 2013 and 2018 surveys.\n\nOR – odds ratio\n\nRegression analysis was performed for the significant independent variables found in each dataset to control for confounding and derive adjusted odds ratio (Table 3). Multivariable logistic regression analysis unadjusted results are shown in Table 3. In the 2007 survey, occupation of the respondent (p=0.015), husband's education (p=0.006), prenatal care utilization (p=<0.001), sex of the previous child (p=0.002), and number of antenatal visits (p=0.001) were significantly associated with newborn PNC within two months.\n\nOR – odds ratio, NS - Not significant in univariable analysis; not included in the multivariable analysis\n\nIn the 2012 survey, the same factors as those of the 2007 survey, namely, occupation of the respondent (p=<0.001), husband's education (p=0.034), prenatal care utilization (p=<0.001), and number of antenatal visits (p=<0.001), also showed significantly positive associations. In addition, factors such as wealth index (p=<0.001), education of the respondent (p=<0.001), and husband occupation (p=0.004) also showed positive associations. However, the number of household members (p=0.042) and total children ever born (p=0.001) were negatively associated with newborn PNC utilization.\n\nIn the 2018 survey, receiving postnatal care within two months of birth was significantly associated with the occupation of the respondent (p=<0.001), education of the respondent (p=<0.001), wealth index (p=0.001), number of antenatal visits (p=<0.001), and sex of the first child (p=<0.011). However, women who had more total children ever born (p=<0.001) were less likely to receive newborn care utilization.\n\n\nDiscussion\n\nThe current study aimed to explore newborn postnatal care determinants from three subsequent rounds of DHS 2006–07, 2012–13, and 2017–18 in Pakistan. Various sociodemographic factors along with household characteristics and utilization of antenatal care services determine the utilization of PNC, in general, from past literature9,10. We also extracted relevant data from three rounds of PDHS pertaining to potential factors, which could affect the utilization of newborn postnatal care in this study as described in the methods section.\n\nThe number of respondents for the three rounds of PDHS was 5724, 7461, and 8287, respectively, for the years 2007, 2013, and 2018, respectively. It was found that the utilization of PNC for mothers and newborn within two months following delivery increased from 13% to 43% in 2013 and the subsequently reduced to 27% in 2018. Similarly, the utilization of PNC within 24 hours increased from 7% in 2007 to 33% in 2013 and reduced to 7% in 2018 (Table 1). This non-linear pattern in service utilization could be due to distinct geographical regions in which the survey was carried out. During DHS 2006–07, data were collected from four regions: Punjab, Sindh, Khyber Pakhtunkhwa (KPK), and Balochistan. In the next round of DHS 2012–13, along with Punjab, Sindh, and Balochistan; three other districts of KPK, Gilgit Baltistan (GB), and Islamabad were included. Similarly, in DHS 2017–18, seven regions, namely, Balochistan, Punjab, Sindh, KPK, GB, Azad Jamu and Kashmir, Islamabad Capital Territory and Fata constituted the sampling frame. The sociodemographic characteristics along with the distribution of health services and quality would have been different, which may have resulted in varied PNC utilization levels across sample11. The study conducted by Iqbal S et al. also indicated variability in PNC service utilization across different regions from where the data were collected10.\n\nAmong all the sociodemographic determinants included in this study, the occupation of the respondent and the utilization of ANC (Table 4) were found to be significantly associated with newborn PNC utilization within two months after delivery across all the three rounds of DHS. It was found that the odds of using PNC was 1.26 times more among women who were employed than among unemployed mothers. Previous studies conducted in Pakistan10 and from other neighboring countries also showed a positive association among mothers with employment12–14 (Table 3). However, wealth index of household15,16, education status of the respondents15,16, and total children ever born16,17 were significantly associated with newborn PNC for two DHS rounds: 2012–13 and 2017–18. Utilization of maternal health services, especially antenatal or prenatal care, was also a strong predictor of PNC throughout all PDHS; it is evident from the literature that ANC is the entry point for the utilization of maternal health services during and after pregnancy18–21. The respondent’s occupation and utilization of antenatal care were found to be associated with newborn PNC from the DHS 2006–07 and 2012–13 data in previous studies8,10.\n\nThis study indicates that the occupation of the respondent and prenatal care services utilization by respondents influenced the utilization of newborn PNC across all the three rounds of the PDHS. Other common factors such as wealth index, education of the respondent, and total number of children ever born also influenced the uptake of newborn PNC services. Another strength of this study is the number of sociodemographic and outcome variables included, which is far higher than those included in previous studies8,10.\n\nHowever, we could not see determinants of newborn PNC utilization due to data unavailability on PNC from the 1990–91 PDHS. Moreover, the data on the reasons for not getting the PNC by the women after delivery were not available.\n\nThere are limitations to the data, which we noticed while conducting this analysis. These limitations may be considered as recommendations for further improving the scope of the DHS. There was no information available on the distribution of health services in the DHS data. This information is important, as differential health service availability and accessibility directly influence PNC utilization, which we could not explore in the current study. In future research, the data could be used to link the availability and accessibility of services with their utilization. The second limitation in data, we noticed, was regarding the quality of PNC, which was not captured in the DHS questionnaire. This question is crucial to explain the reducing uptake of newborn PNC services, especially in 2017–18. The DHS also did not contain any information on the domains for which PNC is provided, which is again important for improving the health of the mother and the newborn.\n\n\nConclusions\n\nThis study reveals that women being employed, utilization of ANC or prenatal services, wealth index, and education of respondents or their husbands increases the uptake of newborn PNC utilization. An increasing number of children ever born to women are less likely to have newborn PNC utilization. Hence, there is a need to address the issues of improving economic status, education, employment of the women, and population control to increase newborn PNC utilization. Similarly, interventions that increase the coverage and quality of ANC services will also increase the utilization of newborn PNC among women in Pakistan.\n\n\nData availability\n\nThe data for this study is owned by the DHS Program. The Individual Recode datasets for the PDHS 2006–07, 2012–13 and 2017–2018 were used for this study and can be obtained here: https://www.dhsprogram.com/data/available-datasets.cfm?ctryid=31\n\nThe electronic data is available from the DHS Program under its terms of use. Before downloading the data, users must register as a DHS user for reasons laid out on the DHS Program website and dataset access is only granted for legitimate research purposes.", "appendix": "Acknowledgements\n\nWe would like to thank Chulalongkorn University and the Higher Education Research Promotion and National Research University Project of Thailand as well as Dr Najma Ghaffar Hospital Quetta, Pakistan, for providing financial support for this publication.\n\n\nReferences\n\nSay L, Chou D, Gemmill A, et al.: Global causes of maternal death: a WHO systematic analysis. Lancet Glob Health. 2014; 2(6): e323–33. PubMed Abstract \| Publisher Full Text\n\nLozano R, Naghavi M, Foreman K, et al.: Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2095–128. PubMed Abstract \| Publisher Full Text\n\nDemographic P: Health Survey (PDHS)(2017-18). National Institute of Population Studies, Islamabad, Pakistan, and United States Agency for International Development (USAID). 2018. Reference Source\n\nWorld Health Organization: WHO recommendations on postnatal care of the mother and newborn. World Health Organization; 2014.\n\nKhan A, Kinney MV, Hazir T, et al.: Newborn survival in Pakistan: a decade of change and future implications. Health Policy Plan. 2012; 27(suppl 3): iii72–87. PubMed Abstract \| Publisher Full Text\n\nSomefun OD, Ibisomi L: Determinants of postnatal care non-utilization among women in Nigeria. BMC research notes. 2016; 9(1): 21. 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PubMed Abstract \| Publisher Full Text\n\nChakraborty N, Islam MA, Chowdhury RI, et al.: Determinants of the use of maternal health services in rural Bangladesh. Health Promot Int. 2003; 18(4): 327–37. PubMed Abstract \| Publisher Full Text\n\nSingh PK, Kumar C, Rai RK, et al.: Factors associated with maternal healthcare services utilization in nine high focus states in India: a multilevel analysis based on 14 385 communities in 292 districts. Health Policy Plan. 2014; 29(5): 542–59. PubMed Abstract \| Publisher Full Text\n\nJat TR, Ng N, San Sebastian M: Factors affecting the use of maternal health services in Madhya Pradesh state of India: a multilevel analysis. Int J Equity Health. 2011; 10(1): 59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRai RK, Singh PK, Kumar C, et al.: Factors associated with the utilization of maternal health care services among adolescent women in Malawi. Home Health Care Serv Q. 2013; 32(2): 106–25. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70514", "date": "10 Sep 2020", "name": "Korravarn Yodmai", "expertise": [ "Reviewer Expertise reproductive health" ], "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 a part of the introduction, the gap of knowledge based on current studies should be identified.\n\nSome details of methods such as target population, where was data collected such as there was collected data from community or health facility. It may important to present the data collecting process (briefly).\n\nInterpretation of the result should explain more detail the magnitude of the result rather than explain just the association.\n\nIn the discussion, the authors should explain more detail about PNC services in each area. What is a barrier of service in those areas, even they may use the same health policy or need to succeed in the same indicators?\n\nAccording to the introduction part, this study has filled the gap of knowledge or not.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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": [ { "c_id": "6043", "date": "21 Oct 2020", "name": "ABDUL GHAFFAR", "role": "Author Response", "response": "In response to comment 1, from Korravarn YodmaiKnowledge gap addedIn response to comment no 2 from Korravarn Yodmai,DHS has standard protocols and data method process is already briefly mentioned, for more details reader are referred to reference below.In response to comment 3, from Korravarn YodmaiMore details added, OR added in results descriptionIn response to comment 4, from Korravarn YodmaiThese data represent entire Pakistan and barrier in different provinces may not be possible to consider in discussion section as we don’t have data about barriers in DHS surveys. Details about PNC services are provided in the introduction and were not included in discussion section due to repetition, and also it is mentioned that currently there is no separate newborn health policy in PakistanResponse to comment 5 from Korravarn YodmaiYes the study fulfilled the knowledge gap, as conclusion describes the findings which are consistent in all three surveys like education of women and utilization of prenatal services" } ] }, { "id": "70513", "date": "07 Oct 2020", "name": "Ejaz Ahmad Khan", "expertise": [ "Reviewer Expertise Systematic reviews", "epidemiology", "burden of disease" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of the manuscript need to aligned with the objective of the study i.e., post natal care and NOT the postpartum case.\n\nAbstract needs to be rewritten in proper academic English with flow.\n\nAuthors leave the conclusion with a question about 2018 data, which they should have had discussed in their discussion section before concluding their remarks.\n\nIntroduction: Pakistan is a Low-middle income country and NOT a low income country as per the World Bank ranking. Introduction needs more robust literature cited.\n\nMethods need to be in the past tense and so should be the results.\n\nResults need to be rewritten in a proper sequence and flow. The DHSs analysed year-wise need to be written together for each background factor.\n\nDiscussion must discuss the most important results as per the objectives of the study, and critique 2018's unexpected results with good literature support.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1061
https://f1000research.com/articles/9-1261/v1	20 Oct 20	{ "type": "Study Protocol", "title": "Protocol for a proof-of-concept observational study evaluating the potential utility and acceptability of a telemedicine solution for the post-anesthesia care unit", "authors": [ "Thaddeus P. Budelier", "Christopher Ryan King", "Shreya Goswami", "Anchal Bansal", "Stephen H. Gregory", "Troy S. Wildes", "Joanna Abraham", "Sherry L. McKinnon", "Amy Cooper", "Ivan Kangrga", "Jackie L. Martin, Jr.", "Melissa Milbrandt", "Alex S. Evers", "Michael S. Avidan", "Thaddeus P. Budelier", "Christopher Ryan King", "Shreya Goswami", "Anchal Bansal", "Stephen H. Gregory", "Troy S. Wildes", "Joanna Abraham", "Sherry L. McKinnon", "Amy Cooper", "Ivan Kangrga", "Jackie L. Martin, Jr.", "Melissa Milbrandt", "Alex S. Evers" ], "abstract": "Introduction: The post-anesthesia care unit (PACU) is a clinical area designated for patients recovering from invasive procedures. There are typically several geographically dispersed PACUs within hospitals. Patients in the PACU can be unstable and at risk for complications. However, clinician coverage and patient monitoring in PACUs is not well regulated and might be sub-optimal. We hypothesize that a telemedicine center for the PACU can improve key PACU functions. Objectives: The objective of this study is to demonstrate the potential utility and acceptability of a telemedicine center to complement the key functions of the PACU. These include participation in hand-off activities to and from the PACU, detection of physiological derangements, identification of symptoms requiring treatment, recognition of situations requiring emergency medical intervention, and determination of patient readiness for PACU discharge. Methods and analysis: This will be a single center prospective before-and-after proof-of-concept study. Adults (18 years and older) undergoing elective surgery and recovering in two selected PACU bays will be enrolled. During the initial three-month observation phase, clinicians in the telemedicine center will not communicate with clinicians in the PACU, unless there is a specific patient safety concern. During the subsequent three-month interaction phase, clinicians in the telemedicine center will provide structured decision support to PACU clinicians. The primary outcome will be time to PACU discharge readiness determination in the two study phases. The attitudes of key stakeholders towards the telemedicine center will be assessed. Other outcomes will include detection of physiological derangements, complications, adverse symptoms requiring treatments, and emergencies requiring medical intervention. Registration: This trial is registered on clinicaltrials.gov, NCT04020887 (16th July 2019).", "keywords": [ "Telemedicine", "Post-Anesthesia Care Unit", "Protocol", "Proof-of-Concept", "Observational Study" ], "content": "Introduction\n\nAfter invasive procedures in the operating room (OR) or other procedure rooms, patients are usually transferred to a post-anesthesia care unit (PACU) for high acuity monitoring. The PACU period is important for patients, especially since they often are still in a vulnerable state1,2. Patients are prone to peri-procedural and post-anesthetic complications including dehydration, anemia, coagulopathy, bleeding, hypothermia, delirium, respiratory depression, airway obstruction, bronchospasm, hypotension, kidney injury, arrhythmias, metabolic acidosis, hypoxemia, glucose and electrolyte abnormalities, atelectasis, and pulmonary edema3,4. These complications must be recognized and appropriately managed by PACU clinicians. Furthermore, PACU clinicians need to identify and manage patients’ adverse symptoms including pain, nausea, urine retention, weakness, and itching, which are common after invasive procedures, whether with or without general anesthesia.\n\nThe ideal PACU environment provides close monitoring and prompt rescue for peri-procedural complications, while also efficiently transferring patients to their next phase of care. For example, when patients deteriorate in the PACU, it is important to recognize this early, intervene appropriately, and arrange transfer to a higher acuity area, such as an intensive care unit, when warranted.\n\nPACU clinicians are responsible for several clinical and organizational tasks5 including patient monitoring and treatment, promoting patient throughput, conducting hand-offs to and from the PACU, and documenting patient care information during the recovery period. As a result, PACU nurses and doctors can feel overwhelmed, and may not always be able to treat symptoms adequately, diagnose physiological derangements accurately, and detect patient deterioration expeditiously. Furthermore, in this high-pressure, high-turnover environment, communication among clinicians is often compromised, resulting in unreliable care coordination. Patient satisfaction with PACU care varies, as the recognition and prompt treatment of symptoms depends on the availability of assigned clinicians.\n\nThe necessity of operating room throughput creates a constant pressure on PACU clinicians to discharge patients rapidly, sometimes before they have recovered sufficiently. This workflow pressure can potentially compromise quality of care and patient safety. Nurses provide the majority of PACU care, typically for no more than two patients at a time during the initial phase of PACU care, in accordance with the American Society of PeriAnesthesia Nurses (ASPAN) guidelines6. Furthermore, physicians with competing responsibilities often provide oversight in the PACU. For example, a physician who has responsibility for patient assessment and management in the PACU is often simultaneously overseeing anesthetic care in operating rooms or other procedural suites. Surgical clinicians also participate in aspects of PACU care, but are often simultaneously engaged in surgical care of other patients. In addition, the coverage and oversight models can vary considerably across different PACUs, and even within the same PACU over the course of a single day. This is in stark contrast to other high acuity patient care settings, such as operating rooms and intensive care units, where roles and responsibilities of various clinicians are well defined, and staffing models are established.\n\nIn this protocol, we describe a proof-of-concept study in perioperative telemedicine that aims to demonstrate the (i) potential utility and (ii) acceptability of integrating telemedicine into the PACU environment. This proof-of-concept study will be conducted in the PACU located in Parkview Tower in Barnes-Jewish Hospital (BJH). If this proof-of-concept proves to be successful, we intend subsequently to show the impact of such a telemedicine solution on safety, quality of care, efficiency, and ultimately postoperative outcomes. Our specific aims for the proposed proof-of-concept study are:\n\nWe hypothesize that clinicians in the telemedicine center for the PACU will:\n\n1a. Detect physiological derangements and complications\n\n1b. Identify adverse symptoms requiring treatment\n\n1c. Recognize situations requiring emergency medical intervention\n\n1d. Determine when patients are ready for PACU discharge\n\n1e. Participate meaningfully in hand-off activity from the OR to the PACU\n\nWe will assess attitudes of key stakeholders towards a telemedicine center for PACU. The key stakeholders will include PACU nurses, anesthesiologists, surgeons, hospital administrators, and PACU-telemedicine center clinicians.\n\n\nMethods\n\nThis proof-of-concept study has been approved and granted a waiver of informed consent for all patients and a waiver of written consent for participants enrolled by the Human Research Protection Office at Washington University in St. Louis (HRPO#201901180) and is registered at clinicaltrials.gov (NCT04020887, 16th July 2019). It is infeasible to conduct this proof-of-concept study without a waiver of consent. Additionally, this study has been determined to involve no more than minimal risk to participants, as study participation would not deviate from or delay current standards of peri-anesthesia care.\n\nThe study will be conducted at Barnes-Jewish Hospital (BJH) in St. Louis, Missouri, a large tertiary care academic medical center.\n\nWe will conduct a single center prospective before-and-after proof-of-concept study to evaluate a telemedicine center for the PACU. Adults (18 years and older) undergoing elective surgery at Barnes Jewish Hospital in St. Louis, Missouri will be enrolled. Approximately 500 patients will be enrolled in this study over a six-month duration, with an estimated 250 patients allocated to each phase of the trial. The first phase is an Observation phase and the next phase is an Interaction phase. More information on these phases is provided below.\n\nBoth the “Good ReseArch for Comparative Effectiveness” (GRACE) checklist7 and PICOTS framework8 (Table 1) were used in designing this study. The conduct and reporting of this observational study will follow the “Reporting of studies Conducted using Observational Routinely-collected health Data” (RECORD)9 statement and the “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE)10 statement guidelines for reporting observational studies.\n\nPACU, post-anesthesia care unit.\n\nTwo bays in Barnes Jewish Hospital (BJH) in St. Louis, Missouri, will be equipped for telemedicine interaction (Figure 1). Video cameras and monitors have been installed in each of these bays to allow for remote monitoring, as well as two-way video communication during the interaction phase. The telemedicine center is staffed by attending anesthesiologists along with certified registered nurse anesthetists (CRNAs), anesthesiology residents, and student registered nurse anesthetists (SRNAs), and is currently providing evidence-based support to clinicians in the operating rooms11–14.\n\nA station in our telemedicine center will be designated for monitoring patients assigned to the two PACU bays during this proof-of-concept study. Patient information flows to the telemedicine center through the electronic health record (EHR), physiological waveform tracings, and direct video observation. A version of AlertWatch® (AlertWatch, Ann Arbor, Michigan) decision-support software, customized for the PACU environment (Figure 2), will assist clinicians in the telemedicine center in performing core PACU-related functions remotely (see Aim 1).\n\nThe assessments in relation to PACU functions will include:\n\n1a. Detection of physiological derangements in PACU patients\n\n1b. Identification of symptoms requiring treatment in PACU patients\n\n1c. Recognition of situations requiring emergency medical intervention\n\n1d. Determination of patient readiness for PACU discharge\n\n1e. Participate meaningfully in hand-off activities\n\nObservation phase (three months). In the first three months (the Observation phase) of this proof-of-concept study, a telemedicine center for the PACU will monitor patients assigned to two PACU bays. Both the telemedicine center and nurses caring for patients in the PACU bays will separately document physiological derangements (Table 2), treatable symptoms (Table 3), or a situation requiring urgent medical intervention (telemedicine center only; Table 4) during the PACU stay. Clinicians in the telemedicine center will assess when the patient meets discharge criteria, based on the modified Aldrete scale15 and their clinical judgment. They will document the time that discharge criteria are met, the modified Aldrete scale score at this time, and any additional relevant information. If clinicians in the telemedicine center judge that they are unable to determine a patient’s readiness for discharge, they will document their reasons (Table 5). Clinical judgment will be used in determining appropriate discharge parameters for patients with pre-existing conditions. The telemedicine center clinicians will document each patient’s information outlined in Table 2–Table 5 directly into REDCapTM (a secure web application for managing online surveys and databases) and AlertWatch. After a patient has been discharged from the PACU, the PACU nurse will fill out a form providing information outlined in Table 2–Table 5. This includes information on physiological derangements, treatable symptoms, and discharge information. This form will be collected by the research team, and the information in the form will be documented in REDCap. During this phase of the study, clinicians in the telemedicine center will not communicate with clinicians in the PACU (nurses or physicians), unless there is a patient safety event.\n\nHR, heart rate; MAP, mean arterial pressure; PACU, post-anesthesia care unit.\n\n*Only the telemedicine center will document the detection of urgent situations. OR, operating room; PACU, post-anesthesia care unit.\n\nBP, blood pressure; PACU, post-anesthesia care unit.\n\nInteraction phase (three months). In the three months following the observation phase, clinicians in the telemedicine center will interact with patients and clinicians associated with the designated PACU bays using audio-visual technology. PACU clinicians and clinicians in the telemedicine center will become a “fused” team, and the telemedicine center will continue to document information on physiological derangements (Table 2), treatable symptoms (Table 3), situations requiring urgent medical intervention (Table 4), and discharge readiness (Table 5).\n\nThe telemedicine center clinicians will assess patients’ discharge readiness throughout their PACU stay. A modified Aldrete scale along with clinical judgment will guide the telemedicine center clinicians in determining readiness for discharge (Table 5). After discharge readiness has been determined by the telemedicine center, the attending anesthesiologist in the telemedicine center will document discharge readiness in AlertWatch and REDCap, and contact the relevant anesthesiologist. The telemedicine center for PACU will document when this information was communicated. At any point clinicians in the telemedicine center might decide to contact PACU clinicians (nurse or physician) if they have specific concerns regarding patients. If the telemedicine center clinicians feel that they cannot adequately assess a patient’s clinical status, they will notify the PACU clinicians. This will be documented together with a relevant explanation (Table 5).\n\nFinal determination and sign-off regarding discharge suitability will be made by the anesthesiologist in the PACU. With this proof-of-concept research project, there will be no change in relation to which clinicians have responsibility for decision making and clinical care. The telemedicine center clinicians will not write any orders in the medical record, and will provide opinions only to physicians and nurses who are responsible for patient care in the PACU. The responsibility to call for help when patients are deteriorating will remain with the PACU nurses, as is the current standard in that environment. The notion is that the telemedicine center will not lead to any decrement in the care that PACU patients are currently receiving from nurses and physicians in that environment.\n\nThe successful integration of the telemedicine center into each of the core PACU functions will be measured in the following ways:\n\nPhysiological derangements – Success will be measured (in the observation phase) by the ability of the telemedicine center clinicians to identify physiological derangements as they are occurring in the PACU. The extent to which the telemedicine center clinicians can identify these physiological derangements will be measured by comparing PACU nurse and telemedicine center assessment surveys16 for each patient (Figure 3).\n\nSymptom identification and management – Success will be measured (in the observation phase) by the ability of the telemedicine center clinicians to identify treatable symptoms as they arise in the PACU. The extent to which the telemedicine center clinicians can identify these treatable symptoms will be measured by comparing PACU nurse and telemedicine center assessment surveys16 for each patient (Figure 3).\n\nEmergency situations – Success will be measured (in the observation phase) by the ability of the telemedicine center clinicians to identify situations requiring emergency medical intervention as they are occurring in the PACU. By construction, any time the telemedicine center feels that an emergency situation is present, preserving patient safety mandates contacting the bedside clinician. During each such contact, the telemedicine center clinician will ask if the PACU nurse was already aware of the situation, disagreed with the assessment, and had already spoken to the supervising physician regarding it. The occurrence of emergency medical situations will be extracted from the electronic health record, and the agreement between telemedicine center and PACU nurse assessments will be quantitated.\n\nPACU discharge – Success will be measured by the ability of the telemedicine center clinicians to identify when patients are ready for discharge (observation phase [without communication] and interaction phase [active communication with patient and PACU clinicians]) (Figure 3). The impact of the telemedicine center on this key function will be examined based on feedback from key stakeholder focus groups (interaction phase; see Aim 2). The difference between sign-out times in the observation and the interaction phases will be compared.\n\nHand-off activity. The telemedicine center clinicians will participate in hand-off activities to and from the PACU. This includes ensuring appropriate transfer of information from operating rooms to the PACU. The telemedicine center clinicians will remotely join the hand-off conversations, and review patients’ medical history and intraoperative course to identify potential missed transfer of information.\n\nDuring the observation phase, the telemedicine center clinicians will observe the hand-off workflow, gain familiarity with the current hand-off routine, and identify possible areas of missed information transfer where the telemedicine center clinicians may have adjunct utility. An example of potential adjunct utility would be communicating the importance of appropriate insulin and glucose management in the PACU for a patient with type I diabetes.\n\nIn the interaction phase of the study, the telemedicine center clinicians will try to fill gaps in information transfer during the hand-off procedure. In addition to remotely joining the hand-off conversation, the telemedicine center clinicians will share pertinent additional patient or procedural information, especially if this could inform the patient’s PACU medical treatment. After the completion of the hand-off procedure, the PACU nurse who interacted with the telemedicine center clinicians will complete a short survey16 to assess the telemedicine center’s involvement in that patient’s transfer of care.\n\nThe successful integration of the telemedicine center clinicians’ hand-off activity will be measured in the following way:\n\nHand-off activity – Success will be measured (in the interaction phase) by the ability of the telemedicine center clinicians to join and contribute meaningfully to the hand-off discussion. The impact of the telemedicine center clinicians on this key function will be determined from feedback from key stakeholder focus groups (see Aim 2) and PACU nurse surveys16. These stakeholders will comment on utility of the telemedicine center’s involvement and provide suggestions for improvement. A binary assessment of hand-off adequacy will be provided by the PACU nurse hand-off survey16. The telemedicine center clinician will use a hand-off content checklist16 to record the number of mandatory items not discussed and number of recommended non-mandatory items discussed. For each of the observation and intervention phases, for 50 randomly selected cases a trained observer (not the participant in hand-off) will use the hand-off communication assessment tool16 of Weinger and others17 substituting the telemedicine center hand-off content checklist. A run-in phase of one month during the intervention will elapse before any of the 50 detailed communication evaluations are performed.\n\nWe will assess the attitudes of key stakeholders in order to identify barriers to and facilitators for implementation of a telemedicine center for the PACU. (Figure 3)\n\nStakeholder focus groups. We will conduct focus groups with stakeholders to gain insights regarding their perceptions of barriers and facilitators related to the above-noted PACU functions before and after the implementation and use of a telemedicine center for the PACU. We will also gather perspectives from the stakeholders on the role and impact of the telemedicine center on their individual and team workflows in the PACU and between units during care transitions. Focus group participants will include nurses, anesthesiologists, surgeons, hospital administrators, and PACU telemedicine center clinicians. Our focus groups will be homogeneous in order to understand the clinician workflow based on their professional role, and their use of the telemedicine center in supporting their role and responsibilities. Each focus group will comprise five to six participants. This will allow in-depth discussions of the workflow problems and unintended consequences caused by the implementation and use of the telemedicine center for the PACU. The focus group sessions will be guided by a semi-structured interview guide focused on the following themes: (1) PACU core functions, (2) PACU patient workflow, (3) PACU clinician activities and tasks, (4) tools and technologies used to support the PACU workflow, (5) major barriers to PACU functions, (6) use of a telemedicine intervention as a potential mechanism to support effective and efficient functioning of the PACU. We plan to conduct 6-8 focus group sessions (four pre-intervention during observation phase, and four post-intervention during interaction phase) or until data saturation is attained.\n\nPatients are allocated to PACU bays according to the discretion of the nurse in charge of the PACU. Currently, approximately two patients per day are cared for in each bay in the participating PACU. Therefore, the telemedicine team will monitor approximately four patients per day over the course of the proof-of-concept study. We estimate that 500 patients will be included in this proof-of-concept study (250 per monitored phase) (Figure 4).\n\nPrimary outcome. This is a proof-of-concept study and will only address surrogate outcomes. The primary outcome (time to PACU discharge readiness) will use two comparison groups. First, historical controls will be drawn from the observation phase. A propensity score for inclusion into the study will be generated as a function of (minimally) surgery performed, day of week, time of day, age, and sex. 3:1 matched control patients will be included. The outcome will be analyzed with interrupted time series methods with flexible functions of calendar time used to adjust for secular trends; the study hypothesis is a non-zero discontinuity at telemedicine implementation. That is, if Yi is the outcome for the ith patient at time ti with covariate vector Xi, while the implementation time is t0, and I() is the indicator function,\n\nwhere f1 and f2 are smooth functions. Other patient factors known to strongly influence PACU length of stay (age, ASA physical status, number of co-morbidities, morbid obesity, obstructive sleep apnea, surgical specialty, primary anesthesia type, history of postoperative nausea and vomiting, preoperative pain, and scheduled case duration) will be included as covariates. The minimization criteria will be least squares or trimmed least squares or other robust criteria if there are substantial outliers. Outcomes will be examined for residual auto-correlation, and if non-negligible, auto-correlation robust standard errors (such as Newey-West errors) and an ARIMA model will be reported. Confidence intervals will be generated by non-parametric bootstrap sampling where possible. No adjustment will be made for matching, but bootstrap methods will respect the matched “units.” P-values will be generated both by likelihood ratio tests and by using non-deployment times as a null distribution; that is, we will run the same analysis looking for discontinuity at times remote from the true implementation time. We will conduct sensitivity analyses with transformations of the outcome variable. We will use an excluded run-in period of one month as a sensitivity analysis. Because hospital length of stay is unlikely to be meaningfully affected by a telemedicine center for the PACU, but does track overall acuity and surgical severity, we will use hospital length of stay as a control time series.\n\nContemporaneous control patients will also be gathered. A propensity score for study inclusion will be generated as a function of (minimally) surgery performed, calendar time, time of day, age, and sex. 3:1 matched control patients will be included. Differences will be analyzed by t-tests using permutation calibration. Confidence intervals on the difference in mean time to discharge readiness will be generated by nonparametric bootstrap. We will include a sensitivity analysis where the interrupted time series method includes historical and contemporaneous control patients with the treatment indicator T for study patients,\n\nBased on data from our EHR, patients are currently in PACU for a mean of 150 min (standard deviation = 65 min) before they are determined to be suitable for discharge. Based on these values, with 250 patients in each phase (observation and interaction), this observational before and after study will have >70% power with an alpha <0.005 and > 90% power with an alpha <0.05 to detect a mean decrease in 20 min (from 150 min to 130 min) to PACU discharge readiness time. Statistical testing will be with appropriate statistical software. Using non-parametric bootstrap of historical data and a 3:1 control sampling ratio, the average standard error on the difference in means under the null hypothesis was 5.5 minutes, giving an anticipated 95% confidence interval width of 22 minutes. A somewhat larger standard error will be encountered when adjusting for covariates or secular trends; however, this suggests that we will be able to resolve differences in PACU readiness times of 20–25 minutes. This difference of approximately a third a standard deviation is usually regarded as a “small-moderate” sized effect.\n\nSecondary outcomes. Hand-off quality assessment from the PACU nurse binary survey response will be analyzed using a logistic regression model adjusting for surgical service, age, and sex. Because observation resources are required for hand-off evaluations, no matching will be performed, and no contemporaneous controls will be gathered. Adjusted differences in rates of inadequate hand-off will be summarized with 95% confidence intervals and model-based p-values. Observed reported hand-off communication quality will be presented as a purely descriptive result.\n\nThe accuracy of physiologic, symptom, and status assessments is less straightforward to analyze. At the heart of the proposal is the belief that telemedicine assistance will detect some abnormalities not caught (or caught later) by the bedside team and detect that the patient has adequate status for PACU discharge before the bedside nurse. Using the bedside assessment as a gold standard is therefore limited. Similarly, although we believe that abnormalities detected by either bedside or telemedicine are unlikely to be false positives, we have no way of assuring that. We also cannot reliably determine the timing of the bedside nurse’s detection of an abnormality, as they may document it much later if they believe it does not require an immediate intervention.\n\nEach status assessment event can occur multiple times for each patient; however, we are unlikely to accurately capture the bedside nurse’s impression of the number of times an event occurred. We will therefore binarize the presence of each assessment type and display confusion matrices (count tabulations) for each assessment type, which we will summarize with Jaccard indicies. The “null hypothesis” that these measures do not agree at all is not meaningful or the subject of this study. As described above, neither is a directional superiority hypothesis possible to evaluate. Final Aldrete scores will be assessed with pearson correlation, and a t-test of the difference in scores presented. Differences in ready-for-discharge times will be summarized as mean and standard deviation, with the null hypothesis of zero mean tested by t-test with a robust standard error.\n\nAgreement of emergency medical status is unlikely to have enough events to be statistically compared. We will present cross-tabulations of (emergency detected by telemedicine center: yes/no) and (PACU nurse: disagree, investigate and agree, already aware, physician contacted). The absolute rate of telemedicine center false positives (team disagrees), true positives (team unaware), true positives (team aware), and false negatives (team aware > 15 minutes prior or t never detects) will be presented with 95% confidence intervals.\n\nMultiple sources will be utilized for data collection from which outcome measures will be extracted. Data from AlertWatch will be automatically logged to a secure database.\n\nPreoperative patient characteristics, comorbidities, surgical and clinical history, as well as perianesthesia information will be captured using Epic Systems software (Verona, WI, USA). Prospective data will be collected from Epic Systems for the datapoints mentioned throughout the proof-of-concept study.\n\nRelevant PACU information outlined in Table 2–Table 5 for patients in this study will be collected and entered into a REDCap database managed by Washington University. Data will not be shared with others outside the research team.\n\nA strength of this study is its pragmatic approach as a real-world study with measurable aims. Feasibility will be determined, and information will be provided regarding logistical implications of establishing a telemedicine solution for the PACU. Many telemedicine solutions have been implemented without considering barriers and facilitators, such as cultural and political obstacles. This study proactively addresses these concerns, which might facilitate future successful implementation and generalization of similar telemedicine initiatives. Specific functions of the PACU have been detailed, and the methods of this study will allow assessment of the ability of the telemedicine center to facilitate the accomplishment of these functions.\n\nThis study also has important limitations. First, as a proof-of-concept, it will only include two PACU bays. Thus, its applicability to a large PACU will not be resolved. Second, PACU clinicians will be aware of the initiative, which could modify their behavior during the conduct of the study. Third, as the study design is observational with a before and after approach, improvements (for example in time to discharge) cannot be causally attributed to the intervention; there could be confounding explanations. Fourth, the current discharge criteria for the PACU do not have a firm evidential foundation (there is no gold standard measure for discharge readiness), and clinician gestalt plays an important role. This limitation can be addressed through development of rigorous, reliable and practical criteria. Finally, as a single center study, results will not necessarily be broadly generalizable.\n\nWe do not anticipate the occurrence of significant adverse events during this study. However, the primary investigator and the study team will review any adverse events identified by the departmental quality improvement program as potentially attributable to this proof-of-concept study. The occurrence of any significant adverse events will be reported to the HRPO, and the study team and HRPO would decide together whether to halt the trial. No formal data-monitoring committee will be used. There will be no audit of trial conduct during the investigation. No interim data analysis is planned for this proof-of-concept trial unless unanticipated safety issues are identified. There are no provisions for post-trial care or compensation to patients enrolled as part of this trial, as the intervention in this proof-of-concept trial involves only the addition of real-time decision-support tools and does not change existing care models.\n\nDissemination of the findings of this study will occur via presentations at academic conferences, journal publications, and educational materials. Data from this study will not be shared with others outside the research team, as this study is a proof-of-concept designed to evaluate the potential utility and acceptability of a telemedicine solution for the post-anesthesia care unit and will only address surrogate outcomes.\n\nThis study transitioned from the observation phase to the interaction phase in September 2020.\n\n\nConclusions\n\nRecovery in the PACU is an important phase in most patients’ surgical course. In this study, we propose a new model for future PACU care. Thought has been given to assess important barriers to and facilitators for the implementation of a telemedicine solution for the PACU. Potential key findings of this study might include decreased length of stay for patients in the PACU, as well as acceptance by identified key stakeholders of the telemedicine solution. Following successful pilot implementation of a telemedicine solution for the PACU, we subsequently intend to expand this model to more PACU bays, and possibly other PACU locations in order to study relevant clinical outcome measures.\n\nThe impact of this this study, and subsequent future studies, may be far reaching. The current PACU model is not well defined. A telemedicine solution for this important recovery environment has the potential to improve safety, clinical outcomes, and quality of care for patients recovering from invasive procedures. A telemedicine solution for the PACU might also provide a suitable solution for PACU environments in under-resourced or remote locations, and decrease healthcare costs for hospital systems.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nFigshare: Supplemental Material for Proof-of-Concept PACU Telemedicine Protocol. https://doi.org/10.6084/m9.figshare.12944489.v116\n\nThis project contains the following extended data in the file ‘PACU_Telemedicine_Supplement.docx’:\n\n- PACU Telemedicine Patient Care Survey – Nurse Version\n\n- PACU Telemedicine Patient Care Survey – Telemedicine Center Version\n\n- PACU Telemedicine OR to PACU Hand-off Survey\n\n- PACU Hand-off Checklist for Telemedicine Center Use\n\n- Handoff Communication Assessment Tool of Weinger and Others\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBelcher AW, Leung S, Cohen B, et al.: Incidence of complications in the post-anesthesia care unit and associated healthcare utilization in patients undergoing non-cardiac surgery requiring neuromuscular blockade 2005– 2013: A single center study. J Clin Anesth. 2017; 43: 33–38. PubMed Abstract \| Publisher Full Text\n\nKellner DB, Urman RD, Greenberg P, et al.: Analysis of adverse outcomes in the post-anesthesia care unit based on anesthesia liability data. J Clin Anesth. 2018; 50: 48–56. PubMed Abstract \| Publisher Full Text\n\nBerg SM, Braehler MR: The Postanesthesia Care Unit. In: Miller’s Anesthesia, Elsevier; 2020; 9: 2586–2613. Accessed December 17, 2019. Reference Source\n\nButterworth IV JF, Mackey DC, Wasnick JD: Postanesthesia Care. In: Morgan & Mikhail’s Clinical Anesthesiology. McGraw-Hill Education; 2018; 6e. Reference Source\n\nStandards for Postanesthesia Care \| American Society of Anesthesiologists (ASA). Accessed December 17, 2019. Reference Source\n\nASPAN Standards. Accessed October 25, 2018. Reference Source\n\nDreyer NA, Schneeweiss S, Mcneil B: A Validated Checklist for Evaluating the Quality of Observational Cohort Studies for Decision-Making Support. 2010; 16. Accessed September 11, 2018. Reference Source\n\nVelentgas P, Dreyer NA, Nourjah P, et al.: Developing a Protocol for Observational Comparative Effectiveness Research: A User’s Guide. Agency for Healthcare Research and Quality (US); 2013. PubMed Abstract\n\nBenchimol EI, Smeeth L, Guttmann A, et al.: [The REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) statement]. Z Evid Fortbild Qual Gesundhwes. 2016; 115-116: 33–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Elm E, Altman DG, Egger M, et al.: The strengthening the reporting of observational studies in epidemiology (STROBE) statement: Guidelines for reporting observational studies. Int J Surg. 2014; 12(12): 1495–1499. PubMed Abstract \| Publisher Full Text\n\nMurray-Torres TM, Wallace F, Bollini M, et al.: Anesthesiology Control Tower: Feasibility Assessment to Support Translation (ACT-FAST)—a feasibility study protocol. Pilot Feasibility Stud. 2018; 4(1): 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFritz BA, Chen Y, Murray-Torres TM, et al.: Using machine learning techniques to develop forecasting algorithms for postoperative complications: protocol for a retrospective study. BMJ Open. 2018; 8(4): e020124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGregory S, Murray-Torres TM, Fritz BA, et al.: Study protocol for the Anesthesiology Control Tower—Feedback Alerts to Supplement Treatments (ACTFAST-3) trial: a pilot randomized controlled trial in intraoperative telemedicine [version 1; peer review: 2 approved]. F1000Res. 2018; 7: 623. Publisher Full Text\n\nKing CR, Abraham J, Kannampallil TG, et al.: Protocol for the Effectiveness of an Anesthesiology Control Tower System in Improving Perioperative Quality Metrics and Clinical Outcomes: the TECTONICS randomized, pragmatic trial [version 1; peer review: 2 approved]. F1000Res. 2019; 8: 2032. Publisher Full Text\n\nAldrete JA: The post-anesthesia recovery score revisited. J Clin Anesth. 1995; 7(1): 89–91. PubMed Abstract \| Publisher Full Text\n\nBudelier T: Supplemental Material for Proof-of-Concept PACU Telemedicine Protocol. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12944489.v1\n\nWeinger MB, Slagle JM, Kuntz AH, et al.: A Multimodal Intervention Improves Postanesthesia Care Unit Handovers. Anesth Analg. 2015; 121(4): 957–971. PubMed Abstract \| Publisher Full Text" }	[ { "id": "73445", "date": "23 Oct 2020", "name": "Alexandre Joosten", "expertise": [ "Reviewer Expertise Technology", "automated systems and artificial intelligence in anesthesia and critical care" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 and innovative proof of concept study ( before -after design) on the application of telemedicine in the PACU. The protocol is clear, well written, original, innovative and will be conduct by renowned experts. Excellent protocol. Abstract and intro are clear Methods are appropriate to conduct the study All tables and figures are very explanatory and illustrative No further comment. Congratulations.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "73442", "date": "10 Nov 2020", "name": "Michael Burns", "expertise": [ "Reviewer Expertise Computer science and medical technology implementation", "database research", "clinical informatics and analytics", "machine learning", "natural language processing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-designed protocol for a proof-of-concept observational telemedicine PACU study. The protocol is broken into two phases: observation and interactive, in which clinicians in the PACU and those in a telemedicine unit will determine and document important patient events. In the observation phase, the telemedicine clinicians will not interact with PACU clinicians unless there is a patient safety event. In the subsequent interactive phase, the telemedicine and PACU teams will interact. Important outcomes include determining readiness for PACU discharge. This protocol will test the utility of telemedicine teams, use of audio-visual communication technologies, and functional implementation of decision-support software. The following potential limitations are for general consideration and this study is scientifically sound without addressing these potential limitations.\nThe study size of 500 patients may limit the number of tested outcomes in the study. Rare events may be difficult to capture.\n\nAssessment surveys may be difficult to accurately identify stakeholder attitudes and may be limiting in determining actionable workflow adjustments.\nOverall this is an excellent study protocol that should provide high-yield results in medical support technology implementation and specific integration of clinical-support technologies to improve PACU functions. Demonstrating the potential utility of a telemedicine center to assist in PACU functions is important (Aim 1), and identifying barriers to implementation (Aim 2) may prove to be the invaluable to those attempting to implement similar technologies in medical practice.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/9-1261
https://f1000research.com/articles/9-1112/v1	09 Sep 20	{ "type": "Method Article", "title": "Inducible TDG knockout models to study epigenetic regulation", "authors": [ "Simon D. Schwarz", "Eliane Grundbacher", "Alexandra M. Hrovat", "Jianming Xu", "Anna Kuśnierczyk", "Cathrine B. Vågbø", "Primo Schär", "David Schuermann", "Simon D. Schwarz", "Eliane Grundbacher", "Alexandra M. Hrovat", "Jianming Xu", "Anna Kuśnierczyk", "Cathrine B. Vågbø" ], "abstract": "Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.", "keywords": [ "Embryonic Stem Cells", "TDG", "Active DNA Demethylation", "Base Excision Repair", "Neuronal Differentiation", "Minigene", "Cre/loxP", "Tamoxifen" ], "content": "Introduction\n\nTo allow for the differentiation to a multitude of cell types during the development of multicellular organisms, stem cells need to respond to a variety of extrinsic and intrinsic developmental cues and integrate these into epigenetically stabilized gene expression patterns during lineage specification. The underlying mechanisms to ensure this necessary epigenetic plasticity are therefore active and highly dynamic in embryonic stem cells (ESC). One of those mechanisms includes the thymine DNA-glycosylase (TDG) and the downstream factors of DNA base excision repair (BER), which were shown to be involved in the dynamic, locus-specific regulation of DNA methylation. Following the activity of dioxygenases of the ten-eleven-translocation (TET) family that iteratively oxidize 5-methylcytosine (5-mC) to formyl- and carboxylcytosine (5-fC, 5-caC), TDG-induced BER provides a mechanism of active DNA demethylation and, thereby, impact gene regulation (Cortázar et al., 2011; Cortellino et al., 2011; Schuermann et al., 2016; Weber et al., 2016). Studying the precise function of TDG (and BER) in epigenetic regulation during differentiation of stem cells, however, is challenging due to its multiple and interwoven interactions with other proteins like transcription factors and epigenetic modifiers (Henry et al., 2016; Jacobs & Schär, 2012; Léger et al., 2014), the deregulation of which may cause a plethora of confounding effects. Also, genetic depletion of TDG itself is not compatible with embryonic development and alters the epigenetic state of cultured cells, causing a difficult-to-control drift of phenotype in long-term cultivated stem cell populations. To reduce and circumvent such confounders in functional studies of TDG (and BER) in murine ESCs, we established and validated a Tdg minigene, introduced it into mice and derived a series of different ESC variants. The minigene carries, among other features, a loxP-flanked coding region of Tdg, allowing for a controlled and fast, 4-hydroxytamoxifen (OHT)-inducible depletion of TDG in the background of a homozygous disruption of the endogenous Tdg. By eliminating phenotypic divergence between TDG proficient and deficient ESC, this versatile model facilitates precise functional and mechanistic investigations into TDG-mediated active DNA demethylation. This novel Tdg minigene introduced into mice as well as ESCs will facilitate future research in the field of active DNA demethylation in vivo and in vitro.\n\n\nMethods\n\nAll animal work was carried out in accordance with the Swiss Animal Welfare Act and the guidelines of the Swiss Federal Veterinary Office (SFVO) or with the UK Animals (Scientific Procedures) Act. Housing, breeding and experimentation of mice has been performed with the approval of the Cantonal Veterinary Office of Basel-Stadt (Licenses 10062-H, 1912) or was covered by a project license to Wolf Reik (80/1896) further regulated by the Babraham Institute Animal Welfare, Experimentation, and Ethics Committee. Mice were housed under specific pathogen free condition in individually ventilated cages under 12 h light/dark cycles at 22 ±2°C and 35-65% relative humidity. Animal and colony health was checked daily and quarterly, respectively. Sterilized diet (Kliba-Nafag Extrudate 3436) and water was provided ad libitum.\n\nTdgtm1Psch mice (http://www.informatics.jax.org/allele/MGI:5487834) were generated from E14 ESC (RRID:CVCL_C320) and backcrossed for more than 10 generations with female C57BL/6JRj mice, obtained at 7-8 weeks of age from Janvier Labs. A Tdg expression cassette (pTCO2-mTDGi.0, Addgene Plasmid #149429) was introduced as single copy into C57BL/6JRj mice by pronuclear injection and crossed into Tdgtm1Psch mice. Genotyping was done by PCR with the HOT FIREPol (Solis BioDyne) on diluted crude DNA preparations (diluted by a factor of five with 10 mM Tris-HCl pH8) by boiling toe clips with 25 mM NaOH/0.2 mM EDTA for 1 h followed by neutralization with 40 mM Tris-HCl. Reactions were performed according to the manufacturer’s recommendation with 35 PCR cycles at 95°C for 30 s, 60°C for 30 s, and at 72°C for 60 s (for details about all used primers and annealing temperatures in PCR reactions, see Extended data Table S1) (Schwarz, 2020).\n\nESCs were derived from male blastocysts from timed matings of male miniTdgtg/tg//Tdg-/- mice with 2 super-ovulated female Tdg+/- mice and cultured on murine immortalized feeder cells in 2i medium with leukemia inhibitory factor LIF (Merck-Millipore) (Ying et al., 2008).\n\nTo introduce the inducible Cre recombinase into the ROSA26 locus, 30 µg of the targeting vector (pROSA26-ERT2CreERT2, Addgene Plasmid #149436) was electroporated into 15×106 mESCs using a gene pulser Xcell (Bio-Rad) at 240 V and 475 μF. Transgenic cells were selected with 8 and 5 µg/ml blasticidin for a week each, before colonies were picked and then amplified without selection pressure. From blasticidin-resistant colonies, genomic DNA was extracted with the “QIamp DNA mini” kit (Qiagen) and screened for a targeted integration at the 3’ junction of the ROSA26 locus by PCR with the Phusion polymerase (NEB) (see Extended data, Table S1) (Schwarz, 2020), applying a general three-step cycling protocol: initial denaturation 95°C for 30 s, 35 times 95°C for 10 s, annealing for 20 s, 72°C for 15–80 s. PCR reactions were analyzed by gel electrophoresis followed by image acquisition with a U:Genius 3 system (Version 3.0.12.0, Syngene).\n\nTo evoke a disruption of the NeoR reading frame by CRISPR/Cas9, two guide RNAs (5’-GCCGATCCCATATTGGCTGCAGG-3’, 5’-GAAGGCGATGCGCTGCGAATCGG-3’, IDT) were designed and transfected with TransIT-X2 (Mirus Bio) into the TDGiKO1 ESCs. RNP assembly was performed according to the manufacturers protocol (IDT). One day after transfection, single cells were sorted with a FACSaria IIIu (BD BioSciences) into 96-well plates coated with inactivated mouse fibroblasts, based on GFP expression from a co-transfected pEGFP-N1 plasmid (Clontech). Screening for the successful disruption of the NeoR gene was done by PCR as described above and assaying the loss of the neomycin resistance with the Cell Counting Kit-8, according to the manufacturer’s instructions (CCK-8, Dojindo).\n\nESCs were cultured under a controlled atmosphere (37°C, 5% CO2, 95% humidity) in serum-free 2i medium with 1000 U/ml LIF, without antibiotics unless indicated otherwise. The 4-OHT (Sigma-Aldrich H7904) was dissolved in DMSO (stock 10 mM) and administered at indicated concentrations for 2 h.\n\nNeuronal differentiation was performed as described before (Bibel et al., 2007; Cortázar et al., 2011; Steinacher et al., 2019). Differentiation towards cardiomyocytes was performed by culturing ESCs in classical ESC medium (ESM) with 15% FCS without LIF in non-adhesive petri dishes (Greiner) for at least 10–14 days.\n\nAll pictures were taken with a DM IL LED Fluo microscope and MC170 HD camera (Leica) at 100x magnification.\n\nProbes for Southern blotting were generated by PCR amplification with Taq DNA Polymerase (Qiagen) from the ROSA26 targeting plasmid using the DIG DNA labeling mix (Roche). Southern blotting was done with 20 µg of genomic DNA, extracted by the “Genomic tip 100G” kit (Qiagen). Probes were hybridized according to the “DIG Application Manual for Filter Hybridization” by Roche. Signals were acquired by exposing the membrane to chemiluminescence detection films (Amersham/GE Healthcare) and digitalized by a CanoScan 8400F scanner with CanoScan Toolbox (Version 4.9.3).\n\nExcision of the miniTdg gene by Cre was assessed by qPCR with the “Rotor-Gene SYBR Green PCR Kit” (Qiagen) on a Rotor Gene 3000 (Qiagen) system. After 95°C for 5 min, qPCR reactions followed a two-step cycling (40x) protocol: 95°C for 5 s, 60°C for 30 s, terminated by ramping from 60 to 95°C in 175 s to generate a melting curve. PCRs to exclude background recombination by Cre were done with NEB Phusion polymerase as described above.\n\nTotal RNA was extracted with the RNAeasy Mini Kit (Qiagen), including on-column DNase digest, and reverse transcribed with RevertAid First Strand cDNA synthesis kit (ThermoFisher) using oligo-dT primers. qPCR was performed as described above with marker-specific primers (see Extended data, Table S1) (Schwarz, 2020).\n\nProtein levels were analyzed by western blotting of 50 µg of NP-40 (or SDS) whole-cell extracts, separated by SDS-PAGE, onto a nitrocellulose membrane (Amersham) and immunodetection with anti-mTDG antibody (rabbit polyclonal L58, Lab P.Schär, dilution 1:10’000, 1 h at 33°C) and anti-GAPDH antibody (Sigma-Aldrich Cat# G9545, RRID:AB_796208, dilution 1:20’000, 1 h at RT). Chemiluminescence signals were detected with a Fusion FX7 system (Software version 16.15.0.0, Vilber).\n\nDNA was extracted and purified with the Genomic tip 100G Kit (Qiagen). High-performance liquid chromatography–tandem mass spectrometry analysis was performed as described before (Weber et al., 2016).\n\nTo test for statistical significance (p ≤ 0.05), we performed two-tailed Student’s t-tests on the indicated number of replicates in Microsoft Excel (Version 16.0.4954.1000)\n\n\nResults and discussion\n\nTo facilitate genetic manipulation of the Tdg gene (~28 kb including the putative promoter), we first constructed a synthetic Tdg minigene (miniTdg, ~11 kb) (Figure 1A). The minigene consists of the endogenous Tdg promoter and terminator sequences flanking the Tdg coding sequence (CDS). It also includes the rabbit β-globin intron at the authentic position of the first intron of the Tdg transcript variant 2 (Genebank NM_172552.4). This intron allows for the expression of the two naturally occurring Tdg splice variants (Um et al., 1998). In addition, the miniTdg contains two loxP sites in the same orientation for Cre-recombinase-mediated excision of the coding region. A plasmid carrying the miniTdg gene (pTCO2-mTDGi.0) was introduced into C57BL/6 mice by pronuclear injection and offspring carrying the minigene were crossed into heterozygous Tdgtm1Psch mutant mice (Kunz et al., 2009). When breeding heterozygous mice for the miniTdg transgene and the Tdg KO allele, the genotypes of offspring followed the expected Mendelian pattern for two loci (Figure 1B). The miniTdg allele segregated with a 3:1 ratio, consistent with a random integration into a single genomic locus. About 25% of born mice with the miniTdg gene were homozygous for the Tdgtm1Psch KO allele while no homozygous TDG KO mouse was obtained without the transgene. This shows that the miniTdg transgene is fully functional in complementing the developmental defects and lethality of homozygous Tdgtm1PschKO embryos (Cortázar et al., 2011; Cortellino et al., 2011). MiniTdg complemented mice were healthy and fertile with normal lifespan.\n\n(A) Synthetic miniTdg gene on the plasmid integrated to a random genomic locus by pronuclear injection. miniTdg consisting of 6 kb endogenous Tdg promoter, the Tdg coding sequence (CDS) including a chimeric splice donor (Tdg exon 1 of transcript variant 2 NM_172552.4, rabbit β-globin intron), to allow for alternative splicing, 3 kb of the 3’ untranslated region (3’ UTR) and terminator (term) of Tdg, and the bovine growth hormone terminator sequence (BGH!). loxP sites are indicated in red. (B) Expected and obtained genotype distribution in offspring (n=116) from crosses of heterozygous mice. (C) Top: Immunoblot for TDG of whole-cell SDS-extracts from derived ESCs with different genotype. Bottom: Loading control by Ponceau staining of the membrane. (D) Accumulation of oxidized 5-mC derivates measured by HPLC-MS/MS in ESCs with the indicated genotype. Shown are means and standard deviation of two independent clones of each genotype. (E) Scheme of the targeting construct pROSA26-ERT2CreERT2 for the ROSA26 locus with the tamoxifen-inducible Cre recombinase (Matsuda & Cepko, 2007). ERT2-Cre-ERT2 expression is under the control of the synthetic cytomegalo-virus/chicken-actin/beta-globin promoter (CAG). Homology arms for ROSA26 targeting are indicated in light grey. A blasticidin resistance cassette (BlaR) for positive selection is under the control of the SV40 promoter and terminator. (F) Predicted restriction pattern of ROSA26 WT and integration events (top) and fragments detected by Southern blotting (bottom). Left: The hybridization probe (P1) locating to the left ROSA26 homology arm detected a genomic fragment of 6.9 kb. The 4.4 kb fragment represents the wild-type ROSA26 allele. Right: Detection of possible off-target events using a second probe (P2) locating to the blasticidin selection marker within the targeting construct. See Underlying data for the raw data behind this figure (Schwarz, 2020).\n\nThree ESC populations were then derived from male blastocysts from crosses of miniTdgtg/tg//Tdg-/- with Tdg+/- mice (Extended data, Table S2: Mm11, Mm01, Mm02) (Schwarz, 2020). In these ESCs, TDG protein levels were similar to those in wild-type ESCs (Figure 1C), indicating that the miniTdg gene is regulated as the endogenous Tdg. To assess functional integrity at the molecular level, we measured the accumulation of oxidized 5-methylcytosine (5-mC) derivates, 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine (5-caC), the substrate for excision by TDG (Maiti & Drohat, 2011; Shen et al., 2013) (Figure 1D). Mass spectrometry showed that TDG deficient ESCs accumulated 5-caC as expected, while ESCs expressing TDG solely from the miniTdg gene showed 5-caC levels similar to those in wild-type ESCs.\n\nTo allow for a controlled excision of the miniTdg coding region, we introduced a tamoxifen-inducible Cre recombinase expression cassette (Figure 1E) into the non-essential ROSA26 locus (Soriano, 1999). Screening 60 blasticidin-resistant colonies by PCR for targeted integration at 3’ ROSA26 locus yielded six clones (TDGiKO1-6). Southern blotting confirmed correct heterozygous integration of the complete ERT2-Cre-ERT2 cassette (Matsuda & Cepko, 2007) for four of the six clones (Figure 1F, left, asterisks). Potential additional off-target integrations were tested and excluded by hybridizing a second probe within the transgene (Figure 1F, right). Original Southern blotting images are available as Underlying data (Schwarz, 2020).\n\nTo ease further genetic manipulations, we eliminated the neomycin resistance gene, which was introduced at the endogenous Tdg locus to generate the original Tdg KO allele (Kunz et al. 2009), by applying a CRISPR-guided Cas9 nuclease. Using two guide RNAs targeting the 5’ and 3’ end of the NeoR gene, we generated two ESC clones (TDGiKO1.1 and TDGiKO1.2) that bear a deletion of 776 and 767 bp, respectively, and are no longer resistant to neomycin (Extended data, Figure S1A/B) (Schwarz, 2020).\n\nTaken together, we established a miniTdg gene that complements expression and function of the endogenous Tdg gene in vivo and in vitro. The functional integrity of the transgene is confirmed by rescuing phenotypes of Tdg KO defects at the systemic and cellular level, namely the rescue of embryonic lethality and suppression of 5-caC accumulation. The introduction of a Cre-recombinase to the ROSA26 locus, without off-targeted events, will allow conditional inactivation of the miniTdg in ESCs.\n\nTo make the inducible TDG depletion (Figure 2A) applicable to differentiation experiments that often depend on complex and strict cell culture procedures, we aimed at establishing a short Cre-induction protocol minimizing any interference with standard differentiation conditions. Titrating 4-Hydroxytamoxifen (OHT) and measuring miniTdg excision by quantitative PCR, we identified OHT concentrations of 1-5 µM in serum-free 2i (Figure 2B) and 5-10 µM OHT in serum-containing medium (Figure 2C) to yield maximal excision efficiencies without impairing cell viability (Figure 2D). While the miniTdg excision product is readily detectable by PCR in different TDGiKO ESCs after OHT treatment, no unintentional background activity of the Cre recombinase is detectable before OHT induction (Figure 2E). Following the kinetics of TDG depletion by SDS-PAGE/immuno-detection, we found that TDG was reduced below detection limit (<2% of endogenous TDG) 24 h after Cre induction for 2 h (Figure 2F and Extended data, Figure S1C) (Schwarz, 2020).\n\n(A) Scheme of the miniTdg cassette before (top) and after (bottom) Cre-mediated recombination. Position of PCR primers are indicated with green arrows. (B) Quantitation of miniTdg excision upon OHT administration in TDGiKO1, TDGiKO4 and parental ESCs without the Cre recombinase (Mm01). Genomic DNA of cells was extracted two days after OHT treatment with indicated concentrations for 2 hours. Copy number of the miniTdg is measured by qPCR using primers 2 and 3, normalized to the nearby terminator region (primers 4/5) and a control locus on chromosome two. Shown are means and standard error from technical quadruplicates per clone. (C) Quantitation of miniTdg excision as in (B), but Cre induction executed in ES medium containing 15% FCS. (D) Phase-contrast images of TDGiKO1 ESCs in 2i medium, two days after OHT treatment for two hours at indicated concentrations. (E) Detection of Cre recombination events (158 bp) by PCR using primers 1 and 3 (A) before and after addition of OHT. (F) Time-course assessment of TDG protein levels expressed from the miniTdg gene in 2i cultivated TDGiKO1 ES cells after Cre induction by 1 µM OHT for 2 h. See Underlying data for the raw data behind this figure (Schwarz, 2020).\n\nWe also assessed the tissue specific induction of Cre-mediated miniTdg excision in vivo. We crossed miniTdg mice with mice expressing Cre-recombinase under the control of a FoxN1-promoter (http://www.informatics.jax.org/allele/key/62500) which drives expression in thymic epithelial cells (TECs), but not in thymic cells (TCs). We observed that the mRNA and protein expressed from the miniTdg are reliably depleted in TECs expressing FoxN1-driven Cre, whereas TDG levels are much less affected in TCs (Extended data, Figure S1D/E) (Schwarz, 2020).\n\nThese data demonstrate the functionality and tight regulation of the inducible miniTdg KO model in murine ESCs as well as in mice. A short-time Cre-induction with 1–5 μM or 5–10 μM OHT (in 2i+LIF or 15% FCS-containing medium, respectively) is sufficient to mediate excision of the miniTdg, resulting in depletion of TDG below detection within 24 hours. This rapid depletion of TDG is presumably facilitated by the cell cycle-regulated proteasomal degradation of TDG via the ubiquitination at its PCNA-interacting peptide (PIP) motif (Hardeland et al., 2007; Shibata et al., 2014).\n\nTo validate the molecular phenotype of TDG depletion in our newly derived TDGiKO ESC, we again assessed changes in levels of oxidized 5-mC derivates, 5-formylcytosine (5-fC) and 5-caC following Cre-mediated miniTdg deletion. One week after depletion of TDG, we measured a 4.4 and 4.2-fold accumulation of the TDG substrates 5-fC and 5-caC in TDGiKO ESCs, respectively, but no changes of 5-hydroxymethyl cytosine (5-hmC), which is not recognized by TDG (Figure 3A). This accumulation reflects well the previously reported measurements in constitutive Tdg KO cells (Shen et al., 2013; Steinacher et al., 2019).\n\n(A) Measurement of oxidized derivates of 5-mC in selected ESCs by HPLC-MS/MS, cultivated in 2i medium + LIF, one week after TDG depletion by 1 µM of OHT for 2 h. Shown are the means and standard deviation of 2-3 biological replica. (B) Scheme of the neural differentiation protocol (Bibel et al., 2007), adapted with a 16 h priming step in ESC medium for ESCs grown in 2i medium. Representative phase-contrast images of TDGiKO1 cells with (+OHT) and without (mock) TDG depletion at the differentiation stages indicated (arrows). (C) Expression of pluripotency and germ layer marker genes were assessed by qRT-PCR in TDGiKO1 cells at the stages of differentiation as marked in (B). Expression was normalized to the housekeeping genes Rps13 and Eef1a1. Shown are means and standard errors of 3 biological replicates. Asterisks indicate p-values of Student’s t-test: p-value < 0.05, **p-value < 0.001. See Underlying data for the raw data behind this figure (Schwarz, 2020).\n\nFinally, we re-tested the pluripotency and differentiation capacity of some of the engineered TDGiKO ESCs clones (Extended data, Table S2) (Schwarz, 2020). When subjecting TDGiKO1 ESCs to all-trans retinoic acid (RA) induced in vitro differentiation towards the neuronal lineage (Bibel et al., 2007), we observed an upregulation of the neural marker Nestin (Figure 3B, C) and, ultimately, the formation of neuron-like cells (Figure 3B). As expected during embryoid body (EB) formation, LIF withdrawal resulted in an induction of lineage-specific genes of all three germ layers and the repression of the pluripotency marker gene Nanog. This indicates the ability of the TDGiKO ESCs to commit to cell types of all germ layers. In support of this, continued cultivation of TDGiKO1 and TDGiKO1.1 in ESM without LIF led to a spontaneous differentiation to cardiomyocytes (mesodermal cell type) forming beating organoids (see Extended data Videos 1 and 2) (Schwarz, 2020). While the early initiation of lineage-specific genes seemed to be unaffected by TDG depletion prior to differentiation, the formation of neuron-like cells (Figure 3B, bottom) was impaired. This deficiency to stably commit to and/or maintain the neuronal lineage was indicated by a low number of neuron-like cells formed and the loss of cells with Nestin expression following RA application. Upon loss of TDG, we furthermore observed a significant difference in the downregulation of the pluripotency marker Nanog as well as and dysregulation of the endodermal marker Gata6 (Figure 3C).\n\nThus, when subjecting the TDGiKO ESCs to in vitro differentiation, we obtained data consistent with previous observations in constitutive Tdg KO ESCs (Steinacher et al., 2019). This highlights the usability of the newly established ESCs in TDG and BER-related research, with the advantage of eliminating clonal divergences between TDG proficient and deficient cells. While here, we focused on the reproduction of known and published TDG KO phenotypes, the fast and reliable depletion of TDG in the TDGiKO ESC model now provides a tool for in-depth execution point analysis of TDG-BER mediated active DNA demethylation during cell differentiation or in any other context of interest. Furthermore, the loxP sites flanking the coding region of the miniTdg gene enable recombinase-mediated cassette exchange to introduce TDG separation of function variants devoid of catalytic function or harbour mutations that ablate posttranslational modifications (Hardeland et al., 2000). Additionally, the availability of mice carrying the miniTdg, which can be combined with Cre-driver mice of interest by crossing, allows the translation of in vitro observations into in vivo settings. All in all, we provide a versatile, well-controlled and validated toolbox (Extended data, Table S2) (Schwarz, 2020) to facilitate functional and mechanistic research on TDG-mediated active DNA demethylation and BER in cell culture and animal models.\n\n\nData availability\n\nZenodo: Inducible TDG knockout models to study epigenetic regulation. http://doi.org/10.5281/zenodo.3979375 (Schwarz, 2020).\n\nThis project contains the following underlying data:\n\nRaw_Data_Figure1.zip. Database excerpt for genotyping of mouse crossings, original pictures of Southern and western blots and MS-quantification of oxidized mC derivates (also for figure 3).\n\nRaw_Data_Figure2.zip. qPCR data of miniTDG excision, original pictures of OHT-treated ESCs and western blots.\n\nRaw_Data_Figure3.zip. Original pictures of differentiating cells and qPCR data of pluripotency and germ line markers.\n\nRaw_Data_Extended_Figure.zip. Sequencing data for NeoR excision, absorption values of G418-treated ESCs, qPCR data for miniTDG excision and original pictures of western blots.\n\nZenodo: Inducible TDG knockout models to study epigenetic regulation. http://doi.org/10.5281/zenodo.3979375 (Schwarz, 2020).\n\nThis project contains the following extended data:\n\nExtended Figure and Tables.pdf. Supplementary information as referenced in the text.\n\nExtended_Video_1_TDGiKO1.mp4. Video of “beating bodies” from an ESC clone.\n\nExtended_Video_2_TDGiKO1.2.mp4. Video of “beating bodies” from a second ESC clone.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe want to express our thanks to the laboratory of Wolf Reik and the transgenics facility at the Babraham Institute (Cambridge, UK) for performing the pronuclear injection. We also thank the animal facility at the DBM Mattenstrasse and the transgenic core facility of the University of Basel for their help in breeding the mouse strains and establishing ESCs. Additional thanks goes to Carlos Mayer from George Holländer’s Lab (DBM) for the sorting of the thymic cells.\n\n\nReferences\n\nBibel M, Richter J, Lacroix E, et al.: Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells. Nat Protoc. 2007; 2(5): 1034–1043. PubMed Abstract \| Publisher Full Text\n\nCortázar D, Kunz C, Selfridge J, et al.: Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability. Nature. 2011; 470(7334): 419–423. PubMed Abstract \| Publisher Full Text\n\nCortellino S, Xu J, Sannai M, et al.: Thymine DNA glycosylase is essential for active DNA demethylation by linked deamination-base excision repair. Cell. 2011; 146(1): 67–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHardeland U, Bentele M, Jiricny J, et al.: Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis. J Biol Chem. 2000; 275(43): 33449–33456. PubMed Abstract \| Publisher Full Text\n\nHardeland U, Kunz C, Focke F, et al.: Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2. Nucleic Acids Res. 2007; 35(11): 3859–3867. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenry RA, Mancuso P, Kuo YM, et al.: Interaction with the DNA Repair Protein Thymine DNA Glycosylase Regulates Histone Acetylation by p300. Biochemistry. 2016; 55(49): 6766–6775. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJacobs AL, Schär P: DNA glycosylases: In DNA repair and beyond. Chromosoma. 2012; 121(1): 1–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKunz C, Focke F, Saito Y, et al.: Base excision by thymine DNA glycosylase mediates DNA-directed cytotoxicity of 5-fluorouracil. PLoS Biol. 2009; 7(4): e91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLéger H, Smet-Nocca C, Attmane-Elakeb A, et al.: A TDG/CBP/RARα ternary complex mediates the retinoic acid-dependent expression of DNA methylation-sensitive genes. Genom Proteom Bioinf. 2014; 12(1): 8–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaiti A, Drohat AC: Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem. 2011; 286(41): 35334–35338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMatsuda T, Cepko CL: Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A. 2007; 104(3): 1027–1032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchuermann D, Weber AR, Schär P: Active DNA demethylation by DNA repair: Facts and uncertainties. DNA Repair (Amst). 2016; 44: 92–102. PubMed Abstract \| Publisher Full Text\n\nSchwarz SD: Inducible TDG knockout models to study epigenetic regulation [Data set]. F1000 Research. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3979375\n\nShen L, Wu H, Diep D, et al.: Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell. 2013; 153(3): 692–706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShibata E, Dar A, Dutta A: CRL4Cdt2 E3 ubiquitin ligase and Proliferating Cell Nuclear Antigen (PCNA) cooperate to degrade thymine DNA glycosylase in S phase. J Biol Chem. 2014; 289(33): 23056–23064. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoriano P: Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999; 21(1): 70–71. PubMed Abstract \| Publisher Full Text\n\nSteinacher R, Barekati Z, Botev P, et al.: SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation. EMBO J. 2019; 38(1): e99242. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUm S, Harbers M, Benecke A, et al.: Retinoic Acid Receptors Interact Physically and Functionally with the T:G Mismatch-specific Thymine-DNA Glycosylase. J Biol Chem. 1998; 273(33): 20728–36. PubMed Abstract \| Publisher Full Text\n\nWeber AR, Krawczyk C, Robertson AB, et al.: Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism. Nat Commun. 2016; 7: 10806. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYing Q, Wray J, Nichols J, et al.: The ground state of embryonic stem cell self-renewal. Nature. 2008; 453(7194): 519–524. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71086", "date": "21 Sep 2020", "name": "Jonathan Sczepanski", "expertise": [ "Reviewer Expertise DNA repair", "base excision repair", "DNA demethylation", "thymine DNA glycosylase" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the method article “Inducible TDG knockout models to study epigenetic regulation” Schwarz et. al establish mice and mESC cell line that allows for inducible KO of TDG. Building on their prior work, the key features of this system is a TDG mini-gene that resides between two loxP-Cre recombinase sequences, along with a tamoxifen-inducible Cre recombinase expression cassette. The authors demonstrate this design achieves near complete (~98%) depletion of TDG upon tamoxifen-induction within 24 h. Importantly, the TDG mini-gene recapitulates the epigenetic and repair functions of endogenous TDG prior to depletion, and cell morphology and pluripotency are not altered until after tamoxifen-induced depletion. These molecular and cellular phenotype studies are particularly well done, and clearly demonstrate agreement between inducible TDG KO ESCs and previously reported constitutive TDG KO ESCs. Overall, this work is very rigorous, and the controls are carefully designed and extensive. I anticipate that inducible TDG KO will be a valuable tool for mechanistic studies aimed at uncovering the role of TDG in DNA epigenetics and repair. I recommend this paper for indexing without further revisions.\nMinor comment: ESC is written \"ECS\" in Figure 3 title.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6034", "date": "19 Oct 2020", "name": "David Schürmann", "role": "Author Response", "response": "We are very grateful to Dr Sczepanski for taking the time to evaluate our manuscript and were pleased to learn about his overall approval and appreciation of our experimental work and data. The spelling mistake in the title of figure 3 was corrected." } ] }, { "id": "71089", "date": "30 Sep 2020", "name": "Joseph Torchia", "expertise": [ "Reviewer Expertise Epigenetics", "Biochemist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThymine DNA Glycosylase (TDG) is a BER protein that is essential for the removal of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-fC) from genomic DNA. TDG knockout is embryonic lethal at E11.5 due to genome wide epigenetic instability. To circumvent embryonic lethality from a deletion of Tdg, the authors synthesized a mini-Tdg allele consisting of an optimized TDG codon arrangement for expression of two naturally occurring TDG splice variants. The authors introduced the mini-Tdg allele in a Tdg -/- background to generate miniTdgtg/tg/Tdg-/- mice, which phenocopy and express TDG similarly to wild-type mice. Furthermore, the TDG codons are flanked by loxp sites to allow for efficient spatial and temporal excision of Tdg in response to tamoxifen. The deletion of TDG in ESC’s results in a global upregulation in 5-caC, and Tdg null ESC’s fail to differentiate in response to retinoic acid both of which are consistent with previous literature. Overall, the paper describes a novel mouse model to study the role of TDG dependent active DNA demethylation in vitro and in vivo. The paper is well written, and the data seems to be convincing. In addition, the experiments appear to be performed to a high standard. However, I feel addressing the following concerns would significantly strengthen the manuscript and increase the novelty of the mouse model:\n\nMajor Comments:\nThe authors need to site appropriate literature and/or provide data to address the claim that epigenetic instability resulting from a loss of TDG leads to complications in long-term ESCs cultures. Multiple studies, such as Hu et al. 20141 (PMID 24529596) and Shen et al. 20132 (PMID 23602152), have rigorously used Tdg -/- mouse ESC’s without any mention of such complications. I feel the argument that the use of such a complicated system to study TDG deletion in MEFs and ESCs needs further clarification.\n\nThere are several published conditional knockout mouse models of TDGthat incorporate loxp sites in the endogenous Tdg allele. These mouse models, and their potential drawbacks, need to be discussed to provide a stronger argument for the use of mini-Tdg allele approach employed in this manuscript.\n\nIts unclear why it was necessary to remove the neo cassette to generate the TDGiKO1.1 and TDGiKO1.2 ESC clones? The use of CRISPR can introduce complications such as off-target mutations and clonal selection that can introduce variability in the model.\nMinor comments and questions:\nFigure 1D, 2B, 2C, 3A are all lacking statistical analysis. Please use the appropriate nomenclature to indicate statistical significance.\n\nPlease describe the sample size for LC-MS/MS experiment outlined in figure 1D and Figure 3A.\n\nFigure 2F: Why is TDG blot not showing a doublet. The SUMOylated TDG band is missing.\n\nWhy was 4-OHT employed at such a high concentration? In my opinion 100 nM to 500 nM should be more than sufficient to induce excision.\n\nWas the mini-Tdg allele integrated in the mouse genome? If so, where did the integration occur?\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6035", "date": "19 Oct 2020", "name": "David Schürmann", "role": "Author Response", "response": "We thank Dr Torchia for the critical reading of our manuscript and his valuable suggestions for improvement, which helped a lot to strengthen our conclusions. We revised the manuscript according to his major and minor comments. Major comments: We understand the reviewers comment that this statement is rather speculative and currently poorly supported by data. We therefore removed it in the revised version. On the other hand, we observed in our experiments with constitutive TDG KO ESCs subtle and rather global differences (e.g. cell division time), which we do not yet fully understand. They could originate from clonal adaptation but also from TDG-driven epigenetic mechanisms. However, to be able to discriminate between these possibilities was the reason for the development of the here presented inducible TDG depletion system. Apart from the not yet ruled out role of TDG as repair enzymes for damaged DNA, the increase of fC and caC might affect DNA-protein interactions and consequently chromatin landscaping. Irrespective of these hypothetical scenarios, where long-term culturing of cells could result in both a genetic as well as an epigenetic drift, we feel that an isogenic system is always the preferable experimental system, even when it seems to be more complicated and laborious. We are aware that several other conditional Tdg allele were generated and available as well, which are genetically easier to handle and could perfectly serve their experimental purposes. In fact, before having started to engineer our complementing miniTdg system, we intended but failed to introduce loxP sites at the extremities of the endogenous Tdg coding region to be able to delete the entire gene, omitting putative dominant truncated transcripts. We suspect that besides a poor accessibility of some regions of the Tdg gene, the multitude of pseudogenes consisting basically of Tdg cDNA sequences interferes with successful genome engineering in certain circumstances as they may act as off-targets, not only for classical gene targeting but also CRISPR/Cas9-based approaches. Having a functional miniTdg gene, in which the entire coding region is flanked by loxP sites, not only allows for an inducible TDG depletion but also enables further engineering steps in a flexible manner by Cre-mediated cassette exchange (although not designed specifically for this purpose). We refer to other available conditional Tdg allele in the revised version of our manuscript. In the last paragraph, furthermore, we point to this advantage and possibility. This was, by the way, one of the reasons why we deleted the neomycin resistance gene (see response to comment 3 below), allowing for the use of this selectable marker for such a replacement strategy. We utterly agree with the reviewer about the potential drawbacks of the CRISPR/Cas9 genome editing approach. Therefore, we added our off-target analysis for the two ESC clones generated in the “Extended data”. In fact, most of the here presented data from cultured cells and in vivo are obtained from the systems without the additional deletion of the neomycin resistance gene (except LC-MS/MS data in Fig. 3A). For the sake of completeness of our system, we included these two ESC clones as they might be of interest when additional selection marker-based transgene integration is intended. These notions may have been presented ambiguously in the first version and are now improved by slightly rephrasing and restructuring of the respective paragraphs. In any case, the possibility to work with the inducible TDG depletion systems ensures an isogenic background, at least for the role of TDG, which could be validated in different in ESC clones. Although one can never completely rule out some issues of clonal selection, we still consider it an advantageous approach. Minor comments and questions: We appreciate reviewer’s pointing to the missing statistical indicators, which we now added. More detailed information about the analysis by LC-MS/MS was added into the figure legends and the respective paragraph in the Methods section. This is a very attentive comment by the reviewer. In contrast to Figure 1, indeed, the SUMO-modified TDG is not apparent in Figure 2F because we used NP-40 soluble protein extraction methods without the addition of specific inhibitors of SUMO proteases (f.e. N-Ethylmaleimide). As de-SUMOylation happens quite fast and efficiently during mild lysis condition even at 4°C, SUMOylated TDG is in our hands only detectable when immediate lysis in Laemmli buffer and/or detergent-based lysis buffers supplement by the inhibitor. We now added the technical information about the extraction method also in the figure legend. We agree with the reviewer that lower 4-OH tamoxifen concentration might work as well and added a comment about suitable Cre induction conditions in the 2nd chapter of the results. Based on an initial literature search, we found that commonly used concentrations of OHT are in the range of 100 nm – 10 µM. To get a maximal flexibility and timely controlled TDG depletion in experimental protocols of differentiation, we aimed at rather short durations of induction. Such single induction 4-OHT treatments of as short as 2 h are rarely found in literature and associated with limited recombination efficiency (e.g. Buelow et al. 2008), while lower concentrations are more frequently used for Cre induction for up to 48 h, sometimes also with repeated administration. For our purpose, we therefore opted for 4-OHT concentrations in the intermediate to high range but avoiding cytotoxic effects. This is indeed a relevant point for a potential user of our system. The miniTdg gene was introduced by pronuclear injection of a plasmid that contained no homology arms for a targeted integration into the mouse genome. Thus, it is a random integration event as stated in the legend of Figure 1. We added now this information also the first result section. Unfortunately, our attempts to identify the genomic locus of integration by conventional molecular methods were so far not successful. Yet, we might get some hints from ongoing and future global NGS analyses, which would be amended to this paper as well as to the MGI entry." } ] }, { "id": "72019", "date": "07 Oct 2020", "name": "Sebastian Bultmann", "expertise": [ "Reviewer Expertise Epigenetics", "embryonic stem cells", "gene editing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript entitled \"Inducible TDG knockout models to study epigenetic regulation\" the authors present new model system for the conditional depletion of TDG. The authors employ a strategy in which a loxP flanked mini gene is introduced into a TDG KO background. In combination with an inducible Cre introduced into the ROSA26 locus the authors convincingly show the rapid end efficient depletion of TDG. Finally, the authors validate their system by investigating the effect of acute TDG depletion on differentiation. In summary, the study presents a clever strategy for induced deleption, is well thought through and scientifically sound.\n\nI have only two minor points the authors should address:\nIs the TDG mini gene randomly integrated into the genome? The authors should make this clear and if possible comment on any observation regarding silencing of the transgene during long-term culture.\n\nThe section \"molecular and cellular phenotypes\" is a little confusing. The authors should make clear in the text when they are talking about induced (TDG depeltion) or uninduced (control) cells. The authors should clearly explain the experimental setup shown in 3B in the text. In particular in the second paragraph of this section the authors start describing the control cells and then suddenly about TDG depleted cells.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "6036", "date": "19 Oct 2020", "name": "David Schürmann", "role": "Author Response", "response": "We thank Dr Bultmann for the evaluation of our manuscript. We appreciate his comments helping to improve it. Accordingly, the following adaptations were made : The first point was also raised by Dr Torchia (reviewer 2) and we describe better the random integration of the miniTdg gene. We also added a note about the transcriptional stability of the transgene in chapter 1 of the Result section. We apologize for the confusing description of our results. We now clearly indicate whether we refer to TDG proficient or deficient cells. We also added an outline of the experimental setup for the neural differentiation." } ] } ]	1	https://f1000research.com/articles/9-1112
https://f1000research.com/articles/8-1217/v1	30 Jul 19	{ "type": "Research Article", "title": "The diversity of Anopheles blood feeding patterns suggests different malaria protection strategies in different localities", "authors": [ "Irfanul Chakim", "Tepanata Pumpaibool", "Irfanul Chakim" ], "abstract": "Background: Malaria is a significant health burden for many countries worldwide. Insecticide-treated bed nets and mosquito repellent are considered effective methods for preventing Anopheles bites. However, changes in the biological properties of the vector have led to a reduction in their effectiveness. Most published studies have only investigated the human population factor, not the dynamics of vector behavior. Therefore, this study aims to investigate the importance of primary vector activity for selecting an appropriate malaria protection strategy. Methods: Initially, active case detection (ACD) was carried out in western and eastern parts of Indonesia, Jambi and Sumba, to confirm their endemicity level. According to the 2016 national health report of Indonesia, Jambi has an annual parasite index (API) of 0.14 and Sumba has an API of 5.41. A series of entomological observations were carried out to compare the biting activity of Anopheles vector in two localities, with a total of 216 houses and catchers (108 in each study site). Results: The results indicated that endemicity at the sub-district level is higher than that at the provincial level. Only Anopheles balabacensi was found to be exophagic. Multiple comparisons found different biting times between the sites, suggesting that early evening (18.00-20.00) is most likely to be the time when mosquitos transmit the Plasmodium parasite in Jambi, while during sleeping hours (21.00-01.00) is the peak biting time of Anopheles mosquitos in Sumba. Conclusions: The study demonstrates the importance of Anopheles species blood feeding patterns in selecting an appropriate malaria protection strategy.", "keywords": [ "Malaria", "Anopheles", "diversity", "blood feeding pattern", "protective strategy" ], "content": "Introduction\n\nMalaria is a disease that is transmitted by female Anopheles vectors. Generally, malaria control is achieved by mass deployment of insecticide-treated bed nets (ITNs), treated with insecticide corresponding to the biological activity of the vector. It has been shown that the distribution of ITNs is responsible for a reduction of 68% in malaria burden in sub-Saharan Africa1. This control method has been widely distributed and a dramatic increase in use has resulted in the mass utilization of ITNs in many countries2,3. Additionally, personal protection has been found to be effective against mosquito bites and its use has led to a reduction in malaria infection4–6. However, frequent daily application is required in order to ensure its effectiveness7–9.\n\nThe efficacy of both protection strategies may be problematic as mosquito behavioral activities differ significantly between locations, as observed in Africa, where the vectors exhibit behavioral plasticity10–15. The shifting behavior of the Anopheles vector is a factor that contributes to reduced ITN effectiveness. The behavioral changes of Anopheles mosquitos are in the form of shifts to exophagic behavior14,15 and biting time modification16. Several findings indicate the ineffectiveness of repellent against malaria infection. The limitations of repellent seem to be related to daily adherence and compliance17,18 and disproportional utilization19. This issue may be due to the assessment of mosquito protection agents being influenced by social desirability rather than the impact that such types of protection have on the biological property of the vector.\n\nThe most effective method of Anopheles biting protection varies between sites and is dependent on the biting activity of the vector. In Uganda, intensive use of ITNs has been suggested due to the biting pattern of Anopheles gambiae, with biting mostly occurring late at night, during the time the human population is asleep20. In contrast, bed nets may not provide proper protection against the same Anopheles species in Burkina Faso due to an early evening biting time21. Limited studies have investigated Anopheles biting patterns in the Indonesian archipelago22. Thus, our study aimed to specifically address the information gap of Anopheles biological properties in Indonesia.\n\n\nMethods\n\nThe sampling was carried out in two localities representing different endemicity areas, namely Jambi province and Sumba Island (Nusa Tenggara Timur Province). Jambi is in Sumatra Island, the western part of Indonesia, geographically situated at 0.45 ° North Latitude, 2.45 ° South Latitude and between 101.10 ° -104.55 ° East Longitude. Sumba Island is situated in the eastern part of Indonesia, with an area of 10,710 km2 and coordinates of 9°40′S 120°00′E. Jambi and Sumba have a total population of 3,515,017 and 685,186, respectively. Jambi has 11 districts with 136 sub-districts and Sumba has four districts with 44 sub-districts. From all of the sub-districts over the sites, the sub-district of each area with the highest number of cases of malaria was selected for our study to be carried out in (Lembah Masurai in Jambi and Kodi Balghar in Sumba). According to the 2016 national health report of Indonesia23, Jambi has an annual parasite index (API) of 0.14 and Sumba has an API of 5.41 (Table 1).\n\nTo investigate the API in each sub-district, a series of parasitological assessments were carried out. This assessment was conducted from November 2017 to July 2018 in Jambi and from May to August 2018 in Sumba. Active case detection (ACD) was carried out daily in each site, performed by a local primary healthcare worker. Only people with a tympanic temperature of more than 37.5°C were included in the study. People were asked to go to the local village office for where the finger prick blood sample was collected. Cases were confirmed by light microscopy and prick blood samples were collected directly onto glass slides. A total of 559 and 500 blood samples were taken from Jambi and Sumba, respectively. Two certified independent microscopists assessed all the slides taken from ACD and determined the parasite species.\n\nThe API of both sites was calculated using the following formula24:\n\n\n\nA series of entomological observations were conducted for comparison of the pattern of blood feeding of the potential vector between the two localities. A 24-day observation was done in each area. The human landing catch (HLC) method was used for obtaining Anopheles vectors22. The HLC method is a standard method for measuring the exposure of humans to mosquito bites as it directly captures mosquitos that land and attempt to feed on collectors25. HLC requires an indoor and outdoor catcher present over 12 hours, from 6 pm to 6 am, to reflect the pattern of Anopheles biting and blood feeding time preference. The catchers were the owners of the houses and were trained on how to conduct the HLC method. In the current study, indoor and outdoor mosquito collection was carried out at each house22. Six houses were selected for daily HLC and there were six days of collection per a week. Inclusion criteria of the houses was as follows: (1) three houses had to have had a malaria infection during the previous one-year period; (2) the other three houses had to have had an absence of malaria infection for at least one-year and had to be in close proximity to the infected houses. The information about malaria infections at each house was obtained by interviewing each house member. In total, there were 216 houses and 216 catchers (108 at each study site). The observation was carried out 24 days in each study site. Random selection was done for repetition (for example, a house which had indoor collection in the first week would have outdoor collection in the next week and change to indoor in the last week and vice versa); thus, each house had the same pattern of an indoor and outdoor collection. The distance between each house was less than two kilometers to avoid biases due to potential differences in mosquito species abundance. All the mosquito species were confirmed by entomological experts from Eijkman Institute for Molecular Biology, Jakarta, Indonesia by dissection and viewing under a light microscope using the Anopheles identification key developed by Rattanarithikul et al.28.\n\nTo analyze the data, descriptive and analytical tests were carried out to analyze the mosquito blood feeding pattern of each site. The analysis provided three types of results: 1) the preferred biting time of Anopheles mosquitos at each site by comparing the number of collected mosquitos in each site using a student t-test statistical method; 2) a comparison of the number of mosquitos collected indoors and outdoors from each location using the Mann Whitney test; and 3) multiple comparisons of biting time by pooled analysis for each location using the Kruskal-Wallis test and Dunn’s multiple comparison test. All the analyses and comparisons were carried out using GraphPad Software version 8.00 (La Jolla California, USA). Relative abundance and human landing rate (HLR) were calculated using the following formulas:\n\n\n\n\n\nInformed consent was obtained from collectors performing HLC. Permission was also received from the owner of the house and the community on both sites. Community permission has been obtained by collectively gathering village residents along with the head of the village in the village office. Written informed consent was also sought for every participant of the parasitological assessment. This study was approved by the ethics commission of Universitas Muhammadiyah Semarang [22/EC/FKM/2017].\n\n\nResults\n\nThe parasitological assessment found a total of 211 cases of malaria in both localities29. Only Plasmodium vivax was found in Jambi, responsible for 71 malaria cases. Participants from Jambi were 60.6% male (43) and 39.4% female (28) with a mean age of 15.5 years, ranging from one to 59 years. In Sumba, three types of Plasmodium were successfully detected during ACD. From a total of 140 malaria cases in Sumba, 92 (65.7%) were Plasmodium falciparum, 43 (30.7%) were Plasmodium vivax, and 5 (3.6%) were Plasmodium malariae. Participants from Sumba were 58.6% male (82) and 41.4% female (58) with a mean age of 10.9 years, ranging from one to 53 years. The calculated APIs of the two study sites were 3.56 and 15.96, respectively (Table 1). The API result of this study is different to the national health report of the Ministry of Health, Indonesia. The API is up to 2.95-25.4-fold higher at the sub-district level, found in this report, than at the provincial level, as stated in the report.\n\nA total of 2,435 Anopheles mosquitos were successfully collected from 216 houses and 216 catchers at the two locations (108 houses and catchers at each study site)29. There was a statistical difference in the total number of Anopheles mosquitos caught between Jambi and Sumba (P value= <0.0001). Jambi had mosquito abundance of 71 and Sumba had 2,364. Four Anopheles species were successfully collected in Jambi, namely Anopheles balabacensis, Anopheles barbirostris, Anopheles maculatus and Anopheles sinensis. An. balabacensis, which belongs to leucosphyrus group, had the highest abundance, as shown with its relative abundance of 78.87 and HLR of 0.52 per person per night, followed by An. maculatus (relative abundance: 18.31 and HLR: 0.12 per person per night), An. barbirostris (relative abundance: 1.41 and HLR: 0.01 per person per night) and An. sinensis (relative abundance: 1.41 and HLR: 0.01 per person per night). In contrast, the dominant Anopheles species in Sumba were Anopheles aconitus and Anopheles sundaicus, with a relative abundance of 40.02 and 58.50 and HLR of 8.76 and 12.81 per person per night, respectively. The other minor species found were An. barbirostris (relative abundance: 0.09 and HLR: 0.02), Anopheles farauti (relative abundance: 0.04 and HLR: 0.01), Anopheles leucosphyrus (relative abundance: 0.04 and HLR: 0.01), An. maculatus (relative abundance: 1.06 and HLR: 0.23), Anopheles subpictus (relative abundance: 0.17 and HLR: 0.04) and Anopheles vagus (relative abundance: 0.09 and HLR: 0.02) (Table 2).\n\nThere was a difference in Anopheles biting time between Jambi and Sumba (Figure 1 and Figure 2). An. balabacensis from Jambi has a peak in biting time during early evening (6 pm), which decreases substantially until midnight, while An. maculatus showed an irregular biting time pattern. On the other hand, there is a similar trend in biting time between An. aconistus and An. sundaicus collected from Sumba; it gradually increased until its peak biting time between 21.00-22.00 and 01.00-02.00; then, it decreased progressively until 05.00-06.00. Additionally, an irregular biting time pattern has also been observed for An. maculatus from Sumba.\n\nHLC, human landing catch.\n\nHLC, human landing catch.\n\nTo investigate the biting preference of Anopheles mosquito, an indoor and outdoor comparison was carried out (Figure 3 and Figure 4). There was a statistically significant finding for the biting preference of An. balabacensis from Jambi; the number of collected mosquitos from outdoor was higher than that of indoor collection. No statistical difference was observed for An. maculatus. A similar pattern was found for An. aconitus, An. maculatus and An. Sundaicus, where there was no difference between indoor and outdoor collection, suggesting that undertaking an indoor or outdoor activity carries the same risk of getting mosquito bites.\n\nHLC, human landing catch.\n\nHLC, human landing catch.\n\nTo investigate the difference in mosquito biting times between Jambi and Sumba, a multiple comparison analysis of pooled mosquito sample data was carried out (Table 3 and Figure 5). Based on the mosquito biting time in Jambi, the number of bites during the early evening (18.00-19.00) was statistically different from other biting times, from 21.00-22.00 to 05.00-06.00 (P value= <0.00001). Additionally, the number of bites at 19.00-20.00 was statistically different from the number at 24.00-01.00 and 05.00-06.00 (P value= 0.0435). In Sumba, the number of bites during the early evening at 18.00-19.00 was statistically different from the other biting times, except for 19.00-20.00, 04.00-05.00 and 05.00-06.00 (P value= <0.0001-0.0069). In addition, the number of bites at 19.00-20.00 differed from the number at 21.00-22.00, 22.00-23.00 and 23.00-24.00 (P value= 0.0088-0.0168): 21.00-22.00 differed from 04.00-05.00 and 05.00-06.00 (P value= 0.0030-0.0052); 22.00-23.00 differed from 04.00-05.00 and 05.00-06.00 (P value= 0.0030-0.0050); 23.00-24.00 differed from 04.00-05.00 and 05.00-06.00 (P value= 0.0059-0.0099);and 24.00-01.00 differed from 05.00-06.00 (P value= 0.0347). These results indicate that in Jambi, the peak biting time is during early evening at 18.00-20.00. In Sumba, the mosquitos started feeding and feeding gradually intensified during the early evening (18.00-21.00), the intensity of the mosquitos was stable until 02.00 and then the mosquito biting intensity declined during the early morning.\n\n* <0.05, <0.01, * <0.001, **** <0.0001.\n\nHLC, human landing catch.\n\n\nDiscussion\n\nAccording to the Malaria Atlas Project30, for API <0.1, Plasmodium falciparum and Plasmodium vivax distributions are similar across the Indonesian archipelago. Plasmodium falciparum is more stable in distribution, where each part of Indonesian archipelago has the same pattern of low to moderate API. Meanwhile, Plasmodium vivax is more intense in the eastern part of Indonesia and unstably distributed in the western part of Indonesia. However, only Plasmodium vivax was found in Jambi, and more diverse Plasmodium species have been observed in Sumba, suggesting a different diversity of Plasmodium species distribution in the two localities. A discrepancy was also found in the calculated API between this study and the basic health report by the Ministry of Health of Indonesia, which might be explained by the different ways of presenting the data. The national health report23 used the provincial population and the larger the area, the larger the population involved in the calculation, as API is calculated by dividing the total cases and the total population. API at a sub-district level is often observed to vary from one district to another and variation between districts is observed at a provincial level31,32.\n\nThere are 20 Anopheles species known to be vectors for malaria in Indonesia. In this study, four and eight species have been found in Jambi and Sumba, respectively. The student t-test suggested a different abundance in the number of Anopheles mosquitos between the two sites. This difference is often explained by environmental conditions. A distinct sampling time may cause this difference in mosquito abundance; however, since rainfall anomalies have been observed in Indonesia, this may not be the case33. Since the existence of Anopheles breeding sites depends on rainfall providing a sufficient water bodies for the mosquitos to lay eggs, rainfall anomalies in Indonesia may lead to to an irregular pattern of mosquito abundance across time and place in Indonesia. The limited number of water bodies or humidity conditions may affect the habitat and abundance of Anopheles mosquitos in Jambi34,35. The difference in mosquito abundance may also reflect the annual incidence rate of malaria infection in different endemic areas. However, no correlation may be found if the correlation of mosquito abundance and annual incidence rate takes into account the species of Plasmodium36.\n\nThe main Anopheles vector and biting preference differs between Jambi and Sumba. An. balabencis, which belongs to leucosphyrus group, is the primary vector in Jambi, as determined from its highest relative abundance and HLR. Moreover, An. aconitus and An. sundaicus are the primary vectors in Sumba, along with other minor Anopheles species found. Only An. balabacensis in Jambi was found to be exophagic, as previously known from the biting preference of this peculiar species37. An. maculatus has been found to be both endophagic or exophagic similar to the finding of Elyazar et al.37. However, previous studies have found that An. aconitus has an irregular pattern of biting preference while An. sundaicus is mainly exophagic37. This study found that there was no significant difference between the indoor and outdoor biting preference of An. aconitus and An. sundaicus, suggesting that these species can be both endophagic and exophagic.\n\nBiting time is essential to understanding the underlying biological properties of mosquitos and to avoid Anopheles bites to control malaria infection. The data obtained suggest different biting times of Anopheles in Jambi and Sumba. Early evening (18.00-20.00) is most likely to be the mosquito feeding time in Jambi, when most people are undertaking activities and are unprotected. However, in the late evening (21.00-02.00), more people in Sumba may get Anopheles bites, reflecting sleeping time, when Sumbanese people may be vulnerable to infection with malaria parasites. This suggests the importance of ITNs for evading malaria infection in Sumba. The biting time of Anopheles in Jambi is similar to that in Halmahera, Maluku Island22. However, the finding from Sumba Island is different from other parts of Indonesia, which shows a gradual increase or decrease in the number of Anopheles mosquitos in accordance with its biting time22. Limited studies have tried to describe mosquito biting patterns in relation to the selection of malaria control strategies20,21. This finding strengthens the previous report that effective malaria prevention depends on local Anopheles vector biting behavior. Anopheles vectors in Jambi share the same behavior as those in Burkina Faso, where bed net protection may not be effective for preventing biting exposure as Anopheles species in the area are dominant in the early evening21. In contrast, similar to Uganda, intensive use of ITNs combined with indoor residual spraying is the most effective protection approach for Sumba Island for avoiding malaria infection20.\n\nBiting preference has previously been known to have an underlying genetic background38. For instance, chromosome inversions of 2Rbc, 2Ra and 3Ra and circadian clock genes are associated with exophagic and endophagic behavior in some Anopheles species39–41. However, genetic background may vary within the genus and among mosquitos within the same species in different locations41. The finding also suggests that differences in Anopheles biting time may be an effect of different genetic backgrounds. Further research might explore this aspect.\n\nThere are some limitations of the current study. There was no intervention included to measure the effectiveness of any type of protection in correlation with the different biting times in each study site. In further research, an intervention approach should be used to find the best protection strategy in locations that may have different Anopheles biting times. Additionally, our collection method was limited to three weeks observational research. A more prolonged study needs to be conducted to reflect yearly fluctuations in local Anopheles biting times.\n\n\nConclusion\n\nIn conclusion, this study suggests four important findings for public health control: (1) API may be significantly lower at the provincial level compared to the sub-district level and varied accordingly, suggesting that malaria foci may be maintained in a locality from a provincial level, especially in areas of low to moderate endemicity; (2) the importance of mosquito abundance information may reflect malaria incidence rate in a location42,43; (3) all Anopheles species, except An. balabacnesis, can be both endophagic and exophagic, suggesting a comprehensive protection approach is required to avoid mosquito bites regardless of being indoors or outdoors; (4) biting time may suggest the use a different prevention approach in each area; for example, people in Jambi may need to use mosquito repellent during activities in the early evening, while ITNs combined with indoor residual spraying may need to be deployed to protect malaria infection during sleeping hours in Sumba.\n\n\nData availability\n\nZenodo: The diversity of Anopheles blood feeding patterns suggest different malaria protection strategies in different localities. https://doi.org/10.5281/zenodo.326982429\n\nThis project contains the following underlying data:\n\n- Supplementary 1.xls (The total number of mosquitos collected, number collected per time period and number collected indoors/outdoors)\n\n- Supplementary 2.xls (The number of mosquitos caught for each species in Jambi and relative abundance and HLC calculations)\n\n- Supplementary 3.xls (The number of mosquitos caught for each species in Sumba and relative abundance and HLC calculations)\n\n- Supplementary 4.xls (Results of all Dunn’s multiple comparisons tests for biting times in Jambi and Sumba)\n\n- Supplementary 5.docx (Flow chart of the HLC collection method)\n\n- Supplementary 6.rar (detailed data of all Anopheles found in Jambi per collection type and collection time)\n\n- Supplementary 7.zip (detailed data of all Anopheles found in Sumba per collection type and collection time)\n\n- Supplementary 8.xlsx (demographic data and parasite species for participants from both study sites)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis research is supported by 90th Anniversary of Chulalongkorn University under the Rachadapisek Somphot fund.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors wish to thank people who have consented to take a part in this study, to Eijkman institute for molecular biology, Jakarta; Health Office of Jambi Province; Health Office of Nusa Tenggara Timur province and those who have helped.\n\n\nReferences\n\nBhatt S, Weiss DJ, Cameron E, et al.: The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. 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PubMed Abstract \| Publisher Full Text\n\nCostantini C, Sagnon N, Ilboudo-Sanogo E, et al.: Chromosomal and bionomic heterogeneities suggest incipient speciation in Anopheles funestus from Burkina Faso. Parassitologia. 1999; 41(4): 595–611. PubMed Abstract\n\nMaliti DV, Marsden CD, Main BJ, et al.: Investigating associations between biting time in the malaria vector Anopheles arabiensis Patton and single nucleotide polymorphisms in circadian clock genes: support for sub-structure among An. arabiensis in the Kilombero valley of Tanzania. Parasit Vectors. 2016; 9: 109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChurcher TS, Trape JF, Cohuet A: Human-to-mosquito transmission efficiency increases as malaria is controlled. Nat Commun. 2015; 6: 6054. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuelbéogo WM, Goncalves BP, Grignard L, et al.: Variation in natural exposure to anopheles mosquitoes and its effects on malaria transmission. eLife. 2018; 7: pii: e32625. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "54515", "date": "22 Oct 2019", "name": "Giles E. Duffield", "expertise": [ "Reviewer Expertise Biological timing of mosquito vectors" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study contributes to a developing insight into the efficacy of malaria control interventions that focus on the mosquito vector and its behaviour. It is fast becoming apparent that the success of interventions such as barrier nets, insecticides and other interventions is highly dependent upon the specific temporal and spatial behavior of the mosquito and its relationship to the patterns of human activity in each locality. Some of this is due to the species composition and in some cases, there is evidence for a shift in the behavior due to previous and/or ongoing selective pressures. The current study provides detailed evidence for distinct differences in the biting behaviours of Anopheles in two different locations in Indonesia. Such differences may be important in explaining differences in rates of malaria transmission, and may be useful in modifying current intervention methods. The manuscript is well written, and easy to follow. It represents a well-executed study and presents interesting data.\n\nMajor Comments:\n\nTable 3 and related results. For the purposes of scientific rigor, I suggest the authors employ multiple means corrected statistics rather than repeated single Students t-tests, such as an ANOVA followed by post hoc multiple means corrected pairwise tests such as Bonferroni, Tukey, Dunnett’s tests.\n\nPage 9. Paragraph 2. Discussion. The authors state that “…circadian clock genes are associated with exophagic and endophagic behavior in some Anopheles species. However, these studies do not report such findings. In fact, Malita et al. (2016)1 states the opposite, that they do not find any association between biting time or biting location and circadian clock gene polymorphisms.\n\nThe authors compare their results with studies of African populations of Anopheles. I think much can also be gleaned from relating the current study with the work conducted in the Solomon Islands and PNG. I am listing a few of those that may be relevant for consideration, although this is not an exhaustive list:\nFrequent blood feeding enables insecticide-treated nets to reduce transmission by mosquitoes that bite predominately outdoors. Russell TL, Beebe NW, Bugoro H, Apairamo A, Chow WK, Cooper RD, Collins FH, Lobo NF, Burkot TR. Malar J. 2016 Mar 10;15:156. doi: 10.1186/s12936-016-1195-8.2\nDeterminants of host feeding success by Anopheles farauti. Russell TL, Beebe NW, Bugoro H, Apairamo A, Cooper RD, Collins FH, Lobo NF, Burkot TR.Malar J. 2016 Mar 10;15:152. doi: 10.1186/s12936-016-1168-y.3\nAnopheles farauti is a homogeneous population that blood feeds early and outdoors in the Solomon Islands.Russell TL, Beebe NW, Bugoro H, Apairamo A, Collins FH, Cooper RD, Lobo NF, Burkot TR.Malar J. 2016 Mar 9;15:151. doi: 10.1186/s12936-016-1194-9.4\nSuccessful malaria elimination strategies require interventions that target changing vector behaviours. Russell TL, Beebe NW, Cooper RD, Lobo NF, Burkot TR. Malar J. 2013 Feb 7;12:56. doi: 10.1186/1475-2875-12-56.5\n\nMore could be discussed by the authors as to the relevance of the specifies specific dominance in each location. For example, much of the temporal biting profile for Anopheles mosquitoes in Jambi can presumably be explain simply by the dominant species at that location being A. balabacensis, which they authors have demonstrated to exhibit an early evening biting profile (Fig. 1). I think this simple species location effect could be stated more clearly as an obvious explanation for the differences in general ‘all-species’ assessment of anopheline biting activity at each location (Fig. 5).\n\nMinor Comments:\n\nAbstract. Methods section. The last sentence is ambiguous. Suggest rewrite as “..vectors in two localities, with a total of 216 houses and 216 catchers (108 at each study site).”\n\nThroughout text, “mosquitoes” is incorrectly spelt.\n\nThroughout text, there is inconsistent italicized “Anopheles”.\n\nIntroduction, paragraph 1. Please define better “personal protection”. Presumably this is the use of repellents, barriers, clothing, head-nets, etc.\n\nFigure 1. Are these SEM or SD bars on the charts?\n\nDiscussion: 2nd paragraph, “The difference in mosquito abundance may also reflect the annual incidence rate of malaria infection in different endemic areas”. Should this not be argued in the opposite direction, i.e. infection rate reflects mosquito abundance?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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": [ { "c_id": "4996", "date": "05 Nov 2019", "name": "irfanul chakim", "role": "Author Response", "response": "Comments by reviewer of the F1000 research: The diversity of Anopheles blood feeding patterns suggests different malaria protection strategies in different localities [Version 1; peer review: Approved with reservations]Author:We thank to the reviewer for his positive feedback. We do agree with these comments.Major commentsNo: 1Comments: We did not use ANOVA as suggested by reviewer due to our data was not normal based on normality test. Therefore, to run a multiple comparison test based on not normal data set, we need to do it by using a non-parametric test. Hence, Kruskall-wallis is a non-parametric test for multiple comparison test and Dunn’s is a non-parametric post hoc test.No: 2Action: We deleted such reference “cicardian clock genes” from the paragraph as suggested by reviewer.No: 3Action: References have been added to the text in the discussion section.No: 4Action: The information has been added in the discussion section.Minor commentsNo: 1Action: Revision has been made according to reviewer suggestion.No: 2Action: We have changed the word “mosquitos” to “mosquitoes” throughout text accordingly.No: 3Action: We have italicized the word “Anopheles” throughout text accordingly.No: 4Action: Information has been added to the text accordingly.No: 5Comment: These are SD bars on the chartsNo: 6Comment:We agreed with the reviewer.Action: Therefore we changed the sentence to “The difference in the annual incidence rate of malaria infection may also reflect mosquito abundance in different endemic areas.”" } ] } ]	1	https://f1000research.com/articles/8-1217
https://f1000research.com/articles/9-1254/v1	19 Oct 20	{ "type": "Research Article", "title": "Nutritional value of the Middle Eastern diet: analysis of total sugar, salt, and iron in Lebanese traditional dishes", "authors": [ "Maha Hoteit", "Edwina Zoghbi", "Mohamad Al Iskandarani", "Alissar Rady", "Iman Shankiti", "Joseph Matta", "Ayoub Al-Jawaldeh", "Maha Hoteit", "Edwina Zoghbi", "Mohamad Al Iskandarani", "Alissar Rady", "Iman Shankiti", "Joseph Matta" ], "abstract": "Background: The expanding burden of diet-related non-communicable diseases in the Eastern Mediterranean Countries requires urgent public health vigilance and actions. This study aimed at establishing a database analysis of total sugar, salt and iron content in Lebanese foods, focusing on traditional dishes. Methods: The collection of food samples was done using stratified sampling techniques. These samples were classified into five strata, taking into account variation by geographical area (Mount Lebanon, Bekaa, Beirut, Tripoli, and Saida). The number of samples per governorate was estimated to be 30 according to the variability in the dishes' composition. Food samples were chemically analyzed for total sugar, salt, and iron. Results: Among all the governorates, all the tested traditional Lebanese dishes contained little total sugar. More than 60% of the samples tested were rich in sodium. The sodium content ranges were 120-720 mg/100 g in Mount Lebanon, 240-960 mg/100 g in Bekaa, 80-520 mg/100g in Beirut, 252-1952 mg/100g in Tripoli and 40-680 mg/100 g in Saida. The highest mean amount of sodium was observed in the dishes Fatayer Sabanikh and Malfouf Mehche (≥ 600 mg/100 g). Furthermore, more than 80% of the samples had poor amounts of iron in all governorates. Conclusion: This study emphasizes the need for multi-cultural education and awareness on food sources of salt and iron, and the health effects regarding high intake of salt and low intake of iron. This study is a stepping stone for further research exploring total sugar, salt and iron content of traditional dishes, as well as potential intake by individuals in the Lebanese population.", "keywords": [ "Total sugar", "salt", "iron", "Lebanese traditional dishes", "governorates" ], "content": "Introduction\n\nIn light of global spread of non-communicable diseases (NCDs), accounting for 70% of the 41 million deaths each year, the probability of premature death, in 2018, caused by NCDs is 91% of all deaths in Lebanon1. The global rise in the prevalence of NCDs is a consequence of shifting dietary patterns specified by a high intake of meals rich in fat, sugar, salt, and low in fiber and micronutrients2. Home cooking and at-home eating have become scarce, while processed foods and prepared meals are increasing and have become a major part of the population's lives2. Furthermore, it is widely believed that urbanization has played a role in producing side effects for NCDs-related health outcomes3. Cardiovascular diseases (CVDs) constitute the primary cause of death in the Middle Eastern Region4. According to the World Health Organization (WHO), based on 2017 data, 17.9 million people die each year from CVDs, representing 31% of all deaths worldwide5. Furthermore, around 23.6 million people are expected to die from many forms of CVDs by 20306.\n\nHigh blood pressure is a dominant factor for CVDs7. A higher intake of dietary salt (equivalent to 5 g salt/day) is associated with high levels of blood pressure, cardiovascular diseases and strokes5. In May 2013, the WHO Member States decided to target a reduction in the global intake of salt by 30% by 20258. A systematic review, published in 2020, on 133 randomized control trials showed a reduction of 1.10 mmHg in systolic blood pressure and 0.33 mmHg reductions in diastolic blood pressure with every 50 mmol reduction in 24-hour sodium excretion. This can lead to a protection against vascular complications and decrease CVD risk9. In addition, there is considerable evidence of the advantages of Mediterranean diet on health10. An umbrella review and meta-analyses of observational and randomized controlled trials showed a strong relation between the adherence to Mediterranean diet and the 50% reduction in mortality from NCDs10.\n\nToday, the major target of nutrition interventions is the energy dense \"added sugars\". The WHO definition for added sugars is \"all monosaccharides and disaccharides added to foods and beverages by the manufacturer, cook, or consumer, and sugars naturally present in honey, fruit juices, and fruit concentrates\"11. In this definition the \"total sugar\" term that is \"naturally occurring or intrinsic sugars, which are stored within the cells of intact fruits, vegetables, or involve lactose in milk or unsweetened dairy products\" was not included11. In 2015, the WHO recommended the reduction of the daily intake of added sugars to less than 10% of the total energy intake12. Additional suggestion of reducing the daily intake of added sugars to less than 5% of total energy intake may promote additional health benefits12. Until today, total sugar has no recommendation for the daily intake13. However, for the aim of achieving the goals set by the Global Action Plan for NCDs 2013–2020, a guideline on total sugar was developed to help in the reduction of the prevalence of diabetes and obesity, and decrease the incidence of premature deaths caused by NCDs by 25% by 202514.\n\nFurthermore, deficiencies in iron lead to the development of anemia, which is one of the most common nutrition problems. As per the WHO, more than 2 billion people are anemic and iron deficiency is one of the main causes15. In addition, there is a high prevalence of 42% of children less than 5 years of age and more than 40% of pregnant women who are anemic15. In 2012, specific dietary guidelines for Arab countries were developed by the WHO and the Arab Center for Nutrition. Only seven countries that represent 29% of the Eastern Mediterranean Region population (namely Afghanistan, KSA, Oman, Lebanon, Qatar and Iran) designated their national food based dietary guidelines16,17.\n\nThe main purpose of this study is to initiate a database analysis of total sugar, salt and iron content in Lebanese foods, focusing on traditional dishes.\n\n\nMethods\n\nThe definition of ‘composite dishes’ is \"dishes consumed at main meals (i.e. lunch or dinner), whose preparation involves culinary skills and contains ingredients from at least three of five main food groups: meat/poultry/fish and eggs; dairy products; fruits and vegetables; starchy foods including legumes; added sweets and fats\"18. The list of Lebanese composite dishes frequently consumed by Lebanese citizens was retrieved from a study done in 2005 on a representative sample of 799 Lebanese adults18, and in line with a study conducted in 2009 where the objective was to compare the consumption of traditional dishes between Lebanon and France19. The Lebanese diet includes a range of foods with often complex recipes, and it is rarely possible to analyze all the types of dishes. In such cases, a laboratory analysis of the traditional dishes and a calculation of some nutrients should be achieved. The names of the dishes most eaten by Lebanese citizens and chosen for this study are shown in Table 1.\n\nSamples of cooked composite dishes were collected (see below). The food samples were bought from five different governorates (Mount Lebanon, Bekaa, Beirut, Tripoli and Saida), taking into account geographical and cultural variations.\n\nA total of 500 g of each dish was collected and used for analysis. According to Greenfield and Southgate, this size is a convenient sample to avoid errors during analysis20.\n\nOur research group collected 500 g of 30 types of traditional dishes from five different central kitchens in the following governorates: Mount Lebanon, Beirut, Bekaa, Tripoli, and Saida to show wide representation. Thus, 150 samples were collected. A list of kitchens that served tradition meals, found on internet, was prepared for each of the governorates. The central kitchens were randomly chosen from the results of the internet search, based on the following criteria: 1) their specialty in cooking home-made dishes; 2) their popularity; and 3) their involvement in supporting women as part of the social entrepreneurship initiatives that are aimed at empowering women.\n\nAfter the receipt of food samples, 500 g of each composite dish was mashed, and then analyzed in the laboratory. The remaining samples were kept frozen at -18°C in tight containers for further analysis.\n\nTotal sugar. An amount ranging between 2 and 10g (depending on expected value of sugar content) of the sample was put in a 250 ml volumetric flask. 125 ml of 50% alcohol solution was placed in steam bath overnight. The volume was made up to 125 ml with 95 % ethanol followed by filtration with filter paper. 100 ml of the filtrate was pipetted into a beaker and evaporated to reduce the volume to 20 – 30 ml. After that, the solution was placed in 100 ml volumetric flask and rinsed thoroughly with water and then added to the flask. Then the solution was made up to volume of 100 ml and mixed thoroughly. Later on, 50 ml of the obtained solution was placed in 100 ml volumetric flask,then a piece of Lithmus paper was added and the total was neutralized with hydrochloric acid (HCL). An addition of 5 ml of HCl was added and the inversion of sucrose was done at room temperature. When the inversion of sucrose was complete, the solution was transferred to a beaker and neutralized with Na2CO3 until a pink color appeared. Later, the solution was returned to 100 ml flask, diluted to volume of 100 ml with water. Filtration was done when necessary, and the determination of reducing sugars in 50 ml of the solution was done by adding 25 ml each of Fehling A and Fehling B solutions and boiling for 4 min. After a red precipitate formed, the solution was cooled and filtered with a Gooch crucible. The mixture was dried in oven and the calculation of the precipitate was achieved (AOAC 906.03, 930.36 and 975.15)21.\n\nThe content of reducing sugars was red from the table, and calculated as % sugars using the formula\n\n\n\nSalt. To measure the salt content, 2–3 g of the food sample was weighteded into a pre-weighed furnace-proof crucible. It was baked until ash in a 600° C furnace overnight.\n\nWhen the sampled had cooled, the ash was dissolved in water and was transferred quantitatively in a 50 ml volumetric flask. After making up to volume of 50 ml, the solution obtained was transferred into an Erlenmeyer flask. Later on, 1 ml of potassium chromate indicator solution was added and the solutions was titrated drop wise with the addition of 0.1 N silver nitrate solutions until the color of the solution changed to a reddish brown. A blank test was carried out in parallel using 50 ml of distilled water instead of the sample solution. The blank value did not exceed 0.2 ml of silver nitrate (AOAC 937.09 & ISO 9297)21.\n\nThe chloride content in the sample was calculated using the formula.\n\n\n\nWhere Vs = volume in ml of silver nitrate\n\nN = normality of silver nitrate\n\nWt = weight of sample used\n\nFinally, the chloride content was used to calculate NaCl. The molecular weight of NaCl is 58.44 and the molecular weight of Chloride is 35.453 g. Thus, to calculate the NaCl in the solution, the Cl should be multiplied by 1.65 (58.44 divided by 35.453).\n\nIron. An amount of 15 g of the edible portions was dried in an air oven at 105°C for more than 2 hours then charred until smoke disappearance. A muffle furnace was used to bake the charred sample at 550°C until a grey-colored ash yielded. 25–30 ml of 3.7% HCl was used to treat the ash, and then the mixture was conveyed to a container and done up to a volume of 50 ml with HCl. For each tested food, two solutions of ash were made ready to be analyzed twice. Each fraction of the ash sample was used for iron measurement using a Varian Atomic Absorption Spectrophotometer model 175 with an air-acetylene flame. Ferric nitrate solution was used as a standard. For each ash solution, at least three readings were obtained, and the average was calculated based on AOAC 975.03, 985.35 &965.0921.\n\nAll the above chemical analyses were conducted at the Industrial Research Institute (Beirut, Lebanon), which is an accredited laboratory for chemical analyses. After food samples were analyzed, stamped laboratory results were received and data was entered on Excel 2019. The data validation process was conducted, and cross checked by two research assistants.\n\nThe recipes of the dishes in this study were selected from those mostly consumed in Lebanon. The dishes were bought and analyzed as they are consumed by customers. The description (ingredients) of the selected recipes is presented in Table 1.\n\n\nResults and discussion\n\nThe composition of 100 g of the Lebanese traditional dishes in the five Lebanese governorates is shown in Table 2.\n\nUnits: Total sugar (Tot S.) in gram, salt (NaCl) in gram, sodium (Na) in milligram, and iron in milligram.\n\nAmong all the governorates, all the tested traditional Lebanese dishes contained little total sugar. The highest total sugar (≥3 g/100 g) was observed in the dishes Hindbe b bzet and Falafel from Mount Lebanon, in Yakhnet Bemieh and Lahm bi ajeen from Bekaa, in Lahm bi ajeen and Foul Moudamas from Beirut, Lahm bi ajeen and Fattoush from Tripoli, and in Lahm bi ajeen and Falafel from Saida (Table 2). As per Table 3, Lahm bi ajeen and Falafel ranked first in the highest mean total sugar.\n\nUnits: Total sugar in gram, salt in gram, sodium in milligram and iron in milligram.\n\nThe sodium (Na) level was high in most dishes (Table 2). The highest mean amount of Na was observed in Fatayer Sabanikh and Malfouf Mehche (≥ 600 mg/100 g) (Table 3). The Na content ranges are 120–720 mg/100 g in Mount Lebanon, 240–960 mg/100 g in Bekaa, 80–520 mg/100g in Beirut, 252–1952 mg/100g in Tripoli, and 40–680 mg/100g in Saida.\n\nOne of the main factors of the richness of these Lebanese dishes in salt is mainly the high amount of salt added to the ingredients. Additional factors could be the use of spices rich in Na, like coriander leaf, cumin, cloves and cinnamon, during cooking, which are commonly used in the Lebanese cuisine22. The Bahraini plates Shawarma Lahme and Shawarma Djeij contained 100 mg of Na23, less than the Lebanese plates (Table 3). Unfortunately, we could not compare the Lebanese traditional dishes to the traditional dishes in other Arab and Middle Eastern countries because few dishes are common between them.\n\nIn general, meat-based dishes contained the highest levels of iron compared with other dishes. This was observed in Mount Lebanon particularly in Chichbarak and Batata w kafta (≥2.5 mg/100 g) (Table 2). However, in all other governorates, the highest level of iron was shown in Fatayer Sabanikh (≥4.5 mg/100g) (Table 2). As for the highest mean level of iron, Table 3 shows that Fatayer Sabanikh and Kafta w Batata ranked first. The Bahraini plates, Shawarma Lahme and Shawarma Djeij, contained the triple amount of iron (>3 mg) compared to the Lebanese plates (around 1 mg of iron)23.\n\nGiven the available evidence about the influence of culture on health and health behaviors, reducing total sugar or salt and improving iron intake cannot be separated from cultural factors influencing nutrition-related-attitudes towards the health benefits of iron intake or the use of salt when cooking foods. Cultural influences play a critical role in framing how people perceive food preparation patterns, dietary habits, and coping with variability in agricultural conditions24.\n\nThe nutrient goal represents the average intake that is compatible with maintaining of good health in individuals25. According to the US Food and Drug Administration (FDA) definition, the daily value (DV) is \"reference values for reporting nutrients on the nutrition labels\". The percentage (%) DV assists the consumer in recognizing how the serving of food and its content in nutrients, fit into their daily diet25. As per FDA regulations, the expression \"high,\" \"rich in,\" or \"excellent source of\" nutrients are used if the food has≥20% of the daily value per reference amount. The terms \"good source,\" \"contains,\" or \"provides\" are used if the food yields 10–19% of the Recommended Dietary Intake (RDI) per reference amount of the nutrient. Foods that carry <10% of the RDI from the nutrient per reference amount are considered as having low amounts25–27.\n\nIn our study, the contribution of each dish to the overall amount of iron, salt and total sugar needed per day was calculated. The calculations are presented in Table 4 and Table 5. The term \"total carbohydrate\" (CHO) includes dietary fiber, total sugar, added sugars and sugar alcohols. As per the updated FDA regulations, the DV of total CHO is 275 g/day (55% of energy intake in a 2000 kcal-diet) and the DV of added sugar is 50 g/day (10% of energy intake in a 2000 kcal-diet)26,27. As per Rapaille et al, and because of the low digestibility of polyols or sugar alcohols, the intake level is restricted to 40–50 g per day in adults28. We hypothesized that based on the above recommendations; the daily total sugar consumption should not exceed 147 g per day. This number was obtained by subtracting 50g (recommendation of total sugar) and 50 g (recommendation of polyols) and 28 g (recommendation of fiber) from 275 g (recommendation of total carbohydrate). As per WHO, the iron intake should be limited to 18 mg/day15 and the salt intake should not exceed 5g/day (1 tsp)29.\n\nThe daily recommended amount of total sugar is 147 g; NaCl is <5 g;iron is18 mg.\n\nWe determined the percent contribution of 100 g of each traditional dish to the mean daily need of total sugar, salt, and iron in a 2000 Kcal-diet. The percentage of contribution of nutrients and minerals in a 2000 kcal-diet tested in 100 g of the dishes is shown in Table 4. Our data showed that all the traditional dishes in the five governorates contain low amount of total sugar (<10% of the DV). As for the salt, the majority (67%) of the dishes contains a high amount of salt; however this differed between governorates. A total of 43% of the dishes from Mount Lebanon and Bekaa, 13% of dishes from Beirut, 86% of the dishes from Tripoli, and 40% of dishes from Saida contained a high amount of salt (>20% of the DV). High sodium intake is associated with hypertension, a modifiable risk factor for CVDs5. In 2013, Powles et al. estimated that 3.13 g per person per day was the daily sodium intake in the Lebanese population30. In addition, a representative survey on 2,543 Lebanese adults showed that the average sodium intake was 2.9 g/day, with high intake in men (3.4 g/day) compared to women (2.4 g/day)31. Around 60% of the adults in Lebanon are considered as exceeding the WHO limit level of Na intake of 2 g/day31. Parallel to these alarming results, another study was conducted to investigate the knowledge, attitudes, and salt-related behaviors in Lebanese consumers. It showed that the majority of Lebanese participants developed discernible behaviors that increase the intake of salt and had limited knowledge of the food sources of salt22. Thus, the adoption of long-term strategies by healthcare providers is required to control adding salt to reduce its negative effects, in Lebanon and elsewhere.\n\nAs for the iron content in the Lebanese traditional dishes, more than 60% of the recipes are deficient in iron (<10% of the DV of iron). Only 6% of the dishes from Mount Lebanon and Bekaa, and 3% of the dishes from Beirut, Tripoli, and Saida contain a high amount of iron in 100 g from each dish. Furthermore, in Table 5, the range of the percentage of DC of iron fluctuates between 4% and 27% indicating low content of iron in these dishes. Pellet and Shadarevian tested some of the same traditional dishes in 1970; their data also showed that all the dishes were also deficient in iron32. More than one third of the region’s population suffers from anemia especially preschool children, pregnant women and women of childbearing age15. Iron deficiency and iron-deficiency anemia have numerous adverse health consequences that affect the health of high-risk groups (women and children)15,33. Iron deficiency and iron deficiency anemia can be caused by numerous factors such as low iron intake, decreased iron absorption, chronic blood loss, and chronic inflammation15,33. There is a high worldwide prevalence range of iron deficiency anemia among pregnant women (14–75%)33. This type of anemia can lead to maternal complications and increased the risk of obstetric hemorrhage in pregnant women34. Furthermore, more than 50% of Lebanese hospitalized children aged between 1 month and 12 years, were anemic moderately or severely35. Taking into consideration that anemia is prevalent in children and women, preventive measures related to good nutritional patterns and supplementation of iron-rich food are recommended. Strategies to tackle iron deficiency involve nutrition education and food fortification as well as targeted interventions for high risk groups, when needed. Wheat and maize flour fortification is a simple, inexpensive and effective strategy for supplying vitamins and minerals36.\n\nThe daily recommended amount of total sugar is 147 g; NaCl is >5 g; iron is 18 mg.\n\nThere have been widespread priority actions that were implemented by the WHO Regional Office for the Eastern Mediterranean Region in response to unhealthy eating patterns among this population and the high prevalence of obesity. In 2016, taking into account the recommendations made by the WHO’s Commission on Ending Childhood Obesity, the WHO Regional Office for the Eastern Mediterranean Region prioritized the importance of dietary patterns modification at a regional level to halt the rise of NCDs in all age categories37. These priority targets and interventions have sufficiently strong evidence based expert analysis38. Furthermore, the WHO Regional Committee for the Eastern Mediterranean Region translated the United Nations political declarations and recommendations into a regional framework for action targeting the endorsement of population and individual measures by WHO Member States39.\n\n\nConclusions\n\nAt the national government level, all the related-health sectors should develop a comprehensive range of policy actions that provide support of healthy diet adherence to ensure that agriculture and food policies meet health recommendations. They should also assess the effects of the agri-food systems on NCDs. At the research level, academia should be engaged proactively in building evidence based, and communicating this data to policymakers, handling and supporting the development of data systems with research donors to enable countries to monitor the implementation of effective policies in conjunction with international health agencies.\n\nThe main limitation of this study is the analysis of nutrients and minerals in limited numbers of Lebanese traditional dishes. Furthermore, since the food samples were purchased from central kitchens, the proportions of ingredients were not described. The ingredients were retrieved from the literature (Table 1). In addition, variations in ingredients and preparation methods among traditional dishes from various governorates in Lebanon were inevitable.\n\nIn conclusion, this study is a stepping stone for studies exploring the total sugar, salt and iron content of traditional dishes, as well as potential intake in the Lebanese population. It emphasizes the need for multi-cultural education and awareness on salt and iron and their impact on health because nutrition education has the power to productively modify behaviors, attitudes and consumption. Healthy choices result from empowering and motivating people by increasing their knowledge. In general, food composition data are essential for taking action to educate and inform the public about nutrition. There remains a need to expand implementation of rules on nutrition labeling and to scale up introduction of simplified front-of-pack nutrition labeling. Widespread adoption of nutrition policies and/or strategies, along with implementation of multisectoral coordination mechanisms, is indicative of strengthened political commitment to improve nutrition in the countries of the Eastern Mediterranean Region.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgments\n\nThe authors would like to thank the laboratory technician Mr. Halim El Bayeh at the Industrial Research Institute and the Research assistants Ms. Nadia Hallak and Mrs. Iman Kheir at the Lebanese University.\n\n\nReferences\n\nNoncommunicable diseases progresses monitor 2020. Geneva: World Health Organization; 2020. Reference Source\n\nPopkin B, Adair L, Wen Ng S: NOW AND THEN: The Global Nutrition Transition: The Pandemic of Obesity in Developing Countries. Nutr Rev. 2012; 70(1): 3–21. 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Int J Food Sci Nutr. 2009; 60(Suppl 4): 285–95. PubMed Abstract \| Publisher Full Text\n\nBatal M, Al-Hakimi A, Pelat F: Dietary Diversity in Lebanon and Yemen: A Tale of Two Countries. Lebanon: American University of Beirut; 2005.\n\nGreenfield H, Southgate DAT: Food Composition Data Production Management and Use. 2nd edition. Rome: Food and Agriculture Organization of the United Nations; 2003. Publisher Full Text\n\nAssociation of Official Analytical Chemists: Official Method of Analysis. AOAC, Washington (DC), 19th edition, 2012.Reference Source\n\nNasreddine L, Akl C, Al-Shaar L, et al.: Consumer Knowledge, Attitudes and Salt-Related Behavior in the Middle-East: The Case of Lebanon. Nutrients. 2014; 6(11): 5079–5102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMusaiger AO: Food composition tables for kingdom of Bahrain. 1st edition. Bahrain: Arab Center for Nutrition. INFOODS regional database center; 2011. Reference Source\n\n2020 Global Nutrition Report: Action on equity to end malnutrition. Bristol, UK: Development Initiatives. ISBN: 978-1-9164452-7-7. Reference Source\n\nhttps://www.fda.gov/regulatory-information/search-fda-guidance-documents/industry-resources-changes-nutrition-facts-label/ accessed on June 10,2020.\n\nhttps://www.fda.gov/food/food-labeling-nutrition/label-claims-conventional-foods-and-dietary-supplements/ accessed on June 10, 2020.\n\nhttps://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=101.13/ accessed on June 10, 2020.\n\nRapaille A, Goosens J, Heume M: Sugar Alcohols. Encyclopedia of Food Sciences and Nutrition. 2003; 5665-5671. Publisher Full Text\n\nWHO: Guideline: Sodium intake for adults and children. Geneva, World Health Organization (WHO), 2012. Reference Source\n\nPowles J, Fahimi S, Micha R, et al.: Global, regional and national sodium intakes in 1990 and 2010: a systematic analysis of 24 h urinary sodium excretion and dietary surveys worldwide. BMJ Open. 2013; 3(12): e003733. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLebanese Action on Sodium and Health. accessed on June 12, 2020. Reference Source\n\nPellett P, Shadarevian S: Food composition tables for use in the Middle East. 2nd edition. Beirut, American University of Beirut, 1970. Reference Source\n\nMirza F, Abdul Kadir R, Breymann C, et al.: Impact and management of iron deficiency and iron deficiency anemia in women's health. Expert Rev Hematol. 2018: 11(9): 727–736.PubMed Abstract \| Publisher Full Text\n\nKhalafallah A, Dennis AE: Iron deficiency anaemia in pregnancy and postpartum: pathophysiology and effect of oral versus intravenous iron therapy. J Pregnancy. 2012; 2012: 630519. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalami A, Bahmad HF, Ghssein G, et al.: Prevalence of anemia among Lebanese hospitalized children: Risk and protective factors. PLoS One. 2018; 13(8): e0201806. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWHO: Recommendations on wheat and maize flour fortification: meeting report-interim consensus statement. Geneva, world Health Organization; 2009. Reference Source\n\nReport of the Commission on Ending Childhood Obesity. Geneva: World Health Organization; 2016.\n\nhttps://extranet.who.int/iris/restricted/bitstream/handle/10665/259519/emropub_2017_20141.pdf;jsessionid=669A113AC9CBCD95934ECDB66AEAFF1A?sequence=1/ acced on July 28, 2020.\n\nFramework for action to implement the United Nations Political Declaration on Noncommunicable Diseases. Cairo: World Health Organization Regional Office for the Eastern Mediterranean; 2015. Reference Source" }	[ { "id": "73294", "date": "02 Nov 2020", "name": "Reema Fayez Tayyem", "expertise": [ "Reviewer Expertise Human Nutrition and policy planning." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a well-written and informative manuscript. This study is targeted to establish and initiate a database analysis of total sugar, salt, and iron content in Lebanese foods, focusing on traditional dishes.\nA few suggestions and corrections have been addressed to improve the quality of the paper. All these corrections are presented in the pdf file attached.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "73820", "date": "26 Nov 2020", "name": "Haleama Al Sabbah", "expertise": [ "Reviewer Expertise Public Health and Nutrition" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks to the journal and authors for giving me this opportunity to review this excellent work.\nThe main objective of this study is to establish a database analysis of total sugar, salt and iron content in Lebanese foods, focusing on traditional dishes. To achieve this objective, the researchers collected the data by using a stratified sampling methodology. The number of samples per governorate was estimated to be 30 according to the variability in the dishes' composition. The presented results are mapped with the objective and assess the total sugar, sodium (Salt) and iron.\nIn order to improve the work of this paper I have the following comments for the authors:\nGeneral\nThere is a need for language editing and proof reading for the consistency in writing and phrasing some of the Arabic food names.\nThe list of References at the end of the paper needs to be checked, delete the repeated references and be consistent in the style of referencing. The majority the references are links to websites and when trying to open some of these links it says that the page is not found (e.g. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/industry-resources-changes-nutrition-facts-label/ accessed on June 10, 2020).\nDetailed comments:\nAbstract:\n“ ...food samples was done using stratified sampling techniques. These samples were classified into five strata, taking into account variation by geographical area (Mount Lebanon, Bekaa, Beirut, Tripoli, and Saida)”.\nThe above statement needs rephrasing as by reading further in the methodology and sampling, it’s clear that the sampling methodology is convenience sampling technique from five regions/ governorate and not a stratified method.\nIt is also not clear in the abstract if the analysis of the samples had been done theoretically or in the lab? You need to add the software used to analyse the data at the end of the methods section. Also, the study design and setting should be added to the methods of the abstract. The total sample size?\nThe following statement needs re-phrasing “... little total sugar” how you define little?” - What is the number/percentage?\nMain Manuscript:\nMethods:\nNeed to be consistent with the methods presented under the abstract and in the main body of the manuscript (e.g. sampling methodology?) “Our research group collected 500 g of 30 types of traditional dishes from five different central kitchens in the following governorates” How many kitchen from each region they used? What are the selection criteria for the kitchen where you take the sample from? Do they know that you are analyzing their food contents for sugar, iron and salt? If they knew that do you think they did changes in the recipe/ ingredients for each dish of food?\n\nIt’s not clear enough how did you do the analysis in the lab for each nutrient. “All the above chemical analyses were conducted at the Industrial Research Institute (Beirut, Lebanon), which is an accredited laboratory for chemical analyses. After food samples were analyzed, stamped laboratory results were received and data was entered on Excel 2019. The data validation process was conducted, and cross checked by two research assistants.”\nThe above paragraph should be revised and integrated with more information about the study design, settings and sampling methodology.\nIt's better to move Table 1 to the annex with more specific and quantifiable ingredients of the recipes for each kitchen you have selected and have samples from.\nResults Total Sugar “Among all the governorates, all the tested traditional Lebanese dishes contained little total sugar.” What is the definition for 'little total sugar'? How did you judge that it is little and according to what? You need a reference to compare and say that it is little.\nTable 3, the mean values in 100g of food is not clear if it’s in gram, milligram or microgram? The unit should be written under each item.\nSalt\nIn the statements under salt “The sodium (Na) level was high in most dishes (Table 2).” and “One of the main factors of the richness of these Lebanese dishes in salt is mainly the high amount of salt added to the ingredients.”\nHow did you judge that the level is high and not low or medium? What is your reference for that and compared to what? You need to have a reference in order to compare and say that it's high otherwise it should be descriptive only without analytical statement that its high.\nThe titles of Table 4 & 5 are too long and some of the information can be inserted in the tables heading or adding them under the table as key information.\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/9-1254
https://f1000research.com/articles/9-1155/v1	18 Sep 20	{ "type": "Research Article", "title": "Bibliometric analysis of road traffic injuries research in the Gulf Cooperation Council region", "authors": [ "Farhan Muhammad Butt", "Murtaza Ashiq", "Shafiq Ur Rehman", "Khurram Shahid Minhas", "Muhammad Ajmal Khan", "Farhan Muhammad Butt", "Murtaza Ashiq", "Khurram Shahid Minhas", "Muhammad Ajmal Khan" ], "abstract": "Background: Despite governmental interventions, the Gulf Cooperation Council (GCC) region continues to experience higher road traffic crash and fatality rates relative to Western nations. This trend suggests a potential disconnect between Road Traffic Injuries (RTI) research and the mitigation measures put in place. Method: Here, we present an in-depth bibliometric analysis to obtain a comprehensive understanding of RTI research in the GCC region. The Web of Science database was used to search and retrieve the relevant articles during the period of 1981-2019. Results: The volume of RTI research increased from 2015–2019, suggesting an increased focus on traffic safety in the GCC region. Saudi Arabia had the highest RTI research productivity level (126 publications); Bahrain had the lowest (7 publications). Inconsistent with its low publication volume, Hammad Medical Corps of Qatar had the highest citation impact score of 16.33. Global collaboration for RTI research was highest between Saudi Arabia and the United States. The most prevalent publication journal for the region was Accident Analysis and Prevention. The most common keywords were “road traffic accidents” and “road traffic injuries”; terms such as “mobile phones”, “pedestrian safety”, “pedestrians”, and “distracted driving” were least common. In the five most productive GCC nations with respect to RTI research (Saudi Arabia, United Arab Emirates, Qatar, Kuwait, and Oman), researchers tended to publish works related to road traffic safety in traffic safety-oriented journals. Conclusions: The quantity and quality of RTI publications in GCC is insufficient to meet the increasing related public health and economic burden in the region. The trends among publication volumes, citations, and impact were inconsistent. There is a lack of research collaboration among the institutions. Most of the research related to RTI is being conducted by researchers with a medical background. Research focusing on pedestrians, cyclists and road user behavior is also inadequate.", "keywords": [ "road traffic injuries", "road traffic accidents", "traffic safety", "bibliometric analysis", "gulf cooperation council" ], "content": "Introduction\n\nRoad traffic injuries (RTI) account for 30% of all deaths worldwide and are the major cause of death among people 15–29 years old1. Figure 1 depicts the leading causes of death expected in 2030 by the World Health Organization (WHO)2. RTI was ranked as the ninth highest contributor to the total global deaths in 2004; by 2030, RTI is expected to rank as the fifth highest contributor2,3\n\n(generated using Microsoft Excel).\n\nThe Gulf Cooperation Council (GCC) region (Saudi Arabia, Oman, the United Arab Emirates, Kuwait, Qatar, and Bahrain) experiences substantially higher road traffic crash and fatality rates relative to Western nations. Across 15 developed European nations, the recorded road traffic fatality rate has decreased from 13.5 deaths per 100,000 populations in the 1980s to 5.5 deaths per 100,000 populations today. In contrast, the road traffic fatality rate in the GCC region has remained relatively constant at 23 deaths per 100,000 population over this same period4.\n\nTable 1 compares the 2013 road traffic fatality rates per 100,000 population and per 100,000 registered vehicles for each of the GCC nations, as well as the United Kingdom and the United States5. In the GCC region, Saudi Arabia and Oman had the highest fatality rates per 100,000 population, followed by Kuwait, Qatar, the United Arab Emirates (UAE), and Bahrain5. The population-based fatality rate in the UAE (10.9 deaths per 100,000 population) was comparable to that in the United States (10.6 deaths per 100,000 population). However, the fatality rate based on vehicle ownership in the UAE (38.2 deaths per 100,000 registered vehicles) was significantly higher than that in the United States (12.9 deaths per 100,000 registered vehicles). The United Kingdom had the lowest fatality rates in terms of both population (2.9 deaths per 100,000 population) and vehicle ownership (5.1 deaths per 100,000 registered vehicles)5.\n\nAs demonstrated in other nations, road traffic crashes and fatalities can be mitigated through a focused program of RTI research that identifies and supports implementation of strategies designed to reduce the magnitude and severity of road traffic crashes and fatalities. Historical RTI research has been conducted in Western nations with a focus on relating aggressive driving behavior and offences to traffic crashes6–8. Early studies showed that more than 75% of all crashes were caused by driver behavior6–11. Although driver behavior clearly constitutes an important risk factor, researchers today caution against trying to identify a single crash cause. Socioeconomic and demographic factors, such as age, sex, marital status, education, training, experience, way of life, emotional status, fatigue, reaction time, vision, vigilance, and driving speed may also affect crash occurrence and should be considered as risk factors in RTI research12.\n\nThe high and sustained number of road traffic crashes and fatalities in the GCC region relative to other comparable Western nations poses serious public health and economic challenges and has been well documented in previous studies13–15. These challenges are exacerbated by the GCC region’s rapidly expanding roadway network and increasing vehicle ownership levels. Unlike demonstrated efforts in Western nations to reduce the magnitude and severity of road traffic crashes and fatalities based on a focused program of research, the high and sustained number of road traffic crashes and fatalities in the GCC region suggests a potential disconnect between local RTI research and any mitigation measures put in place.\n\nTo address this potential disconnect, we performed a bibliometric analysis of RTI research in the GCC region using publication data from the Web of Science indexing and abstracting database for 1981–2019. We considered research productivity, institutional and individual authorship, bibliographic coupling and global collaboration, publication journals and publications, and keywords and associated topical trends for the GCC region. In addition, we performed a three-factor analysis using GCC nation, publication journal, and keyword parameters to obtain a more comprehensive understanding of RTI research in the GCC region. The results from this study can inform governmental authorities in the GCC region of research investment needs and academic researchers of research deficiencies and impactful publishing venues. An informed and collective focus on RTI research in the GCC region will help to ensure broader public safety.\n\n\nMethods\n\nIn this study, we performed a bibliometric analysis of RTI research in the GCC region. Bibliometric analysis is a statistical method that collects, analyzes, and extracts various metrics related to research productivity, institutional and individual authorship, publication journals, publications, keywords, and more from published literature. We used data from the Web of Science (WOS) indexing and abstracting database. The WOS database was chosen to extract the relevant data on RTI. Web of Science is the most authentic and reliable indexing and abstracting global database of scholarly literature. Additionally, it also provides full bibliometric data with a simple extraction process, which is suitable for comprehensive bibliometric analysis. The following search query was used in the topic field of the advance search option of the Web of Science Core collection database on November 13, 2019.To get maximum relevant records we used all keywords related to RTI. We searched using the following search terms in the topic field (that is title, keywords and abstract) of the WOS database. These search terms were selected based on literature review and author keyword analysis in different databases. The authors tried to include all search terms for retrieval of entire spectrum of the literature related to study objectives.\n\nTS=(road safety* OR road injury* OR traffic accident* OR vehicle accident* OR road trauma* OR road casualty* OR traffic casualty* OR traffic safety* OR traffic camera* OR seat belt* OR seatbelt* OR traffic fine* OR car accident* OR bike accident* OR motorbike accident* OR motorcycle accident* OR motorcycle crash* OR airbag* OR air bag* OR road fatality* OR child restraint OR road death* OR traffic enforcement OR pedestrian safety OR road crash*) AND AD=(Saudi Arabia OR Bahrain OR Oman OR Kuwait OR Qatar OR UAE OR United Arab Emi- rates OR Abu Dhabi OR Ajman OR Dubai OR Fujairah OR Ras al-Khaimah OR Sharjah OR Umm al-Quwain).\n\nThis initial query returned 571 publications. The authors did not apply any filter related to languages and time period to achieve a comprehensive view of RTI research. We refined this initial query by excluding editorial materials, notes, meeting abstracts, letters, or corrections as they are not peer reviewed. Research articles, conference papers, review papers, book chapters, and books were included in this study. The complete bibliographic information (title, abstract, author, date of publication, source of publication, keywords, citation count etc.), of the remaining publication records were exported to MS Excel (Microsoft 365) for scrutinizing of these records. Two authors read the title and abstracts of all the records. The third author again repeated this process to ensure the relevancy and reliability of the data. This practice helped the authors to discard 259 irrelevant or duplicate records. Finally, a total of 312 records were selected for data analysis. The final dataset for this study included 312 publication records comprising 230 journal articles and 82 conference papers16. Data analysis was performed using a combination of spreadsheet and bibliometric analysis software packages such as VOSViewer (version 1.6.15) and Biblioshiny (R Package). The author keyword analysis, bibliographic coupling, and three factor analysis was done with the help of VOSViewer. Global collaboration and topical trends were generated by using Biblioshiny.\n\n\nResults\n\nFigure 2 depicts the annual RTI research productivity in terms of publications and citations in the GCC region. The earliest RTI research publication in the GCC region appeared in 1981 and was cited 19 times. Following that first publication, RTI research productivity gradually increased but remained relatively low. The volume of RTI research substantially increased in 2015–2019, suggesting an increased recent focus on traffic safety in the GCC region. In 2015, 28 publications were cited 196 times. The maximum number of publications (42) occurred in 2017.\n\n(generated using Microsoft Excel).\n\nTable 2 summarizes RTI research productivity in terms of publications, citations, and citation impact for each nation in the GCC region for 1981–2019. The GCC nations with the highest number of publications included Saudi Arabia (126 publications), the UAE (84 publications), and Qatar (50 publications). Bahrain had only 7 publications.\n\nPublications originating in Saudi Arabia were cited 890 times and had a citation impact of 7.06. Despite lower publication volumes, publications originating in the UAE and Qatar had higher citation impact (7.79 and 7.76, respectively).\n\nTable 3 lists the most productive institutions for RTI research in the GCC region. Four institutions were in Saudi Arabia, three were in the UAE, two were in Qatar, and one was in Kuwait.\n\nThe UAE University had the highest RTI research productivity with 40 publications cited 545 times with a citation impact of 13.64. King Saud University and Qatar University also had a high number of publications (37 and 29 publications, respectively). The Hamad Medical Corporation and Al Ain Hospital had just 15 and 12 publications, respectively, but their citation impact ranked highest (16.33 and 14.58, respectively).\n\nTable 4 lists the most productive individual authors for RTI research in the GCC region. Only three authors had 10 or more publications: F.M.Abu-Zidan (17 publications), K. Shaaban (15 publications), and H.O. Eid (10 publications).\n\nThe 17 publications by F.M. Abu-Zidan were cited 313 times with a citation impact of 18.41. Select authors such as K. Shaaban had a relatively high publication volume (15 publications) but a low number of citations (68 citations) and a low citation impact (4.53). Conversely, A.F. Hefny and M. Grivna had relatively low publication volumes (7 and 6 publications, respectively) but high numbers of citations (168 and 137 citations, respectively) and high citation impact (24.00 and 22.83, respectively).\n\nBibliographic coupling is a measure of subject matter commonality among different publications and occurs when two publications reference a common third publication. Figure 3 depicts the bibliographic coupling among different nations. The circle size and colors denote bibliographic coupling levels and different coupling clusters, respectively.\n\n(generated using VOSViewer software).\n\nNations with three or more publications were included; 23 of 45 nations met this criterion. Nations with the highest bibliographic coupling activity included Saudi Arabia (110 documents, 774 citations, and a 5,197 total link strength); the UAE (83 documents, 655 citations, and a 3,731 total link strength); and Qatar (50 documents, 385 citations, and a 3,290 total link strength).\n\nFigure 4 depicts the historic global collaboration in RTI research among nations. In total, 232 instances (i.e., publications) of global collaboration involving GCC nations were identified. These instances ranged from a single collaboration between two nations to more than 11 repeated collaborations between two nations. The highest levels of collaboration have occurred among Saudi Arabia and the United States (11 instances), Qatar and the United States (9 instances), Egypt and the UAE (8 instances), Saudi Arabia and Tunisia (8 instances), and Qatar and the United Kingdom (7 instances).\n\n(generated using Biblioshiny Software R Package).\n\nAs a supplemental investigation, we considered the number of individual authors on each RTI research publication. Figure 5 shows the number of publications as a function of the number of authors for publications originating in the GCC region. Select publications had more than 10 individual authors, although this was relatively infrequent. Most RTI research publications originating in the GCC region had no more than five authors; the highest number of publications (74) had three authors.\n\n(generated using Microsoft Excel).\n\nTable 5 lists the most prevalent publication journals for RTI research in the GCC region. Only five journals had 10 or more publications: Accident Analysis and Prevention (25 publications), Saudi Medical Journal (20 publications), International Journal of Injury Control and Safety Promotion (15 publications), Traffic Injury Prevention (12 publications), and Annals of Saudi Medicine (10 publications). Accident Analysis and Prevention also had the highest number of citations (533) and citation impact (21.32) among these journals.\n\nTable 6 lists the 20 most commonly cited RTI research publications originating in the GCC region. Four publications have been cited more than 50 times: “Causes and Effects of Road Traffic Accidents in Saudi Arabia”17 (95 citations); “The Driver Behavior Questionnaire in Arab Gulf Countries: Qatar and United Arab Emirates”18 (60 citations); “Pedestrian-vehicle Crashes and Analytical Techniques for Stratified Contingency Tables”19 (58 citations); and Seat belt Utilization in Saudi Arabia and its Impact on Road accident Injuries”20 (52 citations). Eight of the most cited RTI research publications appeared in Accident Analysis and Prevention.\n\nFigure 6 depicts the most common keywords listed by authors in RTI research publications originating in the GCC region. The circle size and colors denote keyword occurrence levels and different coupling clusters, respectively.\n\n(generated using VOSViewer software).\n\nKeywords that were listed four or more times were included; 33 keywords met this criterion. The most common keywords in RTI research publications were traffic safety (24 occurrences), road traffic injuries (17 occurrences), and road safety (17 occurrences). Terms such as pedestrian safety (5 occurrences), pedestrian (5 occurrences), mobile phone (5 occurrences), driver behavior (4 occurrences), in- jury prevention (4 occurrences), and distracted driving (4 occurrences) were least common.\n\nSupplemental to the keyword analysis results presented previously, we considered topical trends in RTI research publications originating in the GCC region from 1992 to 2018. Figure 7 depicts these results. In 1992–2007, RTI research publications were limited to just six topics: Saudi Arabia, hospital, Riyadh, region, accident, and mortality. In 2008, the breadth of topics began to increase. In 2009–2019, the most common RTI research publication topics included road, traffic, safety, and accidents.\n\n(generated using Biblioshiny Software R Package).\n\nAs a final step in this study, we performed a three-factor analysis using GCC nation, publication journal, and keyword parameters to obtain a more comprehensive understanding of RTI research in the GCC region. Figure 8 presents the results from this analysis.\n\n(generated using Biblioshiny Software R Package).\n\nRTI research originating in the five most productive GCC nations (Saudi Arabia, the UAE, Qatar, Kuwait, and Oman) was most commonly published in just four journals: the International Journal of Injury Control and Safety Promotion, Accident Analysis and Prevention, Traffic Injury Prevention, and the Saudi Medical Journal. The focus of this research was tied to just three keywords: traffic safety, driving, and road safety.\n\n\nDiscussion\n\nResearch related to RTI and road traffic awareness first appeared outside of the GCC region in 1926 and 1966, respectively21. Comparatively, the first publication related to RTI in the GCC region did not appear until 1981, confirming a lag in RTI research activity among GCC nations relative to other developed nations.\n\nFollowing that first publication in 1981, RTI research productivity gradually increased in the GCC region but remained relatively low until 2015–2019, when the volume of RTI research substantially increased. A similar trend was reported in India; publication volumes related to road traffic injuries in India increased significantly after 201022. These collective results suggest an increased recent focus on traffic safety in the GCC region by policymakers, planners, and academic researchers. This increased research productivity can also be attributed to an increased number of print and online publication journals, increased attention to this topic within the scientific community, increased networking and collaboration capabilities, and an increased focus from governmental and specialized agencies such as World Health Organization (WHO).\n\nDisparities in RTI research productivity were observed between the GCC region and other developed nations as well as within the GCC region. The GCC nations with the highest number of publications included Saudi Arabia (126 publications), the UAE (84 publications), and Qatar (50 publications). This finding is consistent with results previously reported by Abdo et al.21 who found that Saudi Arabia had the highest number of publications related to road traffic safety and road traffic awareness relative to all other GCC nations. Saudi Arabia has a higher population and more research institutions than other GCC nations.\n\nTrends among publication volumes, citations, and impact were inconsistent. The 126 publications originating in Saudi Arabia were cited 890 times and had a citation impact of 7.06. Despite lower publication volumes (84 and 50 publications, respectively), publications originating in the UAE and Qatar had higher citation impact (7.79 and 7.76, respectively).\n\nRegarding the publishing institutions, the UAE University had the highest RTI research productivity with 40 publications, which were cited 545 times with a citation impact of 13.64. King Saud University and Qatar University also had a high number of publications (37 and 29 publications, respectively).\n\nAgain, trends among publication volumes, citations, and impact were inconsistent. Although the Hamad Medical Corporation and the Al Ain Hospital had just 15 and 12 publications, respectively, the citation impact for these publications ranked highest (16.33 and 14.58, respectively). These results suggest that the most impactful RTI research publications are originating from medical institutions rather than from universities. Universities should therefore focus on improving the quality of their research to increase its impact.\n\nRegarding individual authorship, only three authors in the GCC region had 10 or more RTI research publications: F.M. Abu-Zidan (17 publications), K. Shaaban (15 publications), and H.O. Eid (10 publications). The 17 publications by F.M. Abu-Zidan were cited 313 times with a citation impact of 18.41.\n\nSimilar to institutions, trends among publication volumes, citations, and citation impact among individual authors were inconsistent. K. Shaaban had a relatively high publication volume (15 publications) but a low number of citations (68 citations) and a low citation impact (4.53). Conversely, A.F. Hefny and M. Grivna had relatively low publication volumes (7 and 6 publications, respectively) but high numbers of citations (168 and 137 citations, respectively) and high citation impact (24.00 and 22.83, respectively).\n\nAs noted previously, bibliographic coupling is a measure of subject matter commonality among different publications and occurs when two publications reference a common third publication. Publications that are closely related in content can be classified into clusters; the connections between the clusters can be described using quantitative network indicators. A visualization of the clustering network can then be generated based on research publication characteristics. Publications spanning different time periods can be clustered and any sub clusters can be merged into a unified visual view23,24. In this study, nations with three or more publications were included; 23 of 45 nations met this criterion. Bibliographic couplings in the RTI research publications originating in each of these 23 countries were classified into five clusters and visually displayed using the VOSViewer software package.\n\nBased on the bibliographic coupling analysis of RTI research publications originating in the GCC region, nations with the highest bibliographic coupling activity included Saudi Arabia (a 5,197 total link strength), the UAE (a 3,731 total link strength), and Qatar (a 3,290 total link strength). This finding is consistent with other RTI research productivity measures. The majority of academic RTI research originated in Saudi Arabia, the UAE, and Qatar. The largest proportion (23,448 publications) originated from Saudi universities. In terms of population size, Qatar was found to be performing well regionally, with a ratio of 1.6 publications to every 1,000 inhabitants. When compared with Western nations, however, RTI research productivity remains relatively low in the GCC region. Long-term transformational visions that emphasize quality education, such as Saudi Arabia’s VISION 2030, may help to bridge the gap between the GCC region and Western nations. The ground realities point to how difficult this task will be. For example, the number of Ph.D. students in Saudi Arabia per 1,000 inhabitants is 0.34 while the number of Ph.D. students in the United Kingdom per 1,000 inhabitants is 2.69.\n\nResearch, by its nature, is a collaborative process. In any bibliometric analysis, the level of research collaboration is an important index to assess the current status of research in a specific field. Considering research collaboration by nation reveals both the degree of international communication as well as the most influential nations in a particular research field. In this study, the highest level of collaboration occurred among Saudi Arabia and the United States (11 instances). This finding is consistent with a previous study’s findings that found that the highest numbers of collaborative publications in the field of Physical Sciences (1980–2014) were produced through partnerships in Saudi Arabia and the United States (23.31%) and Saudi Arabia and Egypt (22.95%)25. Similarly, the highest numbers of collaborative publications related to Health Sciences in Saudi Arabia were produced through partnerships between Egypt (16.5%) and the United States (16.3%)26. These collective findings highlight the strong collaborative and intellectual ties Saudi Arabia maintains with respective academic communities in the United States and Egypt. However, these ties should be further strengthened not only among the GCC nations but also with other developed countries such as Australia and Germany. Australia, the United States and Germany are currently leading the world in terms of best practices, regulations and road testing of connected and automated vehicles (CAVs)27, which represent the future of transportation and traffic engineering and safety research.\n\nWe also considered the number of individual authors on each RTI research publication. Select publications had more than 10 individual authors, although this was relatively infrequent. Most RTI research publications originating in the GCC region had no more than five authors; 60 publications had a single author, 54 publications had two authors, and 74 publications had three authors. The trend depicted in Figure 5 shows a decline in publication collaborations involving four or more authors in the GCC region. Instead, publication collaborations involving a smaller number of authors has been favored. Also, the number of single author publications was high. Efforts to improve collaboration among individual authors from different departments and institutions would improve both the quality and impact of research originating in the GCC region.\n\nAmong the most prevalent publication journals for RTI research in the GCC region, Accident Analysis and Prevention had the highest number of publications (25), number of citations (533), and citation impact (21.32). Of the 20 most commonly cited RTI research publications originating in the GCC region, eight appeared in Accident Analysis and Prevention.\n\nThe most commonly cited RTI research publications originating in the GCC region related to traffic safety, and to a lesser extent, traffic awareness. A number of the commonly cited RTI research publications specifically addressed medical issues associated with these topics. This finding is consistent with findings previously reported by22. Of the 20 most commonly cited RTI research publications originating in the GCC region (Table 6), only two publications considered pedestrians (the most vulnerable road users): one published in Accident Analysis and Prevention19 and one published in Traffic Injury Prevention28. None of these publications considered more contemporary or emerging topics, such as the role of mobile phones in road traffic injuries.\n\nConsistent with the publication-related findings, the most commonly listed keywords in RTI research publications originating in the GCC region were road traffic accidents (41 occurrences) and road traffic injuries (40 occurrences). Terms such as traffic safety (27 occurrences), road safety (20 occurrences), and road traffic crashes (19 occurrences) were also commonly listed. Terms such as mobile phones (5 occurrences), pedestrian safety (5 occurrences), pedestrians (5 occurrences), and distracted driving (4 occurrences) were least common.\n\nTo detect changes over time in RTI research focus in the GCC region, we considered topical trends from 1992 to 2018. In 1992–2007, RTI research publications were focused on the medical aspects of the traffic safety; hospital and mortality were common topics in RTI research publications. In 2008, the breadth of topics began to increase. In 2009–2019, the most common RTI research publication topics included road, traffic, safety, and accidents. This trend can be attributed to the increasing RTI burden in the GCC region, which experiences ever-increasing road traffic crashes and deaths on the roads each year despite the development of both in-vehicle and highway-based safety system technologies. Traffic safety awareness among governmental traffic planning department staff and academic researchers has increased due to improved international data collection and sharing practices and the periodic Global Status Reports on Road Safety in 20131, 20155, and 201829 published by the WHO.\n\nThis aspect of the investigation not only revealed the historic RTI research focus in the GCC region, but also revealed important deficiencies that can guide future research. For example, public safety in the GCC region may benefit from future research focused on pedestrian behavior and safety or the effects of mobile phone usage and distracted driving.\n\n\nLimitations and future research directions\n\nIn this study, we performed a bibliometric analysis of RTI research in the GCC region using data from the Web of Science indexing and abstracting database for 1981–2019. This data was not independently verified to determine if each of the publication records considered for analysis originated from the GCC region. Other databases such as Scopus, PubMed, or Google Scholar may produce a different set of publication records using similar search criteria. Although this comparison was beyond the scope of this study, future work may attempt to verify this study’s findings using data from these alternate sources.\n\nMore broadly, future RTI research should focus on developing preventive actions to increase and promote traffic safety rather than only addressing the medical aspects of traffic injuries in the GCC region. This bibliometric analysis has revealed minimal research related to human behavior factors and pedestrians and cyclists who are most vulnerable for road traffic injuries in the GCC region. Traffic safety researchers and practitioners in the region should focus on these research areas, which show significant publication potential. Researchers should also focus on improving communications between departments and institutions to facilitate sharing and usage of updated data among researchers. This latter effort will also encourage collaboration among authors. As noted previously, efforts to improve collaboration among individual authors from different departments and institutions would improve both the quality and impact of research originating in the GCC region.\n\n\nConclusions\n\nTo address the potential, disconnect between RTI research and any traffic safety mitigation measures put in place in the GCC region we performed a bibliometric analysis using publication data from the Web of Science database. We considered research productivity, institutional and individual authorship, bibliographic coupling and global collaboration, publication journals and publications, and keywords and associated topical trends for the GCC region. In addition, we performed a three-factor analysis using GCC nation, publication journal, and keyword parameters to obtain a more comprehensive understanding of RTI research in the GCC region.\n\nThe first publication related to road traffic injuries in the GCC region did not appear until 1981, confirming a lag in RTI research activity among GCC nations relative to other developed nations. More recently in 2015–2019, the volume of RTI research substantially increased, suggesting an increased focus on traffic safety in the GCC region. This increased focus was not uniform across the GCC region. Disparities in RTI research productivity were observed within the GCC region. The GCC nations with the highest and lowest publication volumes included Saudi Arabia and Bahrain, with 126 and 7 publications, respectively. The UAE University had the highest RTI research productivity with 40 publications, but the Hamad Medical Corporation and Al Ain Hospital had the highest citation impact (16.33 and 14.58, respectively). Because most of the impactful RTI research publications originated from medical institutions rather than from universities, universities must focus on improving the quality of their research to increase its impact. Only three individual authors had 10 or more RTI research publications including F.M. Abu-Zidan (17 publications), K. Shaaban (15 publications), and H.O. Eid (10 publications). Most RTI research publications originating in the GCC region had three collaborative authors, and to a lesser extent, a single author. Efforts to improve collaboration among individual authors from different departments and institutions would improve both the quality and impact of research originating in the GCC region.\n\nAmong the most prevalent publication journals for RTI research in the GCC region, Accident Analysis and Prevention had the highest number of publications (25), number of citations (533), and citation impact (21.32). Of the 20 most cited RTI research publications originating in the GCC region, four publications have been cited more than 50 times. Eight of the most cited RTI research publications appeared in Accident Analysis and Prevention. The most commonly listed keywords in RTI research publications were road traffic accidents (41 occurrences) and road traffic injuries (40 occurrences). Terms such as mobile phones (5 occurrences), pedestrian safety (5 occurrences), pedestrians (5 occurrences), and distracted driving (4 occurrences) were least common. This aspect of the investigation revealed the historic RTI research focus as well as important deficiencies that can guide future research in the GCC region.\n\nAcross several of the bibliometric parameters, trends among publication volumes, citations, and impact were inconsistent. For example, a high volume of publications did not always result in a high number of citations or a high citation impact.\n\nThis study demonstrated the merit of applying bibliometric analysis to guide decision-making related to RTI research. The collective results of this study indicated that—despite the recent increase in RTI research productivity in the GCC region—the quantity and quality of RTI research publications originating in the GCC nations is insufficient to meet the increasing burden of RTI in the region. More effort is needed to increase and promote traffic safety. The collective results from this study can inform governmental authorities and policy makers in the GCC region of research investment needs and support cost- effective resource allocation. In addition, the results from this study can inform academic researchers of research deficiencies and impactful publishing venues. An informed and collective focus on RTI research in the GCC region will help to ensure broader public safety.\n\n\nData availability\n\nFigshare: Bibliometric analysis of road traffic injuries research in the Gulf Cooperation Council region\n\nhttps://doi.org/10.6084/m9.figshare.1285557216\n\nThis project contains the following underlying data:\n\n312 Publications Record CSV.csv. (Results of all 312 publication records used in this study)\n\nRTI_Gulf22Dec19.xlsx. (Excel data sheets and Charts data)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWorld Health Organization: Global Status Report on Road Safety 2013: Supporting a Decade of Action. Geneva, Switzerland: WHO; 2013. Reference Source\n\nWorld Health Organization: Global Status Report on Road Safety 2009. Geneva, Switzerland: WHO, 2009. Reference Source\n\nStaton C, Vissoci J, Gong E, et al.: Road Traffic Injury Prevention Initiatives: A Systematic Review and Metasummary of Effectiveness in Low and Middle Income Countries. PLoS One. 2016; 11(1): e0144971. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Madani HMN: Fatal crashes in GCC countries: comparative analysis with EU countries for three decades. Safety and Security Engineering V Rome, Italy; September 2013; 134: 471–82. Publisher Full Text\n\nWorld Health Organization: Global Status Report on Road Safety 2015. Geneva, Switzerland: WHO, 2015. Reference Source\n\nBener A, Breger E, Al-Falasi AS: Risk-taking behaviour in road traffic accidents. Journal of Traffic Medicine. 1995; 23: 65–70. Reference Source\n\nMesken J, Lajunen T, Summala H: Interpersonal violations, speeding violations and their relation to accident involvement in Finland. Ergonomics. 2002; 45(7): 469–83. PubMed Abstract \| Publisher Full Text\n\nParker D, Reason JT, Manstead ASR, et al.: Driving errors, driving violations and accident involvement. Ergonomics. 1995; 38(5): 1036–48. PubMed Abstract \| Publisher Full Text\n\nJonah BA: Accident risk and risk-taking behaviour among young drivers. Accid Anal Prev. 1986; 18(4): 255–71. PubMed Abstract \| Publisher Full Text\n\nKontogiannis T, Kossiavelou Z, Marmaras N: Self-reports of aberrant behaviour on the roads: errors and violations in a sample of Greek drivers. Accid Anal Prev. 2002; 34(3): 381–99. PubMed Abstract \| Publisher Full Text\n\nLawton R, Parker D, Stradling SG, et al.: Predicting road traffic accidents: The role of social deviance and violations. British Journal of Psychology. 1997; 88(2): 249–62. Publisher Full Text\n\nGregersen NP, Bjurulf P: Young novice drivers: Towards a model of their accident involvement. Accid Anal Prev. 1996; 28(2): 229–41. PubMed Abstract \| Publisher Full Text\n\nBener A, Jadaan KS: A perspective on road fatalities in Jeddah, Saudi Arabia. Accid Anal Prev. 1992; 24(2): 143–8. PubMed Abstract \| Publisher Full Text\n\nBener A, Abu-Zidan FM, Bensiali AK, et al.: Strategy to improve road safety in developing countries. Saudi Med J.2003; 24(6): 603–8. PubMed Abstract\n\nBener A, Omar AOKh, Ahmad AE, et al.: The pattern of traumatic brain injuries: A country undergoing rapid development. Brain Inj. 2010; 24(2): 74–80. PubMed Abstract \| Publisher Full Text\n\nButt FM, Ashiq M, Rehman SU, et al.: Bibliometric Analysis of Road Traffic Injuries Research in the Gulf Cooperation Council Region. 2020. http://www.doi.org/10.6084/m9.figshare.12855572\n\nAnsari S, Akhdar F, Mandoorah M, et al.: Causes and effects of road traffic accidents in Saudi Arabia. Public Health. 2000; 114(1): 37–9. PubMed Abstract \| Publisher Full Text\n\nBener A, Ozkan T, Lajunen T: The Driver Behaviour Questionnaire in Arab Gulf countries: Qatar and United Arab Emirates. Accid Anal Prev. 2008; 40(4): 1411–7. PubMed Abstract \| Publisher Full Text\n\nAl-Ghamdi AS: Pedestrian–vehicle crashes and analytical techniques for stratified contingency tables. Accid Anal Prev. 2002; 34(2): 205–14. PubMed Abstract \| Publisher Full Text\n\nBendak S: Seat belt utilization in Saudi Arabia and its impact on road accident injuries. Accid Anal Prev. 2005; 37(2): 367–71. PubMed Abstract \| Publisher Full Text\n\nAbu Abdo AM, Akash B, Mohsen MS, et al.: Scopus Based Analysis of Related Literature to Traffic Safety and Awareness in the Arab Countries. International Journal of Applied Engineering Research. 2016; 11(6): 4076–4080. Reference Source\n\nSharma N, Bairwa M, Gowthamghosh B, et al.: A bibliometric analysis of the published road traffic injuries research in India, post-1990. Health Res Policy Sys. 2018; 16(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu J, Li X, Wang S: What have we learnt from 10 years of fintech research? a scientometric analysis. Technological Forecasting and Social Change. 2020; 155: 120022. Publisher Full Text\n\nLiao H, Tang M, Luo L, et al.: A Bibliometric Analysis and Visualization of Medical Big Data Research. Sustainability. 2018; 10(1): 166. Publisher Full Text\n\nShehatta I, Mahmood K: Research Collaboration in Saudi Arabia 1980–2014: Bibliometric Patterns and National Policy to Foster Research Quantity and Quality. LIBRI. 2016; 66(1): 13–29. Publisher Full Text\n\nUl Haq I, Ur Rehman S, Al-Kadri HM, et al.: Research Productivity in the Health Sciences in Saudi Arabia: 2008-2017. Ann Saudi Med. 2020; 40(2): 147–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee D, Hess DJ: Regulations for on-road testing of connected and automated vehicles: Assessing the potential for global safety harmonization. Transportation Research Part A: Policy and Practice. 2020; 136: 85–98. Publisher Full Text\n\nAl-Shammari N, Bendak S, Al-Gadhi S: In-Depth Analysis of Pedestrian Crashes in Riyadh. Traffic Inj Prev. 2009; 10(6): 552–9. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Global Status Report on Road Safety 2018. Geneva: WHO, 2018. Reference Source" }	[ { "id": "71718", "date": "22 Sep 2020", "name": "Elhashemi M. Ali", "expertise": [ "Reviewer Expertise Traffic Safety", "Naturalistic Driving Studies", "Big Transportation Data Analysis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 has a great sound of knowledge since it summarized many research efforts to obtain a comprehensive understanding of Road Traffic Injuries (RTI) and presented them in a Bibliometric analysis. The article flow is easy to follow and it was written well.\nThe reviewer has some comments that might add to the paper and these comments are as follows:\nIn the method section: can the authors present the TS equation in is a flow chart, a figure, or a table instead of this long equation of research terms?\n\nThe sentence \"The authors did not apply any filter related to languages and time period to achieve a comprehensive view of RTI research\" need to be revised as there is already a time period between 1981 and 2019 that the article focuses on.\n\nThe \"three-factor analysis\" need to be introduced and explained in details.\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": [ { "c_id": "6014", "date": "16 Oct 2020", "name": "Shafiq Ur Rehman", "role": "Author Response", "response": "1. The flow chart has been added (see figure 2)2. We did not apply any filter of time span while searching our query. The year 1981 was the date of first publication of the topic and 2019 was last publication as a result of our search query.3. The detail about three factor analysis has been added." } ] }, { "id": "71716", "date": "28 Sep 2020", "name": "Uneb Gazder", "expertise": [ "Reviewer Expertise Traffic engineering" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a comprehensive bibliographic analysis of the research related to road safety in GCC countries. All important trends have been analyzed in detail and their possible reasons have been discussed. The authors have also highlighted important issues related to past trends and suggested future improvements. The following corrections are required to be made in the manuscript before accepting it for indexing:\nFigure 1 is not cited before its appearance.\n\nData in table 1 should be updated.\n\nThere are repetitions in the discussion section, for e.g., an explanation of bibliographic coupling and collaboration that have already been done in the previous sections. They should be removed from the discussion.\n\nLimitations should be included in the introduction or method section.\n\nThe authors have mentioned important findings in the discussion related to research trends and their potential future directions (such as pedestrian accidents). These points should be reiterated in the conclusion. Details about the number of publications, authors, etc. can be omitted or reduced in conclusions.\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?\nNot applicable\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? Partly", "responses": [ { "c_id": "6015", "date": "16 Oct 2020", "name": "Shafiq Ur Rehman", "role": "Author Response", "response": "1. Figure 1 is already cited in the text before its appearance. Kindly refer to the Introduction section First paragraph (Lines 3-4). 2. The data used in table 1 is per the Statistical survey of 2013 and reported in the Global Status Report on Road Safety 2015 published by WHO in the Country Profiles (pages 77-255). The reason for not using the WHO 2018 report is that the data for Kingdom of Bahrain is not available in it and for the consistent comparison with other GCC countries it could not be used. Reports links: Reference Source (2015) Reference Source (2018) 3. The mentioned paragraphs have been removed from the discussions section (Lines 309-318). 4. We have moved the limitations in the method section 5. The findings are reiterated in the conclusions (Lines 454-464). Details about the number of publications, authors are also reduced from the conclusions." } ] }, { "id": "71722", "date": "07 Oct 2020", "name": "Ashar Ahmed", "expertise": [ "Reviewer Expertise Road Safety Modelling", "Accident Investigation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper provides a review of the Road Traffic Incident (RTI) research publication frequency across Gulf Cooperation Council region. The country with the highest RTI research productivity, most productive institution for RTI research, most productive author, most prevalent journal for RTI publication, topics of RTI research over the period of time, and future research directions in the GCC region were explored. It was found that the majority of the research was related to medical aspects then engineering aspects of RTI. The findings of this study are very critical which makes the paper extremely worthy of publication.\n\nThe following comments will improve the manuscript:\nThe authors claim that RTIs are the 9th leading cause of deaths. As per the WHO Global Status Report on Road Safety 2018, RTIs are the 8th leading cause of deaths around the world. This may be updated in the ‘Introduction’ section.\n\nIn Table 5 the word ‘behavior’ is in a separate row. It is part of the name of the journal and should be in the preceding row.\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?\nNot applicable\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": [ { "c_id": "6016", "date": "16 Oct 2020", "name": "Shafiq Ur Rehman", "role": "Author Response", "response": "1. This is updated in the Introduction section (line 5).2. It is corrected in Table 5." } ] }, { "id": "71721", "date": "09 Oct 2020", "name": "Muhammad Adnan", "expertise": [ "Reviewer Expertise Transport Planning", "Road Safety", "Transport Modelling", "Big data." ], "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 nice effort from the authors, however, I see that some of the interpretations and conclusions of the results are not appropriate. Please find my specific comments below:\n\nIntroduction: Para 4, line 3: Historical RTI research....to traffic crashes. What do you mean here by historical RTI research.. Sentence seems a bit confusing.\nPara 5, line 5: Unlike demonstrated efforts....put in place. Could you please explain here what were those demonstrations and how you have concluded here that there is a disconnection in GCC.\n\nResults: Sub-section : Three factor analysis...\nWhy 3 factors are considered? I see the importance of nations and keywords but not able to comprehend inclusion of journal (what exactly it will reflect it is not clear to me).\nDiscussion: Para 1: Could you please discuss here what was the reason why before 1981 there wasn't any research study related to RTI? Is it because the RTI problem was not that significant or was no attention given by academics or no funding was available or there wasn't any research culture or academic performance was not graded based on research activity...which is clearly the case now.\n\nPara 4: why articles from KSA have low impact factor compared to UAE and Qatar? Could you elaborate on the possible reasons for this?\nPara 6: medical journals usually have way higher impact factors and have large audience compared to non-medical journals (planning and engineering-based journals). I do not agree on the conclusion you made here that universities are not producing high-quality research just based on the citations impact.\n\nPara 8: My comment here is similar to what I mentioned above, authors need to categories, you cannot ignore the content and make your conclusion on citations.\nPara 12, Line 1: Select publications had... The sentence should start with …Selected...\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? Partly\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?\nPartly\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1155
https://f1000research.com/articles/9-1251/v1	16 Oct 20	{ "type": "Research Article", "title": "Evaluation of the color stability of two different posterior tooth colored restorative materials", "authors": [ "Chandrakanth Majeti", "Ravichandra Ravi", "Bhargav Kambhampati", "Roopesh Borugadda", "Srividya Athkuri", "Abhijeet K. Kakani", "Chandrakanth Majeti", "Bhargav Kambhampati", "Roopesh Borugadda", "Srividya Athkuri", "Abhijeet K. Kakani" ], "abstract": "Background: The aim of this in vitro study was to evaluate the color stability of esthetic restorative materials (Cention N, Solare Sculpt) after exposure to different staining solutions (coffee, green tea and Diet Coke). Methods: Cylindrical specimens of both materials (n=40/material) were prepared using 4x8 mm metal molds. They were further divided (n=10) based on the beverages in which they are immersed. The color of each sample was recorded immediately after sample preparation and at 60 days after the staining procedure. Color changes were then analyzed statistically. Results: Color differences (ΔE) were statistically significant between Cention N and Solare sculpt in all beverages with Cention N showing highest staining after 60 days. Among all the beverages, coffee showed the highest level of staining. Conclusions: Staining beverages caused significant discolorations for both test materials. Cention N showed greater color variations with all beverages compared to Solare Sculpt. Coffee showed the highest staining with both materials, followed by Diet Coke then green tea.", "keywords": [ "Beverages", "color stability", "staining susceptibility", "spectrophotometer" ], "content": "Introduction\n\nThe use of tooth-colored restorative materials has been on the rise in the recent times. Patient awareness and improvement in the properties of composite materials have led the clinicians to use these materials, even in the posterior regions. But strength and other properties like staining have remained as a challenge with the newer materials that are being introduced1.\n\nRecently developed esthetic materials like Cention N and Solare Sculpt have been reported to have improved properties for use as posterior restorative material. One important aspect of esthetic restorative materials is color. Interactions between the components of a restorative material play a key role in their color stability. Composite resin materials are mainly discolored due to the intrinsic or extrinsic factors. The intrinsic discoloration of the composites can be determined by the photo initiator, quality of resin matrix and by the inorganic filler, in the same way the extrinsic discoloration is caused especially by the colorants present in beverages and foods through adsorption and absorption2. In the present contemporary society there is a high prevalence of consumption of coffee, green tea and soft drinks, causing discoloration of the tooth-colored restorations3.\n\nMany studies4–6 have focused on comparing the physical properties of Cention N with composite materials and others. To our knowledge, no study has compared the staining ability of Cention N with Solare Sculpt.\n\nThus, the present study aimed to evaluate the staining susceptibility of a recently developed nanohybrid composite (Solare Sculpt) and Cention N after immersion in artificial saliva, coffee, green tea and Diet Coke using a spectrophotometer. The null hypothesis tested in this study was that there would be no difference in staining susceptibility among the materials tested and no difference in staining ability of all the beverages used.\n\n\nMethods\n\nSolare Sculpt and Cention N (Shade A2) were the materials used in the study. Detailed descriptions of the products used are presented in Table 1. A total of 80 metal molds 4 mm thick and 8 mm in diameter were prepared for each specimen. Each restorative material was prepared as per manufacturer instructions and placed in the metal rings to prepare the 80 samples (n=40 for each material). The ring was then placed between glass slabs to remove the excess and to obtain a flat surface. All the samples were light-cured with a blue phase light-curing unit (Ivoclar Vivadent, Schaan, Liechtenstein, UK) for 20 seconds on both sides. Cured specimens were then removed from the mould and were finished and polished.\n\nFinishing was done with fine-grit Sof-Lex flexible disks (3M ESPE, MN, USA), and then polished using rubber cups (Shofu dental products, San Marcos, USA) at low speed. All samples were stored in distilled water for 24 h after curing. All the samples of both materials were then divided according to the beverage in which they are immersed (n=10):\n\n1. Artificial saliva (Yash Pharma Laboratories, Uttarakhand, India) (control group)\n\n2. Coffee (Bru coffee, Hindustan Unilever Limited Mumbai, Maharashtra)\n\n3. Green tea (Lipton Green tea, Hindustan Unilever Ltd., Mumbai, India)\n\n4. Coke (Hindustan Coca-Cola Bevarages Pvt. Ltd., Pune, India)\n\nAfter finishing and polishing and before immersion, color measurements were conducted as baseline values using a spectrophotometer (Easy shade compact spectrophotometer, VITA, Zahnfabrik H,Bad Sackingen, Germany. Model no; DEASYCS22). After taking the baseline values, the individual samples were transferred into small bottles and labeled according to the type of restorative material and the staining solution (as described below).\n\nControl group. An artificial saliva bottle (Entod Pharmaceuticals, Yash Pharma Laboratories, Uttarakhand, India) was used for dispensing into the containers, at 10 ml per container that were labeled as artificial saliva group.\n\nCoffee. The coffee was prepared by dissolving 10 grams of coffee powder (Bru coffee, Hindustan Unilever Limited Mumbai, Maharashtra) in 500 ml of boiling distilled water. After 10 min of stirring, the solution was filtered through a filter paper. The filtered solution was then dispensed into the containers, at 10 ml per container that were labeled as coffee group.\n\nGreen tea. The green tea was prepared by using two tea bags (Lipton Green Tea, Hindustan Unilever Ltd., Mumbai, India) according to the manufacturer’s instructions. First, 500 ml of water was boiled then allowed to cool down to 80°C as measured by the Lab Thermometer (Ludwig Schneider, Wertheim, Germany). The green tea bags were then steeped in the 80°C water for 1 minute, and the bags were then removed from the water. This prepared green tea was then dispensed into the containers, at 10 ml per container that were labeled as green tea group.\n\nDiet Coke. A 300 ml Diet Coke can (Hindustan Coca-Cola Bevarages Pvt. Ltd., Pune, India) was used for dispensing into the containers, at 10 ml per container that were labeled as the Diet Coke group.\n\nAfter the baseline analysis, the test group specimens were placed in bottles with their respective staining solutions for one hour every day up to 60 days, while the control group specimens were placed in the artificial saliva for 24 hours every day up to 60 days. After each day’s one-hour staining period, the stained test specimens were removed from their respective staining solution, rinsed in distilled water and transferred back to the bottles with artificial saliva (E SALIVA, Yash Pharma Laboratories, Uttarakhand, India) for the remaining time.\n\nAfter the 60 days of immersion period the specimens were rinsed with distilled water for five minutes and blotted dry with absorbent paper and the final color of all specimens were measured as described above. The CIE 1976 Lab* color system4 was used to calculate the color differences. The following formula was used to calculate the color differences (ΔE) at various time intervals:\n\n\n\nWhere ΔE represents the mean color change, ΔL the mean of baseline L values, Δa the mean of baseline a values and Δb the mean of baseline b values.\n\nUnpaired t-test was used to compare the color change values between the two main groups and analysis of variance (ANOVA) was used to compare the color change values between their sub-groups. Data was analyzed using SPSS version 20 software. All statistical analysis was performed at 95% level of confidence, with the significance level established at P < .05.\n\n\nResults\n\nThe color change values (ΔE) observed between the two groups and subgroups can be seen in Table 2 and Table 3. The p-value obtained between the groups shows statistically significant difference between the two materials and between the sub-groups of each group.\n\nBoth the groups stained in artificial saliva showed the least color change after 60 days, while those stained in the coffee showed the highest color change, followed by Diet Coke and green tea. Among the two materials, Cention N had exhibited more staining than Solare Sculpt in all immersions (Table 2 and Table 3).\n\n\nDiscussion\n\nSuccess or failure of an esthetic restoration primarily depends on the color stability of the material7. The extent of discoloration in vitro depends on various factors, including the composition of the material itself, test method, curing time and device, aging conditions, etc.\n\nIncomplete polymerization of the resin composites leaves unreacted monomers, which leads to discoloration of composites by aging and subsequent reactions with other substances3,4. To avoid this, all samples were stored in distilled water for 24 h after curing8.\n\nDiscoloration can also occur through water sorption and food intake. Since the foods and beverages consumed contain a variety of coloring agents, they can alter the color of the resin composites through absorption and/or adsorption of colorants during the long period of exposure. The staining solutions used in this study are common in the diet, and some have the known potential to stain tooth-colored restorative materials. According to Guler and others, storage of samples for 15 days in coffee solution simulates the coffee consumption of an individual for at least 1 year8. Thus, in the present study 60 days of storage was done, equating to about 48 months of beverage consumption.The staining protocol (quality and quantity and concentration of solutions prepared, and also daily changing of staining solutions) were strictly followed to mimic the in vivo conditions. The surface roughness of the restorative surfaces can also affect the discoloration potential because the more the rougher the surface, the greater the surface area for contact with the coloring agents7.\n\nThat is the reason all the samples were finished and polished with fine-grit Sof-Lex flexible disks and rubber cups.\n\nThere are two categories for color evaluation, one is visual and other is through the use of instruments. Instrumental assessment can potentially eliminate subjective errors in color assessments which may be observed visually. Therefore, in the present study a digital spectrophotometer was used for assessing the color change9.\n\nIn the present study following immersion in the test beverages, all restorative materials exhibited a color change of ΔE > 1.5, and those immersed in tea and diet coke exhibited a color change of ΔE >3.310–12. The results indicated that both the groups stained in artificial saliva had shown the least color change (ΔE < 1) while those stained with coffee showed the highest color change (ΔE > 5).\n\nOf the two materials tested, Cention-N had exhibited greater staining than Solare Sculpt after 60 days. The final matrix of Cention N comprises of 12–40% monomers that may leach out; alternatively, fluoride release from the material may be the reason for color change over a period of time.\n\nAmong the subgroups, the coffee solution caused the highest degree of staining followed by Diet Coke and green tea. The least staining was seen with the artificial saliva solution. The higher discoloration caused by the coffee solution to the composites has been well established. Coffee contains yellow colorants with low polarity. The discoloration caused by coffee might be due to both adsorption and absorption of these colorants13,14. The low polarity of these colorants results in slower discoloration. The absorption and penetration of the colorants into organic phase of materials may be due to the compatibility of the polymer phase with the yellow colorants in the coffee15,16. Diet Coke did not produce as much as discoloration as coffee, potentially due to its lack of yellow colorants16–18..\n\nThe specimens immersed in green tea gave ΔE values of between 2 and 2.5 for all the specimens tested. Green tea has a thin green color. In most of the specimens, the green colorant had no greening effect on the specimens, regardless of the composite. This trend suggests that the tested green tea solution did not contain tannins and the concentration of green colorant must be too low to be effective17.\n\nSpecimens immersed in artificial saliva showed no visible color change. For ΔE values, the slight change observed might be due to the adsorbed mucin of the artificial saliva on to the composite surfaces.\n\nComposite resins that can take up water are also competent to take in other fluids containing pigments, leading to discoloration, with water mainly acting as a medium for stain penetration into the resin matrix17,18. This is the reason discoloration is seen in both groups. To our knowledge this is the first study to compare Cention N with a composite (solare Sculpt) in terms of staining. As such, further studies are required for further substantiation.\n\n\nConclusion\n\nWithin the limitations of the present study, the null hypothesis, that there would be no difference in staining susceptibility among tested materials, was rejected. Staining beverages caused significant discoloration for both test materials. Cention N showed higher color variations with all beverages compared to Solare Sculpt. Coffee showed the highest degree of staining with both materials, followed by Diet Coke and then green tea.\n\n\nData availability\n\nFigshare: Evaluation of color stability of two different posterior tooth colored restorative. https://doi.org/10.6084/m9.figshare.12966680.v419.\n\nThis project contains the following underlying data:\n\nBOOK 2.xlsx. (Raw L, a and b colour values from each specimen.)\n\nstudy raw data values.docx. (Mean colour change values for each specimen.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nKielbassa AM, Glockner G, Wolgin M, et al.: Systematic review on highly viscous glass-ionomer cement/resin coating restorations (Part I): Do they merge Minamata Convention and minimum intervention dentistry? Quintessence Int. 2017; 48(10): 9–18. PubMed Abstract \| Publisher Full Text\n\nYap AU, Teo JC, Teoh SH: Comparative wear resistance of reinforced glass ionomer restorative materials. Oper Dent. 2001; 26(4): 343–348. PubMed Abstract\n\nVargas MA, Kirchner HL, Diaz-Arnold AM, et al.: Colour stability of ionomer and resin composite restoratives. Oper Dent. 2001; 26(6): 166–71.\n\nIftikhar N, Devashish, Srivastava B, et al.: A Comparative Evaluation of Mechanical Properties of Four Different Restorative Materials: An In vitro Study. Int J Clin Pediatr Dent. 2019; 12(1): 47–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSujith R, Yadav TG, Pitalia D, et al.: Comparative Evaluation of Mechanical and Microleakage Properties of Cention-N, Composite, and Glass Ionomer Cement Restorative Materials. J Contemp Dent Pract. 2020; 21(6): 691–695. PubMed Abstract\n\nMeshram P, Meshram V, Palve D, et al.: Comparative evaluation of microleakage around Class V cavities restored with alkasite restorative material with and without bonding agent and flowable composite resin: An in vitro study. Indian J Dent Res. 2019; 30(3): 403–7. PubMed Abstract \| Publisher Full Text\n\nGuler AU, Yilmaz F, Kulunk T, et al.: Effects of different drinks on stainability of resin composite provisional restorative materials. J Prosthet Dent. 2005; 94(2): 118–124. PubMed Abstract \| Publisher Full Text\n\nKalantari MH, Ghoraishian SA, Mohaghegh M: Evaluation of accuracy of shade selection using two spectrophotometer systems: Vita Easy shade and DegudentShadepilot. Eur J Dent. 2017; 11(2): 196–200. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeibrock M, Rosentritt R, Lang M, et al.: Colour stability of visible light-curing hybrid composites. Eur J Prosthodont Restor Dent. 1997; 5(3): 125–130. PubMed Abstract\n\nSahadev CK, Bharath MJ, Sandeep R, et al.: An-in vitro comparative evaluation of marginal microleakage of Cention-N with Bulk-Fill SDR and Zirconomer: A confocal microscopic study. Int J Sci Res. 2018; 7: 635–8. Reference Source\n\nGuler AU, Yilmaz F, Kulunk T, et al.: Effects of different drinks on stainability of resin composite provisional restorative materials. J Prosthet Dent. 2005; 94(2): 118–124. PubMed Abstract \| Publisher Full Text\n\nIazzetti G, Burgess JO, Gardiner D, et al.: Color stability of fluoride-containing restorative materials. Oper Dent. 2000; 25(6): 520–5. PubMed Abstract\n\nRosentritt M, Esch J, Behr M, et al.: In vivo. color stability of resin composite veneers and acrylic resin teeth in removal partial dentures. Quintessence Int. 1998; 29(8): 517–22. PubMed Abstract\n\nDietschi D, Campanile G, Holz J, et al.: Comparison of the color stability of ten new-generation composites: an in vitro study. Dent Mater. 1994; 10(6): 353–62. PubMed Abstract \| Publisher Full Text\n\nImparato JC, Garcia A, Bonifacio CC, et al.: Color stability of esthetic ion-releasing restorative materials subjected to pH variations. J Dent Child (Chic). 2007; 74(3): 189–93. PubMed Abstract\n\nGupta G, Gupta T: Evaluation of the effect of various beverages and food material on the color stability of provisional materials - An in vitro study. J Conserv Dent. 2011; 14(3): 287–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIftikhar N, Srivastava DB, Gupta N, et al.: A Comparative Evaluation of Mechanical Properties of Four Different Restorative Materials: An In vitro Study. Int J Clin Pediatr Dent. 2019; 12(1): 47–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaz F, Khan AS, Kader MA, et al.: Comparative evaluation of mechanical and physical properties of a new bulk-fill alkasite with conventional restorative materials. Saudi Dent J, 2020. Publisher Full Text\n\nMajeti C, ravi r, Kambhampati B, et al.: Evaluation of color stability of two different posterior tooth colored restorative. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12966680.v4" }	[ { "id": "115414", "date": "16 Dec 2021", "name": "Rubens Nisie Tango", "expertise": [ "Reviewer Expertise Dental materials", "prosthodontics and dental education" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study design of the present manuscript on the color stability of esthetic restorative materials upon immersion in staining solutions is very simple. However, as presented it can not be accepted for indexing and neither can be reproduced. Please, see the comments as follows:\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly. Many studies have shown a better correlation between DeltaE 00 and interpretation of color change. Additionally, important information about handheld spectrophotometers has already been published. Thus, please provide information on the limitations of Easyshade and on color change interpretation according to CIEDE 2000 (Paravina et. al1,3). Which is the equivalence of 1h/60 days of immersion in real life?\n\nIs the study design appropriate and is the work technically sound? Are sufficient details of methods and analysis provided to allow replication by others? Are all the source data underlying the results available to ensure full reproducibility? No. Important information of sample preparation is lacking: the light intensity of LCU; polishing time, pressure and handpiece rpm per step - wet or dry; how was optimal polishing determined? How were specimens stored in the containers, individually? Were the measured surfaces in contact with the bottom of the container? How many measurements were obtained per specimen?\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No. It would be more accurate to use 2 way ANOVA (materials and staining solutions), because variability of the whole sample is not considered when performing isolated statistical tests. Please, also provide adequate post hoc test.\n\nAre the conclusions drawn adequately supported by the results? No, conclusions are based on statistical tests that should be changed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "121099", "date": "08 Mar 2022", "name": "Neda Eslami", "expertise": [ "Reviewer Expertise Orthodontics" ], "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 and scientifically- written article. It compares color stability of two different types of composites when immersed in three routine drinks (coffee, Kola, green tea). They found that coffee induced more staining compared to other drinks, and difference between the two composites were statistically significant.\nThe authors did not explain if they refresh the staining medium every day. There are many similar articles in literature. Please justify the importance of your research.\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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1251
https://f1000research.com/articles/9-357/v1	13 May 20	{ "type": "Brief Report", "title": "Access to treatment in prison: an inventory of medication preparation and dispensing approaches", "authors": [ "Nguyen Toan Tran", "Dominique Pralong", "Anne-Dominique Secrétan", "Audrey Renaud", "Gérard Mary", "Arnaud Nicholas", "Elisabeth Mouton", "Clémence Rubio", "Célestine Dubost", "Francesco Meach", "Anne-Claire Bréchet-Bachmann", "Hans Wolff", "Dominique Pralong", "Anne-Dominique Secrétan", "Audrey Renaud", "Gérard Mary", "Arnaud Nicholas", "Elisabeth Mouton", "Clémence Rubio", "Célestine Dubost", "Francesco Meach", "Anne-Claire Bréchet-Bachmann", "Hans Wolff" ], "abstract": "The preparation and dispensing of medication in prisons or jails are critical for individuals to access their treatment. This process is resource-intensive for healthcare professionals and may violate principles of confidentiality, autonomy, respect, and dignity if non-qualified staff are involved. However, there are no published best practices on the topic. This report aims to bridge this gap by presenting the results of a mapping exercise on different models of medication preparation and dispensing. Authors call upon healthcare professionals to enrich this live document to inform health services research further and improve access to prescribed medications for people experiencing incarceration.", "keywords": [ "Access to medication", "preparation", "dispensing", "detention", "prison", "autonomy", "confidentiality", "dignity" ], "content": "Background\n\nIndividuals experiencing incarceration carry a high burden of physical and mental health conditions1–4. Clinical services operating in prisons and jails are vital in offering non-pharmacological and pharmacological interventions to treat, care for, and support incarcerated persons. Once prescribed, medications require coordinated preparation and dispensing for individuals to access their treatment on time. The report of the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) published in 1992 recommended that there should be appropriate supervision of the pharmacy and the distribution of medicines. Further, the preparation of medicines should always be entrusted to qualified staff (pharmacist/nurse, etc.)5. Therefore, medication preparation and dispensing should only engage qualified healthcare professionals. This process is notably intensive and can take away resources from other clinically meaningful activities, such as individual patient visits and health promotion and prevention activities. In smaller detention facilities (less than 100 occupants), which usually have limited healthcare staff, prison officers or even prisoners can be involved in medication preparation and dispensing6. Such practices violate the principles of confidentiality, autonomy, respect, dignity, and quality of care. CPT experts raised such concerns during recent visits in different European countries, where they observed a lack of respect for the 1992 recommendations of the CPT7. For instance, prison officers and incarcerated individuals were found in Greece to work as orderlies (i.e., persons trained in first aid and selected healthcare tasks, such as the dispensing of medications, under the supervision of nurses)8. In Norway, although nurses were present daily, custodial officers had the duty to dispense prescribed medications9.\n\nBest practices related to medication preparation and dispensing in prison, and in particular in smaller facilities, could help inform the organization of healthcare service delivery that complies with quality of care, confidentiality, and other human rights principles. There is, however, a paucity of publication on the subject. The objective of this paper is to present a live inventory of different approaches to medication preparation and dispensing in prisons.\n\n\nMethods\n\nFirst, we looked for published literature on different modalities of medication preparation and dispensing. On 15 August 2019, we searched PubMed and Google Scholar for publications studying different approaches using search strings combining medical subject headings (MeSH) terms related to medication preparation, dispensing, and prison with terms related to best practices (i.e., pharmaceutical preparations AND prisons AND practice guidelines as topic). The review of titles and abstracts yielded no relevant articles, prompting us to extend our search to the grey literature by using Google Search, at no avail. The lack of relevant publications underscored the paucity of research on this important operational aspect of health services management in prisons.\n\nSecond, we conducted a focus group discussion among our clinical staff from the Division of Health in Prison, which operates at the post-trial detention facility of La Brenaz in Geneva, Switzerland. On 22 August 2019, the focus group discussion, which involved four female nurses, two male nurses, two internal medicine specialists (one female, one male), and a female psychiatrist, resulted in a preliminary mapping. We captured the mapping onto a whiteboard to facilitate participants’ inputs before categorizing the information in a Word document table (Table 1). We consolidated the initial results with inputs from healthcare colleagues who could not attend the focus group discussion. The mapping drew from our work experience in prisons and visit to other facilities in Switzerland and various countries in Europe and North America. It was also informed by quality of care and operational considerations with a focus on reducing errors10 and promoting key human rights principles, such as autonomy, confidentiality, respect, and dignity11.\n\nThe Cantonal Ethical Review Board of Geneva granted ethical approval for the study (2017-01379). All participants consented to participate in the study and have the data published.\n\nAll the available data is presented in this paper.\n\n\nResults\n\nTable 1 summarizes different models of medication preparation and dispensing with the right column giving comments on the quality of care and operational considerations as well as human rights principles. Within the same facility, various modalities may coexist, depending on staff availability and medication type. Medication can be prepared manually or via an automated and computerized system by a range of health cadres at different locations, including clinics within the facility, pharmacies inside or outside the facility, or prison officers’ quarters if officers carry such a duty. Medication tablets can be given within blister packs or deblistered (intact or crushed), while liquids or creams remain in their original tubes or bottles or are transferred into plastic containers. Dispensing can be the responsibility of clinical staff, prison officers, fellow incarcerated individuals, educators, or teachers. Medication can be given in hand or left inside the cell, a personal locked medication boxes, or a cupboard for self-service. Finally, patients can take their medication under direct supervision or unsupervised.\n\n\nDiscussion\n\nThis report aimed to present an inventory of different medication preparation and dispensing models in carceral settings with a focus on whether they respect quality of care and key human rights principles. Ensuring access to medication while conforming to prison security requirements and taking into account concerns about trafficking, theft, and misuse, particularly of prescribed psychoactive substances12, needs a pragmatic and well-adjusted operational approach. We acknowledge the fact that our inventory is not exhaustive – this was the beginning of an effort to bridge the gap in published best practices on the topic. Therefore, we call upon prison health services managers, providers, and researchers to enrich this live document with their own experience and observations. This continuously enriched inventory can provide a foundation for further operational research and cost-effectiveness studies. The emerging best practices can help inform the design of new medication delivery systems that can contribute to improve the efficiency of healthcare services in prisons as well as empower individuals to safely, timely, and confidentially access and manage their prescribed treatment.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nFazel S, Baillargeon J: The health of prisoners. Lancet. 2002; 377(9769): 956–65. PubMed Abstract \| Publisher Full Text\n\nDudeck M, Drenkhahn K, Spitzer C, et al.: Traumatization and mental distress in long-term prisoners in Europe. Punishm Soc. 2011; 13(4): 403–23. Publisher Full Text\n\nFazel S, Bains P, Doll H: Substance abuse and dependence in prisoners: a systematic review. Addiction. 2006; 101(2): 181–91. PubMed Abstract \| Publisher Full Text\n\nFazel S, Seewald K: Severe mental illness in 33,588 prisoners worldwide: systematic review and meta-regression analysis. Br J Psychiatry. 2012; 200(5): 364–73. PubMed Abstract \| Publisher Full Text\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment: Health care services in prisons - 3rd General Report of the CPT. Council of Europe. 1993. Reference Source\n\nMarshall T, Simpson S, Stevens A: Health care in prisons: a health care needs assessment: University of Birmingham Birmingham. 2000. Reference Source\n\nTran NT, Wolff H: Upholding confidentiality in the preparation and distribution of medication in prisons: implementing recommendations of the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment [version 1; peer review: awaiting peer review]. F1000Res. 2020; 9: 87. Publisher Full Text\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment. Report to the Greek Government on the visit to Greece carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 14 to 23 April 2015; Para 80, 81, 85. Strasbourg: CPT. 2016. Reference Source\n\nEuropean Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment. Report to the Norwegian Government on the visit to Norway carried out by the European Committee for the Prevention of Torture and Inhuman or Degrading Treatment or Punishment (CPT) from 28 May to 5 June 2018; Para 90. Strasbourg: CPT. 2019. Reference Source\n\nStern MF, Greifinger RB, Mellow J: Patient safety: moving the bar in prison health care standards. Am J Public Health. 2010; 100(11): 2103–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUG: United Nations Standard Minimum Rules for the Treatment of Prisoners (the Nelson Mandela Rules). UN GA Res. 2015; 70: 175. Reference Source\n\nElger BS, Shaw DM: Confidentiality in prison health care–A practical guide. Emerging Issues in Prison Health: Springer. 2017; 183–200. Publisher Full Text" }	[ { "id": "63450", "date": "16 Jun 2020", "name": "Saman Zamani", "expertise": [ "Reviewer Expertise Public Health specialist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 brief report titled \"Access to treatment in prison: an inventory of medication preparation and dispensing approaches\" is an important note for further dialogue among public health experts in this field.\n\nThe study team appropriately started their inquiry with the health care providers and analyzed/documented important qualitative insight from the initial respondents.\nIt is important that the qualitative interviews and discussion to continue engaging the beneficiaries, inmates so to understand their perspectives and finding a practical arrangement for proper health service provision while ensuring respect to their rights. I would suggest this great and primary interviews to be continued among inmates and non-health staff of the prison.\n\nGreat work and thanks for all efforts and sharing the insight.\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? Not applicable\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": [ { "c_id": "5973", "date": "01 Oct 2020", "name": "Nguyen Toan Tran", "role": "Author Response", "response": "Dear Reviewer,We are grateful for your review and for your suggestion regarding the continuous engagement of people experiencing incarceration, which we have now added to our manuscript. With sincere appreciation" } ] }, { "id": "69115", "date": "02 Sep 2020", "name": "Lamiece Hassan", "expertise": [ "Reviewer Expertise Prescribing practices in prisons", "forensic psychiatric epidemiology", "qualitative research." ], "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 brief report addresses the topic of medication preparation and dispensing in prisons. The authors draw on a focus group discussion and their own previous experiences to map approaches to medication preparation, dispensing and administration, providing a brief synopsis of different models.\nAs the authors admit - this approach was not intended to be exhaustive; rather, this contribution marks a preliminary effort to gather more comprehensive evidence of experiences and practices, with the hope of eventually informing best practice. Nonetheless, while I accept and endorse their focus on this important topic, I do have some concerns about the quality of the methods and analysis reported. Attention to these matters would improve the scientific contribution of this paper, even at this early stage.\nFirst, the authors stated they looked for relevant literature in the published and grey literature and found nothing of relevance. Having authored reports and published papers commenting on pharmacy services and access to medication in prison myself (e.g. medicines reconciliation practices on entry to prison, in possession medication), I find this a bold statement and difficult to reconcile with my own knowledge of the literature. Perhaps it was their choice of keywords, geographical focus, date range or inclusion criteria that explains these limited results, but as currently written I am concerned that they have missed relevant literature commenting on pharmacy services in prison that might be directly or indirectly relevant to framing the topic.\nSecond, even for a brief scientific report there is very little information about the focus group discussion. There are limited details about the conduct and approach to analysis that would normally be required to satisfy notions of replicability (e.g. Was a topic guide used? Who moderated the group? Was it recorded, transcribed, coded and/or otherwise thematically analysed?). No reference is provided for the mapping approach mentioned and no direct quotations are provided so it is difficult to assess the quality of this approach as an empirical method for data collection and analysis.\nThird, despite my reservations about how it was produced, I do think the table summarising different models of medication preparation and dispensing could potentially be a useful direction to pursue. However, as it is currently presented in a static format, it is not clear how this will become a ‘live document’ (as the authors envisage) that others can actively contribute towards in future. If the paper is published here, this presents an important opportunity to publicise their work and enable others to contribute examples (perhaps of literature and practice). Will/can the authors capitalise on this by including a mechanism for submitting examples e.g. a link to a live document that others can actively add to (e.g. Google docs or a survey)?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "5974", "date": "01 Oct 2020", "name": "Nguyen Toan Tran", "role": "Author Response", "response": "Dear Reviewer,We are grateful for your review and have addressed the three important points you raised as follows.With sincere appreciation*****First, on the literature search:We were also surprised by the findings of our search on 15 August 2019. On 25 September 2020, we ran again the search on PubMed using the same combination of terms as described in our report—again, without any limitations (date, language, geography, etc.): (prisons[MeSH Terms]) AND (\"Practice Guidelines as Topic\"[Mesh]) AND (\"pharmaceutical preparations\"[MeSH Terms] OR pharmaceutical preparations[Text Word]) There was 1 result, but of no relevance: Brown L, Takeuchi D, Challoner K. Corneal abrasions associated with pepper spray exposure. Am J Emerg Med. 2000 May;18(3):271-2. doi: 10.1016/s0735-6757(00)90120-7. PMID: 10830682. We agree that our choice of terms and combinations may have explained the limited results. As our work on this topic moves forward, we will make sure to look into alternative search strategies.Second, on the qualitative component: We have now described this step more explicitly in the manuscript. It now reads as follows:Second, we conducted a focus group discussion among our clinical staff from the Division of Health in Prison, which operates at the post-trial detention facility of La Brenaz in Geneva, Switzerland. On 22 August 2019, the Head of the unit facilitated a focus group discussion, which involved four female nurses, two male nurses, two internal medicine specialists (one female, one male), and a female psychiatrist., resulted in a preliminary mapping. We captured the mapping onto a whiteboard to facilitate participants’ inputs before categorizing the The discussion was guided by the care continuum of medication preparation, distribution, and self-administration and the “4Ws + H” lens (what, where, when, who, and how). We did not record the discussion but directly captured participants’ inputs on a whiteboard to help visualize the emerging mapping and catalyze additional contributions. Photographs of the whiteboard were taken and used to transcribe and further categorize the information in a Word document table ( Table 1). We consolidated the initial results with inputs from healthcare colleagues who could not attend the focus group discussion and validated the content of the table with participants of the focus group discussion and the Division Chief. The mapping drew from our work experience in prisons and visit to other facilities in Switzerland and various countries in Europe and North America. It was also informed by quality of care and operational considerations with a focus on reducing errors 10 and promoting key human rights principles, such as autonomy, confidentiality, respect, and dignity 11 .Third, on the table:We opted for F1000Research as it allowed two features to update our table, which we have now added to the manuscript: First, the comment section enables readers to enrich the table Second, the paper can be resubmitted with an updated version [Update]: “[Update] is a new version, often after the article is indexed and/or the peer review is considered complete, in which authors can add small developments relevant to the research discussed in that article” – see https://f1000research.com/for-authors/article-guidelines-new-versions Our addition reads as follows:Therefore, we call upon prison health services managers, providers, and researchers to enrich this live document with their own experience and observations by adding their contributions directly in the section entitled “Comments on this article” located at the bottom of the online page of the article (an updated version will be uploaded once information saturation is reached)." } ] } ]	1	https://f1000research.com/articles/9-357
https://f1000research.com/articles/9-485/v1	01 Jun 20	{ "type": "Study Protocol", "title": "Identifying and understanding factors that affect the translation of therapies from the laboratory to patients: a study protocol", "authors": [ "Manoj M. Lalu", "Joshua Montroy", "C. Glenn Begley", "Tania Bubela", "Victoria Hunniford", "David Ripsman", "Neil Wesch", "Jonathan Kimmelman", "Malcolm Macleod", "David Moher", "Alvin Tieu", "Lindsey Sikora", "Dean A. Fergusson", "Joshua Montroy", "C. Glenn Begley", "Tania Bubela", "Victoria Hunniford", "David Ripsman", "Neil Wesch", "Jonathan Kimmelman", "Malcolm Macleod", "David Moher", "Alvin Tieu", "Lindsey Sikora", "Dean A. Fergusson" ], "abstract": "Background: The process of translating preclinical findings into a clinical setting takes decades. Previous studies have suggested that only 5-10% of the most promising preclinical studies are successfully translated into viable clinical applications. The underlying determinants of this low success rate (e.g. poor experimental design, suboptimal animal models, poor reporting) have not been examined in an empirical manner. Our study aims to determine the contemporary success rate of preclinical-to-clinical translation, and subsequently determine if an association between preclinical study design and translational success/failure exists. Methods: Established systematic review methodology will be used with regards to the literature search, article screening and study selection process. Preclinical, basic science studies published in high impact basic science journals between 1995 and 2015 will be included. Included studies will focus on publicly available interventions with potential clinical promise. The primary outcome will be successful clinical translation of promising therapies - defined as the conduct of at least one Phase II trial (or greater) with a positive finding. A case-control study will then be performed to evaluate the association between elements of preclinical study design and reporting and the likelihood of successful translation. Discussion: This study will provide a comprehensive analysis of the therapeutic translation from the laboratory bench to the bedside. Importantly, any association between factors of study design and the success of translation will be identified. These findings may inform future research teams attempting preclinical-to-clinical translation. Results will be disseminated to identified knowledge users that fund/support preclinical research.", "keywords": [ "Translational failures", "promising therapies", "bench-to-bedside research", "systematic review", "case-control study" ], "content": "Introduction\n\nAdvances in discovery research form the backbone for the development of novel therapeutics. Such translation relies on a “process of turning observations in the laboratory, clinic, and community into interventions that improve the health of individuals and the public”1. The process is often visualized as a linear model (Figure 1). In reality, however, it comprises a spectrum that masks the complex, iterative and interdisciplinary research and development steps that engage multiple stakeholders2,3. As a result, the successful translation of promising preclinical discovery research into human studies (T1) is rare4, and the resource-intensive efforts to evaluate discoveries in sufficient detail to allow them to be available for patients (T2) takes decades rather than years5.\n\nWhile discovery research has inherent value, funding agencies around the world are increasingly focused on improving the success of “bench to bedside” translation (T0-T2), thereby enhancing the potential impact of biomedical research budgets. Certainly, there is great potential for improvement; it is estimated that the US alone invests 28 billion dollars per year on research that cannot be reproduced6. Return on investment is poor even in more successful areas. For example, the internal rate of return for health benefits of cardiovascular disease research has been calculated to be 9%7. This means that every $1.00 invested into cardiovascular research provides the equivalent of 9¢ per year in health benefits. Importantly, a clinically translated therapy – one which has obtained regulatory approval – does not mean patients will have access to it. An additional hurdle in the translation into practice (T3) and community (T4) access is whether or not insurance companies and healthcare systems will pay for the therapy. The willingness to pay is largely dependent on a cost-comparative analysis of the potential therapy as well as the needs and resources of the jurisdiction. As this is a critical aspect in the translation spectrum and widespread use of a therapy, it has been thoroughly investigated8.\n\nInvestigation of translation in the earlier phases of the translational spectrum – from basic research (T0) to clinical trials in humans (T2) – has uncovered striking deficits when moving from one phase to another9. These inefficiencies ultimately contribute to a 5–10% rate of successful translation of preclinical “bench” research to approved therapies. This means that approximately 90% of promising discoveries may not directly contribute to improved human health5,10,11. Moreover, translation failures expose clinical research participants to potential harms of investigational products that fail on safety or efficacy grounds12,13. The extent of translational failures diverts scarce funding away from developing interventions that are more likely to benefit patients. Given the recent focus on research waste14–17, it is abundantly clear that bench-to-bedside translation needs solutions to become more efficient.\n\nThree highly cited studies investigating this issue in publicly-available data have been previously published. Contopoulos-Ioannidis et al.18 traced the translation of published, highly promising in vivo basic science findings into clinical applications from 1979 to 1983 in six basic science journals. They identified 101 promising therapies and in 2003 (i.e. ~20 years since studies published), 27 entered clinical trials and only 5 were clinically licensed (i.e. only 5% translated into an approved therapeutic). Hackam and Redelmeier11 searched for studies published in seven leading journals between 1980 and 2000 that were cited >500 times and investigated highly promising preclinical agents. Their inclusion criteria resulted in 76 studies of interventions, of which 42 (55%) entered clinical trials and only 8 (10%) were approved for clinical use. These authors attempted multivariable analyses to identify predictors of translation; however, the small sample size “limited power to discern individual predictors of translation.” In the most recent investigation of this issue, Morris et al.19 (2011) performed a pseudo-systematic search to identify studies that had quantified time lags in the development of health interventions. They found time lags of ~20 years for interventions to translate, though this depended on how each study defined if translation occurred. Importantly, they concluded that the “knowledge of time lags is of limited use to those responsible for R&D…who face difficulties in knowing what they should or can do to reduce time lags.” In other words, identifying factors associated with successful translation of preclinical research into clinical trials may be a more practical metric than time lags for decision makers.\n\nIn considering this previous research, it is still currently unknown if/how rates of translation from preclinical research to the therapy’s evaluation in clinical trials are associated with study rigor (i.e. internal validity, risk of bias), and external validity (i.e. reproducibility over a range of conditions), among other factors affecting the quality of the preclinical evidence such as construct validity (i.e. the experimental model appropriately represents the clinical condition and setting). Therefore, the aim of this project is to use established knowledge synthesis methods to identify the prevalence of bench-to-bedside translation, map contemporary trends in translation, and identify key modifiable factors associated with successful and unsuccessful bench-to-bedside translation.\n\n\nObjectives\n\nWe will perform a cross-sectional study to estimate the rate of publicly available bench-to-bedside translation. Specifically, we will identify published preclinical1 research of promising therapies and track how far along the translational spectrum they have progressed.\n\nRegulatory approval of the therapy will be considered successful translation. If approval has not yet been obtained, we will consider a therapy that translated from the preclinical phase (T0) to a Phase II clinical trial (T2) or further, a successful bench-to-bedside translation.\n\nWe will next perform a case-control study to identify factors associated with therapies that have been successfully translated from the preclinical study in which it was identified, to a positive clinical trial evaluating treatment efficacy. Specifically, we will consider a therapy to be ‘successfully’ translated if it 1) has obtained regulatory approval, or 2) has a significant, positive finding in a clinical trial evaluating efficacy (presumably a Phase II trial) at the latest stage trial the therapy has advanced to (i.e. the therapy will not be considered successful if it demonstrated efficacy in an earlier trial and subsequently showed null or negative findings in a later efficacy trial).\n\n\nMethods\n\nAny modifications from this protocol will be included in the final report.\n\nThe first objective of this study is to estimate the rate of translation from preclinical research in animal models to success in phase II clinical studies of therapeutic efficacy. This will be achieved through a cross-sectional study of preclinical investigations. In order to identify and select relevant studies for inclusion, we will employ methods typically used in systematic reviews20. The objective focuses on two questions. What is the current rate of preclinical to clinical translation of promising basic science findings? What are the basic characteristics and design features of the promising therapies identified?\n\nEligibility criteria. We will identify preclinical studies published in Science, Nature, and Nature Medicine. These journals were selected because they are considered leaders across all domains of preclinical research and are anticipated to publish work that may impact human health. Our selection also reflects the philosophy adopted by the largest previous evaluation of bench-to-bedside research5.\n\nEligible articles will be those published between 1995 and 2015. This timeframe allows a significant time-lag (although not necessarily the full ~20-year time lag described previously). This timeframe will also allow for an evaluation of (in an exploratory manner) whether bench-to-bedside translation has improved in terms of both time-lags and the proportion of therapies advancing to later phases of clinical trials or gaining regulatory approval. Importantly, we anticipate that the journal selection and year range will also provide an adequate sample size to perform the case-control study outlined in Objective #2.\n\nInclusion criteria\n\ni. Population:\n\nArticles that describe a preclinical, interventional study: any article that includes in vivo non-human animal experiments that has not been tested for the same purpose in humans prior to the study.\n\nii. Intervention and outcome:\n\nA ‘promising therapy’, defined by the following:\n\na. Any therapy introduced to the animal model (i.e. pharmacologic and non-pharmacologic therapies, vaccines, antibodies, etc.), which was still at the developmental stage and did not have a prior application in humans for the specific indication.\n\nb. Novel uses of existing therapies (e.g. treatment of a different disease) will also be included.\n\nc. The intervention must induce an outcome that positively benefits an indicator of health of the animal (e.g. an immunotherapy that shrinks solid tumor size in the animal model) and/or investigators state that the therapy should be translated clinically based on their findings.\n\niii. Comparison:\n\nAny comparison to the intervention will be acceptable\n\nExclusion criteria\n\ni. Editorials, commentaries, reviews, news articles, articles that focus on a mechanism of action, pathophysiology, or diagnosis, and articles on agricultural or veterinary applications that would not be feasibly applied to humans.\n\nii. Investigators explicitly state that translation should not be attempted based on the study.\n\niii. Ex vivo, in vitro, and human clinical trials.\n\nInformation sources and search strategy. All included journals are indexed in MEDLINE, therefore this database will be searched from January 1st, 1995, to December 31st, 2015. A validated animal filter will limit results to animal studies21. The search strategy will be developed and finalized with the help of an information specialist who has expertise in the design of systematic searches (L. Sikora). The search strategy can be found in Extended data22.\n\nStudy selection process. The literature search results will be uploaded to Distiller Systematic Review Software (DistillerSR®, Evidence Partners, Ottawa, Canada). DistillerSR is an audit-ready, cloud-based software program that allows for transparent and reproducible work required for an accurate review. Two reviewers will independently screen the titles and abstracts from the search results using the predefined eligibility criteria. A calibration exercise will be performed on the first 50 studies to refine the screening question prior to formally commencing the screening process. Two review authors will assess the eligibility of the full-text articles. Discrepancies between the reviewers will be resolved by discussion or with a third-party member if consensus cannot be established. Reasons for excluding studies will be recorded.\n\nData collection process and data items. Standardized forms designed in DistillerSR will be used to extract all study characteristics (see list below). Following a pilot to refine the forms and a calibration exercise to ensure high inter-rater agreement (i.e. above 80%), data will be extracted independently by two reviewers. Disagreements between reviewers will be resolved by discussion or with a third-party member if a consensus cannot be reached. The following data will be collected from eligible preclinical articles, and many of these elements will be incorporated for analyses listed in Objective #2. Data items i–v will be collected from all eligible preclinical articles. Data items vi–x will only be collected from articles selected as either cases or controls in Objective #2:\n\ni. Funding: academic/governmental/charitable vs. biotechnology and/or pharmaceutical companies (defined as reported industry affiliation by an author, financial support, or provision of the therapy being studied)\n\nii. Study characteristics: study title, first and corresponding authors’ name, publication year, journal of publication, country of corresponding author, and the number of authors and affiliated institutions\n\niii. Study population: animal species, sex, age, presence of comorbid illnesses\n\niv. Type of model: disease being studied, name of animal model(s) used, number of different models used\n\nv. Interventions (promising therapy): name of intervention, dose-response tested, anticipated application (e.g. preventative, therapeutic, or both)\n\nvi. Preclinical outcomes: primary outcome (or, if none declared, main in vivo outcome highlighted by authors), death, adverse events\n\nvii. Risk of bias (internal validity): the Cochrane Risk of Bias tool to evaluate preclinical in vivo studies20\n\nviii. External validity (range of experimental conditions tested): more than one animal model used (species or disease model/co-morbid model), inclusion of male and female animals, and multiple treatment doses\n\nix. Completeness of reporting: an operationalized version of the National Institutes of Health Preclinical Reporting guidelines will be used23.\n\nx. Article metrics: The H-index of the corresponding author(s) and the number of times the paper has been cited\n\nIdentifying clinical translation of promising bench findings. After identifying the set of promising potential therapies, the stage of clinical translation that each therapy has achieved will be identified. In collaboration with an information specialist (L. Sikora), a piloted algorithmic search strategy that identifies the current state of clinical use will be used. Backward citation analysis (identifying and examining the studies cited in an article) and forward citation analysis (identifying studies that cite an original article or work after it had been published) will be used. The search strategy consists of searching known databases for citation analysis (i.e. Scopus, ISI Web of Science, Google Scholar). Other resources will also be searched including grey literature sources, clinical trial registries, clinical practice guidelines, and conference proceedings. The full draft algorithm is listed in Extended data. This approach will be used to determine the furthest stage of clinical testing and whether the agent has received regulatory approval.\n\nOutcomes. The primary outcome of interest will be the successful clinical translation of the identified promising therapies defined as 1) having obtained regulatory approval for the therapy, or 2) the conduct of at least one Phase II trial (or greater) for efficacy, with a statistically significant result favoring the treatment (and no later trials with negative or null results). As per the Federal Drug Agency (FDA) in the United States, the purpose of Phase II trials is “efficacy as well as side effects.”24 A positive finding will be defined as a statistically significant result demonstrating therapeutic superiority compared to placebo/no treatment, or established interventions; or stated non-inferiority compared to currently established interventions in an appropriate designed and conducted clinical study. This primary outcome has been selected as it indicates replication of the findings from the animal models of the preclinical study in the human subjects of the clinical trial.\n\nThe secondary outcome is the furthest clinical advances of all promising therapies from Phase I through to regulatory approval. For this outcome we will consider any clinical trial that has been performed and/or published. Whether favorable results were obtained for each identified trial (which we will call a “positive” trial) will also be reported. As above, a positive result will be defined by a statistically significant result demonstrating superiority compared to placebo/no treatment, or established interventions; or stated non-inferiority compared to currently established interventions. In determining regulatory approval status, while the dominant regulatory agencies are often regarded as the FDA (US), European Medical Agency (Europe), and Pharmaceuticals and Medical Devices Agency (Japan), approval by any regulatory agency will be accepted. This outcome will be presented as a proportion of studies that advanced to the clinical trial stage. Results will also be stratified by year of publication: 24–20 years ago (to provide a comparison to the landmark article by Contopoulos-Ioannidis et al.5), 20–10 years ago, and <10 years ago.\n\nData synthesis and analysis plan. The total number of studies screened, assessed for eligibility, and included in the review (with reasons for exclusion) will be reported according to the PRISMA guidelines for reporting systematic reviews25. For each included study, the characteristics outlined above will be described, with frequencies and proportions reported. For the primary outcome, the time from the preclinical paper publication date, to first positive Phase II trial will be calculated and presented. Likewise, for the secondary outcomes, the time to furthest advance (Phase I, II, III, IV, or approval), as well time to first positive trial will be calculated using medians and interquartile ranges. Kaplan-Meier curves will also be constructed for all time to event analyses. All analyses will be performed using SAS (Version 9.4).\n\nFor all promising therapies, a descriptive analysis of all study characteristics and its research trajectory (i.e. furthest level of human experimentation) will be presented. This will present the cohort of successful and unsuccessful promising therapies published in highly influential journals, as well as the ability to evaluate secular trends in translation.\n\nRisk of bias assessment. Risk of bias will be assessed in duplicate by two independent reviewers as high, low or unclear for six domains of bias identified by the Cochrane Risk of Bias tool20, with the addition of two domains suggested to influence the risk of bias in preclinical studies26. Disagreements will be resolved first by discussion and subsequently by consulting a third-party member, if needed. Graphic representations of risk of bias within and across studies will be conducted using RevMan 5.3 (Cochrane Collaboration, Oxford, United Kingdom).\n\nA case-control study will be performed to evaluate the association between preclinical study characteristics and the incidence of translation. Translated therapies gaining regulatory approval or into a Phase II clinical trial (or later) will be defined as cases; unsuccessfully translated therapies will serve as controls. This study will help identify factors of internal and/or external validity, which are associated with “failed” versus “successful” translation. A case-control design was chosen as the most efficient methodological design due to i.) the rarity of preclinical to clinical translation and ii.) the ability to study multiple factors that may contribute to translational success.\n\nData source, identification and matching of cases and controls. Upon completion of the cross-sectional study outlined in Objective #1, a comprehensive roster of promising basic science findings published in top-tier journals between 1995 and 2015 will be generated. From this dataset, cases and matched controls will be selected:\n\nCases: Promising discoveries that became successfully translated will be used as cases in our study. ‘Successfully translated’ will be defined in two ways as mentioned above. In keeping with the primary outcome in Objective #1, successful translation will be defined as having 1) obtained approval from a regulatory agency and/or 2) demonstrated positive findings in a Phase II clinical trial of efficacy - indicating replication of the findings from the preclinical study. As in Objective #1, a positive result will be defined by a statistically significant result demonstrating superiority compared to placebo/no treatment, or established interventions; or stated non-inferiority compared to currently established interventions.\n\nControls: Highly promising basic science findings which were not successfully translated will serve as controls. Unsuccessful translation will be defined as either a highly promising basic science finding without 1) having obtained regulatory approval, AND 2) any clinical trial (i.e. no trial identified by literature search and/or identified in clinical trial registries such as clinicaltrials.gov) or going to trial and failing for any reason (e.g. failing in safety in Phase I; or efficacy in Phase II). If possible, up to 4 matched controls per case will be selected. This 4:1 ratio represents the most statistically efficient ratio while minimizing bias introduced to the study27,28. Controls will be frequency matched based on year of publication (+/- 5 years for each case, disease of interest, and general area of biomedical research (i.e. cardiology, cancer, immunotherapy, etc.)). These stratifying factors are not predictor variables but have been chosen to ensure the comparability of cases and controls in the sample. Following matching, controls will be randomly selected using a computer-generated algorithm for those cases with >4 matched controls.\n\nVariables. The primary analysis will investigate the association between a set of variables thought to influence translation, and successful translation. It is hypothesized that the following four sets of variables are of high importance:\n\ni. Internal validity: Similarly to clinical studies, preclinical studies that lack methodological rigor usually demonstrate the largest measures of efficacy29–31. However, this lack of methodological rigor may reduce the likelihood that these studies can be translated. In our study, risk of bias will be assessed by applying the Cochrane Risk of Bias tool20 – modified for suitability to preclinical studies. We will assess six domains identified by this tool (sequence generation, allocation concealment, blinding of personnel, blinding of outcome assessors, incomplete outcome data, and selective outcome reporting), as well as two additional risk of bias domains (conflict of interest and sample size calculation) for a total of eight risk of bias domains. Again, this checklist will be operationalized into a binary outcome; if the majority (i.e. >4) of items on the checklist are met the study will be considered to have high internal validity.\n\nii. External validity: This will be assessed by the experiment(s) within the preclinical study being performed across a range of conditions: the presence of dose response relationship, more than one animal model (species or method of disease induction) used, male and female animals (or the appropriate sex for sex specific diseases, e.g. prostate or ovarian cancer), and co-morbid animals32. This outcome will be dichotomized, (i.e. if two or more of these four criteria are met, the study will be considered to have high external validity).\n\niii. Funding source: Evidence suggests that industry involvement may lead to faster translation5. This relationship will be examined by determining if the preclinical study involving the promising therapy was industry, government, or philanthropy funded (with attention also given to whether industry funding was from pharmaceutical or biotechnology companies).\n\niv. Completeness of reporting: The National Institutes for Health preclinical reporting guidelines will be used to assess completeness of reporting. We have previously operationalized this list into 21 yes or no questions. This outcome will be dichotomized as high or low completeness of reporting when the majority of items (≥11) are reported upon or not. As a pre-planned sensitivity analysis, we will also evaluate the preclinical studies as high completeness of reporting when they report on over two thirds of the checklist items (≥14).\n\nFor the exposure items of validity (internal and external) and reporting the individual items in each checklist will not be weighted.\n\nExploratory analysis:\n\ni. Individual components of internal validity: We will assess two selected risk of bias domains (sequence generation and blinding of outcome assessor) individually.\n\nii. Individual components of external validity: We will assess all components of external validity individually, rather than as an aggregate outcome.\n\niii. Number of authors and institutions: We will use the number of co-authors and the number of affiliated institutions on the preclinical paper as an approximation for the degree of collaboration within the preclinical study. With this, we plan to evaluate how the degree of collaboration within a preclinical study affects translation.\n\niv. H-index: We will use the corresponding author’s H-index as an approximation for investigator seniority. With this, we plan to evaluate how the corresponding author’s seniority affects preclinical to clinical translation.\n\nv. Number of citations: We will use the number of citations associated with the preclinical paper as an approximation for the degree of dissemination. With this we plan to evaluate how the degree of the preclinical study’s dissemination affects translation.\n\nData analysis and sample size\n\nSample size. In a preliminary screen of Science and Nature, 482 promising therapies (184 Science; 298 Nature) were identified. Application of a conservative estimate suggests that the addition of the third journal (Nature Medicine) will provide an additional 150 promising therapies, for a total of 632. In the previous report citing a 5% translation rate18, 19 of 101 (19%) promising therapies reached the clinical trial stage and demonstrated favorable results. Methodological differences between this study and the previous report may lead to a decrease in the percentage of promising therapies demonstrating favorable results at phase II. A conservative estimate of half the rate from the previous report (9.5%) suggests that roughly 60 of the promising therapies from our sample would demonstrate promising results at phase II or later. Generally, it is recommended that a regression model contain 10 events per variable33,34. Considering the four variables listed above, we will require 40 cases in order to be adequately powered to perform our proposed analysis.\n\nIf an adequate sample size of 40 cases is not reached, we will continue searching for promising preclinical therapies in order to identify additional phase II or later translated therapies. The CAMARADES group has used a machine learning algorithm35 to identify a cohort of over 100,000 reports of in vivo research available on PubMed Central. From this we will randomly select reports of in vivo preclinical research and assess these for clinical translation. The number of studies randomly selected will depend on the number of additional case required. We will sample iteratively until our predetermined minimal set of 40 cases is obtained.\n\nAnalytic plan. A comparison of baseline variables (aside from matching factors) in the cases and controls will be assessed using frequency distributions and univariate descriptive statistics including measures of central tendency and dispersion. Multivariable conditional logistic regression will be performed to determine the adjusted association of our factors to the outcome variable. Odds ratios with accompanying 95% confidence intervals will be calculated. A sensitivity analysis will be performed excluding cases that had a positive clinical trial but had not received regulatory approval. All analyses will be performed using SAS (version 9.4). Data will be presented as odds-ratios and 95% confidence intervals in forest plots.\n\nOur focus on leading journals (Science, Nature and Nature Medicine) may be perceived as a limitation. Given the usually positive findings and higher visibility of studies published therein, this may lead to an over-estimate in the bench-to-bedside translation. Although this selection of journals may represent an over-estimate of translation, we also believe it represents the ‘best-in-class’ of preclinical research. Alternatively, the reliance upon publicly available data may result in an under-estimate of the bench-to-bedside transition as it is possible that preclinical work from within industry that does not proceed to a clinical trial may not be published. Without examining this omitted data, we may not be presenting a fully accurate assessment of the state of preclinical to clinical trial translation.\n\nAnother limitation is that components of internal validity have historically been poorly reported in bench research36, which may not be an accurate reflection of what investigators actually did. A third limitation is that the dichotomization of variables in the case-control could be regarded as an oversimplification of complex concepts. However, our exploratory analyses will allow us to investigate a larger number of factors in more granular detail. Furthermore, our assessment on the external validity and how this may affect translation rests on the assumption that the rationale for initiating a clinical trial is based solely on one preclinical study, rather than several studies from different labs under a range of conditions. In reality, external validity would be best established through multiple preclinical experiments rather than one with a high level of external validity.\n\nAnother important aspect affecting the quality of preclinical evidence is construct validity. Studies have demonstrated that poor construct validity affects reproducibility and downstream clinical translation37,38. Though a checklist to assess construct validity has been developed32, after pilot testing this tool we found that it was infeasible for our study, as it required content expertise for every promising therapeutic to be used appropriately. Thus, another limitation of this study is that we are unable to evaluate the construct validity of the preclinical studies and assess how this aspect affects the translation into clinical trials.\n\nLastly, it should be acknowledged that the translational spectrum involves many complex steps and various phases. We have chosen to focus on the translation of the in vivo animal experiment stage within T0 research; to the stage of therapy evaluation in clinical trials, within T2 research. When we deem a successful translation from preclinical to clinical research, we do not evaluate all stages of preclinical nor clinical research. Furthermore, successful translation to a Phase II clinical trial does not imply that the therapy has been successfully translated to practice or regulatory approval. With this limitation in mind, we will focus on preclinical issues that may affect early translation, rather than potential issues with the clinical trials that subsequently evaluated the therapies in human subjects. We anticipate a future study that will critically analyze the translational clinical trials identified in Objective #1 to address issues associated with those trials.\n\nWe will have a one-day, in-person meeting between the investigators and identified knowledge users (Stem Cell Network and BioCanRx, two Government of Canada funded Networks of Centres of Excellence focused on the development of novel therapeutics). Over the one-day meeting we will examine data and results generated by the project. In addition, each knowledge user will speak about their organization’s experiences and perspectives on bench-to-bedside translation. The meeting will end with a discussion to identify next steps to be taken by stakeholders to improve translation.\n\nWe anticipate the publication of two key papers submitted to appropriate peer-reviews journals. The first will describe key findings of the systematic review and present the results of our case-control study. The second will be a policy piece to describe how our findings may affect stakeholders across the spectrum of bench-to-bedside research. The results of this study will be presented at relevant national and international scientific meetings to promote knowledge transfer.\n\nIf amendments are required for this protocol, the date of each amendment will be provided with a description for rationale for the change in this section.\n\n\nStudy status\n\nWith our systematic search strategy, we have searched the journals Science, Nature, and Nature Medicine and are currently screening articles to identify promising therapies as per Objective #1 of this study.\n\n\nDiscussion\n\nThis study is an innovative project that will use knowledge synthesis methods to identify the incidence of successful translation, map contemporary trends in translation, and identify key modifiable factors associated with successful and unsuccessful bench-to-bedside translation. Additionally, the proposed study will generate the largest dataset to date of promising preclinical therapies and then apply a case-control design to investigate the association of validities with successful or failed translation. This evidence is required to implement a “knowledge-to-action”39–41 cycle to improve bench-to-bedside translational research.\n\nCurrent lags in translation are unknown. Some of our own efforts (and others) at translation suggest that these lags between preclinical findings and first-in-human trials may be shortening26,42–45. This reduction may reflect increased funding towards translational activities, improved research rigor, as well as regulatory changes that have accelerated translation efforts (e.g. FDA’s four unique approaches of Fast Track, Breakthrough Therapy, Accelerated Approval, and Priority Review). If this hypothesis is supported by the data, all stakeholders need information about the factors that support improvements in development timelines.\n\nThe effect of evolving preclinical reporting guidelines on translation is unknown. In the last decade, funders, journals, and other stakeholders have endorsed reporting guidelines36,46 that improve transparency and potentially improve the ability to forecast which interventions may translate successfully. These guidelines have been developed in response to multiple publications, which demonstrated that the majority of basic science findings are irreproducible47–49. Previous studies of bench-to-bedside translation were conducted prior to these guidelines being widely endorsed; thus, a current study is needed to determine if increased awareness and emphasis regarding preclinical reporting and design has improved translation.\n\nTaken together, there is a strong need and justification for the proposed study. This study will provide a contemporary understanding of bench-to-bedside translation, which is required to tailor experimental designs to improve translational efficiency, including the education and uptake of available study design and analysis methods.\n\n\nData availability\n\nNo data is associated with this article.\n\nOpen Science Framework: Identifying and understanding factors that affect the translation of therapies from the laboratory to patients, https://doi.org/10.17605/OSF.IO/WGZ3T22.\n\nThis project contains the following extended data:\n\n- Representative search strategy in PubMed\n\n- Data selection items\n\n- Algorithm to identify clinical translation of promising bench findings\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Footnotes\n\n1 The term ‘preclinical’ has variable definitions, depending on the context. For the purposes of this study, our group has chosen to focus on preclinical interventional in vivo animal experiments, relevant to the betterment of human health and/or treatments for human disease.\n\n\nReferences\n\nNational Center for Advancing Translational Sciences: Translational Science Spectrum. 2018. Reference Source\n\nBonter K, Breckenridge Z, Lachance S, et al.: Opportunities and challenges for the cellular immunotherapy sector: a global landscape of clinical trials. Regen Med. 2017; 12(6): 623–636. PubMed Abstract \| Publisher Full Text\n\nBubela T, Bonter K, Lachance S, et al.: More Haste, Less Speed: Could Public-Private Partnerships Advance Cellular Immunotherapies? Front Med (Lausanne). 2017; 4: 134. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIoannidis JP: Evolution and translation of research findings: from bench to where? PLoS Clin Trials. 2006; 1(7): e36. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLalu MM, Hunniford V, Ferguson D, et al.: Identifying and understanding factors that affect the translation of therapies from the laboratory to patients: a study protocol - Extended Data. 2020. http://www.doi.org/10.17605/OSF.IO/WGZ3T\n\nFergusson DA, Wesch NL, Leung GJ, et al.: Assessing the Completeness of Reporting in Preclinical Oncolytic Virus Therapy Studies. Mol Ther Oncolytics. 2019; 14: 179–187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nU.S. Food and Drug Administration: The Drug Development Process. Step 3: Clinical Research. 2019. Reference Source\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. J Clin Epidemiol. 2009; 62(10): 1006–1012. 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PubMed Abstract \| Publisher Full Text\n\nHirst JA, Howick J, Aronson JK, et al.: The need for randomization in animal trials: an overview of systematic reviews. PLoS One. 2014; 9(6): e98856. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMacleod MR, O'Collins T, Horky LL, et al.: Systematic review and metaanalysis of the efficacy of FK506 in experimental stroke. J Cereb Blood Flow Metab. 2005; 25(6): 713–721. PubMed Abstract \| Publisher Full Text\n\nHenderson VC, Kimmelman J, Fergusson D, et al.: Threats to validity in the design and conduct of preclinical efficacy studies: a systematic review of guidelines for in vivo animal experiments. PLoS Med. 2013; 10(7): e1001489. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nConcato J, Peduzzi P, Holford TR, et al.: Importance of events per independent variable in proportional hazards analysis. I. Background, goals, and general strategy. J Clin Epidemiol. 1995; 48(12): 1495–501. PubMed Abstract \| Publisher Full Text\n\nPeduzzi P, Concato J, Kemper E, et al.: A simulation study of the number of events per variable in logistic regression analysis. J Clin Epidemiol. 1996; 49(12): 1373–9. PubMed Abstract \| Publisher Full Text\n\nBannach-Brown A, Przybyła P, Thomas J, et al.: Machine learning algorithms for systematic review: reducing workload in a preclinical review of animal studies and reducing human screening error. Syst Rev. 2019; 8(1): 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKilkenny C, Browne WJ, Cuthill IC, et al.: Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010; 8(6): e1000412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott S, Kranz JE, Cole J, et al.: Design, power, and interpretation of studies in the standard murine model of ALS. Amyotroph Lateral Scler. 2008; 9(1): 4–15. PubMed Abstract \| Publisher Full Text\n\nReynolds JC, Rittenberger JC, Menegazzi JJ: Drug administration in animal studies of cardiac arrest does not reflect human clinical experience. Resuscitation. 2007; 74(1): 13–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCanadian Institutes of Health Research: Guide to Knowledge Translation Planning at CIHR: Integrated and End-of-Grant Approaches. 2012. (Cat. No. MR4-11/2012E-PDF). Reference Source\n\nCanadian Institutes of Health Research: Integrating Ethics and the Knowledge-To-Action Cycle. 2018. Reference Source\n\nStraus S, Tetroe J, Graham ID: Knowledge translation in health care: moving from evidence to practice. John Wiley & Sons, 2013. Reference Source\n\nTaljaard M, Ward MR, Kutryk MJ, et al.: Rationale and design of Enhanced Angiogenic Cell Therapy in Acute Myocardial Infarction (ENACT-AMI): the first randomized placebo-controlled trial of enhanced progenitor cell therapy for acute myocardial infarction. Am Heart J. 2010; 159(3): 354–360. PubMed Abstract \| Publisher Full Text\n\nGranton J, Langleben D, Kutryk MB, et al.: Endothelial NO-Synthase Gene-Enhanced Progenitor Cell Therapy for Pulmonary Arterial Hypertension: The PHACeT Trial. Circ Res. 2015; 117(7): 645–654. PubMed Abstract \| Publisher Full Text\n\nEiger BioPharmaceuticals: A Phase 2, Randomized, Double-BLInd, Placebo-Controlled Study of UBEnimex in Patients With Pulmonary ARTerial HYpertension (WHO Group 1) (LIBERTY). ClinicalTrials.gov. 2018. Reference Source\n\nTian W, Jiang X, Tamosiuniene R, et al.: Blocking macrophage leukotriene b4 prevents endothelial injury and reverses pulmonary hypertension. Sci Transl Med. 2013; 5(200): 200ra117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Institutes of Health: Principles and Guidelines for Reporting Preclinical Research. 2016. Reference Source\n\nIoannidis JP, Allison DB, Ball CA, et al.: Repeatability of published microarray gene expression analyses. Nat Genet. 2009; 41(2): 149–155. PubMed Abstract \| Publisher Full Text\n\nPrinz F, Schlange T, Asadullah K: Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov. 2011; 10(9): 712. PubMed Abstract \| Publisher Full Text\n\nBegley CG, Ellis LM: Drug development: Raise standards for preclinical cancer research. Nature. 2012; 483(7391): 531–533. PubMed Abstract \| Publisher Full Text" }	[ { "id": "69397", "date": "17 Aug 2020", "name": "John Holcomb", "expertise": [ "Reviewer Expertise injury", "resuscitation" ], "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 embarked on an important project. They will evaluate the issues related to translation of preclinical data into clinical practice. I have several comments.\nThe authors should clearly state the narrow focus of their work, largely in the drug development arena. They seem to exclude other research areas such as devices and blood products. Please inform the reader what focus areas are included and excluded.\n\nThe authors have included only several journals to review. Given the ability to use electronic databases, I would suggest they broaden their search.\n\nLastly, the definition of clinical translation is not completion of phase 2 studies, but rather regulatory approval and adoption into clinical practice. Defining adoption itself is difficult, and should be discussed.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "6017", "date": "16 Oct 2020", "name": "Manoj Lalu", "role": "Author Response", "response": "We thank you for your careful assessment of our protocol and their supportive comments. We have addressed your queries below and within the revised manuscript. The authors have embarked on an important project. They will evaluate the issues related to translation of preclinical data into clinical practice. I have several comments. The authors should clearly state the narrow focus of their work, largely in the drug development arena. They seem to exclude other research areas such as devices and blood products. Please inform the reader what focus areas are included and excluded. We apologize for the lack of clarity on our inclusion criteria. Our search and strategy for selecting therapies is designed to consider all interventions that have therapeutic benefit. We added blood products and devices in the examples of ‘promising therapies’ in the inclusion criteria. A ‘promising therapy’, defined by the following: a. Any therapy introduced to the animal model (e.g. pharmacologic and non-pharmacologic therapies, vaccines, antibodies, blood products, implants/devices, etc.), which was still at the developmental stage and did not have a prior application in humans for the specific indication. The authors have included only several journals to review. Given the ability to use electronic databases, I would suggest they broaden their search. Our group of investigators considered this very issue at length. Ultimately, there simply is no perfect sampling strategy to address the objectives of the study. We selected the three journals as they are highly influential and considered ‘best-in-class’ by many. The number of articles resulting from the search of the three included journals is 20,341. With our available resources, it has taken close to one year to screen all abstracts and full texts in duplicate. Adding more journals would not be feasible with our finite resources. We added the following to the limitation section of the discussion: Furthermore, our sample is limited to three journals, which may reduce the scope of our work. As automation of meta-research projects gains traction (and tools are validated) broader searches will certainly be possible in the future. However, given that this process currently relies on personnel we are limited to the current strategy. Lastly, the definition of clinical translation is not completion of phase 2 studies, but rather regulatory approval and adoption into clinical practice. Defining adoption itself is difficult, and should be discussed. We agree that full clinical translation is not the completion of a Phase II trial and we also do agree that ultimate clinical translation is the clinical adoption of a therapy. In the current study we are focussed on the translation of preclinical research to first studies demonstrating efficacy in clinical trials. Full clinical translation to approved therapies will also be tracked and we will certainly be reporting on this metric as well. We have added/modified a sentence in the manuscript to further explain our intention: Therefore, the aim of this project is to use established knowledge synthesis methods to identify the prevalence of translation from preclinical research (bench) to the first clinical evaluations of efficacy (bedside), map contemporary trends in translation, and identify key modifiable factors associated with successful and unsuccessful bench-to-bedside translation. We added the following sentence in the analysis plan of Objective 1: Additionally, we will calculate the proportion of successful Phase II trials that received regulatory approval. We also added and modified the following in our limitations section: Lastly, it should be acknowledged that the translational spectrum involves many complex steps and various phases. Though ultimate clinical translation is the approval and adoption of a therapy, we will focus on a specific stage in the translational spectrum 4. We have chosen to measure the translation of the in vivo animal experiment stage within T0 research to the first evaluation of the therapy’s efficacy in clinical trials, within T2 research." } ] }, { "id": "69847", "date": "23 Sep 2020", "name": "Jonathan A Lal", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReference 4 is too old (from 2006) to claim that translation from preclinical to human trials is rare. This should be updated. Reference 6 is 5 years old, should work, but if more recent data is available, perhaps replace. Reference 7 is too old to be valid in today's economic development, please update.\n\nFor methods it says any changes will be included in the final report. It is to be noted that the peer review is limited to the manuscript itself.\n\nEligibility criteria limited to 3 prominent journals limits the scope as JAMA and other such high impact factor (preclinical/clincal) journals are left out. At least 5 journals could have been taken into consideration to justify the sample size.\n\nWhat would be interesting to see is the ratio between successful Phase II trails and those successful Phase II trails which actually reach regulatory approvals.\n\nFurthermore, regulatory approval may not still guarantee widespread adoption. The latter of which could be due to other factors which remain to be explored, including competition. A comment towards this end would be advised.\n\nIn limitations it mentions preclinical to Phase II, and eventually analysing the preclinical problems till Phase II. Phase III and/or Phase IV excluded, regulatory touched upon, it seems this should be made clear at the start of the manuscript, that the focus is till Phase II and not bedside directly.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "6018", "date": "16 Oct 2020", "name": "Manoj Lalu", "role": "Author Response", "response": "We thank you for your careful assessment of our protocol and their supportive comments. We have addressed your queries below and within the revised manuscript. Reference 4 is too old (from 2006) to claim that translation from preclinical to human trials is rare. This should be updated. Updated citation to: Seyhan, A.A. Lost in translation: the valley of death across preclinical and clinical divide – identification of problems and overcoming obstacles. transl med commun 4, 18 (2019). https://doi.org/10.1186/s41231-019-0050-7 Reference 6 is 5 years old, should work, but if more recent data is available, perhaps replace. Updated citation to: Hutchins BI, Davis MT, Meseroll RA, Santangelo GM (2019) Predicting translational progress in biomedical research. PLoS Biol 17(10): e3000416. https://doi.org/10.1371/journal.pbio.3000416 Reference 7 is too old to be valid in today's economic development, please update. We agree with the reviewer that this is an old reference. We believe this may not have been the strongest argument to present in the introduction, thus we have removed the sentences on return on investments. For methods it says any changes will be included in the final report. It is to be noted that the peer review is limited to the manuscript itself. Changed sentence to: Any deviation from this protocol will be documented in the manuscript that reports the results of this study, upon its completion. Eligibility criteria limited to 3 prominent journals limits the scope as JAMA and other such high impact factor (preclinical/clincal) journals are left out. At least 5 journals could have been taken into consideration to justify the sample size. Our group of investigators considered this very issue at length. Ultimately, there simply is no perfect sampling strategy to address the objectives of the study. We selected the three journals as they are highly influential and considered ‘best-in-class’ by many. The number of articles resulting from the search of the three included journals is 20,341. With our available resources, it has taken close to one year to screen all abstracts and full texts in duplicate. Adding more journals would not be feasible with our finite resources. We added the following to the limitation section of the discussion: Furthermore, our sample is limited to three journals, which may reduce the scope of our work. As automation of meta-research projects gains traction (and tools are validated) broader searches will certainly be possible in the future. However, given that this process currently relies on personnel we are limited to the current strategy. What would be interesting to see is the ratio between successful Phase II trails and those successful Phase II trails which actually reach regulatory approvals. We certainly agree with this suggestion. This is something we will report on in the manuscript that summarizes the results of this study. We added the following sentence in the analysis plan of Objective 1: Additionally, we will calculate the proportion of successful Phase II trials that received regulatory approval. Furthermore, regulatory approval may not still guarantee widespread adoption. The latter of which could be due to other factors which remain to be explored, including competition. A comment towards this end would be advised. We also agree with this comment and have addressed this limitation in our discussion: Furthermore, successful translation to a Phase II clinical trial does not imply that the therapy has been successfully translated to practice or regulatory approval; and regulatory approval may not guarantee widespread adoption. With this limitation in mind, we will focus on preclinical issues that may affect early translation, rather than potential issues with the clinical trials that subsequently evaluated the therapies in human subjects or barriers that affect both regulatory approval and adoption into practice. In limitations it mentions preclinical to Phase II, and eventually analysing the preclinical problems till Phase II. Phase III and/or Phase IV excluded, regulatory touched upon, it seems this should be made clear at the start of the manuscript, that the focus is till Phase II and not bedside directly. We would argue that Phase II is considered bedside as the evaluation is being completed on patients. However, we do agree that this is quite different from full clinical adoption and therefore should not be conflated with widespread approval. As such we have modified the following in our manuscript: Lastly, it should be acknowledged that the translational spectrum involves many complex steps and various phases. Though ultimate clinical translation is the approval and adoption of a therapy, we will focus on a specific stage in the translational spectrum 4. We have chosen to measure the translation of the in vivo animal experiment stage within T0 research; to the evaluation of the therapies’ efficacy in clinical trials, within T2 research." } ] } ]	1	https://f1000research.com/articles/9-485
https://f1000research.com/articles/9-211/v1	26 Mar 20	{ "type": "Research Article", "title": "Risk factors for composite adverse outcomes of postpartum haemorrhage in a low-resource setting: a single-centre cross-sectional study in Mpilo Central Hospital, Bulawayo, Zimbabwe", "authors": [ "Solwayo Ngwenya" ], "abstract": "Background: Primary postpartum haemorrhage continues to cause considerable global maternal morbidity and mortality. The aim of this study was to determine the risk factors for composite adverse outcomes in postpartum haemorrhage using multivariable logistic regression. The findings could potentially be used to anticipate and prevent composite adverse outcomes in postpartum haemorrhage. Methods: This was a retrospective cross-sectional study carried out at Mpilo Central Hospital, a government tertiary referral centre, covering the period 1 July 2016 to 30 November 2019. Participants were included in the study if they had a diagnosis of postpartum haemorrhage. Those variables that had a p<0.2 from the univariate logistic regression analyses were considered for multivariable logistic regression. The association between independent variables and the dependent variable was assessed using odds ratio with 95% confidence intervals, to identify independent risk factors for composite adverse outcomes in PPH. Results: The independent risk factors for composite adverse outcomes in postpartum haemorrhage were place of dwelling (AOR 4.57, 95% CI 1.87-11.12, p=0.01), prior history of a Caesarean section (AOR 2.57, 95% CI 1.10-6.00, p=0.03), APH (AOR 5.45, 95% CI 2.23-13.27, p<0.0001), antenatal haemoglobin level (AOR 19.64, 95% CI 1.44-268.50, p=0.03), and delivery by Caesarean section (AOR 10.21, 95% CI 4.39-23.74, p<0.0001). Blood loss was also an independent risk factor for composite adverse outcomes in postpartum haemorrhage with the following blood loss; 1001-1500 ml (AOR 9.94, 95% CI 3.68-26.88, p<0.0001), 500-1000 ml (AOR 41.27, 95% CI 11.32-150.54, p<0.0001), and 2001 ml (AOR 164.77, 95% CI 31.06-874.25, p<0.0001). Conclusions: This study found that the independent predictors for composite adverse outcomes in PPH were rural dwelling, prior history of a Caesarean section, antenatal haemoglobin level, delivery by Caesarean section, and blood. In low- and middle-income countries, such information should help in increasing clinical vigilance and preventing maternal deaths.", "keywords": [ "Postpartum haemorrhage", "risk factors", "composite adverse outcomes", "low-resource settings" ], "content": "Introduction\n\nPrimary postpartum haemorrhage (PPH) is defined as a cumulative blood loss from the genital tract of ≥500 mL or more following a normal vaginal delivery or ≥1,000 mL or more following a cesarean section within 24 hours of delivery evidenced by a rise in the pulse rate, and falling blood pressure1–3.\n\nIn 2017, approximately 810 women died from causes related to pregnancy and childbirth, and 94% of all maternal deaths occurred in low and lower middle-income countries4. In a systematic analysis, Say et al. found that low- and middle income countries accounted for 480,000 maternal deaths (32%) compared with 1200 (8%) in the developed regions5. PPH is the leading cause of maternal deaths in SSA6.\n\nThe multi-country Survey on Maternal and Newborn Health reported the prevalence of PPH as 1.2%, with higher rates in developing countries than developed ones7. Other studies in Sub-Saharan Africa (SSA) reported rates of 1.6% in Zimbabwe, 16.6% in Southern Ethiopia, 9% in Uganda and 23.6% in Cameroon. 1,3,8,9, respectively. Ford et al. reported increasing PPH rates from 6.1% in 2003 to 8.3% in 2011 (p<0.0001) in Australia10.\n\nTwo-thirds of women with PPH having no identifiable risk factors11. Recognized risk factors for PPH include previous PPH, twin gestation, large baby, induction of labour, prolonged labour, operative delivery, preeclampsia, caesarean delivery, grand multiparity, maternal age 35 or above, and postdates1,8,12–15.\n\nTort et al. used a multivariable logistic mixed model to identify factors that were significantly associated with PPH maternal death13. However, in this study PPH maternal death or serious morbidity were used as composite adverse outcomes.\n\nThe aim of this research was to documents risk factors for poor composite adverse outcome in PPH. This could help clinicians identify which women with PPH are at risk of composite adverse outcomes and increase further the clinical vigilance associated with the management of PPH thereby preventing deaths.\n\n\nMethods\n\nThis was a retrospective cross-sectional study carried out at Mpilo Central Hospital, a government tertiary referral centre, covering the period 1 July 2016 to 30 November 2019. Mpilo Central Hospital is situated in the township of Mzilikazi in Bulawayo. Bulawayo is the second largest city in Zimbabwe after the capital city Harare, with a population of 653,337 as of the 2012 census16. Participants were included in the study if they had a diagnosis of postpartum haemorrhage within 24 hours of delivery at Mpilo Central Hospital. Women that delivered outside the hospital were excluded from the study.\n\nThe independent variables included socio-demographic factors, mode of delivery, fetal characteristics, blood loss, laboratory tests, causes of PPH and the management of PPH.\n\nThe main outcome of interest for the study was the composite adverse outcome which included maternal death or serious morbidity (either of hypovolaemic shock or haemoglobin <4 g/dL or massive blood transfusion >4 units or hysterectomy or admission to ICU or coagulopathy or major organ dysfunction), similar to the Delphi consensus study on PPH17.\n\nThe Cochran sample size formula was used to calculate the sample size as follows; n0 =z2pq/e2\n\nwhere n0 =sample size\n\nz = is the selected critical value of desired confidence level\n\np = is the estimated proportion of an attribute that is present in the population\n\nq = is 1-p and e is the desired level of precision\n\nAssuming the maximum variability, which is equal to 50% (p = 0.5) and taking 95% confidence level with ±5% precision, the calculation for the required sample size was as follows;\n\np = 0.5 and hence q = 1-0.5 = 0.5, e = 0.05; z = 1.96\n\nSo, n0 = (1.96)2(0.5) (0.5)/(0.05)2\n\n= 384.16\n\n=385\n\nData collection was done using a paper data collection tool (see Extended data)18 that was used to collect secondary data from the labour ward delivery registers, and mortality registers. Hospital case notes were retrieved the clinical data were extracted.\n\nData were cleaned, coded and entered into a Microsoft Excel spreadsheet, then exported to SPSS Version 20 (IBM, Armonk, NY, USA) for analysis. Descriptive statistical analyses were performed and presented as frequencies and percentages for categorical variables. Bivariate correlations of association between main independent variables and the outcome measures were performed using Pearson 2-tailed chi-square test. A p value of <0.05 was considered to be statistically significant, and these were considered for the univariate logistic regression. Those variables that had a p<0.2 from the univariate logistic regression analyses were considered for multivariable logistic regression. The association between independent variables and the dependent variable was assessed using odds ratio with 95% confidence intervals, to identify independent risk factors for composite adverse outcomes in PPH, holding other variables constant and adjusting for co-variates. The Hosmer-Lemeshow goodness-of-fit was used to check if the model fitted well. A p< 0.05 was taken as statistically significant.\n\nThe Ethics Committee at Mpilo Central Hospital made a ruling for all retrospective studies to go ahead in the institution from 2016 onwards as long as the data remained anonymous; the committee waived the requirement for patient consent. No ethical issues will arise during the study as all the data will remain anonymous with no identifying personal data. Minutes of the Committee’s inaugural meeting held on the 13th October 2016 set out the requirements of all the studies at the institution.\n\n\nResults\n\nA total of 386 cases of PPH were recorded during the period 1 July 2016 to 30 October 2019. The summary of maternal and fetal characteristics are shown in the Supplementary Tables in the Extended data18. Deidentified results are available for each patient as Underlying data18.\n\nTable 1 and Table 2 show the results of the multivariable logistic regression. Rural women were 4.6 times more likely to be statistically significantly associated with composite adverse outcomes compared to women from urban areas (AOR 4.57, 95% CI 1.87-11.12, p=0.01).\n\nNot enough data for regression analysis\n\nVacuum, forceps not enough data for regression analysis\n\nWomen with a prior history of a Caesarean section were statistically significantly associated with composite adverse outcomes in PPH. Such women were 2.6 times more likely to be statistically significantly associated with composite adverse outcomes in PPH, compared to women without such history (AOR 2.57, 95% CI 1.10-6.00, p=0.03).\n\nAPH was statistically significantly associated with composite adverse outcomes in PPH. Women who presented with APH were 5.5 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared to women who had no APH (AOR 5.45, 95% CI 2.23-13.27, p<0.0001).\n\nAntenatal haemoglobin count was also statistically significantly associated with composite adverse outcomes in PPH. Women with haemoglobin counts of 0–5.99 g/dL were 19.6 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared with women with haemoglobin counts of 11 g/dL and above (AOR 19.64, 95% CI 1.44-268.50, p=0.03).\n\nDelivery by Caesarean section was statistically significantly associated with composite adverse outcomes in PPH. Women who had a Caesarean section were 10.2 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared to women who delivered vaginally (AOR 10.21, 95% CI 4.39-23.74, p<0.0001).\n\nBlood loss was statistically significantly associated with composite adverse outcomes in PPH. The odds rose significantly higher as the amount of blood loss increased. Women who lost 1001–1500 ml of blood were 9.9 times more likely to be statistically significantly associated with composite adverse outcomes, compared to women that lost 500–1000 ml (AOR 9.94, 95% CI 3.68-26.88, p<0.0001). The odds rose to 41.3 times more like to be associated with composite adverse outcomes in those women who lost 1501–2000ml compared to those women who lost 500–1000 ml (AOR 41.27, 95% CI 11.32-150.54, p<0.0001). Whereas women who lost 2001 ml and above were 164.8 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared to women who lost 500–1000 ml (AOR 164.77, 95% CI 31.06-874.25, p<0.0001).\n\n\nDiscussion\n\nPPH rates have been reported to be rising in both low-income and high-income countries11,19. This means that PPH will remain an important global subject. The strength of this research is that it involves a large homogenous group of patients with PPH, in SSA where PPH continues to contribute significantly to global mortality and morbidity.\n\nRural women were 4.6 times most likely to be statistically significantly associated with composite adverse outcomes compared to women from urban areas (AOR 4.57, 95% CI 1.87-11.12, p=0.01). National governments need to made healthcare accessible to rural women so that the Sustainable Development Goals on Maternal Mortality to reduce global maternal mortality ratio to less than 70 per 100,000 live births by 20304 could be achievable.\n\nWomen with a prior history of a Caesarean section were statistically significantly associated with composite adverse outcomes in PPH. These women are not only at risk of developing a PPH (OR 3.15, 95% CI 1.02-10.3)20, but the women were 2.6 times most likely to be statistically significantly associated with composite adverse outcomes in PPH, compared to women without such history (AOR 2.57, 95% CI 1.10-6.00, p=0.03). The means that women with a prior history of a Caesarean section should receive extra clinical vigilance.\n\nAPH was statistically significantly associated with composite adverse outcomes in PPH. Women who presented with APH were 5.5 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared to women who had no APH (AOR 5.45, 95% CI 2.23-13.27, p<0.0001).\n\nWomen with haemoglobin levels of 0–5.99 g/dL were 19.6 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared with women with haemoglobin levels of 11 g/dL and above (AOR 19.64, 95% CI 1.44-268.50, p=0.03). Anaemia should be screened for antenatally and women should receive treatment so that they enter labour with normal haemoglobin counts.\n\nWomen who had a Caesarean section were 10.2 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared to women who delivered vaginally (AOR 10.21, 95% CI 4.39-23.74, p<0.0001). Women would have had Caesarean sections should be closely monitored post-operatively.\n\nWomen who lost 2001 ml of blood and above were 164.8 times more likely to be statistically significantly associated with composite adverse outcomes in PPH compared to women who lost 500–1000ml (AOR 164.77, 95% CI 31.06-874.25, p<0.0001). The amount of blood loss was found to be related to adverse maternal outcomes19. Prompt, effective management of PPH.19, should be the aim to reduce the amount of blood loss and prevent the development of composite adverse outcomes.\n\n\nLimitations\n\nThe major limitation of this study is that it was a retrospective, single-centre study that used secondary data. This could limit the generalizability of its findings to other centres of low-resourced settings.\n\n\nConclusions\n\nThe independent predictors for composite adverse outcomes in PPH were rural dwelling, prior history of a Caesarean section, antenatal haemoglobin level, and delivery by Caesarean section. Blood loss was also an independent predictor for composite adverse outcomes in PPH. Crucially, this new information should help in increasing clinical vigilance and preventing maternal deaths especially in low- and middle-income countries were PPH mortality is of high prevalence. Regular on-site training of staff can focus on drilling on these important issues and can improve outcomes21.\n\n\nData availability\n\nMendeley Data: Composite adverse outcomes in primary PPH. https://doi.org/10.17632/wjtn8rmgcc.318.\n\nThis project contains the following underlying data:\n\nPPH-Data-Share (XLSX). The raw de-identified data gathered from each patient examined in this study.\n\nde-identified individual-level data for all patients.\n\nMendeley Data: Composite adverse outcomes in primary PPH. https://doi.org/10.17632/wjtn8rmgcc.318.\n\nThis project contains the following extended data:\n\nData Collection Sheet-PPH (DOCX).\n\nSupplementary tables - PPH mortality (DOCX).\n\n∘ Table 1: Maternal and fetal characteristics.\n\n∘ Table B: Socio-demographic characteristics of study patients.\n\n∘ Table C: Present risk factors for PPH\n\n∘ Table D: Fetal birth weight, blood loss and causes of PPH\n\n∘ Table E: Management and outcomes in PPH\n\n∘ Table F: Bivariate correlations between independent variables and composite adverse outcome.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nNgwenya S: Postpartum hemorrhage: incidence, risk factors, and outcomes in a low-resource setting. Int J Womens Health. 2016; 8: 647–650. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDutta DC: Textbook of Obstetrics. Including Perinatology and Contraception. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd. 7ed. 2013.\n\nKebede BA, Abdo RA, Anshebo AA, et al.: Prevalence and predictors of primary postpartum hemorrhage: An implication for designing effective intervention at selected hospitals, Southern Ethiopia. PLoS One. 2019; 14(10): e0224579. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaternal mortality. World Health Organisation. Accessed 30 November 2019. 2019. Reference Source\n\nSay L, Chou D, Gemmill A, et al.: Global causes of maternal death: a WHO systematic analysis. Lancet Glob Health. 2014; 2(6): e323–e333. PubMed Abstract \| Publisher Full Text\n\nLazarus JV, Lalonde A: Reducing postpartum hemorrhage in Africa. Int J Gynaecol Obstet. 2005; 88(1): 89–90. PubMed Abstract \| Publisher Full Text\n\nSheldon WR, Blum J, Vogel JP, et al.: Postpartum haemorrhage management, risks, and maternal outcomes: findings from the World Health Organization Multicountry Survey on Maternal and Newborn Health. BJOG. 2014; 121 Suppl 1: 5–13. PubMed Abstract \| Publisher Full Text\n\nOnonge S, Mirembe F, Wandabwa J, et al.: Incidence and risk factors for postpartum hemorrhage in Uganda. Reprod Health. 2016; 13: 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalle-Ekane GE, Emade FK, Bechem NN, et al.: Incidence and Risk Factors of Primary Postpartum Haemorrhage after Vaginal Deliveries in the Bonassama District Hospital, Cameroon. Int J Trop Dis Health. 2016; 13(2): 1–12. Publisher Full Text\n\nFord JB, Patterson JA, Seeho SK, et al.: Trends and outcomes of postpartum haemorrhage, 2003-2011. BMC Pregnancy Childbirth. 2015; 15: 334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIfeadike CO, Eleje GU, Umeh US, et al.: Emerging trend in the etiology of postpartum hemorrhage in a low resource setting. J Preg Neonatal Med. 2018; 2(2): 34–40. Publisher Full Text\n\nNyfløt LT, Sandven I, Stray-Pedersen B, et al.: Risk factors for severe postpartum hemorrhage: a case-control study. BMC Pregnancy Childbirth. 2017; 17(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTort J, Rozenberg P, Traoré M, et al.: Factors associated with postpartum hemorrhage maternal death in referral hospitals in Senegal and Mali: a cross-sectional epidemiological survey. BMC Pregnancy Childbirth. 2015; 15: 235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTraore Y, Teguete I, Bocoum A, et al.: Management and Prognosis of Early Postpartum Hemorrhage in African Low Setting Health. Open J Obstet Gynecol. 2018; 8: 1–9. Publisher Full Text\n\nTemesgen MA: Magnitude of Postpartum Hemorrhage among Women Delivered at Dessie Referral Hospital, South Woll, Amhara Region, Ethiopia J Women’s Health Care. 2017; 6: 391. Publisher Full Text\n\nZIMDAT: Census Report. 2012. Reference Source\n\nMeher S, Cuthbert A, Kirkham JJ, et al.: Core outcome sets for prevention and treatment of postpartum haemorrhage: an international Delphi consensus study. BJOG. 2019; 126(1): 83–93. PubMed Abstract \| Publisher Full Text\n\nNgwenya S: “Composite adverse outcomes in primary PPH”, Mendeley Data, V3. 2020. http://www.doi.org/10.17632/wjtn8rmgcc.3\n\nFadel MG, Das S, Nesbitt A, et al.: Maternal outcomes following massive obstetric haemorrhage in an inner-city maternity unit. J Obstet Gynaecol. 2019; 39(5): 601–605. PubMed Abstract \| Publisher Full Text\n\nEkin A, Gezer C, Solmaz U, et al.: Predictors of severity in primary postpartum hemorrhage. Arch Gynecol Obstet. 2015; 292(6): 1247–54. PubMed Abstract \| Publisher Full Text\n\nCrofts JF, Mukuli T, Murove B, et al.: Onsite training of doctors, midwives and nurses in obstetric emergencies, Zimbabwe. Bull World Health Organ. 2015; 93(5): 347–51. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "66260", "date": "13 Aug 2020", "name": "Jaffu O. Chilongola", "expertise": [ "Reviewer Expertise Infectious diseases", "Immunology", "Molecular Biology" ], "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 surprising that this manuscript has only one author. This means the lone author did everything from conceiving the ideas, data collection, analysis, writing the manuscript. It has to be confirmed that NO one else has participated in this work.\n\nAge ranges in table 1 variables are too narrow to provide useful information on the outcome variable.\n\nThe analyses are weak perhaps because the design was also weak. When A low post delivery Hb level is considered as a risk for PPH it brings the confusion what would otherwise be expected. Similary, when Ruptured uterus is tested for its association with PPH. Many of the predictors are obviously inherently related to the outcome variable. It is unclear what information this manuscript attempting to bring forth.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\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? No", "responses": [ { "c_id": "6027", "date": "15 Oct 2020", "name": "Solwayo Ngwenya", "role": "Author Response", "response": "Reviewer 1 It is surprising that this manuscript has only one author. This means the lone author did everything from conceiving the ideas, data collection, analysis, writing the manuscript. It has to be confirmed that NO one else has participated in this work. I can confirm that I am the sole author. Response: I have done many studies of this magnitude before as a sole author. Age ranges in table 1 variables are too narrow to provide useful information on the outcome variable. Response: I have decided to leave them as they are as this didn’t not affect the logistic regression. The analyses are weak perhaps because the design was also weak. When A low post delivery Hb level is considered as a risk for PPH it brings the confusion what would otherwise be expected. Similary, when Ruptured uterus is tested for its association with PPH. Many of the predictors are obviously inherently related to the outcome variable. It is unclear what information this manuscript attempting to bring forth. Response: Comments noted." } ] }, { "id": "64325", "date": "26 Aug 2020", "name": "Michael Johnson Mahande", "expertise": [ "Reviewer Expertise Reproductive Health", "Maternal Newborn & Sexual Adolescent Health" ], "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\nRisk factors for composite adverse postpartum haemorrhage in a low rsources setting: a single-centre cross sectional study in Mpilo Central Hospital Bulawayo, Zimbabwe.\nSection Comment, question, suggestion.\nAbstract\nThe abstract background should consist a few statements that explain the meaning and aim of the study not the model used. The model used (multivariable logistic regression) can be explained in the methods section.\nIntroduction Background\nNo comment.\nMethodology\nWhy did authors calculate sample size while the study utilized data that were retrospective collected? I expected the study could use all available data and power could be calculated instead of the sample size.\nResults\n\nIt could be more important if the tables for social demographic characteristics and clinical characteristics were included in the manuscript.\n\nIn presenting numbers in the table is better to have the standard decimal points to be presented especially when presenting p-values.\n\nConsider combining single and divorced in marital status as divorced have few participants for regression analysis.\nDiscussion\nNONE.\n\nStrengths and limitations\nDespite being a study involving a single center, what is the strength of this study?\nConclusion\nNo comment.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "69904", "date": "18 Sep 2020", "name": "Eba Abdisa", "expertise": [ "Reviewer Expertise women health", "mental health", "clinical condition", "child health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle: Risk factors for composite adverse outcomes of postpartum hemorrhage in a low-resource setting: a single-center cross-sectional study in Mpilo Central Hospital, Bulawayo, Zimbabwe\nThe title seems interesting, particularly it tried to address the problem of the low-income countries, but it should be arranged as \"Risk factors for Composite adverse outcome of postpartum hemorrhage in Mpilo central hospital, Bulawayo, Zimbabwe: Cross sectional study\" - this is because this country is known to be in low resource by default; no need to say \"in a low resource setting\".\n\nAbstract:\nBackground: \"Primary postpartum hemorrhage continues to cause considerable global maternal morbidity and mortality.\"\nRewrite the above statement as \"Globally, primary postpartum hemorrhage continues to cause considerable maternal morbidity and mortality\".\nConclusion: how is such information useful for preventing death? What are possible methods? Who will use this information? You must state some of them in the background section. From all independent factors, which one is difficult to prevent? Be specific when you conclude your findings.\n\nIntroduction:\n\nYour citation in paragraph one: you didn’t take the information from the primary results, rather you cited the secondary result(paper). Please try to address the first research finding and cite it correctly\n\nInstead of ‘say et al, ford et al, Tort et al’, use ‘researcher(s), report(s), finding(s)…\n\nAre the 810 women's death related to PPH? You must be specific.\n\nWhat makes the increment of PPH from 6.1%-8.3% in Australia? Please present some evidence findings for the reason that the researchers stated.\n\nIn paragraph 5, you said, ‘Tort et al used multivariable mixed mode’, please state only what the study assessed and what it got, and avoid the methodology the researchers used.\n\nPoor composite adverse outcome (para. 5): you must clearly define it.\n\nSignificance of the study: the last paragraph stated the importance of this study. But do you think that your findings might help only clinicians? What about your study population? What about the policy makers?\n\nYour introduction section seems shallow.\n\nAPH: write the full version.\n\nMethods:\nWhy did you only use a single study setting? Please justify.\n\nWhat if the women delivered outside by developed PPH? Why did you exclude such a risk group? Giving birth out of healthcare setting will even be expected to the complication. Please say something.\n\nDetailed description is expected regarding your study area: number of deliveries per year/month, number of skilled birth attendants (midwifery, physicians & others).\n\nData collection: this seems shallow. It was not clear how you selected your study group (sample size). How did you collect data of 385? Did you start from a specific year? Example random vs nonrandom?\n\nData analysis: software package that you used to enter data(excel). In this case, it seems difficult to manage missed data and mistakes. Why don’t you use Epidata, epi-info…?\n\nWhy did the committee waive the requirement for patient consent?\n\nDiscussion: It seems well organized.\nBut consider to compare your findings with others' past international/national findings and put your opinion when you get the difference.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-211
https://f1000research.com/articles/9-1247/v1	15 Oct 20	{ "type": "Research Article", "title": "The discounted value of human lives lost due to COVID-19 in France", "authors": [ "Joses Muthuri Kirigia", "Rose Nabi Deborah Karimi Muthuri", "Lenity Honesty Kainyu Nkanata", "Newton Gitonga Muthuri", "Rose Nabi Deborah Karimi Muthuri", "Lenity Honesty Kainyu Nkanata", "Newton Gitonga Muthuri" ], "abstract": "Background: This study estimates the total discounted value of human lives lost (TDVHL) due to COVID-19 in France as of 14 September 2020. Methods: The human capital approach (HCA) model was used to estimate the TDVHL of the 30,916 human lives lost due to COVID-19 in France; i.e., assuming a discount rate of 3% and the national average life expectancy at birth of 83.13 years. To test the robustness of the estimated TDVHL, the model was rerun (a) using 5% and 10% discount rates, while holding the French average life expectancy constant; and (b) consecutively substituting national life expectancy with the world average life expectancy of 73.2 years and the world highest life expectancy of 88.17 years. Results: The human lives lost had a TDVHL of Int$10,492,290,194, and an average value of Int$339,381 per human life lost. Rerun of the HCA model with 5% and 10% discount rates decreased TDVHL by Int$1,304,764,602 (12.4%) and Int$3,506,938,312 (33%), respectively. Re-calculation of the model with the world average life expectancy decreased the TDVHL by Int$7,750,187,267 (73.87%). Contrastingly, re-estimation of the model with the world’s highest life expectancy augmented TDVHL by Int$3,744,263,463 (35.7%). Conclusions: The average discounted economic value per human life lost due to COVID-19 of Int$339,381 is 8-fold the France gross domestic product per person. Such evidence constitutes an additional argument for health policy makers when making a case for increased investment to optimise France’s International Health Regulation capacities and coverage of essential health services, and safely managed water and sanitation services.", "keywords": [ "Coronavirus disease", "COVID-19", "France", "Gross Domestic Product", "Value of human life" ], "content": "Introduction\n\nFrance is one of the seven major advanced economies (G7 countries). The country has an estimated population of 64.994 million; a total gross domestic product (GDP) of Int$3,161.335 billion; and GDP per capita of Int$41,637.729 in 20201. In 2018, approximately 10,918,992 (16.8%) of the population lived below France’s poverty threshold of €1,008 per month of disposable income2. France has an inequality-adjusted human development index of 0.808 and a Gini coefficient of 32.73.\n\nBy 14 September 2020, there were 29,182,605 coronavirus disease-19 (COVID-19) cases in the world, including 928,281 deaths, 21,027,161 recovered cases, and 7,227,163 active cases4. Europe had a total of 4,080,753 COVID-19 cases, including 212,545 deaths, 2,245,583 recovered cases, and 1,622,625 active cases. On the same date, France had conducted a total of 10 million COVID-19 tests that revealed a total of 381,094 COVID-19 cases, which included 30,916 deaths, 89,059 recovered cases, and 261,119 active cases4. France bore 9.3% of total cases and 14.55% of total COVID-19 deaths in Europe. France’s densities of 5,836 COVID-19 cases and 473 deaths per million population were higher than Germany’s densities of 3,117 cases and 112 deaths per million population.\n\nFour factors might explain the relatively large number of COVID-19 deaths sustained by France. First, there was more than two months’ delay in country-wide implementation of public health interventions that could have prevented (or slowed) transmission and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There is evidence that COVID-19 was already spreading in France by late December 20195. However, the government only banned in mid-March 2020 gatherings of more than 100 people; the opening of non-essential public establishments; anchoring in inland and territorial waters of ships carrying more than 100 passengers; opening public establishments; opening schools and institutes of higher education; all religious gatherings; and embalming of dead bodies6.\n\nSecond, the average of 13 International Health Regulations (IHR) core capacity score for France was 82 (on a scale of 0 to the target of 100) in 20197,8, denoting an overall IHR capacity gap of 18. As shown in Table 1, the country had IHR capacity gaps of 33 in legislation and financing, 20 in zoonotic events and the human-animal interface, 20 in food safety, 27 in laboratory, 20 in human resources, 27 in national health emergency framework, 7 in health service provision, 20 in risk communication, and 60 in points of entry9. The latter gap denotes suboptimal capacity at ports/airports/ground crossings for coordination and communication of pandemic surveillance; and appropriate medical diagnosis, isolation and care of ill travellers. The French points of entry capacity score of 40 were lower than the average score for the World Health Organization (WHO) European Region (EUR) of 61 by 52.2%.\n\nSource: World Health Organization9.\n\nSecond, as shown in Table 2, generally the health system indicators for France are better than the EUR averages.\n\nSource: WHO1,10–12. Note: N/A - means not available.\n\nThe density of 32.67 medical doctors per 10,000 population was lower than the average of 34.1 for EUR by 4.38%. Nursing and midwifery personnel, dentists, and pharmacist densities in France were 29.12%, 14.54%, and 36.09%, higher than EUR averages. The French density of radiotherapy units per million population of 7.51 was 48.07% higher than the EUR average. The current health expenditure (CHE) per capita of Int$5,011.2 in France was 41.67% higher than the EUR average of Int$2,923. The out-of-Pocket expenditure as a percentage of CHE of 9.38% in France was 224%% higher than the EUR average of 30.4%. The universal health coverage (UHC) service coverage index (UHC SCI) for France was 78%, signifying a gap in coverage of essential health services of 22%12. The UHC SCI component of reproductive, maternal, new-born and child health; infectious diseases; non-communicable diseases; and service capacity and access had gaps of 4, 29, 44 and 4, respectively.\n\nAbout 14,298,680 (0.22%) of the population’s health spending was higher than 25% of total household income, which reflects a very high risk of catastrophic and impoverishing health care expenditures. About 97.85% and 88.37% of the French population, respectively, use safely-managed drinking-water and sanitation services11; signifying that 1,397,371 (2.15%) and 7,558,802 (11.63%) people do not have access.\n\nThe type of economic evidence reported in this paper could be an essential input when health policy-makers make a case for increased investment in optimizing the IHR capacities, coverage of essential health services, and coverage of safely managed water and sanitation services to more effectively prevent and manage the current COVID-19 pandemic and future public health emergencies13–21.\n\nA few macroeconomic studies have estimated the impact of the COVID-19 pandemic on business conditions in France22. However, there is a shortage of information on the value of human lives lost due to the pandemic. This study estimates the total discounted value of human lives lost (TDVHL) due to COVID-19 in France as of 14 September 2020.\n\n\nMethods\n\nThe study relied totally upon the analysis of secondary data contained in the International Monetary Fund (IMF), WHO, Worldometer, and Santé Publique France databases that are freely available to the public. Therefore, ethical clearance was not required.\n\nThis investigation of the value of human life was conducted on the cumulative number of persons who died of COVID-19 by 14 September 2020 in France. The study was a cross-sectional study. All the 30,916 COVID-19 people reported to have died from COVID-19 as of 14 September 2020 in France were included in the study.\n\nThis study applied the human capital approach (HCA), initially suggested by Adam Smith in 177623, to estimate the monetary value of human life. The Organisation for Economic Co-operation and Development24 defines human capital as “The knowledge, skills, competencies and attributes (including stock of health) embodied in individuals that facilitate [the] creation of personal, social and economic wellbeing” (p.18).\n\nDeath from COVID-19 (or any other disease or injury) extinguishes the potential of a person to tap into one’s stock of human capital either for personal development and enjoyment of leisure or to enhance societal cultural and socioeconomic wellbeing. A person’s capacity for personal development, enjoyment of life (or flourishing)25, loving, religious practice, and performing expected societal roles ends upon death. It is also true that death halts individuals’ spending on the consumption of goods and services, investments, government services (including payment of fees and taxes), and imports permanently. In other words, death terminates an individual’s potential contribution to the creation of national output or GDP. Following the death of a human being at any stage of life, society losses not only the statistical person’s contribution to GDP but also other intangible contributions, e.g. child’s joy to parents, love to family and friends, companionship, fellowship, comradeship, sharing of knowledge (written or tacit) and social values.\n\nWeisbrod26 suggested measuring lost human capital as a result of premature death from any cause in terms of the deceased person’s discounted future earnings net of their consumption26. In line with our past research13–21, the current study uses net GDP per capita (i.e. GDP per capita of France minus current health expenditure per person) to value human lives lost due to COVID-19 in France.\n\nThe TDVHL in France (TDVHLFRANCE) due to COVID-19 is the sum of DVHL among persons aged 0–14 years, 15–44 years, 45–64 years, 65–74 years, and 75 years and over13–21. Formally13–21:\n\n\n\nWhere: DVHLi is the discounted value of human lives lost due to COVID-19 in ith age group; i=1 is age 0–14 years, i=2 is age 15–44 years, i=3 is age 45–64 years, i=4 is age 65–74 years, and i=5 is age 75 years and over; ∑i=1i=5 is the sum of the discounted values of human lives lost in age groups denoted by number 1 to 5.\n\nThe DVHLi in each of the five age groups was calculated using the following formula13–21:\n\n\n\nWhere: ∑t=nt=1 is the sum from the first year of life lost (t=1) to the last years of life lost (t=n); Y1 is the GDP per capita of France; Y2 is the current health expenditure per person in France; Y3 is the average life expectancy at birth in France; Y4 is the average life expectancy at the onset of death in the ith age group; Y5 is the total number of COVID-19 deaths in France as of 14 September 2020; Y6 is the proportion of total COVID-19 deaths borne by those in the ith age group. The baseline for the analysis is 2020.\n\nThe data analysed in this paper and the sources are contained in Table 3.\n\nThe human capital model was analysed using Excel 2016 software (Microsoft, New York). The study reported in this paper replicates steps that were developed and applied in our recent valuation of human life studies related to COVID-1913–15,17–21.\n\nStep 1: Estimation of net GDP per capita (NGDPC) as the difference between per capita GDP (PCGDP) and current health expenditure per capita (CHEPC) for France. Thus, NGDPC = PCGDP – CHEPC = (Int$41637.729 – Int$5011.20068359) = Int$36,626.53.\n\nStep 2: Estimation of the undiscounted years of life lost (UYLL) from COVID-19 in France between December 2019 and 14 September 2020.\n\n(a) Calculation of average ages of onset of death (AAOD) from COVID-19 for each of the five age groups. This entailed taking simple averages for each age group, e.g. for AAOD for 0–14 age group = (0+14)/2 = 7 years.\n\n(b) Calculation of YLL by one person who died of COVID-19 in the age group as the difference between national average life expectancy for France and the AAOD for the specific age group. For example, YLL by a person dead in the age group 0–14 years = national average life expectancy for France (83.13 years) minus AAOD for the group (7 years) = 76.13 years. Thus, the YLL for one person who died in each of the five age groups was obtained similarly (see Table 4).\n\n(c) Total UYLL in each age group = UYLL per deceased person in age group multiplied by the number of persons who died in an age group. For example, total UYLL in 0-14 age group = 76 years’ x 4.5974025974026 persons dead = 349.40 undiscounted YLL.\n\nStep 3: Discounting of the years of life lost (DYLL).\n\n(a) A discount rate of 3% was chosen because it has been used in our previous COVID-19 related economic studies13–21, the economic evaluation of public health problems in Africa30, the World Health Report 200031, the burden of disease32, and the World Bank Disease Control Priorities study33.\n\n(b) Calculation of the discount factors applying the discount factor formula: (1(1+r)t). The discount factor for first YLL = (1(1+0.03)1) = 0.970873786407767; discount factor for second YLL = (1(1+0.03)2) = 0.942595909133754; discount factor for third YLL = (1(1+0.03)3) = 0.91514165935316; …, discount factor for the final YLL (which is 76.13 years for 0–14 years) = (1(1+0.03)76.13) = 0.105772050189903.\n\n(c) Calculation of DYLL per deceased person in age group. DYLL in year 1 = discount factor in year one x UYLL in year one = 0.970873786407767 × 1 = 0.970873786407767. DYLL in year 2 = discount factor in year two × UYLL per person in year two = 0.942595909133754 × 1 = 0.942595909133754. DYLL in year 3 = discount factor in year three × UYLL per person in year three = 0.91514165935316 × 1 = 0.91514165935316. DYLL in the last YLL (e.g. 76th year for 0–14 years) = discount factor in 76th year × UYLL per person in 76th year = 0.105772050189903 × 1 = 0.105772050189903.\n\n(d) Estimation of total DYLL per deceased person in age group is equivalent to the sum of discount factors from year one to the last year of life.\n\n(e) Total DYLL in each age group = DYLL per deceased person in age group multiplied by number of persons who died in age group. Therefore: UYLL in 0–14 age group = 29.80759833 × 4.597402597 = 137.04 discounted YLL (see Table 5).\n\nStep 4: Estimation of the total number of COVID-19 deaths in age group (COVID-19Dj) equals the total number of COVID-19 deaths in France (TCOVID-19D) multiplied by the proportion (PROP) for that age group. For example, number of COVID-19 deaths in age group 0–14 years = TCOVID-19D × PROP = 30,916 × 0.000148706 = 4.597402597. The number of COVID-19 for each age group are in Table 6.\n\nStep 5: Estimation of the discounted economic value of human lives lost due to COVID-19 in each age jth group = NGDPC × DYLLj × COVID-19Dj. For instance, DVHL for age group 0-14 = Int$36,626.53 × 29.80759833 × 4.5974025974026 =Int$5,019,209.\n\nStep 6: Calculation of the share of TDVHL accruing to the 13 regions and five territories of France29 through multiplication of the TDVHL by proportion of COVID-19 deaths sustained by specific region and territory.\n\nStep 7: A one-way sensitivity analysis was performed to evaluate the effect of changes in discount rate and the average life expectancy on the estimated TDVHL. This entailed recalculating the HCA model with (a) 5% and 10% discount rates13–21 and (b) the world average life expectancy of 73.2 years and the world highest average life expectancy of 88.17 years, i.e. the average life expectancy of Hong Kong women4. The model was reanalysed while holding all other parameters constant.\n\n\nResults\n\nThe cumulative 30,916 human lives lost from COVID-19 by 14 September 2020 in France resulted in a total of 363,781.74 undiscounted years of life lost; which was equivalent to a total of 286,466.96 discounted years of life lost.\n\nTable 7 depicts the distribution by age group of the TDVHL of the 30,916 human lives lost due to COVID-19 in France by 14 September 2020.\n\nThe human lives lost to COVID-19 had a TDVHL of Int$10,492,290,194, and an average value of Int$339,381 per human life lost. Out of the TDVHL, 0.05% was borne by persons aged 0–14 years, 3.13% by 15–44 years, 21.48% by 45–64 years, 21.87% by 65–74 years, and 53.47% by 75 years and above. Around 46.48% of the TDVHL accrued to persons aged 15 and 74 years. The average TDVHL decreases with increase in age of the deceased, e.g. the average TDVHL for 0–14-year-olds is three-fold that of 75-year-olds and above.\n\nDistribution of the total discounted value of human life by regions and territories. Figure 1 depicts the share of TDVHL across the 13 regions and five territories of France.\n\nAbout 80.3% of the TDVHL accrued to only five regions of France, i.e. Auvergne-Rhône-Alpes, Bourgogne-Franche-Comté, Grand Est, Hauts-de-France, and Ile-de-France. Grand Est, Hauts-de-France, and Ile-de-France regions alone accounted for 66.18% of the TDVHL. The territories combined made up less than 1% of TDVHL.\n\nTable 8 presents the effects of the application of 5% and 10% discount rates on the TDVHL due to COVID-19 in France.\n\nRerun of the HCA model, alternately, with discount rates of 5% and 10% resulted in decreased TDVHL by Int$1,304,764,602 (12.4%) and Int$3,506,938,312 (33%), respectively. The average values per human life lost declined by Int$42,204 and Int$113,434 in turn.\n\nTable 9 displays the impact on the TDVHL of substituting the national life expectancy with the world and world's highest average life expectancies.\n\nReplacement of the national life expectancy of 83.13 years with the world average life expectancy of 73.2 years in the HCA model led to decreases in the total and average TDVHL of Int$7,750,187,267 (73.87%) and Int$250,685, respectively. Contrastingly, application of the world’s highest life expectancy of 88.17 years augmented total and average TDVHL by Int$3,744,263,463 (35.7%) and Int$121,111\n\n\nDiscussion\n\nThe 30,916 human lives lost to COVID-19 in France by 14 September 2020 had a TDVHL of Int$10,492,290,194, which is equivalent to 0.332% of France’s GDP.\n\nThe average value was Int$339,381 per human life lost, which is 8-times the GDP per capita for France in 2020.\n\nRerun of the HCA model with discount rates of 5% and 10% decreased TDEVHL by Int$1,304,764,602 (12.4 %) and Int$3,506,938,312 (33%), respectively.\n\nReanalysis of the HCA model with the world average life expectancy dwindled the TDVHL by Int$7,750,187,267 (73.87%). Instead, a recalculation with the world highest average life expectancy of 88.17 years augmented TDVHL by Int$3,744,263,463 (35.7%).\n\nThe sensitivity analysis revealed that growth in discount rate triggers contraction in the TDVHL, and an upsurge in average life expectancy amplifies the TDVHL. The two findings are consistent with those of our previous studies conducted in Brazil14, Canada15, China16, Germany17, Iran13, Spain18, Turkey19, the United Kingdom (UK)20, and the United States of America (USA)21.\n\nThe China16 and Spain18 average values of Int$356,203 and Int$470,798 per human life loss associated with COVID-19 were 4.96% and 38.72% higher than the French average of Int$339,381. On the other hand, the French average economic value per human life of Int$339,381 was higher than those of Brazil of Int$99,62914, Canada of Int$231,21715, Germany of Int$132,96017, Iran of Int$165,18713, Turkey of Int$228,51419, the UK of Int$225,10420, and the USA of Int$292,88921 by 70.64%, 31.87%, 60.82%, 51.33%, 32.67%, 33.67%, and 13.70%, in that order. Our previous studies have attributed the differences to variations in underlying population age distributions, the YLL, the GDP per capita, and the per capita health spending13–15,17–21.\n\nEvidence on the economic value of human lives losses associated with COVID-19 may be useful to the Ministry of Public Health when advocating within the Government of France for sustaining or increasing investments into the national health system, disease surveillance and response system (including IHR core capacities), and other systems (e.g. water and sanitation) that tackle social determinants of health in the pursuit of the United Nations Sustainable Development Goal 3 to “Ensure healthy lives and promote well-being for all at all ages” and Goal 6 to “Ensure availability and sustainable management of water and sanitation for all” (p.14)34. Of course, the economic evidence reported in this paper is meant to complement the International Bill of Human Rights obliging the Government of France to assure every citizen’s realization of the right to life (Article 3) and to “..a standard of living adequate for the health and well-being of himself and of his family, including food, clothing, housing and medical care and necessary social services… (Article 25)” (p.76)35.\n\nComprehensive studies on the multidimensional impact of COVID-19 on household’s wellbeing.\n\nWide-ranging studies on the multi-sectoral impact of COVID-19 pandemic once the pandemic is eradicated.\n\nConsumer choice behaviour analysis in respect to uptake of various COVID-19 prevention interventions, e.g. handwashing with soap, use of safely managed drinking water and sanitation, use of face masks, and patronage of alcohol bars during COVID-19.\n\nEconomic evaluations of cost and consequences of preventive interventions (including personal hygiene, physical distancing, safely managed human waste disposal, contact tracing, vaccines), diagnostics, and potential treatments for COVID-19. Where feasible, cost-effectiveness, cost-utility, and cost-benefit analyses ought to be designed and conducted alongside ongoing and envisaged clinical, and effectiveness randomized trials30,36.\n\nFirst, HCA has been criticized for valuing non-market contributions to society at zero dollars37. For instance, traditional HCA values YLL among children below 14 years38, retired (62 years and above)38, homemakers (not employed outside the home), unemployed, and severely handicapped. In order to avoid discrimination against these vulnerable groups, which goes against the 1948 United Nations Universal Declaration of Human Rights35 and the Constitution of the World Health Organization39, YLL at all the age groups were valued at equal net GDP per capita.\n\nSecond, the current study did not compare the costs and benefits of a raft of alternative preventive community-level interventions implemented by the French Government and citizens to limit transmission of COVID-19, e.g. bans on gatherings of more than 100 people, all religious gatherings, all travel, ships carrying more than 100 passengers, and embalming; closure of most public establishments, all schools and institutions of higher learning; and mandatory mask-wearing in public places40. It was also outside the scope of the current study to appraise the costs and benefits of various options for diagnosis of COVID-19, contact tracing, quarantine, and management of persons who test positive for COVID-19 .\n\nFinally, Stiglitz, Sen and Fitoussi41 have criticized GDP for not measuring economic wellbeing (or quality of life), ignoring income inequalities, and disregarding environmental damage caused by production processes.\n\n\nConclusion\n\nThe discounted value per human life loss of Int$339,381 is 8-fold the GDP per person of France. Such evidence constitutes an additional argument for health policy makers when making a case for increased investment to optimize IHR capacities, and coverage of essential health services, and safely managed water and sanitation services. The other rationales for increased investments in the development of resilient health-related systems include the fact that a pandemic, such as COVID-19, can trigger health systems and socioeconomic crises of significant magnitudes42; and also the fact that every human being has the right to life35.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nThe authors owe deep gratitude to Jehovah Shalom for inspiration and sustenance during the life course of the study. The paper is dedicated to the citizenry and health workers in France for the chivalrous fight against COVID-19. The views expressed are exclusively those of authors and should not be attributed to institutions of affiliation.\n\n\nReferences\n\nInternational Monetary Fund (IMF): World Economic Outlook Database. [cited 2020 May 13]. 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IOSR J Dent Med Sci. 2020; 19(9): 45–53. Publisher Full Text\n\nKirigia JM, Muthuri RNDK, Nkanata LHK, et al.: The pecuniary value of human life losses associated with COVID-19 in Brazil. IOSR J Pharm. 2020; 10(8): 45–51. Reference Source\n\nKirigia JM: The dollar value of human life losses associated with COVID-19 in Canada. Pharm Biomed Res. 2020. (in press). Reference Source\n\nKirigia JM, Muthuri RNDK: The fiscal value of human lives lost from coronavirus disease (COVID-19) in China. BMC Res Notes. 2020; 13(1): 198. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKirigia JM, Muthuri RNDK: The financial value of human life losses associated with coronavirus disease in Germany. Acad J Econ Studies. 2020. (in press). Reference Source\n\nKirigia JM, Muthuri RNDK: The discounted money value of human lives lost due to COVID-19 in Spain. Journal of Health Research. 2020; 34(5): 455–460. Publisher Full Text\n\nKirigia JM, Muthuri RNDK, Nkanata LHK: The monetary value of human life losses associated with COVID-19 in Turkey [version 1; peer review: 2 approved]. Emerald Open Res. 2020; 2: 44. Publisher Full Text \| Free Full Text\n\nKirigia JM, Muthuri RNDK: The present value of human lives lost due to COVID-19 in the United Kingdom (UK). Pharm Biomed Res. (in press). Reference Source\n\nKirigia JM, Muthuri RNDK: Discounted monetary value of human lives lost due to COVID-19 in the USA as of 3 May 2020. IOSR J Dent Med Sci. 2020; 19(5): 51–54.\n\nBanque de France: Update on business conditions in France at the end of 2020. Paris: Banque de France; 2020. Reference Source\n\nSmith A: An inquiry into the nature and causes of the wealth of nations. 4th Edition. Edited by S.M. Soares. Lausanne: Metalibri Digital Library; 2007. Reference Source\n\nOECD: The well-being of nations: the role of human and social capital. Paris: OECD; 2001. Reference Source\n\nCulyer AJ: Commodities, characteristics of commodities, characteristics of people, utilities and the quality of life. In: The Humble Economist: Tony Culyer on Health, Health Care and Social Decision Making; Culyer, A.J., Cookson, R.A., Claxton, K.P. (Eds.); York Publishing Services Ltd, York, UK. 2012; 55–66. Reference Source\n\nWeisbrod BA: The valuation of human capital. J Political Econ. 1961; 69(5): 425–436. Reference Source\n\nWorld Health Organization [WHO]: Global Health Expenditure Database. [cited 2020 Sept 14]. Reference Source\n\nWorldometer: Life Expectancy of the World Population. [cited 2020 Sept 14]; Accessed 14 September 2020. Reference Source\n\nSanté publique France: COVID-19: Point épidémiologique hebdomadaire du 10 septembre 2020. [cited 2020 Sept 16]. Reference Source\n\nKirigia JM: Economic Evaluation of Public Health Problems in sub-Saharan Africa. Nairobi: University of Nairobi Press; 2009. Reference Source\n\nWHO: The World health report 2000: health systems: improving performance. Geneva: WHO; 2000. Reference Source\n\nMurray CJL: Quantifying the burden of disease: the technical basis for disability-adjusted life years. Bull World Health Organ. 1994; 72(3): 429–445. PubMed Abstract \| Free Full Text\n\nJamison DT, Breman JG, Measham AR, et al.: Disease control priorities in developing countries. Oxford: Oxford University Press; 1993.\n\nUnited Nations: Transforming our world: the 2030 Agenda for Sustainable Development. General Assembly Resolution A/RES/70/1. New York: UN; 2015. Reference Source\n\nUnited Nations: International Bill of Human Rights: A Universal Declaration of Human Rights. General Assembly Resolution A/RES/217 (III)A. New York: UN; 1948.\n\nDrummond MF, Sculpher MJ, Torrance GW, et al.: Methods for the economic evaluation of health care programmes (3rd Edition). Oxford: Oxford University Press; 2007.\n\nMooney GH: The Valuation of Human Life. Macmillan: London, UK. 1977.\n\nFrench Republic: Code du travail (2014). [cited 2020 Sept 18]. Reference Source\n\nWHO: Basic documents. Geneva: WHO; 2014. Reference Source\n\nWikipedia: COVID-19 pandemic in France. [cited 2020 Sept 18]. Reference Source\n\nStiglitz JE, Sen A, Fitoussi JP: Mismeasuring our Lives: why GDP doesn't add up. New York: The New Press; 2010. Reference Source\n\nMaliszewska M, Mattoo A, van der Mensbrugghe D: The potential impact of COVID-19 on GDP and Trade: a preliminary assessment. Policy Research Working Paper 9211. Washington: World Bank Group; 2020. Reference Source" }	[ { "id": "73179", "date": "03 Dec 2020", "name": "Patricia Akweongo", "expertise": [ "Reviewer Expertise Health Equity", "Economic Evaluation", "Health Systems", "Health financing", "Human resource for health", "malaria", "gender and violence", "etc" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSpecific Comments for the revision of the paper\nIntroduction Paragraph 4 line 1: Second, the average of 13 International Health Regulations (IHR) core capacity score for France was 82 (on a scale of 0 to the target of 100) in 20197, Comment: The authors estimate the average IHR core capacity score for France as 82 . It is unclear if this was an average of 13 items scored in Table 1 or how the authors estimated that. For summing up the 13 items in Table 1 under France the average is 74 instead of 82? This method for arriving at the 82 needs to be explained.\n\nIntroduction Paragraph 4 line 4-7: The French points of entry capacity score of 40 were lower than the average score for the World Health Comment: This should read \"The French points of entry capacity score of 40 was lower than the average score for the World Health\"\n\nIntroduction Paragraph 4 line 4-7: Comment: The authors indicate that France point of entry capacity score was 40 whereas that of EU was 61 representing 52.2%. It is unclear how the authors arrived at this percentage (52.2%)\nIntroduction: Paragraph 3-4 Comment: In paragraph 3 the authors mentioned four factors might explain the relatively large number of COVID-19 deaths sustained by France. The first being the more than 2 months delay with restrictions (Paragraph 3), the second being the IHR capacity scores (Paragraph 4) and then it repeats the second factor as in Table 2 as the general health system determinants. This is quite confusing as one loses track of the four factors that accounted for the state in which France found herself with respect to Cover-19. The authors will need to revise this to make it clear what these 4 factors are.\nIntroduction Paragraph 3 line 2 First, there was more than two months’ delay in country-wide implementation of public health interventions that could have prevented (or slowed) Comment: This should be revised to read \"First, there was more than two months’ delay in country-wide implementation of public health interventions that could have prevented or slowed \" (since prevention is not the same as slowed)\nMethods Step 3: Discounting of the years of life lost (DYLL). Comment:The authors indicate here that the discount of 3% is chosen based on their previous work but they cite other articles as references as well. Thus, the article should rather indicate that \"(a) A discount rate of 3% was chosen because it has been used in previous COVID-19 related economic studies13–21, \"\nStep 6: Calculation of the share of TDVHL accruing to the 13 regions and five territories of France29 through multiplication of the TDVHL by proportion of COVID-19 deaths sustained by specific region and territory. Comment:In the introduction of the study it was not indicated that 13 regions and 5 territories of France will be used to show the share of these to the TDVHL. It thus pops up here with a previous mention of that in the introduction or even in the methods where information on the study site could be described. The characteristics of these territories may affect the outcomes of the study.\nStep 7: A one-way sensitivity analysis was performed to evaluate the effect of changes in discount rate and the average life expectancy on the estimated TDVHL. Comment: The authors chose to use the world average life expectancy and the world highest average life expectancy of 88.13 for women from Hong Kong for the sensitivity. Given that the authors began comparing France with the rest of EU, I was expecting that the authors will first conduct the sensitivity analysis using average life expectancy for the Europe before comparing with the rest of the world.\n\nDiscussions Contrasting of study findings with those from other countries: Comment:The authors refer only to their previous studies in comparing and contrasting their findings but I think the paper will benefit greatly by comparing and contrasting the findings with other studies in this same field as well.\nRecap of findings Reanalysis of the HCA model with the world average life expectancy dwindled the TDVHL by Int$7,750,187,267 Comment:This should rather read\" Reanalysis of the HCA model with the world average life expectancy reduced the TDVHL by Int$7,750,187,267\n\nLimitations of Study For instance, traditional HCA values YLL among children below 14 years38, retired (62 years and above)38, homemakers (not employed outside the home), unemployed, and severely handicapped. Comment: This sentence above is incomplete with the use of \"For instance....\". Authors should consider revising this.\nSecond, the current study did not compare the costs and benefits of a raft of alternative preventive community-level interventions implemented by the French Government and citizens to limit transmission of COVID-19. Comment: This sentence above should read \"Second, the current study did not compare the costs and benefits of a raft of alternative community-level interventions implemented by the French Government and citizens to limit transmission of COVID-19.\nGeneral Comment:This paper highlights issues that might explain the years of lives lost due to Covid-19 in France that may appeal to policy makers to make great investments in interventions needed to reduce deaths lost and increase GDP. More elaborately in this paper is the clear and robust description of the analytical methods for calculating the lives lost. This step-by-step methodological approach could easily be replicated in many countries and sites to produce more evidence that will guide policy makers in the prioritizing investments in Covid-19 to curb the pandemic.\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": [] }, { "id": "83336", "date": "28 Apr 2021", "name": "Neda Soleimanvandiazar", "expertise": [ "Reviewer Expertise Health service utilization", "Inequality in Health services", "Inequality in Health", "Social Determinants of Health", "Social support", "Social Network", "Social Health in the Elderly", "AIDS & HIV", "High risk Behaviors", "Drug use and Substance use" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\nComment: It would be better if the authors, in the introduction of the study explain the reason for the selection of 13 regions and 5 territories of France and as well how to select the desired regions (for example the features of these regions) that are given in the results section.\n\nIntroduction: Paragraph 3\nComment: The reason for the explanation of the four factors that caused the relatively large number of deaths due to COVID-19 sustained by France is not specified in the third paragraph by the authors.\n\nMethods: Step 3 Discounting of the years of life lost (DYLL).\nComment: The authors mentioned that the 3% discount rate was chosen because it has been used in our previous COVID-19 related economic studies, but it would be better to explain in a sentence about choosing a 3% discount rate with reference (e.g. In European countries, the discount rate is usually considered 3%, reference).\n\nDiscussions Contrasting of study findings with those from other countries:\nComment: The discussion is not very well written. In the discussion, the authors merely cite the findings of the economic value per human life of their previous studies in this field and do not provide an explanation of the possible reasons for the differences in the economic value of human life between different countries. It is recommended that the discussion section of the article be upgraded.\nSuggestions for further economic studies:\nComment: Some suggestions are not in line with the results of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/9-1247
https://f1000research.com/articles/9-213/v1	27 Mar 20	{ "type": "Software Tool Article", "title": "AlignmentViewer: Sequence Analysis of Large Protein Families", "authors": [ "Roc Reguant", "Yevgeniy Antipin", "Rob Sheridan", "Christian Dallago", "Drew Diamantoukos", "Augustin Luna", "Chris Sander", "Nicholas Paul Gauthier", "Roc Reguant", "Yevgeniy Antipin", "Rob Sheridan", "Christian Dallago", "Drew Diamantoukos", "Augustin Luna" ], "abstract": "AlignmentViewer is a web-based tool to view and analyze multiple sequence alignments of protein families. The particular strengths of AlignmentViewer include flexible visualization at different scales as well as analysis of conservation patterns and of the distribution of proteins in sequence space. The tool is directly accessible in web browsers without the need for software installation. It can handle protein families with tens of thousands of sequences and is particularly suitable for evolutionary coupling analysis, e.g. via EVcouplings.org.", "keywords": [ "alignment viewer", "MSA", "JavaScript", "protein alignments", "web-based", "tool" ], "content": "Introduction\n\nMultiple sequence alignment (MSA) analysis (e.g., analysis of sequence patterns, subfamilies, specificity residues, evolutionary couplings) and visualization allows researchers to extract information and gain a better understanding of protein families. MSA is a basic step in many protein analysis workflows, including 3D structure prediction (Marks et al., 2011), structure detection in flexible (‘disordered’) domains (Toth-Petroczy et al., 2016), function prediction (Tamames et al., 1998) and intracellular localization (Goldberg et al., 2014).\n\nA number of useful tools exist for the visualization of protein MSAs, such as, MView, Wasabi, AliView, MSAViewer and Jalview (Brown et al., 1998; Larsson, 2014;Veidenberg et al., 2016; Waterhouse et al., 2009; Yachdav et al., 2016). MView was one of the first online browser-based MSA viewers, with alignments formatted as an HTML document. Wasabi is a web-based tool particularly useful for phylogenetic analysis and incorporates phylogeny-aware alignment methods. Another desktop application, AliView, has features such as sorting, viewing, removing, editing and merging sequences from large nucleotide sequence datasets. MSAViewer is an interactive MSA visualizer in JavaScript that implements basic features of viewing, scrolling and motif selection. Jalview is a Java-based desktop tool accessible through websites using an embeddable applet, but unfortunately the technology for these applets is no longer supported in most browsers.\n\nAlignmentViewer (Reguant, 2020) complements these MSA tools and provides the following features: (i) in-browser and serverless execution, (ii) visualization of very large MSAs, (iii) visualization of conservation patterns, (iv) sequence filtering, (v) logo display, (vi) pairwise sequence identity map, (vii) sequence space exploration by UMAP dimensionality reduction, and (viii) display of top-ranked evolutionary couplings (Hopf et al., 2019).\n\nAn earlier version of this article can be found on bioRxiv (DOI: https://doi.org/10.1101/269720); additional features have been implemented since the earlier version.\n\n\nMethods\n\nAlignmentViewer (Reguant, 2020) is a web-based tool written in JavaScript with minimal system requirements. AlignmentViewer runs on most modern web browsers such as Chrome, Safari, and Firefox regardless of operating system. AlignmentViewer is developed with the D3 library (d3js.org) to produce dynamic and interactive data visualizations, with performance (speed) for large alignments a major consideration. The tool is entirely client-based, running inside a web browser without the need for server-side computation.\n\nUsers can access AlignmentViewer and all its features directly from alignmentviewer.org, but its serverless execution enables anyone to quickly start a local copy for online or offline use. Hyperlinks for lookup in background databases, such as Uniprot or PFAM, are made directly from the client. Alignments can be passed to AlignmentViewer also via a URL query (e.g., https://alignmentviewer.org/?url=https://alignmentviewer.org/example/1bkr_A.1-108.msa.txt), enabling seamless integration from external web services via a simple link (e.g. the EVcouplings, evcouplings.org, web server (Hopf et al., 2019) offers visualization of computed alignments via a link to AlignmentViewer). The tool has been thoroughly tested with many large alignments. An alignment with, e.g., 50,000 sequences (about 13 MB of memory) loads in the Safari browser within one minute; further speedup is planned. Based on this and other small tests we can safely state that any computer that is able to run any modern web browser will be able to run any alignment that requires visualization.\n\n\nUse case\n\nFigure 1 shows the main functionalities from AlignmentViewer (Reguant, 2020) explained in more detail in the next subsections. The top sub-figure shows the msa view with the sequence logo and the alignment capturing most of the attention. This view lets the user examine in depth the alignment. Each amino acid position is represented in sequence logo and the height shows users the information content of each position, in bits. Then, from left to right we show the pixel view, a part of the stats view, and the sequence space (with annotations). The pixel view gives an overview display of the alignment to enable a coarse view of the alignment for better visualization and pattern identification. The all versus all sequence identity sub-figure in Figure 1 (part of the stats view) displays allows users to identify possible clusters in the alignment based on sequence identity. The bottom right sub-figure of Figure 1 displays the sequences clustered by similarity (see section Sequence space) highlighted by user-provided annotations to aid in interpretation of the clusters.\n\nBars above the sequence alignment quantify residue conservation. The alignment consensus logo (just below the bar chart) is based on the amino acid frequencies. Lower left: pixel view of the alignment especially useful for large families; lower middle: protein-protein sequence similarity matrix graded by percentage identity; lower right: distribution of sequences in sequence space (UMAP projection), colored by species groups.\n\nAlignment details. The msa view page has summary information: number of sequences, conservation and gap counts for each position, a sequence logo, and the residues in one letter code. By default, columns with gaps in the reference sequence (first row) are omitted in order to facilitate visual focus on sequence patterns relative to a protein of interest and to avoid extremely gapped alignment views typical of many MSA presentations. The amino acids are colored using a conventional coloring scheme, adopted from Mview, based on amino acid properties.\n\nSequence attributes and sorting. Sequences in the alignment can be sorted using one of four different methods: (i) the original order provided by the user, (ii–iii) by % sequence identity between a particular sequence and the reference (top) sequence, relative to the first or the second (gaps not counted), and (iv) by user-provided (upload annotations tab) sequence weights or other attributes, such as alignment profile scores (e.g., HMM bit scores). Sequences can be filtered by sequence identity relative to a reference sequence or by percentage of gaps.\n\nThe pixel view (image view website tab) leads to an overview of the entire depth and breadth of an MSA. The amino acid letters are represented by small rectangles of pixels, retaining the amino acid type coloring (image view tab). This striking visual impression can reveal patterns of conservation and variation, especially for large alignments. This is very useful to gain an intuitive view of sequence properties, noise at the uncertain edges of a protein family, as well as subfamily distributions. The coloring scheme can be by (1) amino acid properties, (2) hydrophobicity (red to blue) or (3) mutational difference (stronger color) in a sequence relative to the reference (first row) sequence.\n\nThe stats view tab leads to plots of statistical properties of the set of sequences in the alignment, including (i) sequence identity relative to the reference sequence, and (ii) min, max, and average of (i); and (iii) a pairwise sequence identity matrix in which each pixel represents the degree of similarity between two sequences, such that a block-diagonal structure of the matrix is indicative of distinct subfamilies, given, e.g., a tree-derived sequence order as user input. The ordering of sequences by phylogeny is (currently) not part of the tool and can be performed using external tools, e.g., Wasabi (Veidenberg et al., 2016).\n\nUsers can upload custom numerical attributes or labels for the sequences in the MSA (upload annotations) or evolutionary couplings between residue positions (load couplings). Adding these attributes allows users to use sequence weights, compare different measures of sequence fitness (e.g., bitscore, sequence identity, statistical energy) or visualize evolutionary coupling constraints for pairs of positions.\n\nUsers can view MSA sequences in two- or three-dimensional sequence space under the “sequence space” tab. Dimensionality reduction is performed using the UMAP algorithm (McInnes et al., 2018), adapted for Javascript as umap-js, using the Hamming distance between sequences. UMAP parameters are set to reasonable defaults, but can also be configured via the settings panel. Sequences are colored by user provided annotations (\"upload annotations\" tab).\n\n\nConclusion\n\nAlignmentViewer (Reguant, 2020) is a lightweight online viewer for biological multiple sequence alignments that focuses on usability and performance. Written in JavaScript, this tool can be used in many browsers. The architecture of AlignmentViewer allows its use without software installation and without an internet connection. The visualization capabilities, analysis features and metrics in AlignmentViewer are useful in many areas of biology, especially evolutionary, structural, synthetic and chemical biology. In the future we plan to add a visualization of species diversity, predicted contact maps, and organization by sequence subfamilies with specificity residues. A standalone version of AlignmentViewer is available at alignmentviewer.org and is in use by external services including EVcouplings.org. AlignmentViewer is an open source project hosted on GitHub, which welcomes engagement of interested members of the community.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nAlignmentViewer website and demo can be found at: alignmentviewer.org/.\n\nSource code available at: github.com/sanderlab/alignmentviewer.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3653774 (Reguant, 2020).\n\nLicense: MIT license.", "appendix": "Acknowledgments\n\nWe thank Debora Marks for constructive discussions.\n\n\nReferences\n\nBrown NP, Leroy C, Sander C, et al.: MView: a web-compatible database search or multiple alignment viewer. Bioinforma. 1998; 14(4): 380–381. PubMed Abstract \| Publisher Full Text\n\nGoldberg T, Hecht M, Hamp T, et al.: LocTree3 prediction of localization. Nucleic Acids Res. 2014; 42(Web Server issue): W350–W355. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHopf TA, Green AG, Schubert B, et al.: The EVcouplings Python framework for coevolutionary sequence analysis. Bioinformatics. 2019; 35(9): 1582–1584. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarsson A: AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014; 30(22): 3276–3278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarks DS, Colwell LJ, Sheridan R, et al.: Protein 3D structure computed from evolutionary sequence variation. PLoS One. 2011; 6(12); e28766. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcInnes L, Healy J, Melville J: Umap: Uniform manifold approximation and projection for dimension reduction. arXiv preprint. arXiv: 1802.03426. 2018. Reference Source\n\nReguant R: Alignmentviewer (Version v1.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3653774\n\nTamames J, Ouzounis C, Casari G, et al.: EUCLID: automatic classification of proteins in functional classes by their database annotations. Bioinformatics. 1998; 14(6): 542–3. PubMed Abstract \| Publisher Full Text\n\nToth-Petroczy A, Palmedo P, Ingraham J, et al.: Structured States of Disordered Proteins from Genomic Sequences. Cell. 2016; 167(1); 158–170.e12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVeidenberg A, Medlar A, Löytynoja A: Wasabi: An Integrated Platform for Evolutionary Sequence Analysis and Data Visualization. Mol Biol Evol. 2016; 33(4): 1126–1130. PubMed Abstract \| Publisher Full Text\n\nWaterhouse AM, Procter JB, Martin DM, et al.: Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009; 25(9): 1189–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYachdav G, Wilzbach S, Rauscher B, et al.: MSAViewer: interactive JavaScript visualization of multiple sequence alignments. Bioinformatics. 2016; 32(22): 3501–3503. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "61767", "date": "09 Apr 2020", "name": "Erik Larsson Lekholm", "expertise": [ "Reviewer Expertise Genomics", "bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Software Tool article, describing a web-based sequence alignment viewer (AlignmentViewer), is well-written and very clearly presented. The introduction nicely motivates the need for this tool, given the many other available alternatives. The main features of the software are clearly explained in the article. The tool in itself seems useful and ran quickly and efficiently when tested on sample data. I have only a few minor comments that may be considered by the authors:\nMinor:\nA little bit of detail may be added to the “Sequence space” section. “Two- or three-dimensional sequence space” is at first a bit confusing, as no information has been given at that stage as to what it means. How is UMAP applied to the Hamming distances? Is each sequence first represented by a vector of Hamming distances, with each element corresponding to one of the included sequences? Maybe this can be clarified with a few additional words.\n\nThe following sentence is somewhat confusing: \"Figure 1 shows the main functionalities from AlignmentViewer (Reguant, 2020) explained in more detail in the next subsections.”\n\n\"This view lets the user examine in depth the alignment.” - should it be “examine the alignment in depth\"?\n\nWhen loading an alignment using a somewhat older version of Safari (12.1.2), the “computing conservation” window does not disappear after the operation finishes. This problem was absent in Chrome.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5469", "date": "30 Apr 2020", "name": "Chris Sander", "role": "Author Response", "response": "Thank you for your comments and suggestions - they are spot on.We will work on taking these into account on the web site and in the next version of the manuscript.Chris Sander - DFCI and HMS" }, { "c_id": "6022", "date": "15 Oct 2020", "name": "Roc Reguant", "role": "Author Response", "response": "A little bit of detail may be added to the “Sequence space” section. “Two- or three-dimensional sequence space” is at first a bit confusing, as no information has been given at that stage as to what it means. How is UMAP applied to the Hamming distances? Is each sequence first represented by a vector of Hamming distances, with each element corresponding to one of the included sequences? Maybe this can be clarified with a few additional words. Response: We agree that a bit more detail is warranted and have updated the manuscript that describes using the hamming distance between individual sequences as the distance metric (parameter) for UMAP. The following sentence is somewhat confusing: \"Figure 1 shows the main functionalities from AlignmentViewer (Reguant, 2020) explained in more detail in the next subsections.”Response: We have updated the manuscript to make it more understandable. The citation to Reguant 2020 was also confusing (it points to the codebase) so we moved that codebase DOI to a single instance at the end of the manuscript. \"This view lets the user examine in depth the alignment.” - should it be “examine the alignment in depth\"?Response: The manuscript has been corrected. When loading an alignment using a somewhat older version of Safari (12.1.2), the “computing conservation” window does not disappear after the operation finishes. This problem was absent in Chrome.Response: Thank you for noticing this bug. We have now corrected the issue and the dialog disappears when using Safari." } ] }, { "id": "61763", "date": "16 Apr 2020", "name": "Alex Bateman", "expertise": [ "Reviewer Expertise Computational biology with specialism in biological databases and analysis of protein families." ], "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 there is a need for Javascript based alignment viewers which can handle large numbers of sequences. So this software is rather timely. However, I think the current implementation seems to still contain significant bugs and the web page requires a round of user experience testing to make it easier to use and interpret the results. Once these improvements are made then I think this has the potential to be a very useful tool.\nManuscript changes:\nPlease fix capitalisation of PFAM to Pfam\n\nSoftware/Website changes:\nThe computing conservation box is annoyingly placed. It does not go away and covers the UMAP view significantly. I really need an estimate of how long it will take for the pairwise identity to load. Some time passes…I didn’t realise I had to click on calculate to get the pairwise identity to show. Please UX test this page to make it easier/clearer for the user to understand what to do.\n\nFor the top graph on the stats view the three colours used for max/average and min identity are hard to distinguish. Please use a broader colour palette. Please add axis legends for this graph and make it clearer what the title of this graph is. The title is very clear for the bottom plot.\nPlease add alternative colouring schemes for viewing the alignments. It makes a surprisingly big difference for experienced users ability to interpret an alignment. I personally like the ClustalX colouring scheme that is widely adopted, but there are other popular schemes it would be nice to incorporate as options.\n“Based on this and other small tests we can safely state that any computer that is able to run any modern web browser will be able to run any alignment that requires visualization”. This is overstating the case. Needs to be toned down. Try testing the software on the ABC transporter family (PF00005) alignment in Pfam. The NCBI alignment contains 2.6 million sequences. If it works seamlessly for that then you can safely state.\nI tried to hook the viewer up to a Pfam alignment which is not very large (840 sequences). I got a box that said Fetching file…and no response beyond that.\nhttps://alignmentviewer.org/?url=http://pfam.xfam.org/family/PF00571/alignment/seed/format?format=fasta&alnType=seed&order=t&case=l&gaps=default&download=0\nalso tried Stockholm format with no luck.\nhttps://alignmentviewer.org/?url=http://pfam.xfam.org/family/PF00571/alignment/seed/format?format=stockholm&alnType=seed&order=t&case=l&gaps=default&download=0\nI also tried with a Pfam family with just 3 sequences. Still with no luck.\nI got the viewer to work by uploading a Pfam alignment in fasta format with 3 sequences. Then I tried uploading the seed alignment for the CBS domain downloaded from Pfam in fasta format. I then got the following error:\nParsing error: sequence #3 has different length.\nThe first few lines of the alignment are included below and the third sequence appears to be the same length as all the others.\n>Q8EHI4_SHEON/79-134 I........H.Q...V........MTR........NP.........V.......TVAPY. ...VS.LDVAS....RT..L.....L....E.....HN..I.....G..CL.P.V.L... .........ENG....D......LV.GIVTWKDLLRAYCA >Q97V95_SULSO/193-248 V........L.D...A.......G.TK........NP.........I.......TINRY. ...YS.ILNAA....KL..M.....I....E.....KR..I.....G..TL.L.V.M... .......E..NQ....K......LV.GIVTERDLMYAYIN >Q6M020_METMP/269-321 V........K.E...I.........MS........PP.........V.......MVSPE. ...AA.LNELI....KG..M.....A....N.........T.....D..RV.Y.V.V... .........DNG....N......IL.GIISKTDIVRTLSI >Q72KI1_THET2/367-424 V........E.G...F.........LA........RA.........V.......VLPPS. ...TP.LSQVE....PR..L..........R.....EA..G.....G..RV.L.V.GE.. .......RAGEG...WR......LL.GIYTRTDLYRSAPK >Q8TX96_METKA/244-298 A........R.N...Y.......L..Q........EM.........V.......VVPPE. ...TP.LHEAL....WE..V.........ID.....KM..S.....D..RI.Y.V.M... ..........DG.....R....KLT.GVVPLIDAVYTLAK >Q836T2_ENTFA/77-132 V........Q.E...I........MSP........PL.................MVAQD. ...TS.IRDAI....TN..L................FMYDV.....G..SL.Y.V.M... ........DEAK....E......LL.GVLSRKDLLRASLN >Q8XIV1_CLOPE/77-131 V........K.D...I.........MS........KP.........V.......TVCEE. ...TM.LHDAI....VH.LF.....L...ND.........V.....G..TM.F.VEN... ..........GG....V......LT.GAVSRKDFLKVAIG\nIf I delete the third sequence and re-upload I get exactly the same error.\nFor info I carried out tests with Firefox for Mac version 74.0 and Safari Version 13.1\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6023", "date": "15 Oct 2020", "name": "Roc Reguant", "role": "Author Response", "response": "Manuscript changes:Please fix capitalisation of PFAM to PfamResponse: The manuscript has been updated to Pfam.Software/Website changes:The computing conservation box is annoyingly placed. It does not go away and covers the UMAP view significantly.Response: This bug has now been fixed - the pop-up will disappear after the alignment has been loaded.I really need an estimate of how long it will take for the pairwise identity to load. Some time passes…I didn’t realise I had to click on calculate to get the pairwise identity to show. Please UX test this page to make it easier/clearer for the user to understand what to do. Response: We have added a button into the middle of the plot to indicate that an action needs to be taken by the user to begin calculating pairwise sequence identity.For the top graph on the stats view the three colours used for max/average and min identity are hard to distinguish. Please use a broader colour palette. Please add axis legends for this graph and make it clearer what the title of this graph is. The title is very clear for the bottom plot.Response: We have updated the color palette to increase the contrast, added axis labels and added a clear title. Please add alternative colouring schemes for viewing the alignments. It makes a surprisingly big difference for experienced users ability to interpret an alignment. I personally like the ClustalX colouring scheme that is widely adopted, but there are other popular schemes it would be nice to incorporate as options.Response: We agree alternative coloring schemes are useful and have added the clustal colors as a drop-down option on the main page as well as in the image view.“Based on this and other small tests we can safely state that any computer that is able to run any modern web browser will be able to run any alignment that requires visualization”. This is overstating the case. Needs to be toned down. Try testing the software on the ABC transporter family (PF00005) alignment in Pfam. The NCBI alignment contains 2.6 million sequences. If it works seamlessly for that then you can safely state.Response: We agree with the reviewer that the phrasing was optimistic and have removed the sentence from the main manuscript.I tried to hook the viewer up to a Pfam alignment which is not very large (840 sequences). I got a box that said Fetching file…and no response beyond that.https://alignmentviewer.org/?url=http://pfam.xfam.org/family/PF00571/alignment/seed/format?format=fasta&alnType=seed&order=t&case=l&gaps=default&download=0also tried Stockholm format with no luck.https://alignmentviewer.org/?url=http://pfam.xfam.org/family/PF00571/alignment/seed/format?format=stockholm&alnType=seed&order=t&case=l&gaps=default&download=0Response: There are two technical limitations inherent to how requests happen in the browser. We take as an example your suggested URL: https://alignmentviewer.org/?url=http://pfam.xfam.org/family/PF00571/alignment/seed/format?format=stockholm&alnType=seed&order=t&case=l&gaps=default&download=0Limitation 1: The alignmentviewer.org website is hosted as a secure site under https. However, the location of the requested alignment (from Pfam) is passed via an insecure connection (http). For security reasons mixing different protocols (also called \"mixed content\", see https://developer.mozilla.org/en-US/docs/Web/Security/Mixed_content) is not allowed.Limitation 2: the string passed to the query parameter URL (aka. what follows \"?url=\") needs to be sanitized before being served. More information about this can be found here: https://en.wikipedia.org/wiki/Query_string#URL_encoding. For clarity, we have updated the example in the main text to be stanitized / encoded. In your example, the alignment lives at \"http://pfam.xfam.org/family/PF00571/alignment/seed/format?format=stockholm&alnType=seed&order=t&case=l&gaps=default&download=0\". Passing the string as-is will confuse the browser that will interpret e.g. \"&gaps=\" as query for alignmentviewer rather than for pfam.xfam.org . Many languages offer built-in functions to sanitize strings to be passed to URL queries (e.g. in a JavaScript frontend application, using the window.encodeURIComponent() function; an interactive website can be found here: https://www.url-encode-decode.com). By doing so, the string proposed for the alignment would result in: \"http%3A%2F%2Fpfam.xfam.org%2Ffamily%2FPF00571%2Falignment%2Fseed%2Fformat%3Fformat%3Dstockholm%26alnType%3Dseed%26order%3Dt%26case%3Dl%26gaps%3Ddefault%26download%3D0\".Conclusion: considering the two inherit browser limitations mentioned above, your requests can be sanitized and fixed as follows:https://alignmentviewer.org/?url=https%3A%2F%2Fpfam.xfam.org%2Ffamily%2FPF00571%2Falignment%2Fseed%2Fformat%3Fformat%3Dfasta%26alnType%3Dseed%26order%3Dt%26case%3Dl%26gaps%3Ddefault%26download%3D0Again, note that for both URLs the protocol has been changed to \"https\" instead of \"http\", and that the string that follows \"&url=\" has been sanitized using Chrome's built-in console and the \"window.encodeURIComponent(x)\" function, where x is the string to be sanitized. We have added some text to the manuscript to highlight these requirements.I also tried with a Pfam family with just 3 sequences. Still with no luck.I got the viewer to work by uploading a Pfam alignment in fasta format with 3 sequences. Then I tried uploading the seed alignment for the CBS domain downloaded from Pfam in fasta format. I then got the following error:Parsing error: sequence #3 has different length.The first few lines of the alignment are included below and the third sequence appears to be the same length as all the others.>Q8EHI4_SHEON/79-134I........H.Q...V........MTR........NP.........V.......TVAPY....VS.LDVAS....RT..L.....L....E.....HN..I.....G..CL.P.V.L............ENG....D......LV.GIVTWKDLLRAYCA>Q97V95_SULSO/193-248V........L.D...A.......G.TK........NP.........I.......TINRY....YS.ILNAA....KL..M.....I....E.....KR..I.....G..TL.L.V.M..........E..NQ....K......LV.GIVTERDLMYAYIN>Q6M020_METMP/269-321V........K.E...I.........MS........PP.........V.......MVSPE....AA.LNELI....KG..M.....A....N.........T.....D..RV.Y.V.V............DNG....N......IL.GIISKTDIVRTLSI>Q72KI1_THET2/367-424V........E.G...F.........LA........RA.........V.......VLPPS....TP.LSQVE....PR..L..........R.....EA..G.....G..RV.L.V.GE.........RAGEG...WR......LL.GIYTRTDLYRSAPK>Q8TX96_METKA/244-298A........R.N...Y.......L..Q........EM.........V.......VVPPE....TP.LHEAL....WE..V.........ID.....KM..S.....D..RI.Y.V.M.............DG.....R....KLT.GVVPLIDAVYTLAK>Q836T2_ENTFA/77-132V........Q.E...I........MSP........PL.................MVAQD....TS.IRDAI....TN..L................FMYDV.....G..SL.Y.V.M...........DEAK....E......LL.GVLSRKDLLRASLN>Q8XIV1_CLOPE/77-131V........K.D...I.........MS........KP.........V.......TVCEE....TM.LHDAI....VH.LF.....L...ND.........V.....G..TM.F.VEN.............GG....V......LT.GAVSRKDFLKVAIGIf I delete the third sequence and re-upload I get exactly the same error.For info I carried out tests with Firefox for Mac version 74.0 and Safari Version 13.1Response: Thank you for pointing out this issue. This bug occurred when refreshing an internal variable, and has now been fixed." } ] } ]	1	https://f1000research.com/articles/9-213
https://f1000research.com/articles/4-1097/v1	21 Oct 15	{ "type": "Research Article", "title": "A hydrophobic proclivity index for protein alignments", "authors": [ "David Cavanaugh", "Krishnan Chittur", "David Cavanaugh" ], "abstract": "Sequence alignment algorithms are fundamental to modern bioinformatics. Sequence alignments are widely used in diverse applications such as phylogenetic analysis, database searches for related sequences to aid identification of unknown protein domain structures and classification of proteins and protein domains. Additionally, alignment algorithms are integral to the location of related proteins to secure understanding of unknown protein functions, to suggest the folded structure of proteins of unknown structure from location of homologous proteins and/or by locating homologous domains of known 3D structure. For proteins, alignment algorithms depend on information about amino acid substitutions that allows for matching sequences that are similar, but not exact. When primary sequence percent identity falls below about 25%, algorithms often fail to identify proteins that may have similar 3D structure. We have created a hydrophobicity scale and a matching dynamic programming algorithm called TMATCH (unpublished report) that is able to match proteins with remote homologs with similar secondary/tertiary structure, even with very low primary sequence matches. In this paper, we describe how we arrived at the hydrophobic scale, how it provides much more information than percent identity matches and some of the implications for better alignments and understanding protein structure.", "keywords": [ "Sequence alignment algorithms", "hydrophobicity scale", "protein homologs", "TMATCH" ], "content": "Introduction\n\nAn understanding of the properties and functions of a protein or a nucleic acid often begins with a search of the sequence against databases of proteins (or nucleic acids) with known properties or functions. The fundamental assumption is that sequence leads to structure which in turn leads to an understanding of the function. Search algorithms have improved and continue to improve. Yet, with proteins in particular, it remains difficult to detect remote homologies in the so called twilight zone where proteins have low percent sequence identities starting around 20–25% and descending to around 10–15%. We describe a hydrophobicity scale that is proving to be an excellent measure of sequence relatedness. A robust estimate of the hydrophobicity based sequence identity can be calculated directly from a global alignment score, which may be directly used in database searches. Proteins with low sequence identities, possessing statistically insignificant similarities by conventional measures, but having similar secondary/tertiary structures, which would not be identified as statistically significant by other methods such as FASTA and Smith-Waterman can be identified as homologous using our new alignment algorithm (unpublished report) through the enhanced information content of our hydrophobicity proclivity scale.\n\n\nApproach\n\nHydrophobicity scales (metrics) as understood in the literature are generally divided into four categories, derived from\n\nExperimental physio-chemical data\n\nLog of a partition coefficient derived from protein structure (e.g. Fraction amino-acids inside vs. outside, fraction amino-acids in contact with water vs. completely buried, etc.)\n\nAmino-acid mutation/substitution rates and\n\nParticipation rates/probabilities of occurrence in folded protein secondary structure\n\nThere are a large number and myriad types of scales that appear in the literature starting from the 1960’s through to the present with a fair amount of variation amongst these scales. The correlation between some of the hydrophobicity scales can be best understood as that derived from the energy of interaction between amino-acids and water or the energetics of partition of amino-acids from water as the reference state and some other environment such as a non-polar solvent or the interior of a folded protein. Hydrophobicity can thus be joined within a single, unified, conceptual framework1,2 Through extensive analysis (primarily using regression and scatter plots), we were able to identify patterns and arrive at metrics describing amino acid properties. We derived a number of additional metrics by differentiating metrics that were intrinsic as opposed to extrinsic, as understood in thermodynamics. Extensive cross correlation with the primary and derived metrics using regression modelling were undertaken to recover the best and most meaningful hydrophobicity metrics. We relied on several different sources for our analysis. For data on amino acid surface areas, we used Rose et al.3. Amino acid mass information was obtained using the AAINDEX accession number #FASG7601014,5. Amino acid volume data was obtained from Creighton6 Amino acid absolute entropy of formation was from the AAINDEX database using accession number #HUTJ7001024,5.\n\n\nMethods\n\nWe arrived at our hydrophobicity scale after exhaustive analysis which included numerous scatter plots and the running of a number of multiple regressions. The question we were trying to answer was - What was the best hydrophobicity scale, or combination of scales, that best represented the role of the different amino acids in proteins?\n\nWe started by first collecting many hydrophobicity indices and physico-chemical indices from the literature and scatter plotted/regressed the hydrophobicity indices against each other, and the harvested physico-chemical properties and their derived intrinsic properties of amino acids. For example when a hydrophobic scale is plotted against the ratio of the surface area per specific volume (volume/molecular weight) of each amino acid we get a scatter plot with a distinct pattern. In such a scatter plot, we can identify one or more sets of linear clusters of amino acids, each set of which is considered to be a “property class”.\n\nConsider Figure 1 where our normalized average hydrophobicity index is scatter plotted against the area per specific volume of each amino acid (shown using their alphabetical representations).\n\nWe can clearly see cross-hatched patterns where for example the amino acids G, A, C, V, I and L are on a straight line (starting from the top left to bottom right). Moving right, we see that S, P, T, M and F are on a straight line (nearly parallel to the line formed by G,A,C,V and I). Continuing further right, we see a third line which crosses several amino acid, followed by an outlier, amino acid R. This series of four lines form what we call Property Class 1. We assign a numerical value of 0 to the line through G,A,C,V and I and a value of 1 to the next line and so on. In the same Figure 1 we can see the formation of Property Class 2 which contains only two series. We arrived at Property Class 3 and Property Class 4 by scatter plotting our normalized average hydrophobicity index against specific absolute entropy (and this is shown in Figure 2) The four property classes we identified respectively in the scatter plots shown as Figure 1 and Figure 2, along with the respective X axes physico-chemical property, correlated very highly (as multiple linear regression factors) with our normalized average of three robust hydrophobic indices’s (shown as ave3H) having an R squared >95%.\n\nProperty class #5 reflects a scatter plot between the delta G of burial of AA secondary groups7 (as Y) and the number of atoms in the respective secondary group6, which resulted in 5 linear series. Each of the linear series numbers (0 through 4) for each AA forms the basis of property class #5. The multiple linear regression of the delta G of secondary group burial with number of secondary group atoms and property class #5 resulted in an R2 of 98.1%. Property classes #6, #7 and #8 were derived from 49 fundamental amino-acid properties and derived scales that are based upon an analysis with Analysis of Patterns (ANOPA)8. Together PC #1 to #8 represents 8 X vectors (listed in Table 3) in the multiple linear regression reported in the third column of Table 2. The property class index vectors are shown in Table 3.\n\nWe were able to find three hydrophobicity scales that were the most robust from the regression cross correlation study. The hydrophobicity proclivitity scale that we report in the present paper are the normalized average of three normalized scales2,9,10.\n\nOur hydrophobic index is the result of an extensive mining of the literature about proteins and amino acid scales/metrics in different environments. Almost all hydrophobicity scales reflect in some way a measure of the energetics of transfer of an amino-acid (or proteins) from one solvent environment (water) to another (folded protein or multiple protein assembly). During our data mining and analysis, three hydrophobicity metrics emerged as the most appropriate since we could relate those scales to multiple fundamental properties of the 20 natural amino acids using multi-variate statistical procedures, thermodynamics and biophysical chemistry considerations2,9,10. Hydrophobicity scales reflect different physical properties of amino-acids, such as metrics derived from amino-acid partitioning patterns (e.g. from the hydrophobic core to the exterior of proteins) or log of partition ratios between water and organic solvents. We found, as widely suggested in the literature, that the free energy of transfer from water to octanol turns out to be a good proxy for the hydrophobic core environment of folded proteins.\n\nWe created a normalized average of the three key hydrophobicity scales (The index i=1 is from Tang2, index i=2 is from Neumaier9 and the index i=3 is from the average of the collected scales in Juretic10). This normalized average of three scales provides a reasonably unbiased estimate of the \"true\" average hydrophobicity relationship amongst the 20 amino-acids (index j, from 1 to 20)\n\n\n\n\n\nThe hydrophobicity scale as calculated using Equation 2 using the scales published in2,9,10 has a number of interesting relationships with key physico-chemical properties of the amino-acids in proteins. For example, this normalized average of these three best hydrophobicity metrics possesses statistically significant linear correlation with many other reliable hydrophobicity metrics derived from multiple literature hydrophobicity scales.\n\nAn example scale, derived from an analysis of 28 literature hydrophobicity metrics, possesses a strong linear relationship (R2 = 0.959) with our normalized average of three hydrophobicity scales, that forms a hydrophobicity proclivity scale, has been published in 1.\n\n\nResults\n\nHydrophobicity scales are typically derived from a measure of the probability that a particular residue will be buried in the core of the protein, away from water. What confounds these calculations is the fact that in most proteins, many of the hydrophobic residues are still exposed to the water (solvent). It is often not clear on how to treat residues that have properties intermediate between hard core hydrophobic and polar residues. The size of the residues and difference between alkyl and aromatic residues also pose some difficulty in the calculation of a hydrophobicity scale. Calculations involving cysteine residues add additional complexity in that some of those residues may be involved in providing proteins structural stability through formation of disulfide bonds. Thus, calculation of contributions to any hydrophobicity index through analysis of where specific residues are in a given protein has been complicated and contributed to the scatter we see in the data. We demonstrate this by examining the normalized average of several popular hydrophobicity scales11–16 versus the probability of an amino-acid solvent-exposed area (SEA)17,18 greater that 30 (shown in Figure 3)\n\nFigure 3 shows that there is indeed a relationship between the hydrophobicity scale and whether or not a particular amino acid is within a protein core or exposed on the surface. We see one tight grouping of amino acids in the figure (I, F, V, L, M, W, A and G) and two loose groupings that include P, T, S, Y, H and N, Q, E, D, K and R. The group at the top right (N, Q, E, D, K and R) include amino acids that are ionic/strongly polar and the central group of amino acids are of intermediate polarity. The tight group of amino acids are primarily amino acids with hydrophobic residues. As we go from the very hydrophobic group to the less hydrophobic group (from the lower left to the top right) the scatter goes up. This scatter is indicative of the increase in water amino acid interaction and of the difficulty of accurately calculating the contribution of any particular residue.\n\nIn Figure 4 we show a scatter plot of our amino-acid hydrophobicity proclivities against the popular Fauchere & Pliska free energy of amino-acid transfer from n-Octanol to water (Gtow) scale7,19. It is common in the literature to see n-Octanol used as a proxy for the typical hydrophobic core of folded globular proteins, consequently the Gtow scale has been widely used as a measure of hydrophobicity. As can be seen above the correlation is quite good at 85.9% linearity (coefficient of determination). The regression of these two scales is used to derive a fitted free energy of transfer and reported in Table 1 and used in our new alignment algorithm. Since Gtow reflects a delta G (energy) of transfer, hydrophobic proclivities can also be seen to relate directly to energy.\n\nThe reasonableness of our hydrophobicity scale is also demonstrated by examining the relationship between our scale and the mean residue depth (dpx) defined as the distance between the interior of a protein amino-acid and the nearest water molecule in the aqueous shell surrounding the protein20,21. In Figure 5 we show that there is a strong relationship (97% linearity) between the dpx metric and our hydrophobic proclivities. The dpx metric is a straight forward geometrical description of the local protein interior and can be expected to provide similar information to the solvent accessible area and buried surface area metrics. The dpx depth and hydrophobic proclivities correlate with amino-acid/protein properties such as average protein domain size, secondary structure, protein stability, free energy of formation of protein complexes, major literature amino-acid hydrophobicity scales, residue conservation, post-translational modifications like phosphorylation, and hydrogen/deuterium amide proton exchange rates7,20,21.\n\nIn Table 2 we summarize the performance of several of the hydrophobicity scales published in the literature. The hydrophobicity scales shown as rows are compared with four important quality metrics that are either amino acid physico-chemical properties or derived from such properties. The quality of inter scale regressions are shown as R2. The performance of each row scale can be observed relative to the other row scales within each of the four columns, where the higher the R2 the better the performance of the row scale with regards to the column scale. There are 13 rows in Table 2 representing 11 hydrophobicity scales, one solvent exposed area scale and one delta G of transfer from water to an organic solvent (Octanol).\n\nOf the 11 hydrophobicity scales in Table 2, 7 are popular scales in practice, three are the constituent scales of our hydrophobicity proclivity scale and our hydrophobicity proclivity scale. These row choices in Table 2 are to illustrate a close relationship between AA hydrophobicity and the transfer of an amino acid to an organic solvent (n-Octanol, column 2), used as a proxy for the internal environment of a folded protein, as well as to compare AA hydrophobicity with an AA Solvent Exposed Area scale (column 1) also representing a folded protein environment. The high R2 between the row dG of transfer to Octanol and the first column AA Solvent Exposed Area (SEA) scale in Table 2 illustrates the aptness of comparing the dG of AA burial in protein \"solvent\" to a solvent-solvent transfer model between water as the reference state and an organic solvent as the transfered or final state. In Table 2, the inclusion of the row SEA is to illustrate the high R2 with the first column SEA illustrating the consistency of folded protein behaviour in SEA scales derived from different data sets. With the Rose AA percent buried row hydrophobicity scale3, simlar lessons can be gleaned as with the row Octanol and SEA scales, as the Rose scale represents the environment of a folded protein. The very high R2 between these three row scales and the last two column regression scales in Table 2 illustrate a strong justification for including these row scales, as protein folding is thereby strongly linked with other physico-chemical properties of amino-acids, as reflected by these two columns. We describe the regression X variables in the 4 columns of Table 2) below.\n\nWe can see that the correlation between our hydrophobicity scale (shown as avg 3H in Table 2) and the Moelbert average amino-acid solvent Accessible Surface Area (ASA) within proteins has an R2 = 84.7%. The ASA is the average area of each amino acid exposed to water in the globular proteins. When our hydrophobicity proclivity scale approaches 1 (i.e. hydrophilic) the ASA goes up as would be expected, with the converse being true as our hydrophobicity scale approaches 0 (i.e. hydrophobic) the ASA goes down22.\n\nThe amino-acid Accessible Surface Area (ASA) has long been suggested as a reasonably accurate proxy for hydrophobicity7,17,22 as is also seen in a related scale, the Solvent Exposed Area > 30 square angstroms17,18. The amino-acid property classes are vector sets of clusters/linear families of curves in multiple linear regression relationships between two (or more) amino-acid physico-chemical properties. The first two columns (ASA and Gtow) represent paired variable linear regressions and the third column (Property Classes #1 to #8) and fourth column (Property Class #1 to #4, AA area/specific volume6 and specific absolute entropy4,5) represent multiple linear regressions.\n\nThe R2 in the first two columns of Table 2 represent linear regression results between the Y (row) vectors and the X (column) vectors. The R2 in the last two columns of Table 2 derive from multiple linear regressions, where the independent (X) variables are vectors of amino-acid property Classes (PC) and/or amino-acid physico-chemical properties, and each row parameter is the dependent variable, respectively. Again, the Property Classes can be thought of as distinct subsets of amino-acids representing multiple linear series/clusters (within scatter plots or multiple linear regressions) of amino-acids in reference regressions associated with X variable vectors from some key physico-chemical metrics plotted against the hydrophobicity proclivity vector scale.\n\nIn Table 2, we see that the F and P Gtow scale performs as well (i.e. high R2) as the best of the hydrophobicity scales within columns 1, 2 and 4, thus, further justifying our selection of the Gtow scale as our baseline standard for a free energy of transfer from an aqueous solvent environment to a non-aqueous solvent. The SEA > 30 A2 does as well as the popular hydrophobicity scales in Table 2 and has good correlation with the F and P Gtow scale in column two and thereby establishes a direct link between the F and P Gtow scale and the free energy of burial of amino-acids in proteins and providing strong evidence justifying a solvent-solvent transfer model for protein folding.\n\nThe Tang Q and Neumeier X scales are the top performing individual hydrophobicity scales as seen in the first two column results, followed on average by the Rose scale. The Juretic Avg scale generally performs as well as the five popular hydrophobicity scales in columns one and two, but more importantly it performs better than any other single hydrophobicity scale except for the Tang Q and Neumeirer X scales in columns three and four. Since we consider columns three and four to be a more rigorous test for a robust, high performance hydrophobicity scale, we see the justification for selecting the Tang Q, Neumeirer X and Juretic Avg as the scales from which to prepare our hydrophobicity proclivity (3H) scale. Our hydrophobicity proclivity scale performs basically as well as the best individual hydrophobicity scales in columns one and two, but it is the top performer in columns three and four. No other hydrophobicity scale that we evaluated on average performed as well (i.e. magnitude of R2) in regression comparisons with amino-acid physico-chemical properties as our hydrophobicity proclivity scale.\n\nIn Table 2 column three is the 8 sets of numbers (vectors), dubbed as property classes and are eight X vectors in the multiple linear regression relationships with the R2 shown in the third column. These eight property class vectors can form multi-linear regression fits with very high R2 with a large number of the physico-chemical properties of the of the 20 amino-acids in our accumulated AA physico-chemical property database, thereby serving as proxy’s for these properties. In Table 2 column four, we see four property class vectors (#1-#4) and two AA physico-chemical property vector scales (surface area/specific volume, specific absolute entropy); column four is included to illustrate the method of construction of the eight Property Class (PC) vectors represented by column 3.\n\n\nDiscussion\n\nThe great organizing principle embodied within the hydrophobicity proclivities (and implied by dpx), is that of a neo solvent-solvent partitioning effect, where the energetics of the solvent shell waters are the dominant effect in the energy balance. As with clathrates (ordered aqueous shells), which form spontaneously with hydrophobic molecules, there is a solvent shell of ordered waters that form spontaneously around solvated globular proteins. However, there is a confounding factor in trying to obtain an accurate hydrophobicity proclivity in that even the most hydrophobic residue will have some average solvent exposed area, so it is reasonable to postulate that there is some functional reason for exposure of some grease to the solvent The presence of hydrophobic surface area causes an aqueous clathrate shell to form at that point perhaps effectively becoming part of the folded structure of the folded protein, possibly as a retaining structural element operating through surface tension and putting the interior of the globular protein under pressure.\n\nThe importance of amino-acid hydrophobicity to the structure and function of globular proteins is critical to the function and survival of cells, a reality that is even reflected in the very structure of the standard genetic code. The amino-acid codons are arranged/coded in such a way as to reflect the underlying hydrophobicity of the respective amino-acids. A careful analysis reveals that the genetic code has a built in redundancy through amino-acid hydrophobicity (in addition to codon redundancy) such that point mutations in a codon that yield a different codon tend to result in an amino-acid with similar hydrophobicity. It has been shown that the underlying amino-acid codon structure has a direct relationship with high quality hydrophobicity scales that are published in the literature23.\n\nA legitimate question about the hydrophobic proclivity scale we have described is why our scale is superior to alignment score matrices such as PAM (Point Accepted Mutation)24, BLOSUM (BLOck SUbstitution Matrix)25 or Gonnet26 that continue to be used for multiple protein alignments and database search alignments. There are indeed several practical and theoretical problems with the use of these log odds score matrices for the alignment of divergent protein sequences. For example, BLAST and several of the major multi-sequence alignment programs like Clustal W use particular BLOSUM matrices as the default. BLAST uses BLOSUM62 as the default. Quotes from select papers have been summarized below to more clearly illustrate these problems.\n\nThe substitution matrices used by the alignment programs are generally log of Bayesian probabilities for two amino-acids I and J of the form:\n\nQij=prob(A/B)=prob(I−>J)prob(IandJ)=prob(I−>J)(prob(I)∗prob(J))\n\nThe probability of occurrence of the 20 primary aminoacids is not the same throughout the domain/kingdoms of life, so this mathematical formulation can cause issues for identifying and aligning homologous proteins.\n\nSuperimposed on the log of Bayesian probabilities formalism are evolutionary models derived from Markov stochastic process evolutionary models (PAM), which implies apriori knowledge of the evolutionary amino-acid substitution rates. Necessarily, if one chooses PAM or BLOSUM, one must choose one of the series of matrices that one believes is appropriate for the approximate evolutionary distance between any two protein sequences under analysis. Obviously, this practice can cause an undue restriction if the evolutionary distance is too great within the protein dataset being aligned. The only assumption that we make with hydrophobicity and our new alignment algorithm is that nature will strongly tend to substitute similar amino-acids in order to preserve the overall function and structure of homologous proteins, and that it is possible to define a hydrophobicity distance to define a fuzzy match between any two amino-acids, which is recognized as a “similarity match.”\n\nWe summarize the salient points regarding alignment matrices with quotes from four select literature articles below.\n\n1. “The most common substitution matrices currently used (BLOSUM and PAM) are based on protein sequences with average amino acid distributions, thus they do not represent a fully accurate substitution model for proteins characterized by a biased amino acid composition”27\n\n2. “We have investigated patterns of amino acid substitution among homologous sequences from the three Domains of life and our results show that no single amino acid matrix is optimal for any of the datasets”28\n\n3. “Many phylogenetic inference methods are based on Markov models of sequence evolution. These are usually expressed in terms of a matrix (Q) of instantaneous rates of change but some models of amino acid replacement, most notably the PAM model of Dayhoff and colleagues, were originally published only in terms of time-dependent probability matrices (P(t)). Previously published methods for deriving Q have used eigen-decomposition of an approximation to P(t). We show that the commonly used value of t is too large to ensure convergence of the estimates of elements of Q. We describe two simpler alternative methods for deriving Q from information such as that published by Dayhoff and colleagues.”29\n\n4. These authors note another interesting problem with the residue substitutions rates use in the Q matrix: “Because different local regions such as binding surfaces and the protein interior core experience different selection pressures due to functional or stability constraints, we use our method to estimate the substitution rates of local regions. Our results show that the substitution rates are very different for residues in the buried core and residues on the solvent-exposed surfaces.”30\n\nTomii et al.5 essentially conclude that in the “evolutionary” limit, alignment/mutation matricies reflect the hydrophobicity and amino-acid secondary group size. For example, when the correlation coefficient between a hydrophobicity scale and a amino-acid secondary group size, and the PAM matricies are plotted against the PAM distance, the correlation coefficient monotonically increases from 0.58 at a PAM near zero, to a PAM distance of 200 where the correlation coefficient reaches an asymtotic limit of about 0.735.\n\n\nConclusion\n\nThe amount of information available to an alignment algorithm is essential to its ability to find matching proteins, especially matches with remote homologies where the percentage identity has dropped off to around 20–25%. In this study we have sought to find an optimalhydrophobicity scale that would reflect the real properties of amino-acids within the context of folded proteins. We contend that hydrophobic proclivities transcend mere statistical trends and reflect the functional necessities of globular proteins by amino acid properties according to a solvent-solvent (water → interior of a folded protein) partitioning model. Within this model the primary driving force is that of water-water attractions that exceed water-amino acid attractions. Hydrophobicity is not a force that repels amino acids from water, but rather that water molecules attract each other more. When hydrophobic amino acids are exposed to water, clathrate shells spontaneously form at those areas, creating an anchored aqueous patch of ordered water molecules with surface tension. Thus, the preferred hydrophobicity scale of hydrophobic proclivities as we have described here provides significant new information to alignment algorithms and in particular our TMATCH algorithm (described elsewhere), optimized to work with our hydrophobicity proclivity scale.", "appendix": "Author contributions\n\n\n\nDC arrived at the hydrophobicity index several years ago after an exhaustive look at the literature and through extensive regression analysis of several published values of amino acid properties in proteins and how they may contribute to the structure of proteins in solution. This paper is the result of a long collaborative effort with KC whose interests were also in understanding protein structure and search algorithms in bioinformatics.\n\n\nCompeting interests\n\n\n\nWe declare that there are no competing interests for DC or KKC that have influenced the content of this article.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe (DC and KKC) appreciate our discussions about proteins and their solution structure with Dr. Joseph Ng (Department of Biological Sciences) and Dr. John Shriver (Department of Chemistry) of University of Alabama in Huntsville.\n\n\nReferences\n\nCornette JL, Cease KB, Margalit H, et al.: Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins. J Mol Biol. 1987; 195(3): 659–685. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeane TM, Creevey CJ, Pentony MM, et al.: Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol Biol. 2006; 6: 29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKosiol C, Goldman N: Different versions of the Dayhoff rate matrix. Mol Biol Evol. 2005; 22(2): 193–9. PubMed Abstract \| Publisher Full Text\n\nTseng YY, Liang J: Estimation of amino acid residue substitution rates at local spatial regions and application in protein function inference: a Bayesian Monte Carlo approach. Mol Biol Evol. 2006; 23(2): 421–436. PubMed Abstract \| Publisher Full Text" }	[ { "id": "14648", "date": "11 Jul 2016", "name": "Charles Carter", "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\nPeer Review Oath: I will be an ambassador for open science. I have benefited substantively from open reviews on several previous occasions, so I believe in its value. I will endeavor to be constructive, while at the same time remaining true to my own scientific values.\nReview This manuscript addresses a worthy problem: improving multiple sequence alignment via the use of enhanced amino acid similarity metrics would enhance our ability to draw inferences from sequences of proteins whose structures, were they known would establish homology, but which owing to divergence have unrecognizably homologous sequences. It seems almost certain that we should be able to do a better job at homology searches if more about how amino acid physical chemistry leads to protein structure. It was for this reason that I agreed to review this manuscript based on the abstract. The authors allude to work they have done that demonstrates the value of the new scales they describe here, but there is essentially no coverage of this central question in this manuscript, which is disappointing and detracts substantially from the value of the paper. The device advocated by the authors is a neologism they call a “hydrophobic proclivity index”. This index is the result of statistical modeling from a variety of different scales of what has been called “hydrophobicity” and their derivatives (with respect to which variables is not described) in order to maximize agreement between the scale and calculations of the exposure of each of the twenty amino acids in folded proteins. The resulting presentation is interesting and potentially relevant, but is deficient its citation of the literature, and in results indicating either their methods or the results to which they allude. I conclude that the although the work described is well-motivated, and may lead to better homology searches, it nevertheless suffers from a variety of methodological and conceptual problems that may in the end compromise the work quite seriously. These are summarized below.\nThe data base: The quest for a single “predictor” for the degree to which each amino acid is exposed on average in folded proteins has a long history. The authors have cited just about every previous attempt to correlate the two variables, but have excluded the one set of experimental data representing the actual physical chemistry of the twenty amino acid side chains, the vapor to water and water to cyclohexane distributions of side chain mimics measured and re-measured by Wolfenden’s group 1-5. Wolfenden has argued persuasively that octanol is a very unsatisfactory reference solvent for a variety of reasons, in part because of the ability of side chains to bring variable amounts of bound water into it from aqueous solution. Omitting the Wolfenden free energies is a grave oversight, because it means that the regression analyses they describe are looking for signal in a variety of data sets that have already been corrupted by similar unsuccessful attempts by previous investigators who have kludged the extant variety of multiple scales. For that reason, any useful result the present authors may have achieved is likely to be idiosyncratic and only indirectly based on physical chemistry. Moreover, the authors provide no evidence of statistical tests that might suggest significance, and the correlations they describe, some of which are more impressive than others, are very likely to be successful only in proportion to the number of parameters from which their models are built and, I suspect, of somewhat circular logic.\nRelating protein structure to amino acid physical chemistry is very probably multi-dimensional. It is very probable that the inability of previous researchers to arrive at a single scale that predicts the accessible surface area in folded proteins arises because the problem itself is multi-dimensional. The authors describe a variety of classification schemes derived from attempts to rationalize scatterplots of amino acid properties. Indeed, they mention that one useful additional classification is likely related to the size of the side chain.\nRecommendation: The authors should read carefully the papers from Wolfenden and Carter 1,6,7 in which those authors describe first the correlation between the free energies of vapor to water distribution coefficients and amino acid side chain volume, and second, their success in predicting Moelbert’s accessible surface areas using a two-dimensional coordinate system one axis of which is the free energies, respectively, of water to cyclohexane and vapor to cyclohexane partition coefficients.\nIn conclusion, what might be of interest in this paper is the TMATCH algorithm and the improvements it brings to homology searches. That is not described at all. Instead, there are a variety descriptions of how a multitude of idiosyncratic hydrophobiciy scales describing amino acid physical chemistry, notably excluding the (only) authentic ones, might be combined into one that predicts exposed accessible surface area by an algorithm that essentially produces a linear combination that is correlated with ASA by hidden, but nevertheless circular reasoning.", "responses": [ { "c_id": "5857", "date": "15 Oct 2020", "name": "David Cavanaugh", "role": "Author Response", "response": "Charlie Carter \"... We should be able to do a better job at homology searches if more about how amino-acid physical chemistry leads to protein structure ... the authors allude to work they have done that demonstrates the value of the new scales they describe here, but there is essentially no coverage of this central question in this manuscript, which is disappointing and detracts substantially from the value of the paper\" This original work is described in 3 manuscripts, including this present paper. The other two manuscripts (theory and applications/results) describing the TMATCH alignment algorithm are almost ready for publication. These three manuscripts were all part of a single monograph that we had decided to partition up owing to length and the fact that the physical chemistry considerations of the current manuscript would be inappropriate for a bioinformatics journal and we needed to get this manuscript published to support another published paper (also available for review). We will incorporate into the present manuscript an appropriate level description of the TMATCH algorithm and actual results from these two manuscripts A fourth manuscripts which goes significantly more deeply into the biophysics and thermodynamics of hydrophobicity and the broader justification for our hydrophobicity proclivity scale from these two points of view. We would like to provide you a copy of these three other manuscripts, which are in an advanced state of preparation, in answer to a number of your points. We agree with you that more summary material needs to be brought into this manuscript. We point out the excellent linear relationship between our hydrophobicity proclivity scale and the dpx average residue depth (from water) scale in figure 5 of the manuscript. The dpx scale is derived from a suite of proteins with actual solved folded structures, hence dpx is in effect a protein structural description of hydrophobicity where the deeper on average that a residue is buried, the more hydrophobic it is. Contrast the dpx concept with the ideas of average solvent exposed are, average buried area, percent buried, etc. When the relationships in figures 3-5 (with supporting literature) are taken together, the hydrophobicity potential driving protein folding is suggested to be the traditional solvent-solvent partitioning model of protein folding where amino-acids partition between aqueous exposure and burial within the hydrophobic core of folded proteins. We believe that this traditional model of the primary driving force for globular protein folding is apt, but needs some updates and modifications that we develop in detail in the “Hydrophobicity revisited: A Molecular Story” manuscript. We suggest in the present manuscript that aqueous clathrates form about the hydrophobic area of amino-acids in an unfolded state (a structural feature) and hydrophobic surface area on folded proteins possess a surface tension like a gas bubble in water. We are suggesting that the physics driving the initial stages of protein folding is the same physics as the coalescing/condensing of bubbles of non-polar species or gas in water are essentially the same physics which cause the mutual attraction of residues based upon the residue hydrophobic area with associated aqueous clathrate. We expand upon this hypothesis in “Hydrophobicity Revisited: A Molecular Story.” Our aim was to try to develop a scale which reliably represented the central hydrophobic tendency (average) as a central/first order effect that allowed a simple but meaningful paired comparison of amino-acids for homology relationships. Adding additional variables/properties would in our opinion have detracted from our goals of simplicity of calculation and utilization of what we believe is the primary first order effect driving protein folding. \"... this index is the result of statistical modeling from a variety of different scales of what has been called 'hydrophobicity' and derivatives ... derivative variables not described ... presentation is interesting and potentially relevant, but is deficient in its citation of the literature and in results indicating their methods or results to which they allude ... well motivated ... suffers from a variety of methodological and conceptual problems ...\" We agree that an expansion of what we meant by derivative variables should be included in the manuscript The derivative variables concept referred to in the manuscript are primarily ratios of molecular properties or directly measureable bulk properties of amino-acids. In this way we derive normalized intrinsic thermodynamical properties versus extrinsic/bulk properties. For example, we find that the ratio of amino-acid surface area to their volume has a relationship to hydrophobicity. Other derivative variables we used could be exemplified by estimating the water cavity volume/surface area using amino-acid volume and surface areas and some assumptions about the aqueous cavity geometry. We will be expanding our reference list of amino-acid scales utilized to better define the amino-acid property scales we utilized in addition to hydrophobicity scales. We will also include the work of Wolfenden et. al. into the manuscript. \"... the authors have cited just about every effort to correlate a single hydrophobicity predictor with amino-acid surface exposure in folded proteins ... but have excluded an important hydrophobicity metric derived from classical physical chemistry methods by Wolfenden and co-workers using water/vapor and water/cyclohexane partition distributions of amino-acid side chain mimics ... Wolfenden has persuasively argued that n-octanol is a very unsatisfactory reference solvent for multiple reasons such as the ability of side chains to drag non-repeatable amounts of water into the n-Octanol solvent ... \" Since this experimental approach is unique, we will add additional material to the manuscript to incorporate these considerations and the work of Wolfenden et. al. and the implications of the work. We agree that the standardization of water concentration within a polar solvent (immiscible with water) capable of hydrogen bonding is difficult in addition to the uncontrolled/variable amount of water dragged into the organic solvent phase in solvent/water partition experiments. We cover these issues at length in “Hydrophobicity Revisited: A Molecular Story,” but we agree that these considerations should be covered to some extent in the present manuscript. We also point out that solvent/water concentration ratios used to calculate free energy of transfers from water to the non-polar phase as a hydrophobicity measure relevant to protein folding suffer a systemic error if the organic solvent is incapable of hydrogen bonding as is the hydrophobic “solvent like” core of folded globular proteins. Our hydrophobicity proclivity scale has no units, but rather is a normalized proclivity. We do show in figure 4 of the manuscript that our hydrophobicity proclivity scale is directly relatable to free energy of solvent-solvent transfer using a popular dGow (water to n-octanol) scale. We also point out that characterizing the amino-acid secondary groups alone does not treat the important contribution of the free energetics of folding by peptide bonds, such as is accounted for by some researchers by treating the 20 amino-acids as guests in the center of tri-peptides in octanol to water free energy studies. \"... omitting the Wolfenden free energies is a grave oversight ... are looking for signal in a variety of data sets that have already been corrupted by similar unsuccessful attempts by previous investigators who have kludged the extant variety of multiple scales ... any useful result the present authors may have achieved is likely to be idiosyncratic and only indirectly based upon physical chemistry ...\" We will include and discuss the work of Wolfenden et. al. due to its novelty and the additional insight that it provides We largely agree with these statements by Dr. Carter regarding the large profusion of hydrophobicity scales in the literature. Our criticisms are a bit more muted in that we recognize and discuss some of the difficulties in trying to reflect the wide range of amino-acid behaviors and the difficulties inherent in trying to define hydrophobicity as a experimentally measurable concept related to the driving forces of protein folding. We have relied upon other experimentally measured amino-acid properties to cross check our hydrophobicity proclivity scale using methods such as Multiple Linear Regression (MLR) as can be seen in table 3. For example, we can draw a good linear correlation between certain hydrophobicity scales, such as our hydrophobicity proclivity scale, and amino-acid reverse phase (C18 column) HPLC retention times (other researchers have drawn this conclusion as well). \"... The authors provide no evidence of statistical tests that might suggest significance ...\" We will provide both an F test from the MLR software we used and a Student’s T test of the MLR (8 amino-acid property class scales) correlation coefficient R. We will show these results in a new table with amino-acid properties versus the R2, T and F significances. The MLR is significant where both the T test and the F test are significant at an alpha of 0.05 or better (actual alpha = 0.050.05 =0.0025 or better). These statistical significance tests are on the MLR results and not the individual MLR coefficients. \"... the correlations they describe ... are very likely to be successful only in proportion to the number of parameters from which their models are built ... suspect some circular logic ...\" The derivative scales as discussed above were derived from fundamental molecular properties or experimentally measured, so there is no concern on that score. The row scale property relationships with the properties in columns 1, 2 and 4 of table two reflect paired comparisons are independent amino-acid scales. The eight property class scales in column three of table 3 reflected in MLR pair wise comparisons in column two are independent, although 4 of the amino-acid property class scales are also reflected in in columns 1, 2 and 4 of table 3. The other 4 amino-acid property class scales come from separate relationships as described in the manuscript. The last 3 amino-acid property class scales in table 4 derived from an ANOPA analysis are based upon 49 amino-acid property scales, none of which appear in the row property scales, thus, are independent. The 8 property class scales in table 4 result in statistically significant MLR relationships with about 50 amino-acid property scales not included in the ANOPA analysis of 49 amino-acid property scales, but the MLR back check of these 49 amino-acid property scales resulted in most of these 49 amino-acid property scales having statistically significant results. Overall, about 150 amino-acid property scales were evaluated in the work. \"... probable that the inability of previous researchers to arrive at a single scale that predicts the accessible surface area in folded proteins arises because the problem itself is multi-dimensional ... authors describe a variety of classification schemes derived from attempts to rationalize scatterplots of amino acid properties ... authors mention useful additional classification likely related to size of AA side chain ...\" We agree that there are multiple factors involved with the free energetics of protein folding and that the available amino-acid property scales and hydrophobicity scales measure different aspects of the relationship of amino-acids to each other and to water. We believe that we should be able to related fundamental molecular properties of amino-acids and water into a meaningful larger picture. In the “Hydrophobicity Revisited: A Molecular Story” manuscript we demonstrate that the controlling factors for our hydrophobicity proclivity energy scale (regression corrected dGow) are: #H-bonds, polar surface area, non-polar surface area, diameter of the side chain, length of the side chain, residue volume, polar area/volume and surface area/entropy. The MLR regression coefficient of determination is 99.995% and is F test significant at 6.4x10-15. However, the point of the present work is to find a single scale that measures a central tendency of “hydrophobicity” with the assumption that residue hydrophobicity (the contrast with water) is the dominant relationship between amino-acids for the purposes of protein alignments. \"... Read Wolfendson and Carter ... AA free energies of vapor to water partition vs. side chain volume ... Wolfenden & Carter accurately predicts Moelbert ASA with 2D plot/linear regression with AA water/vapor and AA water/cyclohexane partition confidents ...\" Agreed. We will cover this material at an appropriate level of detail given the objective of the present manuscript. \"... need description of TMATCH and homology searches ... use multitude of idiosyncratic hydrophobicity scales describing AA physical chemistry ... notably excluding the only reliable/authentic physical chemistry scale describing AA by Wolfendsen and Carter ... concern that the hydrophobicity proclivity scale produces a linear combination that correlates well with the Moelbert ASA scale by hidden , but still circular reasoning ...\" We will provide the TMATCH theory and applications manuscripts to supplement the material available for review. We will also include an appropriate amount of material from these two TMATCH manuscripts into the current manuscript. The Juretic average, Rose percent buried and Neumaier X hydrophobicity scales are independent of each other and the Moelbert ASA scale, therefore there can be no circular or self-referential reasoning/logic involved. Our point is that these three hydrophobicity scales are the best performers that we found and that a normalized average of these three scales will be a top performer as a robust, central tendency hydrophobicity scale. As seen above from the results in the “Hydrophobicity Revisited: A Molecular Story” manuscript, our hydrophobicity proclivity scale can be partitioned into the sums of distinct amino-acid molecular properties and as such the scale is directly relatable to first principles, which by definition are not circular in nature. We believe we have demonstrated that our hydrophobicity scale embodies the central tendency (i.e. first order effect) of the hydrophobicity phenomenon. We believe that the manuscript improvements requested Dr. Jeroncic will obviate any additional concerns with circular reasoning/analysis. \"Seven references to papers where Wolfendsen and/or Carter are one of the authors\" Agreed. Thanks." } ] }, { "id": "14649", "date": "25 Jul 2016", "name": "Ana Jerončić", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors aimed to develop a hydophobicity scale which optimally reflects properties of amino acids residues/amino acids that are relevant for folded proteins. The long term goal was to use such a scale to estimate hydrophobicity-based protein sequence relatedness from a global alignment in order to improve identification of structural homologs with less than 25% sequence identity.\nThe authors have identified eight properties (so called property classes) of amino acids relevant for folded proteins by recognizing distinct patterns in a series of scatter-plots that plotted many hydrophobicity indices and other physico-chemical properties of amino acids/ amino acid residues against each other. Apparently, different property classes were visually recognized in scatter-plots after the “linear cluster of amino acids”-fingerprint was found (clusters of amino acids with similar physico-chemical properties that are aligned along distinct imaginary lines in a scatter-plot).\nThe final set of plots from which eight property classes were derived included: scatter-plots of the hydrophobicity scale that was developed by the authors versus a) the area per specific volume of each amino acid (property classes 1 and 2) or b) the specific absolute entropy (classes 3 and 4); c) the plot of delta G of burial of amino acid secondary group versus number of atoms in a group (class 5); and finally, ambiguously defined “classes #6, #7 and #8 were derived from 49 fundamental aminoacid properties and derived scales that are based upon an analysis with Analysis of Patterns (ANOPA)”.\nIt is this set of eight property classes that the authors have used as dependent variables in multiple linear regression models (MLR) of candidate hydrophobic scales. The measure of goodness of fit of a MLR model (R2 value) was used to identify optimal hydrophobic scale(s) as according to the authors the MLR’s R2 value represents “rigorous test for a robust, high performance hydrophobicity scale”. The rationale for such assumption was that for all tested hydrophobicity scales, R2 value of MLR models were higher than R2 values of simple regression models using either Moelbert’s average aminoacid solvent Accessible Surface Area (ASA) or Fauchere & Pliska free energy of amino-acid transfer from n-Octanol to water (Gtow) as the dependent variable.\nHowever, I disagree with the authors on the MLR R2 rationale as I have concerns about appropriateness of data analysis (see below). In addition, the reporting in the manuscript should be substantially improved.\nMajor comments\nData analysis Comment 1 Throughout the paper description of MLR models is very confusing and it is not clear what models were actually run (what was the dependent and independent variables; also the estimated coefficients and statistical significance of independent variables were not shown for a single model). As already stated, it seems that MLR models were mainly used to model different hydrophobic scales using eight property classes as independent variables. However on the page 7 the authors state “The amino-acid property classes are vector sets of clusters/linear families of curves in multiple linear regression relationships between two (or more) amino-acid physico-chemical properties.” which is very confusing – it seems that in addition to eight property classes, a few additional independent variables (amino-acid physico-chemical properties) were also included in a model or it was the multivariate regression model that was used (if yes – what were dependent variables)?\n\nIf indeed the authors used MLR models as claimed, this means that for a model with 20 observations (20 amino acids) at least eight independent variables were included in a model. Moreover, as majority of these property classes were actually ordinal variables, the number of independent variables included in MLR model should have been even higher (due to introduction of dummy variables). Consequently the sample size of these models was far too small to estimate model’s parameters precisely, and the reported R2 was actually quite inflated (due to an overfitted model but also a large number of independent variables that the authors did not adjusted for when comparing simple linear regression models and MLR ). In addition, there was a problem of multicollinearity between independent variables which additionally inflated MLR’s R2 (based on the Table 2 Kendall tau coefficient between i.e. PC2 and PC4 is 0.82, P<0.001). Therefore the main result of this paper which is based on assumption that the property classes of amino acid residues identified through ‘linear-clusters’ represent “real properties of amino-acids within context of folded proteins” is not based on validated assumption.\n\nComment 2 The authors have used a measure of goodness of fit of a regression model, R2 to compare strength of linear relationship(s) modelled in different regression models (including simple and multiple linear regression models). However, R2 is an overused statistics for linear regression analysis and additional metrics are required to get the whole picture. In particular, it is a Pearson correlation coefficient between paired data (i.e. two hydrophobicity scales) that quantifies the degree to which two variables are related and is a proper statistical measure of the strength of a linear relationship. Linear regression models find the best-fit line that predicts dependent variable from independent variable(s) with R2 actually representing squared Pearson correlation of the fitted values and the observed values.\nReporting Comment 3 A reader should know precisely which scatter plots were screened for “linear-cluster” pattern. This means that the entire set of hydrophobic scales and other physicochemical properties of amino acid residues/amino acids that were collected from the literature and used for generation of these plots should be listed in the paper. Also, it should be specified how many scatter plots were finally generated (in example: N(N-1)/2 where N – number of hydrophobic scales or physicochemical amino acid properties that were collected, ...).\n\nComment 4 Since the eight property classes of amino acid residues are the most important novelty of this paper, the process of their identification should be clearly described in a sufficient detail. In particular:\nWhat was the reasoning behind the assumption that the property classes of amino acid residues identified through ‘linear-clusters’ represent “real properties of amino-acids within context of folded proteins”. Or there was no assumption and the fact that the regression models of all hydrophobicity scales exhibited the highest R2 values when these property classes were used as independent variables actually justified such interpretation. If latter was the case, such reasoning would not be justified (see comments on multiple linear regression analysis) The authors should describe the method they used to identify linear clusters on a plot (i.e. visual identification, followed by analysis of amino acid physicochemical/biochemical properties in clusters and regression-analysis of clusters that confirmed the cluster status or something else) How did the authors end up with the final set of 6 (or 3?) scatter plots from which they have derived their property classes? Were “linear-clusters” identified only in these plots or did the authors select the final plots based on relevance of plotted variables in folded proteins. If latter was true – what was the criteria they used to identify the most relevant scatter-plots All property classes including the classes #6, #7, and #8 should be precisely defined. The description “classes #6, #7 and #8 were derived from 49 fundamental amino acid properties and derived scales that are based upon an analysis with Analysis of Patterns (ANOPA)” is unacceptable. Which of 49 fundamental amino acid properties and their derived scales were used, and how, to identify property classes from #6 to #8. Scatter-plots that were used for generation of classes from 5 to 8 should be shown.\n5 - Comments on the reporting style\nThe Introduction section is quite short – the authors should elaborate more on relevant physico-chemical properties of amino acids and their importance in protein folding in this section. There are parts of the Introduction in the Results section (the first paragraph) and the Discussion section (alignment matrices). The hydrophobic scale that was chosen as the optimal one was normalized average of three published hydrophobicity scales that were found “most robust in correlation analyses” with robustness vaguely defined in the Methods section as associations to “multiple fundamental properties of the 20 natural amino acids using multi-variate statistical procedures, thermodynamics and biophysical chemistry considerations”. It is just latter, at the very end of the Results section that one can find out that “robust” scales are actually those whose MLR models using property classes as dependent variables exhibited highest R2 values. The Methods section should be written more clearly. Table 2 – The labelling of Table 2 should be improved as authors keep explaining what is presented in which column of the Table 2 throughout the Results section.", "responses": [ { "c_id": "5858", "date": "15 Oct 2020", "name": "David Cavanaugh", "role": "Author Response", "response": "Ana Jeroncic \"... Ambiguously defined classes #6, #7 and #8 were derived from 49 fundamental amino-acid properties and derived scales that are based upon an analysis with Analysis of Patterns (ANOPA) ...\" The 49 AA properties are represented by: 2 sequence frequency scales 6 secondary structure propensity scales 8 hydrophobicity scales 4 free energy scales (in water, protein folding/unfolding) 9 HPLC retention time scales 4 probability of an AA inside a folded protein core or on the outside 7 molecular property scales 9 physical (measured) property scales The ANOPA method is a pattern recognition, pattern projection method. The ANOPA procedure projects n-space pattern point/vectors into a 3 dimensional object which is a cylinder. The axis of the cylinder, which is termed the relation vector, is formed from the pattern point centroid (averages of each AA property for all points) to an outgroup average point (averages of each AA property for a pair of selected points). The outgroup pair are selected on the basis of a histogram of the pattern point Euclidian distances from the pattern point centroid. Two pattern point distances are calculated with respect to the relation vector from a projection of each point onto the relation vector yielding the distance along the relation vector and the distance from the relation vector. The angle of rotation of each pattern point projection onto the relation vector is calculated. Thus, a cylindrical coordinate system is formed which is then converted into rectilinear coordinates. The X’ and Y’ rectilinear points are formed from each pattern point’s pattern projection distance times the cosine and sine respectively of the pattern projection angle of rotation. The Z’ point is simply the pattern projection intersection distance along the relation vector. There is a relatively flat and linear structure to the higher order correlation structure in the 49 AA property pattern space. The 3D ANOPA X’ coordinate has a strong linear relationship (R2=95.13%) with the angle of rotation Ar pattern point/vector relation vector projection vectors. Similarly, the 3D ANOPA Y’ coordinate has a strong linear correlation (R2=99.86%) with the d2 distances of the pattern points/vectors from the relation vector. Each of the 3D ANOPA coordinates have meaningful interpretations/associations with amino-acid properties. The X’ coordinates have 3 good linear series when scatter plotted with AA refractivity [179] and AA mass/(area/volume) [192], and it has 4 linear series with the Tanford hydrophobicity scale [197]. The AA refractivity scale has a strong linear relationship with the AA mass/(area/volume) derived scale, except that Glycine is off the regression line. The Y’ coordinates have 2 linear series when scatter plotted against the DNA/RNA numerically encoded (0-63) lexical table (UCAG, UCTG) averages and 3 linear series when scatter plotted against the AA property derived scale 2H-B-DB. In the latter scale H is our hydrophobicity proclivity scales, B is a beta sheet proclivity scale derived from several published statistical scales and DB is the double bend proclivity scale derived from several literature statistical scales. The 2H-B-DB scale reliably predicts (a number of proteins were sampled) the presence (or propensity) of alpha helices (value>0) and beta sheets (value<0) with sinusoidal trends (running average of 3) as seen with the primary sequence AA ordinal numbers plotted along the X axis. Since the Y’ coordinate has a strong relationship with the 2H-B-DB scale, which itself has a strong relationship with protein secondary structure we see that the Y’ coordinate also has a relationship with protein secondary structure. We also can infer that there is a definite relationship between protein secondary structure and the DNA code, which in itself is related to AA hydrophobicity that we have documented in the hydrophobicity paper and the fact that the secondary structure proclivity scale by definition is related to hydrophobicity. Finally, the 3D ANOPA coordinate Z’ strongly correlates with all of the AA hydrophobicity scales (and some of the AA HPLC scales) in our database, both those used in the 49 property ANOPA analysis and those that were not used in this analysis. \"... MLR R2 value represents a rigorous test for a robust, high performance hydrophobicity scale ... The rationale for such an assumption was that for all tested hydrophobicity scales, the R2 values of the Multiple Linear Regression (MLR) were higher than the R2 values of the simple binary regression models ... I disagree with the authors on the MLR R2 rationale as I have concerns about the appropriateness of the data analysis\" The argument presented is not that the MLR regression was superior to that of the binary regression because of higher correlation coefficients, although possibly that may be true, but rather that the AA property class MLR represented more information in that it represented the behaviors of amino-acids in a larger series of contexts and properties. Also, the argument implied by having a MLR with the 8 property class scales is that for each context the amino-acids partition into sub-sets and that these different context sub-sets join together to determine amino-acid behavior in protein folding, interactions with water, interactions with membranes, secondary structure and electronic behaviors associated with AA-AA interactions. Moreover, we argue that within any given regression that the higher the correlation coefficient, the stronger the evidence for the superiority of a given hydrophobicity scale within that regression criterion. Top performance of a hydrophobicity scale within several regression relationship criterions based upon different property information leads to a performance/robustness conclusion based upon the consilience of the evidence. We use the coefficient of determination (R2) rather than the correlation coefficient because it is a much more conservative statistic than the correlation coefficient (R) and can meaningfully be interpreted as the percent linearity between the dependent variable and the X independent variable(s) in the regression. Concerns might be raised about two points in the property class MLR, which are the possibility of inter-correlation between X variables and the loss of degrees of freedom in the regression statistics (over-fitting). We deal with these concerns in several fashions. First off, we calculate a T test significance only for the regression itself and not for any individual X variable regression coefficient since the Y variable regression performance itself is what is being measured. Secondly, the correlation coefficient T test (null hypothesis being that R=0) number of degrees of freedom (20) is reduce by 2 for each X variable coefficient in the regression and by 1 for the Y axis intercept. In the AA property class MLR we calculate the resulting degrees of freedom in the regression and correlation coefficient T test as 20-(28+1) =3. We allow for some dimensionality reduction due to inter-X pair correlation as long as the dimensionality reduction (i.e. percent reduction in the number of states with discrete variables) is not large and new information is being brought forth between each pair of X variables. Where there is no new information adduced by inclusion of a X variable we find that the magnitude of the correlation coefficients are reduced and significantly reduced by the squaring process to get the coefficient of determination. Furthermore, we demand that any set of property class vectors produce large significant coefficient of determinations with a large number of disparate AA property scales in our database. The latter criterion produces a very, very high degree of statistical confidence that the collection of property class scales can serve as a highly robust basis set for MLR vector regressions that can evaluate the robustness/significance of individual AA property scales. \"... the reporting in the manuscript should be substantially improved ...\" We agree and are revising the manuscript accordingly. \"Throughout the paper the description of the MLR models is very confusing ... it is not clear what models were actually run ... what was the dependent and independent variables ... what was the estimated regression coefficients and their statistical significance ...\" We have addressed these comments to some extent above. Additionally we will say that we will move all of the related discussion of regression methodology into the methods section where the discussion will make more sense. The key point is that the MLR X’s are not single variables, but rather are column vectors with each column vector having values for each amino-acid. In this context we equate the concept of a scale with the concept of a column vector. Having made this distinction, the concept of a Multi-Linear Regression (MLR) is still valid and simply represents a broader mathematical context. Our use of the MLR methodology with the 8 property class scales is to evaluate a correlation relationship as whole, so the entire regression correlation is what is used/important and the statistical significance of individual variable regression coefficients is meaningless. If the MLR method was being used to fit a variable for the purpose of extrapolating new Y values with scale values outside of the basis set, such as with new amino-acids, then the statistical significance of variable coefficients would be germane. \"eight property classes ... vector sets of clusters/linear families of curves in multiple linear regression relationships between two (or more) amino-acid physico-chemical properties .. which is very confusing ... seems like more independent variables are being added than the eight property class variables ...\" One must keep in mind that the MLR method that we are using uses vectors (scales) of dimensionality of 20 (one for each AA) and not individual variables. When doing AA property scale pair scatter plots between each property scale selected for our database we often find clustering behavior which indicates that the amino-acids are partitioning into distinct sub-sets and each amino-acid can be assigned a numerical value for the ordinal number of the sub-set into which it partitions. The clustering relationships uncovered for any particular pair of two AA property scales is generally driven by molecular geometry, secondary group geometry, numbers and types of atoms/inter-atomic bonding in the secondary group, molecular size, molecular mass, molecular volume, molecular surface area and entropy distribution about the molecule. No given pair of AA property scale relationships represent the full range of these molecular properties and the nature of the interaction with water and cellular membranes. The key point though is that there are distinct sets and sub-sets, which when numerically encoded can jointly describe each amino-acid as a row vector of some dimensionality M. We have found through extensive analysis that M is a very good size. Within each property pair scatterplot where clustering occurs, we can have different patterns such as unstructured clusters, multiple quasi-parallel linear clusters or multiple linear clusters that intersect at some point (often at glycine or alanine). Generally, there is a geometric ordering that allows the assignment of a meaningful ordinal number. To reiterate, a property class scale (column vector) represents a relationship between property scales and the values assigned within the scale to each amino-acid are their respective sub-set/cluster ordinal number. Property class scales assembled in this way can be used as one of the basis set vectors to be used to evaluate the performance and reliability of any individual amino-acid property scale. We also need to note that the three property class vectors defined between the 3D ANOPA coordinate system planes represent the correlation based dimensionality reduction from a pattern space of dimension 49 to a pattern space of dimension 3. \"... majority of these property classes were actually ordinal variables ... more actual variables should have been added owing to the introduction of dummy variables ... sample size of these models too small to estimate model parameters precisely ... \" In fact all of the numbers within the 8 property class scales are ordinal numbers representing real facts of clustering and the distinct geometrical ordering of the clusters. The practice of using assigned numbers to sets/sub-sets of objects, especially if the numbers can represent some form of ordering, is widely used in multi-variate statistical procedures such as MLR regression, discriminant analysis, Principal Component Analysis (PCA)and procedures cognate to PCA, as is found in fielids such as numerical taxonomy, computational biology and other fields that numerically encode state data. The authors have enjoyed great success in using discrete data to represent state data in a number of contexts over a number of years. Since the MLR regressions are not used for predictive purposes, but rather to express the degree of correlation and relatedness as expressed in the correlation coefficient and its T test based P value, the concept if regression coefficient statistical significance is a non-sequitur. \"... R2 quite inflated ... over fitted model ... large number of independent variables that the authors did not adjust for compared to simple linear binary \"regression models ...\" The authors would point out that additional variables do not inflate R2 values unless there is a very serious inter-variable correlation between a large sub-set of the X variables. To the contrary, we find that working with the amino-acid property scales that we have assembled that there is generally an artificial deflation of R2 relationships. Therefore, we do not agree that the introduction of variables into an MLR artificially inflates a MLR correlation versus a binary regression correlation. We adjust for these concerns in three ways: use of the coefficient of determination statistic is very conservative and we screen out a number of potentially significant relationships thereby; we use a T test to assess the statistical significance of the whole regression relationship and reduce the number of degrees of freedom of the T test accordingly as described above; we uncover the specific physical meanings reflected in each property scale relationship cluster to ensure that the physical-chemical factors at play are distinct and bring new information to the table. \"... problem of multicollinearity between independent variables ... inflated MLR R2 ... table 2 PC2 and PC4 had a Kendall tau coefficient of 0.82, P<0.001\" We note that property classes 1 and 2 devolve from the relationship between the hydrophobicity proclivity scale and specific absolute entropy and that property classes 3 and 4 devolve from the relationship between the hydrophobicity proclivity scale and the specific volume as seen in figures 2 and 1 respectively. Both the specific absolute entropy and the specific volume are thermodynamic intrinsic property scales formed by the normalization of the amino-acid molecular volumes and molecular absolute entropy by their molecular masses, which create two secondary or derived scales from fundamental molecular properties that have the molecular mass in common. Furthermore, both the total entropy and the volume are related in some way to the molecular mass, so dividing by the molecular mass in both cases offset that dependence and leaves an intrinsic average property. Property class 2 partitions into two linear subgroups on the basis of polar and non-polar amino-acids. Property class 4 partitions into 4 linear subgroups on the basis of size, relative amounts of non-polar area and the presence/absence of a relatively strong or weak polar group. While property class 4 does reflect the polar/non-polar distinction to some extent, it does not reflect a strong and clear partition of AA by polarity or non-polarity, therefore new information is introduced by the addition of property class 4. We note that there are 2*4 possible combinations of states between property classes 2 and 4, but in fact there are actually 5 states formed giving a dimensionality reduction of 1-5/8 =37.5%, which we consider to be acceptable. We do not see strong enhancement of MLR correlation coefficients with any statistically significant AA property regression with the 8 property class scales. Given the nature of the interrelationship of fundamental molecular properties based upon AA molecular size, molecular geometry, entropy/entropy density, mass/mass density, surface area or electronic configuration/hybridization, some inter-property class correlation is inevitable and must be tolerated to make any progress in understanding amino-acid physico-chemical properties and protein folding. See the additional relevant discussion above. \"... main result of this paper ... based upon assumption that amino acid property classes forming linear clusters ... within context of folded proteins ... is not a valid assumption\" We agree that the 8 property class scales are one of the major results of this study, but we do not agree that it is the only one. We do not assume that the 8 amino-acid property class scales are a major determinate of protein folding per se, but rather we assume the fundamental molecular properties of each amino-acid in the context of a folded protein environment or the context of contact with a water environment are what determine protein folding. The assemblage of the 8 amino-acid property class scales that we report reflects a wide spread clustering pattern (sometimes in linear series) in 2D scatterplots and 3D ANOPA scatterplots between two or more scales. The 8 amino-acid property class scales we have assembled for our present study reflect the best of the clustering patterns forming unique subsets of amino-acids in different contexts/molecular properties with regard to hydrophobicity scales, but also with a number of other amino-acid physico-chemical property scales, secondary structure statistical propensity scales and molecular property scales. To the extent that any given hydrophobicity scales correlate with, and were determined from folded proteins, reflecting secondary/super-secondary structure of folded proteins, then the 8 property classes that we report are relevant to folded proteins. The 8 property classes that we report have statistically significant (P<0.05) MLR regressions with 124 amino-acid property scales in our assembled database, which implies that the amino-acid clustering behaviors reflected in these numerical scales can largely be reproduced with the clustering behaviors of the amino-acids. While the idea of linking of hydrophobicity to protein folding is an important idea, the finding that nature has selected the 20 natural amino-acids on the basis of distinct sub-sets/clustering in several different joint property dimensions is a significant finding in and of itself, perhaps leading to criteria for the engineered selection of un-natural amino-acids that could provide unique properties to active enzymes/proteins. \"... R2 is an overused statistic for linear regression analysis and additional metrics are required to get the whole picture ...R2 actually represents the square of the Pearson correlation coefficient\" We do not agree with the contention that the R2 statistic is trivial and meaningless from overuse. Rather we point out that R2 is a more conservative statistic than is the correlation coefficient and rolls off much more quickly, as well as having an important interpretation as the percent linearity that directly indicates the relative scatter of the regression calculated Y’s with respect to the actual Y’s. \"... A reader should know precisely which scatter plots were screened for a 'linear-cluster' pattern ... which hydrophobic scales and other physico-chemical properties of amino-acids were used for the scatter plots ... how many scales used\" There were approximately 175 amino-acid property scales used for our study, of which maybe 15-20 were derivative scales prepared from ratios of fundamental molecular properties such as volume and mass, which would represent intrinsic versus extrinsic scales in the thermodynamical sense. About another 10-15 scales represent averages of literature reported amino-acid property scales. Please note that linear clusters were not the other patterns seen. There were some non-linear patterns and simple clusters for example. Also note that the scales selected represent a very wide range of amino-acid properties including secondary structure statistical propensity scales, fundamental molecular properties like surface area/mass/volume, bulk properties such as index of refraction/melting point/pK-C, HPLC retention times, free energy in water, solvent/water partitioning, average fraction occurring in proteins, average amino-acid burial in proteins/amino-acid exposure wot water in folded proteins, hydrophobicity scales, NMR parameters, Rf parameters, several literature Principal Component Analysis/Factor Analysis parametric scales and other miscellaneous property scales. Of the 175 amino-acid property scales (and about ~200 total scales) 124 scales had statistically significant (P<0.05) MLR regression correlation with the 8 property classes that we have reported. \"... the eight amino-acid property classes most important novelty of this paper ... what is the reasoning that AA property classes represent real AA physico-chemical properties in the context of folded proteins ... rather was the assumption that high R2 from MLR justifies assertion of relatedness to physico-chemical properties ... a high R2 not a valid basis for assumption ...\" We agree that the 8 amino-acid property scales are novel and an important finding. We disagree with the comments on the MLR regression correlation and assert that high MLR R2 values are both statistically and physically valid measures for reasons discussed above. The high and statistically significant coefficients of determinations are spread over many types of amino-acid physico-chemical property scales, including structural statistical propensity scales and hydrophobicity scales. We find that there is a strong relationship between amino-acid HPLC retention times and a number of the higher quality hydrophobicity scales in our assembled database, hence there is a direct link between a measureable amino-acid bulk property and amino-acid partitioning behavior in the structure of folded proteins. \"... the authors should describe the method that they used to identify linear clusters on a plot ... follow up analysis of physico-chemical/biochemical properties of clusters ... regression analysis ...\" We did describe most of the points commented here in the original manuscripts, but agree that the descriptions need to be expanded upon. There were a number of linear clustering patterns, amongst other patterns, that can be described as multiple quasi-parallel series, multi-linear series that intersect at a given amino-acid serving as a quasi or virtual origin (e.g. Karplus 1997), quasi-parallel cross hatched linear patterns (e.g. figures 1 & 2 in the manuscript). We ran, although we did not report a regression correlation coefficient on each series to assess its relative linearity. Where we found what we discerned as significant relationships, we evaluated those putative relationships from the point of view of physical reality as expressed by fundamental molecular properties and potential relationships with water, especially with aqueous clathrate membranes. \" ... how did the authors end up with the final set of scatter plots from which AA property classes were derived ... were they linear clusters ... did authors select scatterplots on basis of plotted variable relevance ... what was the criteria used to select most relevant scatterplots ...\" Our primary and starting premise was to look for single linear or non-linear patterns in all amino-acids in the scatterplot of any two given scales. Along the way we found that multiple linear series occurring in scatterplots between many pairs of amino-acid properties, which was a pattern we could not ignore. Many of these multi-family linear series ranged between clearly discernable to very high quality; the latter end of the range being what we concentrated on. We also cross checked to see if sister AA property scales in the same class such as hydrophobicity resulted in similar patterns, such as which we see in figures 1 and 2 and in table 2 of the manuscript. Once the linear/non-linear (single and multi-family) patterns were found a detailed review of these patterns were made to find underlying physico-chemical and biological reasons as well as statistical generalizations. The most relevant scatter plots were selected based upon their quality (visual and linear/non-linear regression) and explanatory power for protein structure and function and reliability/specificity for protein alignments. \"... all property classes including #6, #7 & #8 should be precisely defined ... derived from 49 fundamental amino-acid properties/derived scales ... Analysis of Patterns (ANOPA) ... need expanded description ...\" We have defined property classes #1 through #5, but we will expand upon these definitions. We will define property classes #6-#8 with plots and physical explanations. We will describe the ANOPA procedure and explain the specific 49 amino-acid property scale ANOPA analysis used in this study. The 49 amino-acid property scales used in the ANOPA analysis are property scales gleaned from the literature and no derivative scales were used for this portion of the overall analysis. We will be putting in a couple of new tables to define the 49 amino-acid properties used in the ANOPA analysis and the 124 statistically significant (by regression correlation) amino-acid property scales \" ... show scatter plots for property classes #5 through #8 ...\" We agree to revise the manuscript accordingly. \"... need expanded introduction ... expand more on AA physico-chemical properties relevant to protein folding ...\" We agree to modify the manuscript according to these comments, except that we note that he scope of this paper is to define our hydrophobicity scale and its application to protein alignments, whereas we take up the challenge of applying this work to protein folding with an extensive analysis in our follow up paper “Hydrophobicity Revisited: a Molecular Story.” This latter manuscript is in an advanced state of preparation and can be provided for review, which is recommended since the treatment of the material in this manuscript is way beyond what we can put into the current manuscript you have reviewed. We will be putting in a couple of new tables to define the 49 amino-acid properties used in the ANOPA analysis and the 124 statistically significant (by regression correlation) amino-acid property scales \"... there is material in the results section (first paragraph) and discussion section (alignment matrices) that belong in the introduction section ...\" We will revise the manuscript per these comments. \"... hydrophobic scale chosen as optimal ... normalized average of 3 hydrophobicity scales ... most robust in correlation analysis ... robustness vaguely defined in methods section as association to multiple, fundamental AA properties using multivariate statistical procedures, thermodynamics and biophysical chemistry considerations ... later find out that this means R2 values derived from MLR models ...\" We have expanded upon what this means in our responses to your review. We will incorporate this material into the manuscript and significantly beef up the methods section to accommodate the spirit of your comments. We also note that the statistical correlations were only part of the rationale for defining a “robust” hydrophobicity scale, which is based upon bringing a coherent theoretical analysis to bear upon this work as well. We will be adding some additional material requested by Dr. Carter that should speak to these comments. We offer to make available for your review the TMATCH theory and application papers to provide additional justification for what constitutes a robust hydrophobicity scale as we use the term. \"The methods section needs to be written more clearly\" We agree and will modify the manuscript accordingly. \"... Need better tag description for table 2 owing to the considerable discussion of table two columns in the manuscript ...\" We agree and will modify the manuscript accordingly." } ] } ]	1	https://f1000research.com/articles/4-1097
https://f1000research.com/articles/9-82/v1	04 Feb 20	{ "type": "Research Article", "title": "Toolkit of methodological resources to conduct systematic reviews", "authors": [ "Marta Roqué", "Laura Martínez García", "Ivan Solà", "Pablo Alonso-Coello", "Xavier Bonfill", "Javier Zamora", "Laura Martínez García", "Ivan Solà", "Pablo Alonso-Coello", "Xavier Bonfill", "Javier Zamora" ], "abstract": "Background: Systematic reviews (SR) can be classified by type depending on the research question they are based on. This work identifies and describes the most relevant methodological resources to conduct high-quality reviews that answer clinical questions regarding prevalence, prognosis, diagnostic accuracy and efficacy of interventions. Methods: Methodological resources have been identified from literature searches and consulting guidelines from institutions that develop SRs. The selected resources are organized by type of SR, and stage of development of the review (formulation of the research question, development of the protocol, literature search, risk of bias assessment, synthesis of findings, assessment of the quality of evidence, and report of SR results and conclusions). Results: Although the different types of SRs are developed following the same steps, each SR type requires specific methods, differing in characteristics and complexity. The extent of methodological development varies by type of SR, with more solid guidelines available for diagnostic accuracy and efficacy of interventions SRs. This methodological toolkit describes the most up-to-date risk of bias instruments: Quality in Prognostic Studies (QUIPS) tool and Prediction model study Risk Of Bias Assessment Tool (PROBAST) for prognostic SRs, Quality assessment of diagnostic accuracy studies tool (QUADAS-2) for diagnostic accuracy SRs, Cochrane risk of bias tool (ROB-2) and Risk of bias in non-randomised studies of interventions studies tool (ROBINS-I) for efficacy of interventions SRs, as well as the latest developments on the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. Conclusions: This structured compilation of the best methodological resources for each type of SR may prove to be a very useful tool for those researchers that wish to develop SRs or conduct methodological research works on SRs.", "keywords": [ "Systematic reviews", "prevalence", "prognostic", "diagnostic accuracy", "efficacy of interventions" ], "content": "Introduction\n\nSystematic reviews (SR) are studies that use a systematic and explicit method to identify, analyse and synthesize empirical evidence, and to answer a specific research question1. Therefore, SRs are key tools to make informed health choices2,3.\n\nAll SRs are based on a specific research question. Classic epidemiological research questions relate to the prevalence of a medical condition, the associated prognosis of the medical condition (including incidence or global prognosis, prognostic factors associated to the condition's incidence or outcome, and risk profiles defined by prognostic models4), diagnostic accuracy of tests that allow us to diagnose the medical condition, and efficacy of interventions to treat the medical condition. SRs can be classified by the type of research question they answer, as shown in Table 1.\n\nThe stages to develop an SR are common to all the types of SRs: 1) Formulating the research question, 2) development of the protocol that explicitly describes the methods to carry out each step of the SR, 3) literature search, 4) risk of bias assessment, 5) synthesis of findings, 6) assessment of the quality of evidence, and 7) report of SR results and conclusions1. Although the different types of SRs share the same structure and follow a similar development process, their methods can be different and more or less complex depending on the type of SR.\n\nNowadays there are numerous methodological resources to conduct reviews, especially for intervention SRs and diagnostic SRs. However, the scattering of these resources and the lack of widely established manuals or recommendations are, in many situations, an obstacle to access them, especially for prevalence SRs and prognostic SRs. Therefore, the objective of this review is to identify and describe the methodological resources available to develop prevalence SRs, prognostic SRs, diagnostic accuracy SRs and efficacy of interventions SRs.\n\n\nMethods\n\nWe consulted the guidelines from the main organizations that establish methods to conduct SRs (Cochrane, Joanna Briggs Institute, European Network for Health Technology Assessment (EUNETHTA), Enhancing the Quality and Transparency of Health Research (EQUATOR) network, Grading of Recommendations Assessment, Development and Evaluation (GRADE)) in order to identify their proposed resources.\n\nAdditionally, we performed a literature search in MEDLINE (accessed through PubMed) in November 2019 using the following search syntax: ((“Review Literature as Topic”[Mesh] OR systematic review[tiab] ) AND (handbook[ti] OR methodolog*[ti] OR manual[ti] OR guide[ti]).\n\nWe also performed ad hoc scientific literature searches to find other resources for each type of SR in relation to the research question structure, the literature search strategy, the risk of bias assessment and the statistical analysis.\n\nWe included the resources available to design prevalence SRs, prognostic SRs, diagnostic SRs and intervention SRs.\n\nWe excluded the methodological resources to develop other types of SRs (methodological, economic evaluation and qualitative research SRs).\n\nThe authors are members of CIBERESP (Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública - Biomedical Research Center Network of Epidemiology and Public Health) and experts in different fields of knowledge (statistics, development of Cochrane reviews, research methodology, information retrieval, development of clinical guidelines). They evaluated the search results, selected the most relevant and accurate resources, and summarized the most relevant information by development stage and type of SR.\n\nThe resources were organised in 7 sections, following the development stages of an SR: 1) Formulating the research question, 2) development of the protocol and review registration, 3) search strategy, 4) risk of bias assessment, 5) statistical synthesis of findings, 6) quality of evidence assessment, and 7) results report and presentation. The resources are presented by type of SR in each section, and an example of their use is included.\n\n\nResults\n\nWe identified several manuals as a result of the bibliographic searches. Joanna Briggs Institute has a specific section in their methodological manual dedicated to the development of prevalence and global prognosis SRs, and another one dedicated to prognostic factor SRs14–16. Other specific publications offer guidelines to develop prognostic factor and aetiology SRs17,18. During the performed search, we did not identify specific methodological manuals to develop prognostic model SRs. Instead, methodological information can be found in the series of publications from the PROGRESS project, in the resource compilation from Cochrane's Prognosis Methods Group and in specific publications19.\n\nFor diagnostic accuracy SRs and efficacy of interventions SRs, the methodological manuals developed by Cochrane Collaboration are available1,20. The recommendations drawn from these are complemented with specific resources that refer to each one of them, as we will see in the following sections.\n\nThe type of SR is determined by the research question, which must be formulated in a structured manner as shown in Table 1. Careful development of the research question is vital, since the SR inclusion criteria will stem from it.\n\nPrevalence review. Prevalence SRs aim to answer the question “How common is a health problem in a specific population?” Prevalence SRs focus on existing cases at a given time, measure the global burden of a health problem, and describe the characteristics of the affected population, the geographical distribution of that problem and its variation among subgroups. The structure of the research question must include the elements of condition, context, population and study design (CoCoPop-S)21, as shown in Table 1. The most adequate study designs to estimate the prevalence would be population registers or cross-sectional studies that include population-representative samples. For instance, Guthold et al. (2018) considers studies based on population surveys as a reliable source of information to obtain global prevalence estimators of insufficient physical activity6.\n\nPrognostic review. SRs of prognosis are mainly based on three types of research questions: 1) “What is the risk of an specific population to have a health problem?”, descriptive question (review of global prognosis) that focuses in new cases occurring within a period of time (incidence), 2) “what factors are associated with or determine a specific outcome?”, an explanatory question (review of prognostic factors), and 3) “are there risk profiles that have higher probability of presenting specific outcomes?”, a result prediction question (review of prognostic models or risk prediction). We have excluded from the aim of this project a 4th type of prognostic question, known as stratified medicine, and that alludes to the use of prognostic information to individualise therapeutic choices in a group of people with similar characteristics4.\n\nStructured questions about global prognosis must specify population, outcome, condition to be predicted, context and time frame to determine the incidence (CoCoPop-S). The study designs that provide more reliable incidence estimates are prospective cohort studies with representative samples15,22. Structured questions regarding either prognostic factors or models must include population; exposure in terms of the prognostic factor or model of interest, including how it is measured, the intensity and the exposure time; outcome, condition to be predicted; follow-up time; and context (PICOT-S or PFO-S)19,21. The best study designs to evaluate prognostic factors or models are also prospective cohort studies. For instance, Westby et al. (2018) published a prognostic factor SR that gives priority to the inclusion of cohort studies and, if none is found, it resorts to including case-control studies, which also explore the association of prognostic factors with the outcome of interest, although with less reliability8.\n\nDiagnostic accuracy review. Diagnostic SRs aim to answer the question “How good is a test to identify or dismiss the presence of a condition or health problem in a particular population, in comparison with a reference test?” The research question can be posed with the elements of population, index test, reference test, diagnosis of interest and study design (PIRD-S)21. The SR approach will depend on the role of the index test in the clinical diagnostic pathway: if it replaces another test, if it will be used in addition to another test to refine the diagnosis, or if it is a triage test previous to other tests23,24.\n\nDiagnostic SRs preferentially include cross-sectional studies, where the participants are evaluated using the index test and/or the reference test to determine if they have the condition of interest. Case-control designs are subject to risk of bias and their inclusion in diagnostic SRs is not recommended25. For instance, Ambagtsheer et al. (2017) include in their SR cross-sectional studies where one or more self-reported frailty screening scales have been compared with one of three reference standards: frailty phenotype, frailty index or comprehensive geriatric assessment10.\n\nEfficacy of interventions review Interventions SRs aim to answer the question “What effect does a specific intervention have on the relevant outcomes in people with a particular health problem, in comparison with a reference intervention?” The research question is posed with the elements of population, intervention, comparator, outcomes of interest and study design (PICO-S)1.\n\nThe randomised clinical trial (RCT) is the most appropriate study design to evaluate the efficacy of an intervention, as it is the design with less risk of bias and that best helps to establish causality. In cases where it is not possible to conduct randomised trials for ethical or organizational reasons, non-randomised trials, before-after studies, time series, cohort studies or case-control studies can be considered for their inclusion in the SR1. For instance, the SR by Johnson et al. (2018) regarding ribavirin for treating Crimean Congo haemorrhagic fever included both RCTs and non-randomised trials to use the available data, given the previous lack of preparedness for experimental research therapeutics in outbreak situations, but concludes that estimates of effect based on the existing literature are highly uncertain due to confounding in non-randomised studies12.\n\nWriting the SR protocol is a fundamental step that must be done before designing an SR. Herein, the stages and methods to be applied during the development of the SR can be pre-specified. Similarly to the requirement of clinical trial registration, the SR should also be registered in order to avoid redundancies and, more importantly, to avoid reporting bias, therefore guaranteeing transparency and rigor during the development of the SR26. Prospective registration of an SR protocol is recommended by the PRISMA guidelines and is associated with higher SR methodological quality27,28. The largest and most well-known SR register is PROSPERO, produced by the Centre for Reviews and Dissemination in York. With PROSPERO, it is possible to prospectively register any type of review, provided that its aim is a health-related outcome. It contains more than 30,000 entries29. All Cochrane SR protocols are published in Cochrane Library and automatically registered in PROSPERO.\n\nDesigning a comprehensive research study for an SR is vital in order to reduce bias when identifying studies, and it is important to describe it in the relevant section within the protocol in a transparent and thorough manner to facilitate its evaluation by third parties and its reproducibility.\n\nMethodological reference standards to design comprehensive searches have been published30,31. In addition, methodological manuals to develop SRs provide guidelines for diagnostic and efficacy of interventions SRs32–34.\n\nThe design of the search strategies does not differ by type of SR, but rather their differences are due to the elements of the research question and the design of studies to be identified. In general terms, electronic searches are designed to identify bibliographic references that use a language similar to the elements of the review's clinical question. To this effect, the strategies are built based on the elements of the structured clinical question. Search algorithms use a combination of natural language and the appropriate controlled vocabulary for each bibliographic database. Validated filters can be applied to these strategies to determine specific study designs that can be useful to identify, among others, clinical trials34,35, or prognostic studies36. However, the use of filters is controversial in diagnostic accuracy studies33,37.\n\nSearch performance will vary depending on the type of studies that are included in the SR. Thus, in intervention SRs, the search results for RCTs are more precise (they have a higher proportion of relevant references among all the references that the search has identified), due to better indexation of this type of studies in bibliographic databases. On the contrary, in SRs that include observational studies, like prognostic SRs, identifying studies is more complex given the variability of designs to be included and its poorer indexation in databases, which results in less specific literature searches that lead to a longer and more complex study selection process17.\n\nSearches must be designed to optimise their sensitivity (the ability to retrieve as many relevant study references as possible), which is a feature that tends to be a detriment to precision, which in SRs ranges on an average of 3%38. To obtain an efficient search with adequate sensitivity, performing searches in MEDLINE and EMBASE is sufficient, as they are the two most frequently used bibliographic databases39, and they are enough to identify most relevant studies for a specific SR40.\n\nSearching in bibliographic databases can be completed with additional strategies, such as checking public trial registers41,42, searching in the reference list of relevant studies43, or cross-searching citations44. Searching grey literature, understood as any document that is not published in biomedical or scientific journals, has a limited impact in efficacy of interventions SRs45, but offers good results in other types of SRs, such as qualitative evaluation SRs46.\n\nIf we take into consideration the methodological and technical challenges that the design and implementation of search strategies pose, involving a medical librarian can be convenient to improve the search quality47–49.\n\nAssessing the risk of bias is a key element in any SR. It helps evaluate and interpret the included studies results, and it is a determinant of the evidence quality of the SR results. The current tools to assess risk of bias are organised by domains, which roughly correspond to the classic epidemiological biases related to each type of research question. The identified tools to assess risk of bias are presented in Table 2, organised by type of SR and by domain of epidemiological bias assessed.\n\nNo risk of bias tool has been identified for global prognosis systematic reviews.\n\nEach of the domains of these tools includes a number of index questions related to specific aspects of study design or development that can lead to a bias in that domain. The tools can be adapted a priori to each review, modifying or deleting questions, or adding new questions specific to the considered research question. The process to assess risk of bias is similar in all the current scales. Firstly, they identify the risk of bias in each domain based on the answers to the questions, and secondly, they integrate these risks in a risk of bias assessment for each health problem, prognostic factor, diagnosed condition or outcome of interest assessed, depending on the type of SR.\n\nPrevalence review. The tool to assess risk of bias by Hoy et al. (2012) is available for prevalence SRs. It assesses internal and external validity aspects in the prevalence study50. The tool comprises 10 questions where a judgement of high or low risk of bias is made. Based on the answers, the researcher makes a subjective assessment of the study’s overall risk of bias as low, moderate or high50.\n\nPrognostic review. There is no scale available to assess the risk of bias in global prognostic studies, although a series of criteria has been proposed to assess risk of bias. These are classified in 1) definition and representativeness of the population, 2) completeness of follow-up, and 3) objective and unbiased measurement of outcome of interest22. However, some authors like Roerh et al. (2018) use a version of the scale to assess risk of bias designed by Hoy et al. (2012), adapted to the assessment of incidence studies considering the duration of the incidence period7.\n\nFor the prognostic factor studies, the tools QUIPS and “RoB instrument for NRS of exposures” were identified51–53. The QUIPS tool helps assess the risk of bias using 31 questions divided in 6 domains. For each domain, a judgement of high, low or unclear risk of bias is made. Before using the tool, one must carefully consider the potential confounders that can lead to bias. Clinical experts in the specific topic of the SR should participate. The tool “RoB instrument for NRS of exposures” evaluates the risk of bias using 32 questions divided in 7 domains, including a key domain regarding confounders and a domain regarding departures from intended exposures. For each domain, a judgement of critical, serious, moderate or low risk of bias is made. An example of the use of the QUIPS scale can be seen in the review by Westby et al. (2018). The authors defined a priori two key confounders (age and infection), which the experts and the literature described as prognostic factors for their condition of interest (venous leg ulcers), and which were simultaneously associated with the prognostic factor of interest in the SR (protease activity biomarker). These two confounders were included in the QUIPS scale in the section of control by confounders8.\n\nWe identified the Prediction model Risk Of Bias ASsessment Tool (PROBAST) for the prognostic model SRs54. This tool assesses the risk of bias using 20 questions divided in 4 domains (participants, predictors, outcome and analysis). For each domain, a judgement of high, low or unclear risk of bias is made. The questions vary according to the aim of the study (development, validation, or development and validation of the prognostic model).\n\nDiagnostic accuracy review The tool QUADAS-2, which evaluates 11 questions divided in 4 domains, is available to assess the risk of bias in diagnostic accuracy studies55. For each domain, a judgement of high, low or unclear risk of bias is made. In addition, the external validity or study applicability in relation to the SR is assessed in each domain.\n\nDiagnostic SRs mainly include observational studies, which are more subject to risk of bias, and therefore adapting the QUADAS-2 tool, modifying or adding specific questions to the SR topic, is virtually a requirement during the protocol stage. For instance, the SR by Martínez et al. (2017) studied the diagnostic accuracy of an imaging test (amyloid PET) that requires complex visual interpretation. For this reason, a question was included in the QUADAS scale to assess whether the test interpretation was performed by trained readers11.\n\nEfficacy of interventions review. For intervention SRs, the Risk of Bias (RoB) 2.0 tool is available to assess the potential bias in randomised clinical trials, and the Risk Of Bias In Non-randomised Studies - of Interventions (RoBiNS-I) tool in non-randomised clinical trials56,57. The RoB 2.0 tool includes 16 questions divided in 5 domains, including a specific domain for randomisation and a domain for deviations from intended interventions56. For each domain, a judgement is made: high or low risk of bias, or some concerns. For instance, in their SR, Ellis et al. (2017) assessed the risk of bias in the evaluation of results separately for the objective outcomes (such as living at home or death) and for the subjective outcomes, showing a lower risk of bias in the evaluation of the objective outcomes13.\n\nThe RoBiNS-I tool assesses the biases that the non-randomised study has when compared with an ideal, pragmatic, unbiased randomised trial, which answers the clinical question of interest (even if this ideal trial may not be feasible or ethical)57. RoBiNS-I has 34 questions divided in 7 domains, including a key domain regarding confounders and a domain for deviations from intended interventions. As in the case of prognostic SRs, there should be an a priori careful consideration of the potential confounders that must be included in the tool to assess individual studies. A judgement of critical, serious, moderate or low risk of bias is made for each domain. A low risk of bias implies that the non-randomised study is comparable to a well-performed randomised trial. For instance, Johnson et al. (2018) excluded from their analyses the non-randomised studies that showed a critical risk of bias according to RoBiNS-I, rejecting 18 out of the 22 included studies12.\n\nSRs may include a section with a quantitative statistical synthesis or meta-analysis, where a combined estimator of the parameter of interest is obtained from the estimators of the individual studies. Table 3 shows the main characteristics of the meta-analysis methods and the main software commands for each type of SR.\n\na Tukey-Freeman or logit transformation. bTransformation for the cumulative incidence. c Hierarchical summary receiver operating characteristic (HSROC) method allows estimation of a Receiver operating characteristic (ROC) curve or sensitivity and specificity indexes.\n\nA necessary previous step to any meta-analysis is the evaluation of the existing clinical and statistical heterogeneity in the set of studies, which will inform us 1) if it is reasonable to perform a quantitative synthesis of findings, 2) what meta-analysis model we should apply, and 3) if additional investigation of the causes of heterogeneity is required, for example, subgroup and sensitivity analyses, or meta-regressions58,59.\n\nWhen it is reasonable to perform a statistical synthesis, there are two main models to conduct a meta-analysis: fixed effects model and random effects model. For practical purposes, the chosen model determines how the studies included in the meta-analysis will be numerically weighed. Both models are based on different assumptions, and they differ in their application and interpretation59.\n\nFinally, there is a variety of resources to conduct meta-analyses, from specific programs to perform meta-analyses (free or paid) to user-defined routines using general statistics packages (SAS, Stata, SPSS), as well as Excel utilities or R libraries. An archive with software and utilities is available from SR Tool Box.\n\nDue to the complexity of the statistical techniques to synthesise results, and the difficulty to standardise methods and decisions to be made during the analysis, it is vital to involve a statistician in the planning and conduct stages of the meta-analysis, especially for prognostic and diagnostic SRs.\n\nPrevalence review. In prevalence SRs, the meta-analysis combines ratios, which are transformed to be meta-analysed using the inverse-variance method59. Siriwardhana et al. (2018) calculated combined frailty prevalence estimates using a random effects model. The authors assessed that there was high clinical heterogeneity between the studies in terms of actual frailty prevalence, geographic setting, frailty assessment method, cut-off points applied and sample age, although this heterogeneity did not rule out performing a meta-analysis5.\n\nPrognostic review. In global prognostic SRs, the meta-analysis combines cumulative incidence ratios or incidence rates, while in prognostic factor SRs, the meta-analysis combines odds ratios or hazard ratios, which can be presented in individual studies as raw estimates or as covariate-adjusted estimations derived from logistic or Cox regression models. If combining adjusted estimates, all of them should be adjusted by a minimum set of common factors17. In prognostic model SRs, the meta-analysis combines estimates of model discrimination and calibration. These indicators can be synthesised separately or jointly using multivariate models19.\n\nPrognostic studies usually show significant variability in terms of design, sample case-mix, measurement instruments, analysis methods and presentation of results17. Therefore, in prognostic factor and model SRs, it is recommended to perform the meta-analysis using the random effects model, and even to use multivariate meta-analysis methods adjusting for relevant factors17. For instance, the SR by Westby et al. (2018) describes how the authors dismissed performing a meta-analysis due to the high risk of bias and the extreme heterogeneity across the included studies in terms of population, measurement of the prognostic factor (cut-off points and analytical methods) and outcome measurement8.\n\nDiagnostic accuracy review. In diagnostic SRs, the meta-analysis combines estimates of sensitivity and specificity of the index test. The meta-analysis in diagnostic SRs shows a higher degree of complexity because the studies may have used different thresholds, both implicit and explicit, to define a positive result in the evaluated test. This leads to a correlation between the sensitivity and specificity indexes, which must be modelled jointly using multivariate methods60. The most common available statistical methods are the bivariate hierarchical model and the HSROC model (Hierarchical summary receiver-operating characteristic)61. Diagnostic SRs tend to combine studies with very heterogeneous results, and it is recommended to use the random effects model by default and perform a comprehensive examination of the sources of heterogeneity using meta-regression20. For instance, the protocol of the SR by Ambagtsheer et al. (2017) expects to estimate an average sensitivity and specificity for the frailty scales, when the included studies have applied the same explicit cut-off points to the considered scales. However, given that they are subjective, self-reported scales, the studies could share the same explicit cut-off point, and yet that cut-off point could correspond to different levels of frailty in the studies (implicit thresholds), which will advise against calculating pooled estimates of diagnostic accuracy10 .\n\nEfficacy of interventions review. In intervention SRs, the meta-analysis combines different measures, depending on the type of outcome: odds ratio or hazard ratio for binary outcomes, mean difference or standardised mean difference for continuous outcomes, hazard ratio for time-to-event outcomes, and incidence rate ratios for outcomes that count number of events.\n\nIn intervention SRs, the I2 estimator has been proposed to assess statistical heterogeneity as a supplement to the assessment of clinical and methodological heterogeneity. This indicator is defined as the percentage of the overall variability that cannot be explained by chance, and has values ranging from 0% to 100%; with higher values indicating higher statistical heterogeneity59. For instance, in the SR by Ellis et al. (2017), the authors established a 70% heterogeneity limit for I2, beyond which a meta-analysis combining the results would not be performed13. Despite its popularity and ease of interpretation, the use of this indicator is not exempt of controversy due to its dependence on the number of studies and sample size; thus, a small statistical heterogeneity could seem substantial only by the effect of a large sample size of the included studies62.\n\nThe quality (also confidence or certainty) of evidence in an SR is the degree of confidence that is held against the fact that an estimate of effect or association is close to the actual value of interest1. Certainty of evidence is evaluated for each one of the key SR outcomes or factors. Certainty in the obtained estimates is classified as high, moderate, low or very low. A level of certainty of evidence is first established from the design of the studies that form the evidence body, which might or might not have an optimal design for the type of considered question. This initial confidence in the evidence body can then decrease in one or two levels if the following is detected: 1) design or execution limitations, 2) inconsistency, 3) indirect evidence, 4) imprecision in estimates, or 5) publication bias63.\n\nThe certainty of evidence is a key element to interpret and communicate results, and as such, it should be included in the sections of results, discussion, conclusion and abstract, using semi-standardised statements64. Additionally, it can be included in a Summary of Findings table, where for each comparison, the key information regarding relative effect and absolute effect magnitude, quantity of available evidence and its certainty is presented65.\n\nWe will now highlight the specific aspects in which the GRADE system adapts to each type of SR.\n\nPrevalence review. There are no formal adaptations of the GRADE system for prevalence SRs, but there is a proposal to assess the quality of the evidence based on this system66. High initial certainty is awarded to survey or cross-sectional study designs with population representativeness that have been properly designed and conducted, while studies with no population representativeness will have lower initial quality.\n\nPrognostic review. There is a GRADE proposal for global prognostic SRs22 and an adaptation for prognostic factor SR67. Guidelines for prognostic model SR are still under development.\n\nIn global prognostic SRs, the study designs that have high initial certainty are longitudinal cohort studies and pragmatic randomised controlled trials with representative samples22. Other observational designs would offer low initial certainty. In prognostic factor SRs, explanatory and confirmatory longitudinal designs offer high initial certainty, while exploratory studies are considered to be of moderate quality67.\n\nIn prognostic SRs, the assessment of the limitations follows the general procedure already described, with two particularities: 1) qualitative assessment of inconsistency, because of low reliability of I2 estimator in the prognostic field22,67, and 2) possibility of increased certainty in the studies that do not show limitations in the quality of evidence, if (i) the estimated effect magnitude is substantial, or (ii) there is an exposure-response gradient67. For instance, the prognostic factor SR by Westby et al. (2018) considered the possibility of increasing the certainty of evidence in the studies presenting no limitations. Due to the exploratory nature of the included studies and their high risk of bias, the certainty was not increased in any case and the evidence obtained in the review was of very low quality8.\n\nDiagnostic accuracy review. The methods to assess quality of evidence in diagnostic SRs are still under development63. The study designs that start with the highest degree of evidence are RCTs and cohort or cross-sectional studies where the index test and the reference standard have been directly compared in all participants68. If the SR included case-control studies, these would offer low-quality initial evidence25.\n\nThere is uncertainty regarding how to assess inconsistency, because heterogeneity is common and hard to quantify in diagnostic SRs, and it often cannot be explained even if multivariate models are adjusted. It is also unclear how to assess inaccuracy when the SR has estimated the SROC curve, or when the role of the test in the clinical pathway gives different weight to the sensitivity and specificity estimates. With regard to the criteria to increase the level of evidence, it is unclear whether they should be applied and how to do it in diagnostic SRs63,66. The uncertainty surrounding the process of assessing the quality of evidence in diagnostic SRs explains why it is not a requirement in Cochrane SRs. For instance, the SR by Martínez et al. (2017) only included a Summary of Findings table with numerical results and an estimation of the absolute effect that the test would have on a hypothetical cohort of individuals53.\n\nEfficacy of interventions review. The GRADE system for assessing the quality of evidence was initially developed for intervention SRs, and it is the indication for which clearer and widely agreed guidelines are available63. In terms of study design, RCTs are initially classified as having high certainty, while all non-randomised or observational studies are classified as having low certainty.\n\nThe assessment of the certainty limitations is well-defined in intervention SRs. Inconsistency can be assessed using the I2 estimator63. Imprecision is assessed taking into account whether the review meets the optimal information size, and whether the confidence interval of the effect estimate allows reaching a conclusion, because either it only includes values consistent with a relevant intervention effect, or it completely dismisses it63. In observational studies that do not have limitations in the quality of evidence, three criteria are considered to increase certainty: 1) the estimated effect magnitude is important or very important, 2) there is an exposure-response gradient, and 3) all possible biases that could reduce the observed effect confirm the obtained conclusions.\n\nFor instance, the SR by Ellis et al. (2017) applied the GRADE system to the included randomised trials, and it concluded that there was high certainty of the effect of the comprehensive geriatric assessment on the efficacy outcomes based on a high number of studies and participants, with a globally low risk of bias, and results consistent among studies. However, the certainty of evidence obtained in cost‐effectiveness was low, due to imprecision and inconsistency of results56.\n\nIt is vital to inform about the methods, results and conclusions of the SRs in a transparent and thorough manner so that their users can interpret, evaluate and apply them. The EQUATOR initiative has developed, and keeps up-to-date, a library with guidelines to communicate the different types of research studies. The PRISMA statement (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) has been proposed in the SR field69. This statement consists of a checklist comprised of 27 items and a flow diagram to present the number of studies considered in the SR. In addition, several extensions focusing on reporting specific aspects of SRs have been developed, such as PRISMA-P for reporting SR protocols70, PRISMA-Abstracts for reporting abstracts71, and PRISMA-Harms for reporting harms outcomes in SRs72.\n\nAlthough the PRISMA statement and the cited extensions are focused on intervention SRs, a specific PRISMA extension has also been developed for diagnostic SRs73. On the contrary, no tools have been identified to communicate prevalence or prognostic SRs. In recent years, clarity and transparency in study communications has improved thanks to the development of checklists for scientific paper publication, although there is still room for improvement74–76.\n\n\nDiscussion\n\nThis review identifies and describes the most relevant methodological resources to conduct prevalence, prognostic, diagnostic accuracy and efficacy of interventions SRs. This review offers a general and comparative perspective of the methodological resources by SR stage, highlighting the differential elements of each type of SR.\n\nThis paper corroborates that developing a rigorous SR is a complex and resource-intensive task77,78. In order to tackle the increasing complexity of SRs and ensure the adoption of rigorous methodology, it is necessary that the reviews are made by multidisciplinary work groups with knowledge and experience in methodology (such as statistical analysis and information retrieval)79,80. In addition, it is important to consider the increasing availability of artificial-intelligence-based technological tools, which make it possible to semi-automate the different steps of the SR development, and thus reduce the time and human resources required to conduct the review81.\n\nOnce the rigorous SR has been developed, ensuring the conveyance of the generated knowledge is essential. In this sense, new formats for synthesis and presentation of SR results are being explored nowadays to help their dissemination and the adoption of their conclusions in clinical practice and healthcare decision-making. For instance, new formats for result presentation and Summary of Findings tables are being proposed, adapted to the profile of their potential users82,83.\n\nThe four types of SRs considered in this paper are fundamental to define preventive activities and public health policies, as well as to make health decisions. However, this research has not considered other types of SRs, such as methodological, economic evaluation and qualitative research SRs, for which it would be convenient to perform similar methodological compilations. Another limitation of this research is the need to keep it up to date, given the speed at which the methods and methodological resources to develop SRs are updated.\n\nOn the other hand, the main strengths of this paper are its transversal approach for the different types of reviews, and the identification of resources for all the stages in the development of an SR. There are few previous publications that offer a transversal perspective of the different types of systematic reviews, and these are focused on a specific stage of the review or on a particular topic. For instance, the work carried out by Munn et al. (2018) defined a typology for SRs, characterised from 10 different types of research questions, and delving into the format of each type of question21. Pollock et al. (2017) review the steps of an SR for 5 types of question, specifically focusing on the particularities of the reviews on stroke rehabilitation84. Muka et al. (2019) offer a structured compilation of resources for each SR stage, but without delving into the specificities of the different types of SRs85. Finally, organising the resources to assess the risk of bias by type of review is a strength and a novelty compared with previous works, which compile the quality assessing tools by type of study design but without linking them to the aim of the study nor the type of systematic review86,87.\n\n\nConclusions\n\nSRs are a key research tool to make decisions in healthcare, public health and medical research. There are methods and resources to develop high-quality reviews to answer most types of clinical questions. This review offers a complete resource guide for prevalence, prognostic, diagnostic and intervention reviews, and is a very useful tool for those researchers that wish to develop SRs or conduct methodological research works in that field.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nMarta Roqué i Figuls is presently working on her PhD with the PhD Programme in Biomedical Research Methodology and Public Health from the Autonomous University of Barcelona.\n\n\nReferences\n\nHiggins JPT, Sterne JAC, Savović J, et al.: A revised tool for assessing risk of bias in randomized trials. In: Chandler J, McKenzie J, Boutron I, Welch V, (editors). Cochrane Methods. Cochrane Database Syst Rev. 2016; 10(Suppl 1). Reference Source\n\nUrrútia G, Bonfill X: Revisiones sistemáticas, una herramienta clave para la toma de decisiones clínicas y sanitarias. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInstitute of Medicine (US) Committee on Standards for Systematic Reviews of Comparative Effectiveness Research, Eden J, Levit L, et al.: Finding What Works in Health Care: Standards for Systematic Reviews. Washington (DC): National Academies Press (US); 2011.2, Standards for Initiating a Systematic Review. 2011. PubMed Abstract \| Publisher Full Text\n\nMarshall IJ, Wallace BC: Toward systematic review automation: a practical guide to using machine learning tools in research synthesis. Syst Rev. 2019; 8(1): 163. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarrasco-Labra A, Brignardello-Petersen R, Santesso N, et al.: Improving GRADE evidence tables part 1: a randomized trial shows improved understanding of content in summary of findings tables with a new format. J Clin Epidemiol. 2016; 74: 7–18. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "59538", "date": "14 Feb 2020", "name": "Miranda Cumpston", "expertise": [ "Reviewer Expertise In presenting my comments", "I acknowledge that my own experience focuses on methods around systematic reviews of the effects of interventions", "and so I cannot comment with expertise on the details of methods described for other review types." ], "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 summarised the current literature around methods for conducting systematic reviews, encompassing reviews of prevalence, prognosis, diagnosis and intervention effectiveness. In a field of methodology where some guidance is collated in well-known handbooks, but other areas are scattered among the methods literature, and where development of new methods is relatively fast-moving, this map of available guidance and methods studies is likely to be very useful to researchers new to systematic reviews or branching out into different review types. The authors are experienced experts in this field, and the review is well-written and clear.\nIn my understanding, this paper aims to fulfil two roles: (1) a toolkit that enables authors of systematic reviews to navigate out to key methods resources, and (2) a brief summary of methods guidance for each review type. It may be helpful throughout the paper, including in the abstract and conclusions, to separate out these two roles more clearly. I have some suggestions to improve the transparency and usability of the paper from each of these perspectives.\nMajor recommendations As a review and toolkit of methodological resources, I would make the following suggestions:\nMethods: The authors could provide more transparent detail on the selection process for included documents, including specifying which organisational websites were searched, how many resources were identified, and if any were excluded. Although this is intended as a mapping, rather than systematic review, some additional detail would help readers to understand how the guidance documents were selected, and why perhaps some resources they may have used before are not listed.\n\nImportantly, more detail could presented on how the authors selected the most “relevant” or “best” resources, especially where there may have been multiple candidate documents. For example, were they most recent, most comprehensive, those endorsed by a credible organisation, most introductory, most rigorous methods? The role of the authors’ expert judgement in these decisions should be stated explicitly. Note that I am not suggesting that a different process should have been used, just suggesting a more detailed description of the judgement process.\n\nResults: As a navigation guide, I found that citing the resources within the text did not tell me everything I wanted to know. Also, the key resources are mixed in the reference list with exemplar reviews and other citations. A table outlining the recommended resources by category with hyperlinks to each resource would be helpful.\n\nAs a reader, I would find it helpful for the authors to draw the distinction between the different types of resources cited. For example, some were synthesised, ‘best practice’ guidance intended for use by authors (such as the Cochrane or JBI Handbooks). Some were reporting guidelines (which often list good practice but are not intended to provide detailed guidance on the conduct of reviews). Some were primary methods studies (e.g. measuring the prevalence or impact of a method, which may describe or evaluate possible methods options but are not intended as guidance on the ‘best’ available methods for authors. All may be useful in different ways.\nAs a brief summary of methods guidance, I would make the following suggestions:\nMethods: It would be helpful to include a brief description of your role in selecting and writing this summary of guidance, for example, stating briefly that key areas of methods were summarised, and how the methods to be highlighted were selected, especially where multiple and potentially conflicting sources were available (e.g. major methods applicable to all reviews, key differences between review types, expert opinion about interesting or important advice).\n\nIt might be helpful to note that the summary of guidance presented is just that, a brief summary, and that authors who are new to systematic reviews should consult the more detailed documents for complete guidance.\n\nResults: It might be particularly helpful to note where there is disagreement in the literature in relation to particular areas of guidance. For example, comments that searching the grey literature is not useful for effectiveness reviews, or that risk of bias tools should be adapted for each review, which may be in disagreement with some of the major guidance handbooks and reporting guidelines. I’m not arguing about these specific items, just noting that some methods choices have been made by the authors, and it may be helpful to make this transparent.\nMinor recommendations\nTitle and abstract: It would be helpful to briefly define the scope of this review, especially in the context of a journal such as F1000Research, which publishes in a wide range of scientific fields. For example, using a term such as ‘systematic reviews in health care’, and noting that the review looks at prevalence, prognosis, diagnosis and health interventions (and not other areas relevant to health such as environmental exposure).\n\nCompeting interests: The authors could note any roles with Cochrane and the GRADE Working Group. These are not necessarily financial conflicts, but may be relevant to the authors’ decision to recommend guidance from these sources (which of course I agree with!).\n\nResults: In the first two paragraphs, you discuss some specific points in relation to resources available for diagnostic and prognosis reviews. As all review types appeared to cite both guidance handbooks and additional primary methods studies, I wasn’t clear on the point you were trying to make in this section.\n\nThere were some key guidance documents that I would have expected to see, although there may be good reasons not to include them, such as:\nGeneral guidance on systematic reviews from the US Institute of Medicine and the Centre for Reviews and Dissemination at the University of York (both are older documents, so this may be the reason). Tools to assess the risk of bias in reviews, such as ROBIS or AMSTAR, which may enable reflection by authors on their choice of methods, in a similar manner to reporting guidelines. Resources on the development of protocols (as distinct from study registration), such as Chapter 1 of the Cochrane Handbook. Guidance on synthesis in the absence of meta-analysis, such as Chapter 12 in the Cochrane Handbook and papers by (Campbell, McKenzie et al. (2020)1) and (Popay, Roberts et al. (2006)2). This may be relevant to synthesis, assessment of heterogeneity and GRADE. Guidance on network meta-analysis, such as Chapter 11 of the Cochrane Handbook and multiple journal articles, as well GRADE guidance and CINeMA. Two papers from the GRADE Working Group (published this week!) on the use of GRADE for diagnostic test accuracy studies published this week.\n\nIn the text on risk of bias and Table 2, is it worth noting that the number of items in RoB 2 varies depending on the effect of interest and the included study designs?\n\nIn Table 3, as there are more variations on the measures to combine (e.g. Ratio of Means), and statistical methods for meta-analysis of efficacy of interventions (including the inverse-variance method, which is mentioned for other types), could it be helpful to note in the table somewhere that these are common characteristics, not an exhaustive list?\n\nWhen discussing fixed effect and random effects models, it may be helpful to note that they differ in relation to assumptions and heterogeneity, as you mention something briefly about analysis using random-effects models later in the paper that relies on understanding this.\n\nOn page 9, under “Efficacy of intervention reviews”, I think hazard ratios are listed in error against binary outcomes as well as time-to-event outcomes. Perhaps you meant risk ratios?\n\nIn the section on assessing certainty of the evidence, it would be helpful to describe the GRADE approach in the first paragraph (it is currently named without description in the third paragraph).\n\nDiscussion: I would also acknowledge under limitations of this review that the selection of resources and summary of guidance were informed by expert opinion, and that others may have selected different resources or made different recommendations.\n\nReferences: 1: I think there may be an error – should this be a reference to the 2019 edition of the Cochrane Handbook, rather than an older paper by Higgins in Cochrane Methods on RoB 2? I assume this based on its use in the Introduction as a general reference about review methods, and also on the availability of general Handbooks in para 2 of the Results.\n\n20: could be based on the references to individual chapters, i.e. Deeks JJ, Bossuyt PM, Gatsonis C (editors), Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy.\n\n63: It’s great to see the Spanish Manual GRADE cited. If I am correct, this is a translation of the 2013 GRADE Handbook (apologies if I am wrong). Would it be helpful to cite some of the more recent GRADE papers as well, as they reflect the most up to date guidance? I note you also cite the paper on using standard language to express GRADE, as well as the recent Cochrane Handbook Chapter, so perhaps this is sufficient.\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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5747", "date": "11 Aug 2020", "name": "Marta Roqué", "role": "Author Response", "response": "Methods: The authors could provide more transparent detail on the selection process for included documents, including specifying which organisational websites were searched, how many resources were identified, and if any were excluded. ANSWER: Thanks for this suggestion. The text lists the main organisational websited we checked to identify guidelines (\"Cochrane, Joanna Briggs Institute, European Network for Health Technology Assessment (EUNETHTA), Enhancing the Quality and Transparency of Health Research (EQUATOR) network, Grading of Recommendations Assessment, Development and Evaluation (GRADE))\", as suggested by the peer reviewer. Also, the text is now more explicit about the process followed to select resources. See answers to comments below regarding this specific issue.More detailed description of the resource selection process has been added to the text (see detailed modifications in the next comments) Importantly, more detail could presented on how the authors selected the most “relevant” or “best” resources, especially where there may have been multiple candidate documents. ANSWER: Thanks for this suggestion. A paragraph was added to the eligibility criteria section \"The resources were selected based on the authors expert judgement, prioritising those resources which were endorsed or part of a guideline from the organisations cited above, and those which were more recent. \" Results: As a navigation guide, I found that citing the resources within the text did not tell me everything I wanted to know. ANSWER: We have added a table in the Appendix where the references and resources considered in the review are classified into guidelines to conduct SR (or chapters of those guidelines), reporting guidelines, primary methods papers and references to examples used in the manuscript. Methods: It would be helpful to include a brief description of your role in selecting and writing this summary of guidance, ANSWER: Thanks, we will be more explicit on this issue. A sentence has been added to the data selection and extraction section \"For each pre-defined section, the authors selected and summarized the methods that were considered to be more rigorous and widely accepted, prioritizing major methods applicable to all reviews over more controversial methods, or methods which required highly specialized knowledge. The text organises the results pedagogically with the aim to highlight key differences between review types, present the key characteristics of each method, and be a comprehensive tool that contains the most relevant advice based on the authors judgement.\" It might be helpful to note that the summary of guidance presented is just that, a brief summary, and that authors who are new to systematic reviews should consult the more detailed documents for complete guidance. ANSWER: Thanks for the suggestion, we have clarified this point in the text. A sentence has been added to the key results section of the discussion, stating \"This project does not aim to be a standalone tool for a researcher to find complete guidance on how to conduct and report a review, but rather it aims to be a signpost pointing out to the resources where researchers may find in depth guidance to develop their reviews.\" Results: It might be particularly helpful to note where there is disagreement in the literature in relation to particular areas of guidance. ANSWER: As far as possible, we have avoided presenting controversial advice, although we are aware that any topic can be approached differently by different researchers or even institutions. It could be quite daunting to be comprehensive in identifying all the controversies in the different steps of conducting a review, as the volume of publications on methods for reviews is extremely large and diverse. Additionally, we think that the discussion or even the identification of issues where controversy exists may fall outside of the project scope, as it might reduce the usefulness to a new researcher which needs to find clear guidance on a topic, even if there are other alternative methods available. We have stressed the subjectiveness and risk of implicit selection biases in the discussion \"An inherent limitation of this project is its methodology based on a selection of resources and summary of guidance informed by expert opinion, which may be susceptible to implicit selection biases or lack of comprehensiveness. \" Title and abstract: It would be helpful to briefly define the scope of this review, especially in the context of a journal such as F1000Research, which publishes in a wide range of scientific fields. ANSWER: Thanks, this is a very useful suggestion.Title has been modified to \"Toolkit of methodological resources to conduct systematic reviews in health care: reviews on prevalence, prognosis, diagnosis and interventions\". The concept has also been introduced in the abstract (\"This work identifies and describes the most relevant methodological resources to conduct high-quality reviews that answer health care questions regarding prevalence, prognosis, diagnostic accuracy and effects of interventions\") Competing interests: The authors could note any roles with Cochrane and the GRADE Working Group. ANSWER: A sentence was added to the Data selection and extraction \"The authors are members of CIBERESP (Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública - Biomedical Research Center Network of Epidemiology and Public Health), hold active roles within Cochrane and the GRADE Working Group,\" Results: In the first two paragraphs, you discuss some specific points in relation to resources available for diagnostic and prognosis reviews. As all review types appeared to cite both guidance handbooks and additional primary methods studies, I wasn’t clear on the point you were trying to make in this section. ANSWER: We realize we didn't make our point clear, that was to illustrate what guideline handbooks had been identified by type of review. We've rewritten both paragraphs aiming to be more clear. \"We identified guidance handbooks, primary studies and reporting guidelines as a result of the bibliographic searches. The resources selected are presented in the Appendix.We have identified methodological guidelines dedicated to the development of prevalence SRs14, global prognosis15, and prognostic factor SRs 16, 17, 18 .During the performed search, we identified methodological manuals to develop prognostic model SRs in the series of publications from the PROGRESS project19, and in the resource compilation from Cochrane's Prognosis Methods Group .\" There were some key guidance documents that I would have expected to see, ANSWER: .Thanks for the bringing forth this issue, which was overlooked in the manuscript. The issue of narrative synthesis of results has been cited in several sections the paper (statistical synthesis, quality of evidence, results report). An explicit mention to narrative synthesis has been added to the Statistical synthesis section \"In those cases when a quantitative synthesis is precluded, the SR will be restricted to a narrative synthesis. A narrative synthesis should not simply summarize the findings from the included studies in order to draw conclusions about the body of evidence, but instead should be a more formal process which includes a formulation of the theory of how the intervention works, why and for whom, the exploration of the relationships in the data, and the assessment of the robustness of the synthesis. 59\". Certainty of evidence from narrative synthesis has been mentioned \"Certainty of evidence can be assessed too when no quantitative synthesis is possible. 65\". Also, the SWiM paper has been cited in the REsults report section\"Additionally, the SWiM guideline is available for reporting intervention SRs where the effects of interventions are synthethised narratively without metanalysis, focusing on the key features of narrative information synthesis (grouping of studies, presentation of data and summary text, and appropriate discussion of limitations of this type of synthesis).\" Network metanalysis have been mentioned in the Statistical methods and Quality of evidence sections. A cite to Chapter 11 in the handbook has been added to both sectionsThanks, we've added the GRADE diagnostic references to the paper. These references were incorporated to the corresponding Quality of evidence section, modifying the paragraphs on diagnostic SR. In the text on risk of bias and Table 2, is it worth noting that the number of items in RoB 2 varies depending on the effect of interest and the included study designs? ANSWER: Thanks, we've added this information. A sentence has been added to the risk of bias section (\"The number of questions may vary, depending on the effect of interest and the design of the study assessed\"). A footnote has been added to Table 2 \"The number of items in the risk of bias tools may vary depending on the effect of interest and the included study designs, as well as the addition or suppresion of index questions by the researchers to tailor the tool to the SR .\" In Table 3, as there are more variations on the measures to combine (e.g. Ratio of Means), and statistical methods for meta-analysis of efficacy of interventions (including the inverse-variance method, which is mentioned for other types), could it be helpful to note in the table somewhere that these are common characteristics, not an exhaustive list? ANSWER: Thanks, we've corrected the oversight of not mentioning the inverse variance method for continuous outcomes in the efficacy of interventions SR, and have clarified in the text that the table is by no means exhaustive. The text introducing table 3 now reads \"Table 3 shows a non-exhaustive compilation of the main characteristics of the meta-analysis methods and the main software commands for each type of SR\". The inverse-variance method has been added to the efficacy of interventions row When discussing fixed effect and random effects models, it may be helpful to note that they differ in relation to assumptions and heterogeneity, as you mention something briefly about analysis using random-effects models later in the paper that relies on understanding this. ANSWER: This is certainly an important point. Although there is already an explicit link between choice of model and heterogeneity in the previous paragraph (\"the evaluation of the existing clinical and statistical heterogeneity in the set of studies, which will inform us /.../ 2) what meta-analysis model we should apply\"), we agree to further stress this point as suggested. We've modified the existing sentence on random and fixed-effects models \"Both models are based on different assumptions regarding distribution of effects and heterogeneity across studies, and they differ in their application and interpretation\" On page 9, under “Efficacy of intervention reviews”, I think hazard ratios are listed in error against binary outcomes as well as time-to-event outcomes. Perhaps you meant risk ratios? ANSWER: You're right, it should have read 'risk ratios' instead of 'hazard ratios'. hazard ratio' substituted by 'risk ratio' In the section on assessing certainty of the evidence, it would be helpful to describe the GRADE approach in the first paragraph (it is currently named without description in the third paragraph). ANSWER: Thanks, we agree. We've added an explicit reference to the GRADE system \"Certainty of evidence is best evaluated with the GRADE system . Certainty in the obtained estimates for each one of the key SR outcomes or factors is classified as high, moderate, low or very low\" Discussion: I would also acknowledge under limitations of this review that the selection of resources and summary of guidance were informed by expert opinion, and that others may have selected different resources or made different recommendations. ANSWER: While we have strived to draw a unbiased selection of the best resources, it is right to point out this potential limitation. We've added the sentence \"An inherent limitation of this project is its methodology based on a selection of resources and summary of guidance informed by expert opinion, which may be susceptible to implicit biases or lack of comprehensiveness\" Reference 1: I think there may be an error – should this be a reference to the 2019 edition of the Cochrane Handbook, rather than an older paper by Higgins in Cochrane Methods on RoB 2? ANSWER: Yes, you are right. References 1 and 56 were interchanged, and have now been corrected. New reference 1 and new reference 56 Reference 20: could be based on the references to individual chapters, i.e. Deeks JJ, Bossuyt PM, Gatsonis C (editors), Cochrane Handbook for Systematic Reviews of Diagnostic Test Accuracy. ANSWER: Thank you, the reference has been modified as suggested Reference 63: It’s great to see the Spanish Manual GRADE cited. If I am correct, this is a translation of the 2013 GRADE Handbook (apologies if I am wrong). Would it be helpful to cite some of the more recent GRADE papers as well, as they reflect the most up to date guidance? I note you also cite the paper on using standard language to express GRADE, as well as the recent Cochrane Handbook Chapter, so perhaps this is sufficient. ANSWER: We wish to keep the original 2013 reference, as the most comprehensive manual on GRADE. However, we will substitute the reference of the Spanish version for the English version. Reference 63 substituted for the reference to the original version" } ] }, { "id": "59540", "date": "28 Feb 2020", "name": "Edward Purssell", "expertise": [ "Reviewer Expertise Paediatrics", "infection control", "systematic review." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have a few suggestions which may be helpful to the authors:\nWhen discussing research methods, it may be worth just mentioning the efficacy/effectiveness issue.\n\nI think that this \"To obtain an efficient search with adequate sensitivity, performing searches in MEDLINE and EMBASE is sufficient, as they are the two most frequently used bibliographic databases39, and they are enough to identify most relevant studies for a specific SR40\" might be a bit of a sweeping statement - are the two databases always sufficient? I think it would be helpful to mention some others as well - unless the authors really do believe that these are enough.\n\n\"involving a medical librarian can be convenient to improve the search quality47–49\". I am not sure that convenient is quite the right word.\n\nBe clear to differentiate risk of bias at the study level from RoB at the review level. For example RoB2 is study-level, ROBIS is review level.\n\n\"For instance, in the SR by Ellis et al. (2017), the authors established a 70% heterogeneity limit for I2, beyond which a meta-analysis combining the results would not be performed13\" - why 70%? This does not sound like a sensible decision making process anyway. Either the authors thought it was worth doing a MA in which case the heterogeneity form parts of the results, or it is not worth doing in which case this would not be a consideration. Also not doing a MA may have the effect of hiding this heterogeneity. There are also ways of investigating heterogeneity that can be illuminating.\n\nMention ROBIS and AMSAR2 as review-level tools.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5748", "date": "11 Aug 2020", "name": "Marta Roqué", "role": "Author Response", "response": "When discussing research methods, it may be worth just mentioning the efficacy/effectiveness issue. ANSWER: We agree that this is an important issue, which merits a full discussion that unfortunately falls outside the scope of this project. The selection of resources done is not dependent on whether the reviewer explores efficacy or effectiveness, and will be useful to the researchers regardless of their intended purpose. However, we have clarified this issue throughout the text by referring to 'effects of interventions' (rather than efficacy of interventions) and we have explicitly commented on it in the discussion.We have substituted 'efficacy of interventions' by 'effects of interventions'. We have added the following limitation \"The selection of resources done is not dependent on whether the reviewer explores questions on efficacy or effectiveness, which are often described as explanatory or pragmatic questions, and will be useful to the researchers regardless of their intended purpose. However, we have not considered the resources to conduct in-depth exploration of effectiveness issues such as reviews of complex interventions or implementation reviews. \" I think that this \"To obtain an efficient search with adequate sensitivity, performing searches in MEDLINE and EMBASE is sufficient, as they are the two most frequently used bibliographic databases39, and they are enough to identify most relevant studies for a specific SR40\" might be a bit of a sweeping statement - are the two databases always sufficient? I think it would be helpful to mention some others as well - unless the authors really do believe that these are enough. ANSWER: Certainly, while MEDLINE and EMBASE are most used databases, there are other databases which can provide complementary information. However, the bibliographic references provided in the text supports the notion that these two databases are enough for an efficient search regardless of topic, and the role of these other databases is mostly complementary, but with little added benefit. To avoid the implicit suggestion that specialized databases cannot generate any added value, a sentence has been added mentioning them. Two sentences have been added \"To obtain an efficient search with adequate sensitivity, performing searches in MEDLINE and EMBASE is may be sufficient especially in intervention reviews\". \" These searches can be complemented with additional searches in other databases such as PEDro (Hyperlink: https://www.pedro.org.au/), which may provide specific information for certain topics. \" \"involving a medical librarian can be convenient to improve the search quality47–49\". I am not sure that convenient is quite the right word. ANSWER: Thanks, we've substituted the term 'convenient' for 'desirable' Be clear to differentiate risk of bias at the study level from RoB at the review level. For example RoB2 is study-level, ROBIS is review level. ANSWER: Thanks for pointing out this issue, we have clarified in the risk of bias section that this project focuses in presenting study-level resources, and that tools for reviews are not included in the manuscript. As we expand later in comment 6, we have been more explicit about the exclusion of overviews in the eligibility criteria section, and we have added a mention to this fact in the discussion section, as a limitation of the review, with an explicit mention of these tools. A sentence has been added \"Assessing the risk of bias of the included studies is a key element in any SR\". We have modified the eligibility criteria (\"We excluded the methodological resources to develop other types of SRs (methodological, economic evaluation and qualitative research SRs, or overviews)\" and the discussion (\"Reviews of reviews (or overviews) were also not considered, and as such, there are a number of review-level resources which have not been discussed, for example the risk of bias assessment tool ROBIS or the methodological tool AMSTAR2\") \"For instance, in the SR by Ellis et al. (2017), the authors established a 70% heterogeneity limit for I2, beyond which a meta-analysis combining the results would not be performed13\" - why 70%? This does not sound like a sensible decision making process anyway. Either the authors thought it was worth doing a MA in which case the heterogeneity form parts of the results, or it is not worth doing in which case this would not be a consideration. Also not doing a MA may have the effect of hiding this heterogeneity. There are also ways of investigating heterogeneity that can be illuminating. ANSWER: We agree with the peer reviewer that the analysis choices of the examples shown may not always be universally shared. We chose the examples based on several considerations, as they had to be useful for the different parts of the manuscript, but they are not necessarily a perfect role model in all their decisions. Readers will need to take into account that these examples are only illustrative, as we are presenting these choices descontextualized, without a full vision of the authors decision making process. Mention ROBIS and AMSTAR2 as review-level tools. ANSWER: Thanks for this suggestion. We restricted our focus to a limited list of SR types, not including overviews (or reviews of reviews). For this reason, only study-level resources for assessing risk of bias have been described, and review-level resources (such as ROBIS or AMSTAR2) are not mentioned. We realize that this issue may not be clear enough, and consequently we have been more explicit about the exclusion of overviews in the eligibility criteria section, and we have added a mention to this fact in the discussion section, as a limitation of the review, with an explicit mention of these tools. We have modified the eligibility criteria (\"We excluded the methodological resources to develop other types of SRs (methodological, economic evaluation and qualitative research SRs, or overviews)\" and the discussion (\"Reviews of reviews (or overviews) were also not considered, and as such, there are a number of review-level resources which have not been discussed, for example the risk of bias assessment tool ROBIS or the methodological tool AMSTAR2\")" } ] } ]	1	https://f1000research.com/articles/9-82
https://f1000research.com/articles/9-1235/v1	14 Oct 20	{ "type": "Opinion Article", "title": "Improving open and rigorous science: ten key future research opportunities related to rigor, reproducibility, and transparency in scientific research", "authors": [ "Danny Valdez", "Colby J. Vorland", "Andrew W. Brown", "Evan Mayo-Wilson", "Justin Otten", "Richard Ball", "Sean Grant", "Rachel Levy", "Dubravka Svetina Valdivia", "David B. Allison", "Danny Valdez", "Colby J. Vorland", "Andrew W. Brown", "Evan Mayo-Wilson", "Richard Ball", "Sean Grant", "Rachel Levy", "Dubravka Svetina Valdivia", "David B. Allison" ], "abstract": "Background: As part of a coordinated effort to expand research activity around rigor, reproducibility, and transparency (RRT) across scientific disciplines, a team of investigators at the Indiana University School of Public Health-Bloomington hosted a workshop in October 2019 with international leaders to discuss key opportunities for RRT research.\nObjective: The workshop aimed to identify research priorities and opportunities related to RRT.\nDesign: Over two-days, workshop attendees gave presentations and participated in three working groups: (1) Improving Education & Training in RRT, (2) Reducing Statistical Errors and Increasing Analytic Transparency, and (3) Looking Outward: Increasing Truthfulness and Accuracy of Research Communications. Following small-group discussions, the working groups presented their findings, and participants discussed the research opportunities identified. The investigators compiled a list of research priorities, which were circulated to all participants for feedback.\nResults: Participants identified the following priority research questions: (1) Can RRT-focused statistics and mathematical modeling courses improve statistics practice?; (2) Can specialized training in scientific writing improve transparency?; (3) Does modality (e.g. face to face, online) affect the efficacy RRT-related education?; (4) How can automated programs help identify errors more efficiently?; (5) What is the prevalence and impact of errors in scientific publications (e.g., analytic inconsistencies, statistical errors, and other objective errors)?; (6) Do error prevention workflows reduce errors?; (7) How do we encourage post-publication error correction?; (8) How does ‘spin’ in research communication affect stakeholder understanding and use of research evidence?; (9) Do tools to aid writing research reports increase comprehensiveness and clarity of research reports?; and (10) Is it possible to inculcate scientific values and norms related to truthful, rigorous, accurate, and comprehensive scientific reporting?\nConclusion: Participants identified important and relatively unexplored questions related to improving RRT. This list may be useful to the scientific community and investigators seeking to advance meta-science (i.e. research on research).", "keywords": [ "Meta-Science", "Science of Science", "Rigor Reproducibility and Transparency (RRT)", "Workshop" ], "content": "Introduction\n\nRigor, reproducibility, and transparency (RRT) are scientific cornerstones that promote truthful, accurate, and objective science (McNutt, 2014). In the context of scientific research, rigor is defined as a thorough, careful approach that enhances the veracity of findings (Casadevall & Fang, 2012). There are several types of reproducibility, which include the ability to evaluate and follow the same procedures as previous studies, obtain comparable results, and draw similar inferences (Goodman et al., 2016; National Academies of Sciences, 2019). Transparency is a process by which methodology, experimental design, coding, and data analysis tools are reported clearly and openly shared (Nosek et al., 2015; Prager et al., 2019). Together, these scientific norms represent the best means of obtaining objective knowledge of the world (Anderson et al., 2010; Allison et al., 2016). The science concerning these norms is a specific branch of meta-science, or “research on research”, led by scientists who promote these values through the education of early career scientists, identifying areas of concern for scientific validity, and postulating paths toward stronger, more credible science (Ioannidis et al., 2015).\n\nSeveral factors compete with the pursuit of rigorous, reproducible, and transparent research. For example, the rate of scientific publication has risen dramatically in the last two decades. Although this is indicative of many important scientific breakthroughs (Van Noorden, 2014), the rate of manuscript retractions due to either researcher error or malfeasance has also increased (Steen et al., 2013). A survey found between 40% and 70% of scientists agreed that factors including fraud, selective reporting, and pressure to publish contribute to the irreproducibility of scientific findings (Fanelli, 2018). These concerns also have the potential to decrease public trust in science, although research on this question is needed (National Academies of Sciences, 2017).\n\nBasic and applied science are undermined when scientists fail to uphold high standards of conduct (Prager et al., 2019). Given that many authors have identified issues or concerns in science, the emerging challenge for scholars in this area is to find workable solutions to improve RRT, rather than simply continuing to illustrate problems related to RRT (Allen & Mehler, 2019). To this end, in October 2019, Indiana University School of Public Health-Bloomington hosted a multidisciplinary meeting of leading scholars to discuss ongoing RRT-related challenges. The purpose of the meeting, which was funded by the Afred P. Sloan Foundation, was to identify new opportunities to advance sound scientific practice, from the early stages of planning a study, through to execution and the communication of findings. This paper presents findings from that meeting.\n\n\nMethods\n\nThe meeting was structured around three areas:\n\n(1) Improving education & training in RRT.\n\n(2) Reducing statistical errors and increasing analytic transparency.\n\n(3) Looking outward: increasing truthfulness and accuracy of research communications.\n\nWe invited participants based on prior contributions to RRT research. Participants included representatives from several leading organizations and Indiana University (IU) faculty, staff, and graduate students who were invited to participate in the meeting and proceedings (Table 1). For their participation in the meeting, invited guests who were not federal employees or IU employees received a $1,000 honorarium.\n\nThe two-day meeting was comprised of nine prepared research talks, moderated panel discussions, and small-group open-forum style sessions related to each of the three previously stated goals.\n\nDay one. On the first day, participants presented 10–12 minute research talks, each of which was followed by a moderated question-and-answer period. Participants discussed questions pertaining to RRT and sought to identify emerging areas of research including novel approaches, testable outcomes, and potential limitations. During the afternoon session, participants were divided into three small-groups to discuss potential research opportunities, moderated by an IU faculty representative charged with compiling notes for record keeping and dissemination.\n\nDay two. On the second day, one representative from each group summarized major points through a brief presentation, which was followed by a question-and-answer session with all participants. This dialogue was intended to clarify ideas raised and to identify fundable research opportunities. The meeting concluded with a call to action by the Dean of the School of Public Health-Bloomington and Co-Principal Investigator of the project (DA), to continue promoting interdisciplinary RRT Science.\n\n\nResults\n\nWe asked the first subgroup to discuss research opportunities related to implementing and testing RRT-guided academic curricula. The group identified elements of current undergraduate and graduate education that contribute to problematic data practices, including possible underlying causes and potential solutions (see Table 2). Three primary education-related questions guided the discussion:\n\n1We present here only two of the most salient challenges.\n\n(1) Can RRT-focused statistics and mathematical modeling courses improve statistical practice?\n\n(2) Can specialized training in scientific writing improve transparency?\n\n(3) Does modality affect the efficacy of RRT-related education?\n\nWith respect to each question the existing and entrenched practices, feasibility of change, and proper audience for interventions were discussed.\n\n1. Can RRT-focused statistics and mathematical modeling courses improve statistical practice?\n\nIncorrect analyses are some of the most common, preventable errors in science (Resnik, 2012). Scholars attribute mistakes to gaps in statistics education (Thompson, 2006). With the rise in data science as a component of scientific exploration, students need more exposure to evidence-based pedagogical approaches to statistics and mathematical modeling (GAISE, 2016; GAIMME, 2016, and NASEM, 2018). Many introductory data science courses include topics from statistics (e.g., contingency tables [chi-square tests], multiple regression, analysis of variance, and the broader general linear model) (Gorsuch, 2015), as well as mathematical modeling approaches and computational algorithms. These topics can be reframed through an RRT lens as modules/domains within existing mathematics or data-science courses or structured as new data-driven courses entirely.\n\nIndeed, participants noted that to improve RRT practices, there are opportunities to design new courses with a direct RRT focus at the undergraduate, graduate and postdoctoral levels (Willig et al., 2018). Courses could include modules related to the identification of errors in published research, proposing solutions to these errors, addressing real-world contexts and demonstrating the importance of careful methodological decision-making (Peng, 2015). Specific assignments could test for and reinforce RRT principles, such as research compendia (i.e. sharable electronic folders containing code, and other exploratory information to validate reported results) (Ball & Medeiros, 2012; King, 1995; Stodden et al., 2015), workflows, which are described later in this paper, and other research projects related to communication and computational reproducibility. The learning practices could be assessed to ensure that students appropriately apply concepts rather than demonstrate rote-formula memorization (Thompson, 2002; Ware et al., 2013). Integrating learning into stages of education where students are concurrently engaged in research can help improve both retention and transfer of the RRT ideas to future scientific settings.\n\n2. Can specialized training in scientific writing improve transparency?\n\nClear scientific writing is necessary to reproduce and build on research findings. To facilitate better writing, scholars have developed curricula to help academics improve writing practice and quality (e.g., Goodson, 2016). However, many academic writing programs focus on personal habit building and development of linguistic mechanics to craft more powerful prose (Elbow, 1998; Kellogg & Whiteford, 2009). In such courses, RRT-related dimensions of writing (such as writing transparently or minimizing ‘spin’) may not be emphasized. Thus, the subgroup discussed how existing writing curricula could incorporate RRT principles, what new writing courses guided by RRT would entail, and research opportunities to test the efficacy of new writing curricula.\n\nParticipants identified several RRT-specific writing principles and discussed how a deeper understanding of the extent to which writing and research are intertwined may increase transparency. Examples included learning about methodological reporting guidelines, writing compelling post-publication peer reviews, and other transparent writing practices. The group also discussed how courses could be developed or redesigned specifically to center on RRT principles. One theme of the discussion was the need for rigorous testing of student learning outcomes associated with novel writing content. However, a primary concern was the identification of the appropriate outcome measures for writing-specific interventions (Barnes et al., 2015) given the subjective and nebulous nature of constructs like writing quality, individual improvement, and writing-related self-efficacy.\n\n3. Does modality affect the efficacy of RRT-related education?\n\nAnother research opportunity discussed by the subgroup related to instructional modality, which refers to the manner in which a curriculum or intervention is experienced by the learner (Perry & Pilati, 2011). These may include traditional face-to-face instruction, synchronous or asynchronous online meetings/trainings, and various hybrid formats (Beall et al., 2014). Understanding the relative benefits of each modality is important in choosing an appropriate intervention. Indeed, educational needs vary among learner groups; for example, what is most effective for undergraduate students may not be effective or feasible for post-doctoral researchers with full-time professional commitments. Broad research questions identified by the group included:\n\na) What modalities exist beyond face-to-face, online, or hybrid instruction?;\n\nb) How can technology push modality beyond online courses and other Massive Open Online Course formats?\n\nc) Which modality is most effective and among which audiences?\n\nIn the context of previously discussed coursework in statistics and writing, participants explored the strengths and weaknesses of various modalities and how interventions could be conducted to test them empirically. There are logistical considerations, such as cost, space, and faculty time, that further complicate the feasibility of these interventions. For example, a face-to-face intervention may offer more tailored instruction to individual learners, while an online intervention may better deliver content to a wider audience. Thus, the subgroup identified several areas for future research, including comparisons of student learning across modalities, strategies for scaling educational content to institutional constraints, and the moderating effects of learner demographics on intervention efficacy.\n\nErrors are “actions or conclusions that are demonstrably and unequivocally incorrect from a logical or epistemological point of view” (Brown et al., 2018). Despite the adage that science is self-correcting, uncorrected errors are prevalent in the scientific literature (Brown et al., 2018; Ioannidis, 2012). Subgroup 2 discussed questions related to reducing and mitigating such errors, including:\n\n(4) Can automation help identify errors more efficiently?;\n\n(5) What is the impact of errors within disciplines?;\n\n(6) Do standardized procedures (i.e., workflows) prevent errors?\n\n(7) How do we encourage post-publication error correction?\n\nThe costs and benefits associated with each question were also discussed (see Table 2).\n\n4. Can automation help identify errors more efficiently?\n\nVarious automated and manual methods have been developed and applied to assess analytic inconsistencies, statistical errors and improbabilities, and other errors (e.g., Anaya, 2016; Baggerly & Coombes, 2009; Brown & Heathers, 2017; Georgescu & Wren, 2018; Labbé et al., 2019; Monsarrat & Vergnes, 2018). An increase in automation (i.e., producing more user-friendly tools and algorithms) has the potential for surveilling the prevalence, prevention, and correction of errors. However, more work is needed to determine the most efficient use of such tools, including their collective abilities to detect field-specific issues that require subject matter expertise (Lakens & Debruine, 2020). For example, the automatic recomputation of some p-values is possible using the program ‘Statcheck’, but only for articles that utilize the American Psychological Association’s (APA) in-text citation style for statistical reporting (Nuijten et al., 2017). Other examples require statistical ratios (Georgescu & Wren, 2018), or integer-based data and sample sizes (e.g., Brown & Heathers, 2017; Heathers et al., 2018), which are both challenging to automate and not recurrent across all fields.\n\nAutomated error detection is currently limited to a narrow range of errors. Other types of errors might be detected by careful readers, such as the ignoring of clustering in cluster-randomized trials (Brown et al., 2015; Heo et al., 2018), misinterpretation of differences in nominal significance, and post-hoc fallacies (Brown et al., 2019; George et al., 2016). The subgroup discussed opportunities to define, and possibly automate, diagnostic checklists, advanced natural language processing, or other computational informatics approaches that would facilitate the detection of these errors. These novel automated measures could be tested empirically for effectiveness.\n\n5. What is the prevalence and impact of errors?\n\nDifferent errors will have varying impacts on study conclusions. While some errors can be easily corrected and reported, some fundamentally invalidate study conclusions. Some general statistical errors have occurred repeatedly across disciplines for decades (e.g., mistaken differences due to “regression to the mean” since at least 1886 [Thomas et al., 2020] and “differences in nominal significance” for decades [Altman, 2002; Thompson, 2002]). Automated methods, such as those outlined above, have been used almost exclusively to illuminate problems but not necessarily correct them (Georgescu & Wren, 2018; Monsarrat & Vergnes, 2018; Nuijten et al., 2017).\n\nTo achieve the goal of error reduction, one must first know how pervasive errors are. Yet, it remains challenging to generalize the detection and correction of scientific errors across disciplines because of field specificity (i.e. the unique nuances and methodological specificities inherent to a specific field of study) (Lohse et al., 2020), the various terminologies used for describing the same models (e.g. ‘Hierarchical Linear’ models vs ‘Multilevel’ models), as well as the seeming need to repackage the same problem as new disciplines arise (e.g. ongoing multiple comparison issues raised anew with the advent of genome-wide association studies, microarray, microbiome, and functional magnetic resonance imaging methods). Thus, this subgroup discussed the value of longitudinal, discipline-specific error surveillance and error frequency estimation to collect empirical evidence about error rate differences among disciplines. Other issues discussed were the identification of better prevalence estimates across fields, and how simulation studies can modify our confidence in the understanding of the prevalence of errors and their generalizability across disciplines.\n\n6. Do error prevention workflows reduce errors?\n\nWorkflows are the various approaches for accomplishing scientific objectives, usually expressed as tasks and dependencies (Ludäscher et al., 2009). The implementation of clear, logical workflows can potentially prevent errors and improve research transparency. Workflows may be of value to catch errors at various stages of the research process, from planning, to data collection and handling procedures, and reporting/manuscript screening (Cohen-Boulakia et al., 2017). Error detection processes within scientific workflows may serve as mechanisms to prevent errors before publication, akin to how text duplication software (e.g. iThenticate) is used prophylactically to catch inadvertent plagiarism. Separately, some research groups implement workflows that require two independent scientists to verify data, analyses, and statistical reporting prior to manuscript publication, with at least one of those individuals being a professional statistician (George et al., 2016). A similar workflow is to establish “red teams”, consisting of methodologists, statisticians, and subject-matter experts, to critique the study design and analysis for errors, offering incentives akin to “bug bounty” programs in computer software development (Lakens, 2020).\n\nThe development and dissemination of research workflows could be modeled after those outlined above, or in other ways such as the use of checklists to complete work systematically. Registrations, reporting guidelines, and other workflow approaches essentially serve as checklists of the plan for a study and what should be reported. Although this subgroup agreed about the importance of preventive versus post-publication workflows and integration of automated methods to detect errors, questions regarding their efficacy remained. For example, how might workflows be generalized across academic disciplines? At what level should standardizing data collection and handling be taught to scientists to maintain data provenance (e.g. Long, 2009)? And can workflows be tested empirically? What is the cost of automated versus manual workflows, versus none at all, at detecting and preventing errors? How do workflows impact productivity?\n\n7. How do we encourage post-publication error correction?\n\nScience cannot self-correct without processes that facilitate correction (Firestein, 2012). Unfortunately, errors in science may be tied with perceived reputation costs, yet it is unclear that correcting errors actually harms a researcher’s reputation (Azoulay et al., 2015). Thus, destigmatizing error correction and likewise embracing the importance of scientific failures may be of value for individual scientists and editors overseeing content through the peer-review process (Teixeira da Silva & Al-Khatib, 2019). Journals and their editors, as gatekeepers of science, are key stakeholders in this culture shift. They may also require practical guidelines to facilitate judgement-free corrections that would be acceptable to editors and reviewers.\n\nError correction should be done in a fair and efficient manner (e.g., Vorland et al., 2020). Although there are several existing standards for publication ethics and norms (e.g., Committee on Publication Ethics [COPE], and the International Committee of Medical Journal Editors [ICMJE]), few have been tested empirically. The subgroup debated how journals and their editors could be part of empirically tested trials on best approaches to facilitate correction and minimize the incurring of additional costs. For example, based on our experiences, journals have few procedures for handling errors separate from typical scholarly dialogue. We believe it is important to examine which procedures are more efficient and fair to authors, whether such procedures can be standardized to enable editors to handle different types of errors consistently and transparently, whether correction mechanisms are sufficient or require additional innovation (e.g. retraction and republication is sufficient or versioning), and how authors can be supported and encouraged in the process. Three such costs that require further study include the actual cost of post-publication error correction across all parties involved (e.g. page charges, salary), how those costs to the scientific enterprise compare to implementing prevention strategies, and the cost-benefit of salvaging a publication containing an error depending on the quality of the collected data versus simply retracting.\n\nThe third working group discussed opportunities for research related to research reporting and dissemination, primarily highlighting the importance of accuracy and truthfulness when communicating research findings (see Table 2). Specifically, this group identified research opportunities tied to the following questions:\n\n(8) How does ‘spin’ in research communication affect stakeholders’ understanding and use of research evidence?\n\n(9) Do tools to aid writing research reports increase the comprehensiveness and clarity of research reports?\n\n(10) Is it possible to inculcate scientific values and norms related to truthful, rigorous, accurate, and comprehensive scientific reporting?\n\n8. How does “spin” in research communication affect stakeholders’ understanding and use of research evidence?\n\nIn addition to conducting research rigorously, investigators should describe their research comprehensively and interpret their findings by balancing the strengths and limitations of their methods and results (Brown et al., 2017). By contrast, researchers might ‘spin’ their results through misleading reporting, misleading interpretation, and inappropriate extrapolation (Fletcher & Black, 2007; Yavchitz et al., 2016). Some evidence suggests that spin is common in reports of clinical trials and meta-analyses (Boutron et al., 2019; Lazarus et al., 2015) and that authors in a variety of research disciplines often draw inappropriate causal inferences (Bleske-Rechek et al., 2015; Casazza et al., 2013; Chiu et al., 2017; Knight et al., 1996; Ochodo et al., 2013). Moreover, spin in popular media (e.g., newspapers) appears to stem from spin in scientific reports (e.g., journal articles) and associated press releases (de Semir et al., 1998; Schwartz et al., 2012; Schwitzer, 2008).\n\nSpin is unscientific, and could have implications for policy and practice (Adams et al., 2016; Boutron et al., 2019; Matthews et al., 2016). Workshop participants discussed the need for more evidence to determine whether and how spin in scientific reports affects other stakeholders such as healthcare and social service providers, service users, policymakers, and payers. Evidence concerning the ways in which stakeholders use and interpret research evidence could inform future efforts to improve research communication (Boutron et al., 2019; Lazarus et al., 2015).\n\n9. Do tools to aid writing research reports increase the comprehensiveness and clarity of research reports?\n\nResearch reports (e.g., journal articles) should describe what was done and what was found (von Elm et al., 2007). Stakeholders need comprehensive and accurate information about research methods and results to assess risk of bias, interpret the generalizability of study results, and reproduce the conditions (e.g., interventions) described (Moher et al., 2011). Reporting guidelines describe the minimum information that should be included in reports of different types of research, yet much evidence suggests that scientific reports do not include this information (e.g., Grant et al., 2013). Some tools to help authors write better reports have been developed, such as the consort-based web tool (COBWEB) (Barnes et al., 2015); some preliminary evaluations suggest that these tools could help authors write better reports.\n\nWorkshop participants identified a need for research to develop and to test tools that could help authors write reports that adhere to existing guidelines. Some tools could be used when writing scientific manuscripts (Turner et al., 2012) while other tools could be used in graduate education (e.g. class assignments, dissertation writing) or continuing education. Guidelines designed to increase authors’ and reviewers’ knowledge of reporting requirements are not commonly adhered to and, thus, have minimal impact on reporting quality (Capers et al., 2015). Participants emphasized the need for new interventions and implementation research that promote guideline adherence.\n\n10. Is it possible to inculcate scientific values and norms related to truthful, rigorous, accurate, and comprehensive scientific reporting?\n\nIn the 1940s, Robert Merton proposed that communism/communality, universalism, disinterestedness, and organized skepticism constitute the ethos of modern science (Merton, 1942). As the National Research Council stated in their report “Scientific Research in Education”, these fundamental principles are enforced by the community of researchers that shape scientific understanding (Shavelson & Towne, 2003). Evidence suggests that most scientists endorse these positive values and norms, but fewer scientists believe that their colleagues behave in accordance with these positive norms (Anderson et al., 2007). Better incentives (Begley et al., 2017; Fanelli, 2010; Nosek et al., 2012) and better methods for detecting scientific errors, might improve scientific practice and communication; yet fundamentally, we will always have to place some trust in the veracity of our fellow scientists (Jamieson et al., 2017).\n\nParticipants agreed that ethics and responsibility are vital across scientific disciplines, yet graduate research often neglects the philosophy of science and the formation of professional identity as a scientist. Instead, training tends to focus on the technical skills needed to conduct experiments and analyze data in specific disciplines (Bosch, 2018; Bosch & Casadevall, 2017). Technical skills are essential to produce good science; to apply them ethically and responsibly, however, it is paramount that scientists also endorse scientific values and norms. Participants identified a need for research to determine how these scientific values could be inculcated in scientists and how scientists should be taught to enact those values in their research.\n\n\nConclusion\n\nScientists slow the pursuit of truth when research is not rigorous, reproducible, or transparent (Collins & Tabak, 2014). To improve the state of science, RRT leaders have long raised concerns about many of the current challenges the scientific enterprise faces by identifying novel strategies intended to uphold and improve scientific validity. Discussions among RRT leaders at Indiana University Bloomington reinforce the value and importance of promoting accurate, objective, and truthful science. The proposal, execution, and evaluation of the ideas presented herein showcases how the collective and interdisciplinary efforts of those investing in the future of science can solve problems in unique and exciting ways.\n\n\nData availability\n\nNo data are associated with this article.\n\nAll participants have provided their permission to be named in this article.", "appendix": "References\n\nAdams SA, Choi SK, Eberth JM, et al.: Adams et al. Respond. Am J Public Health. 2016; 106(6): e8–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllen C, Mehler DMA: Open science challenges, benefits and tips in early career and beyond. PLoS Biol. 2019; 17(5): e3000246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAllison DB, Brown AW, George BJ, et al.: Reproducibility: A tragedy of errors. Nature. 2016; 530(7588): 27–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltman DG: Poor-Quality Medical Research: What Can Journals Do? 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "77096", "date": "28 Jan 2021", "name": "Judith A Hewitt", "expertise": [ "Reviewer Expertise infectious diseases", "animal models of infectious diseases", "translational research", "scientific rigor", "reproducibility" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 concise and well written summary of a meeting of ~30 people on the vitally important topic of rigor, reproducibility & transparency. The meeting discussion questions were very well formulated, though with the small size of the meeting and the limited number of invited participants outside of the university host, it is difficult to say whether the discussions, presented in a very succinct format of key challenges, is representative of all of the issues or viewpoints on the topic. Nevertheless, this appears to have been a good discussion that raised significant challenges. I would have preferred to see a bit more focus on solutions, as the challenges raised are all daunting.\nSpecific Comments:\nIntroduction: Regarding the statement that 40-70% of scientists agreed on factors contributing to irreproducibility, the original citation be used, (Baker, 2016; added1). Also, reference to the funder for the meeting is very much appreciated - but it is \"Alfred P Sloan\" not \"Afred\". In the last sentence, \"\"through to execution\" is unwieldy - either \"through\" or \"to\" works but no need for both.\nMethods: I very much appreciate the list of participants and acknowledgement of honorariums - kudos on the transparency! I also appreciate knowing who participated in the small groups, but it would have been nice to see the agenda or titles of the Day One research presentations. Were those research or meta-research presentations? Also, \"small-groups\" should not be hyphenated, in fact you could just say three groups and let the reader come to their own conclusion about size; \"breakout\" is another useful term.\nResults: Subgroup 1, first paragraph: the following wording could be more precise by changing \"three primary education-related questions\" (where primary modifies education and not questions) to \"three primary questions, education-related,\" or something similar. Precision of language is one of the articulated goals of training and communication in this article!\nQ5, 2nd paragraph: I disagree with the first sentence, \"To achieve the goal of error reduction, one must first know how pervasive errors are.\" I think any reduction in errors is a win, even without understanding the entire landscape, and needing to fully understand the landscape before attempting solutions is just kicking the can down the road. It's the \"measurement\" of error reduction or assessing progress toward a particular goal (which is not articulated) that requires knowing the pervasiveness first, and I agree that is extremely difficult to measure.\nQ7, 2nd paragraph, last sentence: I question whether understanding \"salary\" costs of error correction is a valid pursuit, whether it's a case of pay now or pay later; page charges are a different matter.\nConclusion: Since the Methods section stated that the meeting ended with a \"call to action\" to continue promoting interdisciplinary RRT science, I wonder if that call to action is accurately summarized? I found a great summary of the discussion but didn't walk away with a clearly articulated call to action in the very brief conclusion.\nGeneral Comments: I tend to agree that the challenges are many and difficult, though the small group discussions are distilled down to two challenges per question. They are mainly framed in negative terms, which is hard to read as a \"call to action\" without more detail. Nonetheless, the challenges raised are important and should be addressed, I'm just left scratching my head on what the next step is for many of these, given how they are stated.\nI note that many of the references are from participants at the meeting, which may reflect the meeting content (difficult to judge without seeing the agenda), but does not necessarily instill in others an unbiased approach; this is perhaps a limitation of a small-meeting-by-invitation and could be formally recognized in the paper. This is not a value judgement on the references, indeed there is some balance, but it is a selected view that focuses on the meeting participants.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "78351", "date": "18 Feb 2021", "name": "Christopher A Mebane", "expertise": [ "Reviewer Expertise I am an environmental scientist who has published on related topics of research rigor", "bias", "and transparency in the environmental sciences." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 “Improving open and rigorous science....” is a report out on a workshop intended to make recommendations on improving rigor, reproducibility, and transparency (RRT) in interdisciplinary science. The idea of peer reviewing a workshop report is a bit of a curious assignment. What’s a reviewer to say? No, those weren’t the best topics to debate at your workshop, please reconvene and discuss something else? Raise questions about whether the article faithfully reports the workshop deliberations and consensus, when the reviewer wasn’t there? As such this review is rather limited. The article reads well and has clearly been well vetted by the authors. The workshop and paper are interdisciplinary, although the focus is strongly slanted toward biomedical research and the health sciences.\nNot all errors are mistakes My only criticism of substance is the use of the term “statistical errors.” Consider replacing it with “statistical mistakes” throughout the manuscript. In many fields, including mine (environmental science), the word “error” could refer to variability in the data, such as “the standard error of the mean.” In other contexts, the word error is often used to describe the limits of precision. DNA and cells replicate with small errors, which over time lead to aging and senescence. In analytical chemistry, deviations from instrument values for calibration or quality control samples may be termed measurement error. Measurement error might refer to the inherent limits of a sensor in the instrument or the combined errors of the method. For example, in a bathymetric survey, errors accrue from inherent limits in the measuring distance as a function of sound through water, temperature changes in the water introduce error, a breeze adding motion to the boat introduces error, plants growing on the bottom muddy the signal increasing error, imprecision in the Earth’s spheroid and canyon walls interfere with the GPS, and on and on. The hydrologist tries to reflect the accumulated error with a margin of error statement on overall accuracy. Those are examples of error – something the scientist always seeks to reduce and to accurately report the uncertainties associated with measurements, modeling, etc., but the presence of error is unavoidable. A mistake on the other hand is a blunder. Attaching the bathymetric sensor backwards, entering the wrong units into the calculations, using a long-wave, deep ocean sensor in shallow water, using the wrong datum, using a poorly suited method, neglecting calibrations, .... Just as with statistical mistakes, the topic of the argument, while there are often different appropriate methods of measurement for just about any scientific setting, some controversial or debatable methods, and some that are just plain wrong. The focus of the authors is on the latter – helping scientists avoid statistical blunders that are just plain wrong. I strongly urge you to call these “statistical mistakes” which is less ambiguous than “errors.” There are supposed to be interdisciplinary RRT recommendations.\nMinor suggestions p7., in subsection titled “5. What is the prevalence and impact of errors,” I thought the second paragraph was particularly dense and probably impenetrable to those not already in the know: : “Thus, [Subgroup 2] discussed the value of longitudinal, discipline-specific error surveillance and error frequency estimation to collect empirical evidence about error rate differences among disciplines. Other issues discussed were the identification of better prevalence estimates across fields, and how simulation studies can modify our confidence in the understanding of the prevalence of errors and their generalizability across disciplines.”\nI think if you could expand on these points with some examples or examples with citations, readers might have better understanding of what is being recommended.\nThat’s all. This was a tightly written report out of the workshop. Thank you for considering my rant about mistakes versus errors, where depending on the field and context, the latter is often a neutral descriptor of uncertainty.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "78363", "date": "19 Feb 2021", "name": "Sheenah M Mische", "expertise": [ "Reviewer Expertise Pathology", "Technology", "Shared Research Resource" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a concise summary of a two day workshop held at Indiana University School of Public Health - Bloomington on identifying key opportunities for rigor, reproducibility & transparency (RRT) in research. This is not a research report, rather a report on the status of scientific research. The meeting attendance was invitation only and IU faculty staff and graduate students were joined by invited participants with recognized expertise in RRT, and reflected in the extensive references. Opportunities were focused in three key areas: 1) education and training, 2) reducing statistical errors while increasing analytical transparency and 3) improving transparency (truthfulness) and accuracy of research communications to promote accurate, objective, and truthful science. The article reads well with a focus on biomedical research.\nSpecific Comments: This manuscript provided an excellent summary of numerous and important challenges facing the research enterprise. There were no applicable outcomes or solutions. Of particular note:\nEducation and training: instructional modality, and understanding the relative benefits of various hybrid formats: there is no debate on the importance of RRT education and training. Both formal and informal forums are critical to research-integrity issues. Bringing scientific integrity issues into the open provides practical guidance for everyone from graduate students to faculty members. Faced with the pandemic, we all have adapted modalities to virtual platforms, emphasizing the importance of instruction regardless of format. Furthermore, formal instruction in rigorous experimental design and transparency is now required for NIH training, career development, and fellowship applications.\n\nReducing statistical errors while increasing analytical transparency and 3) improving transparency (truthfulness) and accuracy of research communications: sharing knowledge is what drives scientific progress—each new advance or innovation in biomedical research builds on previous observations. Experimental reports must have sufficient information to validate the original results and be verified by other researchers to be broadly accepted as credible by the scientific community. While statistics is a necessary for data interpretation by clinical researchers, psychologists, and epidemiologists whose conclusions depend wholly on statistics, the interpretation of data in papers published in the biological sciences does not always require sophisticated statistical analyses; rather, diligent data reporting and transparency is essential.\nConclusion: The authors summarize with “proposal, execution, and evaluation of the ideas presented herein showcases how the collective and interdisciplinary efforts of those investing in the future of science can solve problems in unique and exciting ways”. While appreciating this forward looking statement, the message is clear: the issue of reproducibility in science is complex and will continue to be debated and discussed in workshops such as this manuscript describes in the coming years. In response to well-publicized allegations of the inability to reproduce published biomedical research there have been numerous declarations of the components of trustworthy research and research integrity such as the Singapore Statement in 2010, the Montreal Statement in 2013, the Hong Kong Principles in 2019 and the European Code of Conduct for Research Integrity in 2017, and U.S. NIH and NSF Federal RRT policies. Ultimately we are all responsible for careful assessment of the rigor of the prior research, rigorous experimental design for robust and unbiased results by application of the scientific method; consideration of relevant biological variables and authentication of key biological and/or chemical resources used to conduct research and the use of numerical identifiers and the RRID syntax to improve communication of critical experimental details within the research community and to the public.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1235
https://f1000research.com/articles/9-1234/v1	14 Oct 20	{ "type": "Case Report", "title": "Case Report: Hemolysis in a G6PD-deficient patient, a dose-dependent effect of metformin", "authors": [ "Yusra Irshad", "Mahum Nadeem", "Usman Khan", "Ezza Tariq", "Aemen Khakwani", "Mahum Nadeem", "Usman Khan", "Ezza Tariq", "Aemen Khakwani" ], "abstract": "Glucose-6-phosphate deficiency is an X-linked genetic disorder, which predisposes erythrocytes to oxidative stress, resulting in hemolysis. It is the most common enzymatic deficiency, and typically affects African, Asian, Mediterranean, and Middle Eastern lineages. It can be induced by some medications, chemicals, and foods. Metformin is an uncommon drug to cause hemolysis in G6PD-deficient patients. We report a case of a 52-year old African American male with G6PD-related hemolysis secondary to toxic metformin accumulation with acute kidney injury (AKI). The patient was a type-2 diabetic and was taking metformin (500mg twice daily) for three years. He presented to the ER with nausea, vomiting, and diarrhea for last three days with severe hemodynamic instability. Labs revealed hemoglobin 15mg/dl, white blood count 28 mm3/L, creatinine 10 mg/dl, blood urea nitrogen 100 mg/dl, bicarbonate 7 mEq/L, lactic acid 17 mg/dl, pH 6.8, pCO2 21mmHg, metformin 41 mcg/ml, albumin-globulin 41. Severe sepsis protocol was activated; IV fluids 30ml/kg bolus and antibiotics were given. CT abdomen revealed colitis. The patient was started on continuous renal replacement therapy. The next day, the patient’s hemoglobin dropped to 12.6 mg/dl. A hemolytic panel was unswerving with hemolysis and G6PD levels reported low at 1.72. The patient improved with antibiotics, but the hemolysis continued. Metformin toxicity induced hemolysis was suspected. The patient’s hemoglobin dropped to 6g/dl and he received blood transfusions. His hemoglobin started to improve with hemodialysis sessions, as metformin levels started to normalize, emphasizing the fact that patient was clearing metformin. Unlike most cases reported, in which hemolysis occurs within days to months of starting metformin, in our patient it occurred due to the cumulative effect of metformin because of the patient’s underlying AKI. This led us to propose that the hemolytic effect of metformin may not only be time-dependent but also dose-dependent.", "keywords": [ "metformin", "glucose-6-phosphate dehydrogenase deficiency", "hemolytic anemia", "acute kidney injury." ], "content": "Introduction\n\nG6PD is an X-linked inherited red blood cell enzymatic disorder and affects 400 million individuals worldwide. It is most frequently seen in individuals with African, Asian, Mediterranean, or Middle Eastern lineage. The disorder can be asymptomatic but varied gene mutations cause different levels of enzyme deficiency, leading to inconstant disease presentation1. The deficiency causes neonatal hyperbilirubinemia, acute hemolysis, and chronic hemolysis. Acute hemolysis occurs due to exposure to an oxidative stressor that can be in the form of an infection, oxidative drug, or fava bean.\n\nNumerous medications have been identified to induce G6PD deficiency induced hemolysis, but metformin is seldom reported as a cause of hemolysis. It is an oral antidiabetic drug and the first-line agent in the treatment of type II diabetes mellitus. Its side effects include lactic acidosis, a serious condition that occurs in case of drug overdose (most commonly seen in patients with liver disease and kidney disease), individuals with low circulating oxygen level in the blood (e.g., congestive heart failure, recent stroke), alcoholism, and dehydration2. Hemolysis is not a side effect of the drug. So far only eight cases have been reported3–10 in the literature where metformin use resulted in either G6PD associated hemolysis4,6 or drug-induced hemolysis3,5,7–10.\n\n\nCase presentation\n\nA 52-year-old African American male with significant past medical history of type II diabetes mellitus, hypertension, presented to the ER with complaints of severe vomiting, nausea, and diarrhea for three days. The patient was taking losartan (50 mg per oral daily) and metformin (500mg twice daily) for the last three years. He was compliant with his medications despite having profuse diarrhea for the last 3–4 days. On examination, the patient was severely hypovolemic and hemodynamically unstable with blood pressure of 76/46 mm of hg.\n\nThe patient was admitted and managed with IV fluids and meropenem 1gm iv BID, as per severe sepsis guidelines. Initial investigations revealed the following labs: hemoglobin 15mg/dl (normal range, 11.9–14.8mg/dl); white blood count 28 mm3/L (3.8–10.4mm3/L); creatinine 16.77 mg/dl (0.70–1.30mg/dl); blood urea nitrogen 100 mg/dl (8-20mg/dl); potassium 5mmol/L (3.5–5mmol/L); blood glucose 78mg/dL (140–200mg/dL); bicarbonate 7 mmol/L (23–28mmol/L); lactic acid 17 mg/dl (6–19mg/dl); arterial blood gas showed severe metabolic acidosis; pH 6.8 (7.38–7.44); pCO2 21mmHg (38–42mmHg); metformin level was 41; and anion gap 41mmol/L (7–13mmol/L). Diagnosis of sepsis and metformin-induced severe lactic acidosis was suspected.\n\nA CT abdomen/pelvis was done, which showed colitis with some diverticulitis. Blood cultures were sent, and the patient was started on meropenem 1 gram, intravenous twice daily, empirically. The patient was emergently started on continuous renal replacement therapy in the light of severe metabolic and lactic acidosis. The next day, the patient developed jaundice, and hemoglobin dropped from 15 to 12.6 mg/dl. Total bilirubin was 3.2mg/dl (0.3–1mg/dl) and indirect bilirubin was 2.5mg/dl (0.2–0.7mg/dl). A hemolytic panel was consistent with hemolysis, and G6PD levels were reported as low at 2.82 (5–15 units/g of hemoglobin). Antiglobulin tests for IgG and C3 were negative with normal C3 and C4 levels. HIV ELISA and Reichlin's profile was negative.\n\nThe patient markedly improved clinically two days’ post-admission with IV antibiotics, emergent hemodialysis with the resolution of sepsis, but the hemolysis persevered and anemia deteriorated. Metformin toxicity induced hemolysis was suspected after reviewing the literature. The patient dropped hemoglobin to the nadir of 6 g/dl on day five of admission with hematocrit dropping to 15%. He underwent packed red blood transfusion considering life threatening low hematocrit and hemoglobin levels. The patient’s hemoglobin levels improved with intermittent hemodialysis as the metformin levels normalized, emphasizing the fact that the patient was clearing metformin from the system. Table 1 shows the downward trend in the metabolic profile of the patient.\n\nThe patient was discharged home on day 11 once his hemoglobin rose to 9.8g/dl and creatinine dropped back to 1.4mg/dl. He remained uneventful after the dialysis. Metformin use was prohibited for the rest of life. In our patient, metformin-induced hemolysis occurred due to dose-dependent toxicity, as previously he was using metformin 1 gram per oral twice daily but developed no side effects.\n\n\nDiscussion\n\nOur patient’s medication history was meticulously studied in order to find any other drug causing hemolytic anemia besides metformin. No other hemolytic risk factors were identified in the patient’s medical history. His blood G6PD levels were reported significantly low at 1.72. The drop in hemoglobin did not improve even with the cessation of antibiotics and dropped consistently until metformin was cleared from the patient’s system by dialysis, confirming that it was a dose-dependent effect of metformin. Our patient had acute kidney injury secondary to sepsis, gastroenteritis and hypovolemia and unfortunately the patient continued to take his metformin during this phase, which resulted in accrual of the metformin levels.\n\nA literature search was performed concerning the side effects of metformin and its connection with G6PD related hemolytic anemia. Metformin-induced hemolysis in G6PD patients is very rare and, to the best of our knowledge, only seven cases are reported in the literature3–10. Discontinuation of metformin resulted in the recovery of most cases, except one which had a fatal outcome (hemoglobin drop of 11.4mg/dl) regardless of metformin discontinuation5. That patient developed fulminant and fatal Coombs-positive hemolytic anemia, which was temporally related to the initiation of metformin treatment in the absence of any other likely cause.\n\nTable 2 describes the demographic variation of individuals and the temporal relationship after the initiation of metformin and the onset of hemolytic anemia from the literature. All the patients developed hemolytic anemia irrespective of the dosage of metformin, but in one patient a definitively higher dosage caused the sudden onset of hemolytic anemia within hours of metformin toxicity secondary to overdosing (45000mg)9 with a drop in hemoglobin of 8.6g/dl. This patient also developed lactic acidosis and underwent continuous renal replacement therapy. In our patient, due to acute kidney injury, hemolytic anemia started after a day with a severe drop in hemoglobin of 9.5g/dl over a course of four days. Hemolysis occurred due to the cumulative effect of metformin because of the patient’s underlying acute kidney injury. Naranjo's adverse score was 5. This leads us to propose that the hemolytic effect of metformin may not only be time-dependent but also dose-dependent. Both of these cases prove that with higher doses and renal impairment (as in our case), the drastic effect of metformin-induced hemolysis was seen together with lactic acidosis.\n\nACLSP, advanced cardiac life support protocol; BT, blood transfusion; CRRT, Continuous Renal Replacement Therapy; Hb, hemoglobin; T.B, total bilirubin\n\nFrom the literature, it can be observed that most patients developed hemolytic anemia within 2–14 days of metformin commencement. Our patient is unique as he developed anemia in his third year of treatment after accumulating toxic level of metformin in the setting of acute kidney injury and lactic acidosis. Patients with North African, Jewish descent had low G6PD activity with a negative anti-globin test, showing the connection between G6PD-induced hemolytic anemia secondary to the use of metformin as seen in two cases prior in addition to our case4,6. Other cases with positive antiglobulin test3,5,7 increased the possibility of drug-induced hemolytic anemia secondary to antibody formation. Supportive treatment was given in all cases, except in the case of metformin poisoning9 and our case, which required continuous renal replacement therapy and blood transfusion. The case with the fatal outcome5 developed cardiac arrest after treatment under advanced cardiac life support protocol.\n\n\nConclusion\n\nMetformin induced hemolysis is an infrequent presentation of hemolytic anemia. Patients with G6PD deficiency can be more predisposed to this side effect, and this should always be kept in the back of mind while reconnoitering causes of hemolysis in G6PD deficiency. Clinicians should be vigilant before starting metformin and renal function test should be periodically done to rule out any acute kidney injury as it triggers the toxic effects of metformin, leading to severe adverse effects like lactic acidosis and hemolysis. Patients should be educated that in case of weakness, pallor, fatigue, and jaundice, they should report to hospital immediately.\n\nThe temporal relationship has been beforehand observed in metformin-induced hemolysis, but the dose-dependent snowballing effect can also give a similar presentation of metformin toxicity. Metformin induced hemolysis could be either due to drug-induced antibodies with normal G6PD levels or by oxidative damage in G6PD deficient individuals with normal or low G6PD levels.\n\n\nConsent\n\nWritten informed consent for publication of the case report along with any associated images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nJennifer E, Frank MAJ: Diagnosis and Management of G6PD Deficiency. Am Fam Physician. 2005; 72(7): 1277–1282. PubMed Abstract\n\nNasri H, Rafieian-Kopaei M: Metformin: Current knowledge. J Res Med Sci. 2014; 19(7): 658–664. PubMed Abstract \| Free Full Text\n\nKirkiz S, Yarali N, Bilir OA, et al.: Metformin induced hemolytic anemia. Med Princ Pract. 2014; 23(2): 183–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPacker CD, Hornick TR, Augustine SA: Fatal hemolyticanemia associated with metformin: a case report. J Med Case Rep. 2008; 2: 300. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlum A, Ghaben W, Slonimsky G, et al.: Metformininduced hemolytic anemia. Isr Med Assoc J. 2011; 13(7): 444–445. PubMed Abstract\n\nMeir A, Kleinman Y, Rund D, et al.: Metformin-induced hemolytic anemia in a patient with glucose-6- phosphate dehydrogenase deficiency. Diabetes Care. 2003; 26(3): 956–957. PubMed Abstract \| Publisher Full Text\n\nKashyap AS, Kashyap S: Haemolytic anaemia due to metformin. Postgrad Med J. 2000; 76(892): 125–126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin KD, Lin JD, Juang JH: Metformin-induced hemolysis with jaundice. N Engl J Med. 1998; 339(25): 1860–1861. PubMed Abstract \| Publisher Full Text\n\nJagia M, Taqi S, Hanafi M: Metformin poisoning: a complex presentation. Indian J Anaesth. 2011; 55(2): 190–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuggiero NA, Kish TD, Lee Mikyung L: Metformin-Induced Hemolytic Anemia in a Patient with Glucose-6-Phosphate Dehydrogenase Deficiency. Am J Ther.2016; 23(2): e575-8. PubMed Abstract \| Publisher Full Text" }	[ { "id": "75380", "date": "07 Dec 2020", "name": "Carmelo J Blanquicett", "expertise": [ "Reviewer Expertise IM/heme/onc" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 highlights a previously-described, but rare occurrence. It would be interesting to determine whether the g6pd variant of the patient was investigated. Was it? Was nephrology consulted to investigate the AKI? Overall, interesting phenomenon is presented, particularly the argument of dose-dependance that occurred due to lack of drug clearance as a result of AKI; however, details of the AKI investigation/workup would be useful to know (UA and microscopy results. etc).\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "100821", "date": "10 Jan 2022", "name": "Shaimaa El-Ashwah", "expertise": [ "Reviewer Expertise Haematology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting case report concerning argue of dose-dependent toxicity especially that the patient was on the same dose for 3 years, however, is there a possibility that infection and dehydration could be risk factors for lactic acidosis and hemolysis?\nIt would be better to determine the G6PD variant\n\nWas there a nephrology consultation concerning AKI? is this AKI on top of CKD as there is a history of HTN and DM?\n\nDouble check values written in abstract & case description and also, units used.\n\nPatient's management by IV fluids and antibiotics was repeated twice in the case description\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1234
https://f1000research.com/articles/9-444/v1	26 May 20	{ "type": "Research Article", "title": "Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal", "authors": [ "Firdus Firdus", "Samadi Samadi", "Abdullah A. Muhammadar", "Muhammad A. Sarong", "Zainal A. Muchlisin", "Widya Sari", "Agung S. Batubara", "Firdus Firdus", "Abdullah A. Muhammadar", "Muhammad A. Sarong", "Zainal A. Muchlisin", "Widya Sari", "Agung S. Batubara" ], "abstract": "Background: The giant trevally, Caranx ignobilis, is a commercially important marine fish in Indonesia. This species was initially cultured in Aceh Province. Previous reports showed that charcoal has a positive effect on survival and feed utilization of the giant trevally. However, the effects of adding charcoal to the diet on gut and intestine biometrics has, to our knowledge, never been described. Methods: Four activated charcoal sources were tested in this study using a completely randomized experimental design; coconut shell charcoal, mangrove wood charcoal, rice husk charcoal, and kernel palm shell charcoal. All treatments were performed with four replications. Juvenile giant trevally (average body weight, 16.52 ± 3.12 g; and average total length, 10.26 ± 0.64 cm) were stocked into the experimental tank at a density of 15 fish per tank. The fish were fed an experimental diet twice daily at 7 AM and 5 PM ad satiation for 42 days. Results: Analysis of variance showed that adding charcoal to the diet had significant effects on the length and width of the foveola gastrica and villous intestine (P < 0.05). The greatest length and width of the foveola gastrica was recorded in fish fed an experimental diet of rice husk charcoal with average values of 311.811 ± 9.869 µm and 241.786 ± 10.394 µm, respectively. The greatest length of intestinal villous was found in fish fed the mangrove wood charcoal diet, with a value of 135.012 ± 5.147 µm, but this length was not significantly different to that in fish fed rice charcoal and kernel palm shell charcoal. However, the greatest width of intestinal villous was recorded in fish fed the control diet (without charcoal; P < 0.05). Conclusion: The optimal sizes of the foveola gastrica and villous intestine were found in fish fed an experimental diet with rice husk charcoal.", "keywords": [ "Foveola gastrica", "villous intestine", "coconut shell", "mangrove wood", "rice husk", "and kernel palm shell" ], "content": "Introduction\n\nTrevally fish are a commercially important group of marine fish in the family Carangidae. A total of 146 species of trevally have been recorded worldwide1. These fish are distributed in tropical, subtropical, and temperate waters2–7. In Indonesia, trevally fish are found in the Aceh waters8,9, East Borneo10, Papua and Wester Nusa Tenggara11,12, and Java13.\n\nGiant trevally, Caranx ignobilis, is among the most popular trevally fish in Indonesia. The population of this species has declined over the years due to overfishing7,14–16. Culture of this fish has been initiated in Aceh Province, Indonesia. However, farmers are faced with a feeding obstacle. Giant trevally in culture systems are currently fed waste fish and a commercial diet (Hi-Pro-Vite, Central Proteina Prima Company). The commercial diet is costly and difficult to obtain in remote areas, and the waste fish supply is very seasonal. Trash fish are limited in nutrients, particularly the essential amino acid composition17. Therefore, it is crucial to formulate a diet for giant trevally using local raw materials with higher protein, that is inexpensive, easy to find, and digestible.\n\nActivated charcoal is commonly added to the diet to increase digestibility and trigger growth in fish. For example, Jahan et al.18 successfully used activated charcoal to increase the digestibility and growth performance of river catfish, Pangasiaodon sp. Other researchers have used charcoal in the diets of fish species, such as Nile tilapia, Oreochromis niloticus19–21, tiger pufferfish, Takifugu rubripes22, Japanese flounder, Paralichthys olivaceus23, African catfish, Clarias gariepinus24,25, gilthead seabream, Sparus aurata26, and sturgeon, Huso huso27. Firdus et al.28 added rice husk charcoal to the diet of giant trevally. However, the effect of charcoal on the morphology of the gut and intestine has not been reported.\n\nOrganogenesis of the digestive system occurs as fish age, and this process is strongly dependent on the quantity and quality of food29–32, which is related to the development of mucosal cells, amplification of apical plasma membranes, and formation of the foveola gastrica and intestinal villi33,34. It has been hypothesized that adding activated charcoal to the diet triggers the digestive organogenesis system process35,36. In this study, we tested four charcoal sources in the diet to evaluate the morphology of the gut and intestine of giant trevally. Information on the gut and intestinal morphology is important to understand the absorption mechanism of nutrients from the diet.\n\n\nMethods\n\nThe study was conducted at the Center for Brackish Water Aquaculture, Ujung Batee, Aceh, Indonesia from February to July 2018. The activated charcoal was characterized at the Integrated Laboratory of Calibration, Universitas Gajah Mada, Yogyakarta, Indonesia. Histological samples were prepared at the Laboratory of Histology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala, Banda Aceh, Indonesia.\n\nA completely randomized experimental design with five different charcoal sources was used in this study. The experimental groups were: (A) the experimental diet without charcoal, (B) the experimental diet with 2% charcoal from coconut shell, (C) the experimental diet with 2% charcoal from mangrove wood, (D) the experimental diet with 2% charcoal from rice husk, and (E) the experimental diet with 2% charcoal from kernel palm shell. All treatments were performed with four replications.\n\nA total of 300 giant trevally juveniles of mixed sex (average body weight, 16.52 ± 3.12 g; total length, 10.28 ± 0.64 cm) were purchased from a local farmer in Lancang Barat Village, Aceh Utara District, Aceh, Indonesia. The fish were acclimatized in ponds (ponds size 2 x 1.8 m and temperature of around 29°C) at the Center for Brackish Water Aquaculture, Ujung Batee for 2 weeks. The fish were fed an experimental diet containing 50% crude protein twice daily at 7 AM and 5 PM at 3% of body weight per day (see Table 1).\n\nNote: (A) diet without charcoal, (B) diet with charcoal from coconut shells, (C) diet with charcoal from mangrove wood, (D) diet with charcoal from rice husks, (E) diet with charcoal from kernel palm shells.\n\nThe raw coconut shells, mangrove wood, rice husks, and kernel palm shells were chopped and ground. Approximately 500 g of the ground materials were placed on aluminum foil and heated in a furnace at 400°C for 1 hour. Nitrogen gas was flowed into the furnace to remove the oxygen. Then, the temperature was decreased to 30°C gradually and held for 1 hour. After 1 hour, the charcoal was removed from the furnace, sieved through a No. 40 mesh, and held in a jar before activating. A total of 100 g of sieved charcoal was taken and mixed with 400 ml of 0.2 M citric acid. The solution was stirred for 24 hours. After 24 hours, the solution was filtered through filter paper. The filtered charcoal was washed with distilled water and dried in an oven at 110°C for 24 hours.\n\nThe experimental diet was formulated from both plant and animal-based protein sources, such as Ebi-shrimp meal, fish meal, blood meal, soybean meal, rice flour, and corn flour. All raw materials were subjected to a proximate analysis before use in the formulation. Three types of amino acids i.e. isoleucine, L-tryptophan, and DL-methionine were also added (Table 1). A total of 2% of the tested charcoal sources was added to the formulation (Table 1). The formulated diets were subjected to a proximate analysis before use in the experiment.\n\nThe fish was captured randomly, measured for body weight and total length, and then distributed into 20 plastic containers (48 × 43 × 70 cm) at a stocking density of 15 fish per container. The water volume in the container was 75 L. The fish were fed an experimental diet twice daily at 7 AM and 5 PM to satiation for 42 days.\n\nGastric and intestinal samples were collected at the end of the study. Three fish from each treatment were taken randomly from the experimental tanks. The fish were anesthetized with 30 mg L−1 clove oil37, and the abdomen of the fish was gently dissected following the procedure of Purushothaman et al.38. The stomach and intestines were removed with scalpel scissors and preserved in 4% formalin for 1 week. Histological sampling was carried using the paraffin method based on Osman and Caceci39. The samples were dehydrated through an alcohol series and cleared in xylol. Subsequently, the gut and intestine samples were embedded in paraffin. The paraffin block was sectioned to 6 µm, and the sections were stained with hematoxylin and eosin. The size (height and width) of villi was determined using a binocular microscope (Zeiss Primo Star, Carl Zeiss Suzhou Co., Ltd., Suzhou, China) which was connected to a CCD camera and computer monitor19. All efforts were made to lessen harm to the animals by complying to the guidelines of ethics animal use in research of Syiah Kuala University.\n\nThe qualitative gut and intestinal morphology data were subjected to one-way analysis of variance followed by Duncan’s multiple range test. The analysis was performed using SPSS ver. 18.0 software. The qualitative (histological) gut and intestinal data were analyzed descriptively. A P-value < 0.05 was considered significant.\n\n\nResults\n\nAdding activated charcoal to the diet significantly affected the length and width of the foveola gastrica and intestinal villi (P < 0.05). In general, fish fed the activated charcoal diets produced better results than those not fed the charcoal (Figure 1 and Figure 2). The best foveola gastrica morphology was obtained with the rice husk charcoal and the mean length and width of the foveola gastrica were 311.811 µm and 241.786 µm, respectively; followed by coconut shell charcoal (257.040 µm and 183.816 µm), kernel palm charcoal (229.969 µm and 169.131 µm µm), and mangrove wood charcoal (229.595 µm and 166.509 µm).\n\n(A) Diet without charcoal, (B) diet with coconut shell charcoal, (C) diet with mangrove wood charcoal, (D) diet with rice husk charcoal, (E) diet with kernel palm shell charcoal.\n\n(A) Diet without charcoal, (B) diet with coconut shell charcoal, (C) diet with mangrove wood charcoal, (D) diet with rice husk charcoal, (E) diet with kernel palm shell charcoal. M, tunica mucosa; SM, tunica submucosa; Mc, tunica muscularis; Le, lamina epithelialis; Lp, lamina propria; m, muscle; Lm, longitudinal muscle fibers; Cm, circular muscle fibers (Cm).\n\nThe greatest length of the villous intestine was recorded in fish fed a diet with activated charcoal than those not fed the activated charcoal (Figure 3). The greatest growth of intestinal villi was determined in the mangrove active charcoal (mean, 135.012 µm) group, but this value was not significantly different from the rice husk or kernel palm shell charcoals (Figure 4). However, the greatest intestinal villi width was obtained in the treatment without activated charcoal (38.341 µm), and this value was significantly different from the other treatments.\n\n(A) Diet without charcoal, (B) diet with coconut shell charcoal, (C) diet with mangrove wood charcoal, (D) diet with rice husk charcoal, (E) diet with kernel palm shell charcoal.\n\n(A) Diet without charcoal, (B) diet with coconut shell charcoal, (C) diet with mangrove wood charcoal, (D) diet with rice husk charcoal, (E) diet with kernel palm shell charcoal. M, tunica mucosa; SM, tunica submucosa; Mc, tunica muscularis.\n\nRaw biometic data, in addition to unprocessed imaged, are available as Underlying data40,41,42.\n\n\nDiscussion\n\nThe results show that adding activated charcoal to the diet of C. ignobilis significantly affected favoela gastrica and intestinal villi biometrics. According to Pirarat et al.19, activated charcoal plays a significant role stimulating the development of epithelial cells of the digestive organs. Activated charcoal in the diet functions as a decontaminating agent to eliminate pathogenic organisms and toxic compounds, such as mycotoxins20. Hence, a longer foveola gastrica and larger intestinal villi provide a larger surface area to absorb nutrients43.\n\nOptimal development of the alimentary tract was recorded in giant trevally juveniles fed the experimental diet containing rice husk charcoal. This was presumably due to the high hemicellulose, cellulose, and lignin contents in the rice husk charcoal. A previous report indicated that rice husk charcoal contains 39% hemicellulose, 44% cellulose, and 30% lignin44, while mangrove wood charcoal has 30% hemicellulose, 36% cellulose, and 28% lignin45, coconut shell charcoal has 19.27% hemicellulose, 33.61% cellulose, and 36.51% lignin46, and kernel palm shell charcoal has 26.27% cellulose, 12.61% hemicellulose, and 42.96% lignin47. Maria and Banu48 and Jamilatun et al.49 reported that the concentration and quality of charcoal depend on the composition of hemicellulose, cellulose, and lignin. The quality of the activated charcoal is higher when these three components increase. According to Jasman50, rice husk contains 85–95% activated charcoal, while mangrove wood has 76% activated charcoal51, kernel palm shell 65% activated charcoal47, and coconut shell has 60% activated charcoal46.\n\nThe microscopic observations showed that the intestinal villi of the fish fed the diet with activated rice husk charcoal had a more pointed shape compared to other treatments, in which the villi tended to be round and blunt. According to Guo et al.52, blunt or rounded villi probably occur due to inflammation in the intestinal mucosa, which is characterized by infiltration of neutrophils into the lamina propria. An increase of intestinal villus size is related to nutrient absorption capacity. According to Nafis et al.53, long mucosal folds increase nutrient absorption and reduce food flow movement due to reduced peristaltic contractions, which provides sufficient time to optimally absorb nutrients. The increase in intestinal villi size is strongly related to the activities of digestive enzymes, such as lactase, sucrase, alkaline phosphatase, and disaccharidase54–57.\n\nThe morphology of the intestinal villi of fish fed a diet without activated charcoal was wider and shorter than that of fish fed the diets with activated charcoal. This was probably due to impaired intestinal mucosal integrity, causing interference in nutrient absorption. According to Choct58, shortening of the intestinal villi is related to the accumulation of intestinal pathogenic bacteria, resulting in increased susceptibility to infection in the intestinal mucosal layer. This causes the digestive organs to form more secretory cells than absorbent cells, which reduces nutrient uptake59,60. The active charcoal likely acts as an adsorbent of metabolic pathogens in the intestine in the form of endotoxins and ammonia to improve intestinal function61.\n\n\nConclusions\n\nThe application of activated charcoal in the diet significantly affected the length and width of the foveola gastrica and intestinal villi of giant trevally, C. ignobilis. The optimal biometrics of the foveola gastrica and intestinal villi were observed in fish fed the experimental diet with activated rice husk charcoal.\n\n\nData availability\n\nFigshare: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal. https://doi.org/10.6084/m9.figshare.12203525.v240.\n\nThis project contains the following underlying data:\n\nDATA BIOMETRIC GUT OF GIANT TREVALLY Caranx ignobilis_Edited (XLSX). (Raw biometric data for the foveola gastrica of all fish examined in this study.)\n\nDATA BIOMETRIC OF INTESTINE OF GIANT TREVALLY Caranx ignobilis_edited (XLSX). (Raw biometric data for the intestinal villi of all fish examined in this study.)\n\nFigshare: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal. hhttps://doi.org/10.6084/m9.figshare.12301124.v241.\n\nThis project contains uncropped, unprocessed images of the intestinal villi of giant trevally.\n\nFigshare: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoaltem. https://doi.org/10.6084/m9.figshare.12269606.v242.\n\nThis project contains uncropped, unprocessed images of the foveola gastrica of the giant trevally.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe thank the Kemenristekdikti for supporting this study. All staff at the Center for Brackish Water Aquaculture in Ujung Batee who assisted with this study are acknowledged. Special thanks to Mr. Boihaqi and Maisyarah Rita for their assistance during the study.\n\n\nReferences\n\nFishbase: Family Carangidae - Jacks and pompanos. 2020. (Accessed on 9 April 2020). Reference Source\n\nWetherbee BM, Holland KN, Meyer CG, et al.: Use of a marine reserve in Kaneohe Bay, Hawaii by the giant trevally, Caranx ignobilis. Fish Res. 2004; 67(3): 253–263. Publisher Full Text\n\nHo JS, Lin CL: Three species of Caligus Müller, 1785 (Copepoda: Caligidae) parasitic on Caranx spp. (Teleostei: Carangidae) off Taiwan. Syst Parasitol. 2007; 68(1): 33–43. PubMed Abstract \| Publisher Full Text\n\nMeyer CG, Holland KN, Papastamatiou YP: Seasonal and diel movements of giant trevally Caranx ignobilis at remote Hawaiian atolls: implications for the design of marine protected areas. Mar Ecol Prog Ser. 2007; 333: 13–25. Publisher Full Text\n\nAmarasinghe US, Wickramaratne IU, Wijeyaratne MJS: Hook selectivity of giant trevally (Caranx ignobilis) and nakedbreast trevally (Carangoides gymnostethus) (Carangidae) caught in the hook-and-line fishery off Negombo, Sri Lanka. Sri Lanka J Aquat Sci. 2011; 16: 11–26. Publisher Full Text\n\nAcuña-Marrero D, Salinas-De-León P: New record of two Indo-Pacific reef fish, Caranx ignobilis and Naso annulatus, from the Galapagos Islands. Mar Biodivers Rec. 2013; 6: 1–5. Publisher Full Text\n\nNeethiselvan N, Karthy A, Mol CB: Gillnet selectivity on the yellow fin Trevally (Caranx ignobilis, Forsskal, 1775) along Thoothukudi coast, Southeast coast of India. J Exp Zool India. 2015; 18(1): 29–37. Reference Source\n\nTimorya Y, Abdullah A, Batubara AS, et al.: Conservation and economic status fishes in the Krueng Sabee River, Aceh Jaya District, Aceh Province, Indonesia. IOP Conf Ser: Earth Environ Sci. 2018; 216: 012044. 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PubMed Abstract \| Publisher Full Text\n\nPryor GS, Royes JB, Chapman FA, et al.: Mannanoligosaccharides in fish nutrition: effects of dietary supplementation on growth and gastrointestinal villi structure in Gulf of Mexico sturgeon. N Am J Aquac. 2003; 65(2): 106–111. Publisher Full Text\n\nLiu H, Guo X, Gooneratne R, et al.: The gut microbiome and degradation enzyme activity of wild freshwater fishes influenced by their trophic levels. Sci Rep. 2016; 6: 24340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÖzel OT, Coşkun I, Çakmak E: Intestine villi morphology of black sea trout (Salmo labrax. Pallas, 1814). LimnoFish. 2018; 4(1): 42–46. Publisher Full Text\n\nInfante JZ, Cahu CL: Ontogeny of the gastrointestinal tract of marine fish larvae. Comp Biochem Physiol C Toxicol Pharmacol. 2001; 130(4): 477–487. PubMed Abstract \| Publisher Full Text\n\nWilson JM, Castro LFC: Morphological diversity of the gastrointestinal tract in fishes. Fish physiology. 2010; 30: 1–55. Publisher Full Text\n\nBurr G, Gatlin D III, Ricke S: Microbial ecology of the gastrointestinal tract of fish and the potential application of prebiotics and probiotics in finfish aquaculture. World Aquaculture society. 2005; 36(4): 425–436. Publisher Full Text\n\nHoseinifar SH, Roosta Z, Hajimoradloo A, et al.: The effects of Lactobacillus acidophilus. as feed supplement on skin mucosal immune parameters, intestinal microbiota, stress resistance and growth performance of black swordtail (Xiphophorus helleri). Fish Shellfish Immunol. 2015; 42(2): 533–538. PubMed Abstract \| Publisher Full Text\n\nJavahery S, Nekoubin H, Moradlu AH: Effect of anaesthesia with clove oil in fish (review). Fish Physiol Biochem. 2012; 38(6): 1545–1552. PubMed Abstract \| Publisher Full Text\n\nPurushothaman K, Lau D, Saju JM, et al.: Morpho-histological characterisation of the alimentary canal of an important food fish, Asian seabass (Lates calcarifer). PeerJ. 2016; 4: e2377. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOsman AHK, Caceci T: Histology of the stomach of Tilapia nilotica (Linnaeus, 1758) from the River Nile. J Fish Biol. 1991; 38(2): 211–223. Publisher Full Text\n\nMuchlisin ZA, Firdus F, Samadi S, et al.: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12203525.v2\n\nMuchlisin ZA, Firdus F, Samadi S, et al.: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoal. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12301124.v2\n\nMuchlisin ZA, Firdus F, Samadi S, et al.: Gut and intestinal biometrics of the giant trevally, Caranx ignobilis, fed an experimental diet with difference sources of activated charcoaltem. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12269606.v2\n\nQuaiyum MA, Jahan R, Jahan N, et al.: Effects of bamboo charcoal added feed on reduction of ammonia and growth of Pangasius hypophthalmus. J Aquac Res Dev. 2014; 5(6): 1–5. Publisher Full Text\n\nMahvi AH, Maleki A, Elslami A: Potential of rice husk and rice husk ash for phenol removal from aqueous systems. Am J Appl Sci. 2004; 1(4): 321–326. Reference Source\n\nVerheyden A, Roggeman M, Bouillon S, et al.: Comparison between δ 13C of α-cellulose and bulk wood in the mangrove tree Rhizophora mucronata: implications for dendrochemistry. Chemical Geology. 2005; 219(1-4): 275–282. Publisher Full Text\n\nMaryono M, Sudding S, Rahmawati R: Preparation and quality analisis of coconut shell charcoal briquette observed by starch concentration. Jurnal Chemica. 2013; 14: 74–83.\n\nSunardi S, Nurliani N: Pemanfaatan arang aktif sekam padi dengan aktivator natrium karbonat (Na2CO3) 5% untuk mengurangi kadar besi (Fe) dalam air ledeng. Jurnal Teknologi Hasil Hutan. 2008; 23: 99–104. Reference Source\n\nMaria NSL, Banu ASS: Activated carbon from rice husk for treating dye waste water. Internasional Journal of Green Chemistry. 2015; 1(1): 1–9. Reference Source\n\nJamilatun S, Setyawan M, Salamah S, et al.: Pembuatan arang aktif dari tempurung kelapa dengan aktivasi sebelum dan sesudah pilorisis. 2015. Reference Source\n\nJasman J: Uji coba arang aktif sekam padi sebagai media filtrasi dalam menurunkan kadar Fe pada air sumur bor di Asrama Jurusan Kesehatan Lingkungan Manado. Jurnal Kesehatan Lingkungan. 2011; 1(1): 49–53. Reference Source\n\nJauhari A: Penanggulangan kadar besi (Fe) air sumur menggunakan arang aktif kayu bakau (Rhizophora mucronata.Lamck) dengan aktivator natrium karbonat. Jurnal Hutan Tropis Borneo. 2009; 28: 321–331.\n\nGuo J, Luo Y, Lua AC, et al.: Adsorption of hydrogen sulphide (H2S) by activated carbons derived from oil-palm shell. Carbon. 2007; 45(2): 330–336 . Publisher Full Text\n\nNafis M, Zainuddin Z, Masyitha D: Gambaran histologi saluran pencernaan ikan gabus (Channa striata). Jimvet. 2017; 1(2): 196–202. Reference Source\n\nSamanya M, Yamauchi K: Morphological changes of the intestinal villi in chickens fed the dietary charcoal powder including wood vinegar compounds. J Poult Sci. 2001; 38(4): 289–301. Publisher Full Text\n\nGilmore MS, Ferretti JJ: The thin line between gut commensal and pathogen. Science. 2003; 299(5615): 1999–2002. Publisher Full Text\n\nYamauchi K: Review of a histological intestinal approach to assessing the intestinal function in chickens and pigs. Anim Sci J. 2007; 78(4): 356–370. Publisher Full Text\n\nŠabatková J, Kumprecht I, Zobač P, et al.: The probiotic BioPlus 2B as an alternative to antibiotics in diets for broiler chickens. Acta Veterinaria Brno. 2008; 77: 569–574. Publisher Full Text\n\nChoct M: Managing gut health through nutrition. Br Poult Sci. 2009; 50(1): 9–15. Publisher Full Text\n\nMcLean E, Donaldson EM: Absorption of bioactive proteins by the gastrointestinal tract of fish: a review. Journal of Aquatic Animal Health. 1990; 2(1): 1–11. Publisher Full Text\n\nRingø E, Olsen RE, Mayhew TM, et al.: Electron microscopy of the intestinal microflora of fish. Aquaculture. 2003; 227(1-4): 395–415. Publisher Full Text\n\nMulyono P, Wibisono W: Kinetika adsorpsi amonia dalam air dengan karbon aktif. Jurnal Media Teknik. 2007; 2: 26–42." }	[ { "id": "63939", "date": "15 Jun 2020", "name": "Srikanta Samanta", "expertise": [ "Reviewer Expertise Aquatic Chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 has communicated a short but interesting study on the beneficial effects of adding different types of charcoal as a dietary component of giant trevally. The addition of charcoal has significantly improved the gastro-intestinal microstructures including foveola gastrica & villous intestine and thereby, has the potentiality to improve the fish nutrition and health. The rice husk was recorded as the best source of charcoal with respect to 3 other sources including coconut shell, mangrove wood, and kernel palm shell. However, some deficiencies have been noticed which needs rectification.\nIn Abstract : This species was---: Better to use The species was---\n\nFour activated charcoal sources were tested in this study -----but in Experimental Design mentioned ----A completely randomized experimental design with five different charcoal sources----\n\nIn Abstract : average total length, 10.26 ± 0.64 cm, In Experimental fish : total length, 10.28 ± 0.64 cm\n\nponds size 2 x 1.8 m. No unit given after 2. It may be 2 m X 1.8 m\n\n(see Table 1) : see may be deleted\n\nTable 1 : CaCo3 : CaCO3\n\nIn Abstract : and 241.786 μm --- In Figure 1 241.768\n\nFigure 2 and 4 : Histological sample ---- may be replaced by ---- Histology\n\nSentence to be modified : Hence, a longer foveola gastrica and larger intestinal villi provide a larger surface area to absorb nutrients43.\n\nSentence to be modified : A previous report indicated that rice husk charcoal contains 39% hemicellulose, 44% cellulose, and 30% lignin44 : Since the addition of 3 parameters are giving values >100%, you may mention \"up to\" otherwise people will not understand.\n\nSentence to be modified/changed : The active charcoal likely acts as an adsorbent of metabolic pathogens in the intestine in the form of endotoxins and ammonia to improve intestinal function61. The sentence to be rewritten to increase clarity.\n\nIt has been noticed that the SE values have gone out of the bar area. Please check about it and reason behind. For the paper, the fish growth parameters may be given as supplements for better clarity.\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": [ { "c_id": "5759", "date": "22 Jul 2020", "name": "Samadi Samadi", "role": "Author Response", "response": "Dear Prof. Srikanta Samanta, Thank you very much for your comment and we will revise the article based on your suggestions. Best regards, Samadi" } ] }, { "id": "65152", "date": "10 Jul 2020", "name": "Abdus Salam", "expertise": [ "Reviewer Expertise Aquaculture" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 seems the manuscript has potentials in the scientific world, and work was done and formatted with care. The abstract is concise but well written.\n\nThe introduction has supported with up to date literature and aim and objectives are well described.\n\nResults supported with details and tables and figures were provided.\n\nThe discussion was also well described and literature supported.\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/9-444
https://f1000research.com/articles/9-1229/v1	13 Oct 20	{ "type": "Opinion Article", "title": "The ELIXIR Human Copy Number Variations Community: building bioinformatics infrastructure for research", "authors": [ "David Salgado", "Irina M. Armean", "Michael Baudis", "Sergi Beltran", "Salvador Capella-Gutierrez", "Denise Carvalho-Silva", "Victoria Dominguez Del Angel", "Joaquin Dopazo", "Laura I. Furlong", "Bo Gao", "Leyla Garcia", "Dietlind Gerloff", "Ivo Gut", "Attila Gyenesei", "Nina Habermann", "John M. Hancock", "Marc Hanauer", "Eivind Hovig", "Lennart F. Johansson", "Thomas Keane", "Jan Korbel", "Katharina B. Lauer", "Steve Laurie", "Brane Leskošek", "David Lloyd", "Tomas Marques-Bonet", "Hailiang Mei", "Katalin Monostory", "Janet Piñero", "Krzysztof Poterlowicz", "Ana Rath", "Pubudu Samarakoon", "Ferran Sanz", "Gary Saunders", "Daoud Sie", "Morris A. Swertz", "Kirill Tsukanov", "Alfonso Valencia", "Marko Vidak", "Cristina Yenyxe González", "Bauke Ylstra", "Christophe Béroud", "Irina M. Armean", "Michael Baudis", "Sergi Beltran", "Salvador Capella-Gutierrez", "Denise Carvalho-Silva", "Victoria Dominguez Del Angel", "Joaquin Dopazo", "Laura I. Furlong", "Bo Gao", "Leyla Garcia", "Dietlind Gerloff", "Ivo Gut", "Attila Gyenesei", "Nina Habermann", "John M. Hancock", "Marc Hanauer", "Eivind Hovig", "Lennart F. Johansson", "Thomas Keane", "Jan Korbel", "Katharina B. Lauer", "Steve Laurie", "Brane Leskošek", "David Lloyd", "Tomas Marques-Bonet", "Hailiang Mei", "Katalin Monostory", "Janet Piñero", "Krzysztof Poterlowicz", "Ana Rath", "Pubudu Samarakoon", "Ferran Sanz", "Gary Saunders", "Daoud Sie", "Morris A. Swertz", "Kirill Tsukanov", "Alfonso Valencia", "Marko Vidak", "Cristina Yenyxe González", "Bauke Ylstra" ], "abstract": "Copy number variations (CNVs) are major causative contributors both in the genesis of genetic diseases and human neoplasias. While “High-Throughput” sequencing technologies are increasingly becoming the primary choice for genomic screening analysis, their ability to efficiently detect CNVs is still heterogeneous and remains to be developed. The aim of this white paper is to provide a guiding framework for the future contributions of ELIXIR’s recently established human CNV Community, with implications beyond human disease diagnostics and population genomics. This white paper is the direct result of a strategy meeting that took place in September 2018 in Hinxton (UK) and involved representatives of 11 ELIXIR Nodes. The meeting led to the definition of priority objectives and tasks, to address a wide range of CNV-related challenges ranging from detection and interpretation to sharing and training. Here, we provide suggestions on how to align these tasks within the ELIXIR Platforms strategy, and on how to frame the activities of this new ELIXIR Community in the international context.", "keywords": [ "Copy Number Variation", "Data analysis", "next-generation sequencing", "whole genome sequencing", "Human Genetics", "Oncogenetics", "Common Diseases", "Federated Human Data" ], "content": "Introduction\n\nIn the late 1950s, Tjio and Levan established that the human karyotype consists of 46 chromosomes1. This was promptly followed by the description of numerical chromosomal abnormalities in Down syndrome and, in less than one year, a new discipline emerged in the field of human genetics: “cytogenetics”2. Shortly after, somatic karyotype alterations were attributed to the identification of “Philadelphia chromosome” in the blood of Chronic Myeloid Leukemia patients3, which were found to be the result of specific underlying chromosomal rearrangements4. From then on, cytogenetics was not only applied to identify heritable chromosomal aberrations, but also established the field of “Cancer Cytogenetics”5, which led to a leap forward in approaching the molecular mechanisms of malignant diseases.\n\nSince these early discoveries, multiple links between chromosomal alterations and diseases have been described. In parallel to those microscopic observations, the identification of the first genomic mutation in humans occurred in 1949 with the discovery of an amino acid change responsible for sickle cell anemia6, while the genetic alteration itself was identified only a few years later by Ingram7. Thanks to the rapidly advancing Sanger sequencing technology8, disease-causing genome variations are now routinely identified and confirmed. However, while both “cytogenetic” and “genomic” alterations in heritable human diseases and cancer are based on alterations of DNA sequence or structure, an epistemological dichotomy remained between the fields of molecular biology and genetics (targeting specific sequence alterations) and cytogenetics (focusing on genetic alterations detected by cytogenetic methods).\n\nIn the late 1980s, the development of DNA labeling techniques using the direct or indirect incorporation of fluorescent dyes9,10 helped to establish the field of “molecular cytogenetics”, in which the hybridization of labeled DNA probes informs about characteristics of chromosomal substrates. Variations of the Fluorescent In-Situ Hybridization (FISH)11 technology include interphase FISH (i.e. the hybridization of specific fluorescent probes on interphase nuclei) and reverse in-situ hybridization techniques8, in which the labeled DNA sample of interest is hybridized against a substrate consisting of normal metaphase chromosomes. While overall these technologies helped to put “the Genetics back into Cytogenetics”12, the development of Comparative Genomic Hybridization (CGH), a dual-color, whole-genome extension of the reverse in-situ hybridization concept was particularly instrumental in the delineation of a type of structural genome variations termed “Copy Number Variations” (CNVs). This category of genome variants encompasses those that range from a few hundred DNA base pairs to such affecting several megabases up to duplications or deletions involving whole chromosomes.\n\nWhile abundant CNVs have been demonstrated to affect virtually every type of cancer13,14, they have also been shown to be a large contributor to inherited genome variation and have been shown to impact both genic and intergenic regions alike15.\n\nSince the recognition of CNVs as contributors to the genomic variation landscape, multiple CNVs have been associated with human traits and diseases. Recently, a review by Srebniak et al.16 reported, in a meta-analysis of data from 10,314 fetuses, that CNVs were associated with an early-onset syndromic disorder in 0.37% (95% CI, 0.27-0.52%) of cases; with late-onset disease in 0.11% (95% CI, 0.05%-0.21%); and with diseases susceptibility in 0.30% (95% CI, 0.14-0.67%). The prevalence of early-onset syndromic disorders caused by CNVs was thus calculated to be 1:27016. In parallel, it has been known for years that CNVs account for the majority of disease-causing variation of some genes, as illustrated for example by the DMD gene, for which up to 74% and 87% of mutations are deletions or duplications of one or more exons, respectively, for Duchenne and Becker patients17. Additionally to their role in syndromic disorders, CNVs have also been associated with common, polygenic diseases such as obesity13,14, and mental disorders15,16.\n\nCNVs are also a major contributor to the somatic mutation landscape in cancer. Specific deletion CNVs may lead to loss of heterozygosity events involving tumor suppressor genes, with a tumor promoting effect demonstrated early on for the somatic loss of wild-type alleles in many inherited cancer syndromes18. In contrast, proto-oncogenes, such as MYCN and ERBB2, are frequently deregulated through chromosomal amplification events, leading to over-expression of the gene products due to a highly increased genomic dosage19. However, while the association between individual duplication and deletion CNVs and tumor-related genes has been determined for many types of cancer, most types of malignancies show recurring CNV patterns with involvement of large genomic regions, beyond a limited number of cancer-associated gene loci.\n\nCNV detection has been performed since the early 1990s using various generations of chromosomal (cCGH)20,21 and array-based (aCGH)22,23 CGH technologies, including high-density oligonucleotide arrays and Single Nucleotide Polymorphism (SNP) genotyping arrays24,25. The current generation of these hybridization technologies is still considered as ‘gold standard’ in order to fulfill the diagnostic needs of clinical cytogenetics laboratories. Their use is highly flexible, as it is easy to design custom arrays, including hundreds of thousands to millions of probes. It is thus possible to use the technology for genome-scale analysis through to region specific analysis.\n\nHowever, these technologies also have limitations: for example, their inability to distinguish chromosomal abnormalities as tandem, inverted, or translocated duplications; and the probe hybridization process itself which may result in poor sensitivity and precision, depending on probe design and the quality of analyzed DNA. In parallel, it is not currently possible to simultaneously achieve a genome-wide level and a high resolution because of the limited density of the array probes. In addition, as was shown by Haraksingh et al., the current generation of arrays still requires careful quantitative comparative analysis for researchers and clinicians to be able to select the appropriate tool for a given application26.\n\nToday, emerging Next-Generation Sequencing (NGS) technologies, especially deep coverage Whole Genome Sequencing (WGS), is quickly becoming the primary strategy for CNV detection in the study of human disease. Because of the ability of this technology to provide a nucleotide level resolution, it can theoretically solve the limitations observed for array-based technologies, and also provide the exact boundaries and localization for a given CNV.\n\nNevertheless, due to the demanding data analysis workflows and high costs of deep coverage WGS experiments, many laboratories have implemented either low coverage WGS, or the Whole Exome Sequencing (WES) based approach for CNV detection (which stands between array-based approaches and deep WGS). Thus, when targeting the classes of inherited and somatic genome alterations subsumed as CNV for research and clinical applications, one faces a heterogeneous field encompassing different experimental technologies and related bioinformatics methods for data analysis, without a clear ‘gold standard’ serving all heterogeneous applications. Despite these technical challenges, the CNV field is of primary importance for human disease diagnosis and research, including the field of cancer genetics.\n\nIn this context, in February 2018, ELIXIR-FR and ELIXIR-ES initiated the creation of the \"ELIXIR human CNV Community\" (hCNV Community) as a starting point for consolidating this field at the European level, and beyond. The first hCNV Community meeting “The future of hCNV in ELIXIR” took place in Hinxton (UK), as a general, strategically focused, meeting to discuss, prioritize, and map the future of hCNV related activities in ELIXIR. In this white paper, we first summarize the main conclusions of the meeting, and then explain possible future directions for the incorporation of human CNV activities across ELIXIR.\n\n\nMeeting: “The Elixir Human Cnv Community”\n\nThe initial face-to-face meeting of the ELIXIR human CNV Community took place on September 28th, 2018 in Hinxton (UK). Attendance was open to any member from any ELIXIR Node, with the limitation of 25 participants at maximum. Remote participation (via teleconference) was offered to those that wished to attend but could not do so in person. There were 21 attendees at the meeting, representing 11 ELIXIR Nodes: EMBL-EBI, France, Germany, Hungary, Luxembourg, Netherlands, Norway, Slovenia, Spain, Switzerland, United Kingdom, and there were also two representatives from the ELIXIR Hub. The meeting started with a presentation from Gary Saunders (ELIXIR Human Data Communities coordinator) who provided a general overview of the current ELIXIR Communities and activities. This initial talk was followed by a series of presentations where each Node summarized their ongoing activities and expertise related to hCNV research. The remainder of the meeting was devoted to an open discussion on the challenges of CNVs and how to address them through activities and tasks building on ELIXIR partners' experience(s). Each activity was mapped to at least one of the five ELIXIR Platforms (Data, Tools, Interoperability, Compute, and Training). In addition, the potential interaction of the hCNV Community with the other ELIXIR Communities and international initiatives was discussed.\n\n\nOutcomes and discussion\n\nThe field of human CNV research is complex and evolving; activities range from CNV detection to their interpretation as potentially pathogenic to common genomic background variation. To simplify this process analysis, seven objectives have been defined by the ELIXIR hCNV Community:\n\nObjective #1: optimal CNV detection pipelines\n\nMultiple publications have reported pipelines to detect CNV using micro-array, WES or WGS data27,28. Nevertheless, as reported by Zare et al.29, who evaluated the performance of various CNV detection tools in cancer, there is a low consensus among the tools in calling CNVs, especially from widely used WES experiments: a moderate sensitivity (50 to 80%); a fair specificity (70 to 94%) and a poor false discovery rate (27 to 60%). Similar results were reported by Yao et al.30 who concluded that read-depth based programs are still immature for WES-based CNV detection with a low sensitivity and an uncertain specificity. Comparable experiences were revealed by participants of the ELIXIR hCNV workshop, and it was concluded that, even if micro-array technologies provide overall better CNV detection parameters, the wide adoption of NGS technologies represents a true challenge for the accurate detection of CNV. Based on these observations, the need for an extensive assessment and benchmarking of existing tools was established as one of the working areas of the hCNV Community. The objective will be to release a set of sensitive and reliable pipelines, optimized and validated to detect CNV from various high throughput datasets. These pipelines will be available either through ELIXIR compute nodes and/or as stand-alone solutions. Considering the actual performances of available systems, it is anticipated that we will provide a portfolio of tools, each being useful for a specific situation: CNV type; detection technology; disease context.\n\nTo reach this objective, three tasks are proposed:\n\nTask #1.1 will establish the list of available pipelines/software as well as partners’ local solutions to detect CNV from gene panels, WES, low and deep coverage WGS, array CGH, and SNP arrays.\n\nTask #1.2 will benchmark these systems using datasets from Objective #2 to select the most sensitive, specific, reliable, and rapid systems for each dataset for germline and somatic CNVs. Note that if no system is efficient enough for some conditions, the hCNV partners will develop new system(s) to address community needs. The CNV field is very dynamic and multiple systems are released monthly. We therefore expect to be able to select a combination of tools that will provide enough efficiency to be used for research purposes and eventually diagnostics.\n\nTask #1.3 will optimize the selected pipelines from Task #1.2 to increase performance on ELIXIR compute nodes and define optimal parameters and guidelines to help end-users to efficiently and reliably detect CNV in various situations through the ELIXIR training platform.\n\nObjective #2: definition of reference datasets\n\nThe ambition is to produce reference datasets of fully validated somatic and germline CNVs representing a wide range of sample types and experimental technologies. The aim is to provide reference materials available to the community for the comparison and evaluation of pipelines and/or NGS technologies and/or for quality assurance. This will include both digital raw data and, potentially, biomaterials.\n\nTo do so, the hCNV Community will establish reference datasets including various CNV (deletions and duplications) of various sizes ranging from a single exon CNV to large genomic rearrangements. Two subsets will be defined for germline and somatic CNVs. These datasets will contain samples with fully validated CNVs by other approaches such as multiplex ligation-dependent probe amplification and quantitative or semi-quantitative PCR. Four tasks are proposed:\n\nTask #2.1 will be dedicated to WES reference datasets.\n\nTask #2.2 will be dedicated to WGS reference datasets.\n\nTask #2.3 will be dedicated to gene panels reference datasets.\n\nTask #2.4 will be dedicated to CGH/SNP microarrays reference datasets.\n\nDuring the strategic workshop in Hinxton, participants discussed the GDPR and its impact on reference human datasets. The participation of lawyers and ethics specialists is therefore needed, and this was proposed to be addressed at a more global level by the ELIXIR Human Data Communities as a whole. In case no human reference dataset could be exchanged at a community level, alternatives using other model organisms have been proposed.\n\nAs previously mentioned, the NGS technologies are rapidly evolving and therefore the reference datasets will need to be regularly updated.\n\nIn the context of tools metrology, i.e. efficiency evaluation and improvement of detection, the Genome in a Bottle initiative has released a set of reference materials which has already been largely used for Short Nucleotide Variation(s) (SNV)28, 29. In a recent paper, Zook et al.31 reported the use of a new reference dataset for germline structural variant detection including CNV32,33. We will therefore adopt these reference samples and use them with all technologies to produce reference datasets.\n\nObjective #3: data exchange formats\n\nInternational collaborative projects require harmonization and standardization of results in order to ensure efficient data aggregation and comparison. Although various international initiatives, such as the GA4GH Genomic Knowledge Standards and Large Scale Genomics Work Streams, are currently addressing aspects of this issue, no robust and exhaustive standard CNV annotation format has emerged so far. To address this issue, the hCNV community identified two tasks:\n\nTask #3.1 will establish the list of existing formats to describe CNVs and list common and specific features.\n\nTask #3.2 will develop recommendations to use a standardized format to report CNVs. If a few alternative formats are frequently used, it will provide bioinformatics resources to convert data into the common data exchange format.\n\nhCNV partners notably discussed the adoption of the VCF format and its current limitations for the CNV field. It was concluded that, although this format is well-known by molecular biologists and could therefore be a starting point, it is less frequently used by cytogeneticists. There is therefore a strong need to improve this format and identify other nomenclatures and widely used formats in other communities. It is being recognized that any development or improvement of standards for CNV annotation should be performed in alignment with existing efforts, notably GA4GH work streams and the ELIXIR Interoperability Platform.\n\nObjective #4: identification of patients with similar genotypes and phenotypes\n\nFinding similar cases at the clinical level is a key component of clinical diagnosis and research to identify disease-causing genes and to explain genotype/phenotype correlations and intermediate clinical phenotypes. Although perhaps straightforward in the case of common diseases, this is much more of a challenge for the millions of patients affected by a rare disease, as routinely each case is similar only to a handful of patients across Europe. Within the Discovery Work Stream of GA4GH there are standards, such as Beacon34, to establish a federated data discovery network that is able to connect databases of genomic variations and phenotypic data using a common application programming interface (API). The GA4GH Driver Project “ELIXIR Beacons” provides a schema definition and API, together with a reference implementation to allow data owners to add their resources to the Beacon Network, as simply as possible (https://beacon-project.io). With the recent addition of CNV representation to the Beacon API and planned ontology-based phenotype queries the Beacon ecosystem represents a prime target for the implementation of (CNV related) genotype-phenotype representation and querying. The hCNV group will work towards enabling its use for the envisioned patient discovery, through the support of extended clinical descriptions including enabling and testing of relevant annotation standards (e.g. HPO, NCIt, additional ontologies). To do so:\n\nTask #4.1 will select ontologies required to efficiently capture phenotypic description useful for data interpretation for any genetic disease.\n\nTask #4.2 will provide lists of common data elements that should be provided in various situations such as rare disease, oncology, or common diseases.\n\nVarious ELIXIR hCNV partners are already strongly involved in the development of ontologies, such as HPO35 and ORDO36 and in the mapping of various ontologies, medical terminologies, vocabularies and nomenclatures. In addition, national databases described in Objective #6, will not only be FAIRified (see below) but also adopt the recommendations from Objective #4 to ensure cross queries and the identification of similar patients.\n\nObjective #5: creation of innovative tools\n\nCNVs often involve large genomic regions encompassing multiple genes. In addition, in recessive diseases, CNVs can alter one allele of a specific locus, whilst the second could be altered by SNV. In many situations, it is therefore difficult to identify the single, or multiple, genes harboring variation that is directly associated with a particular phenotype.\n\nThe hCNV Community will develop innovative tools to: annotate CNV; facilitate their interpretation through a combinatorial approach; and help to pinpoint key genes in regions of interest. The following tasks have been identified and will most likely evolve as more data become available and as technologies evolve:\n\nTask #5.1 will define CNV annotations including: type; genotype; genes and transcripts; expression level; exons; regulatory elements; breakpoints/ fusion fragments for WGS only (allowing detection of tandem duplications vs. inverted duplications and translocations).\n\nTask #5.2 will develop a specific pipeline to interpret duplications as tandem, inverted or translocation duplications may result in very different phenotypes.\n\nTask #5.3 will develop specific bioinformatics tools to select candidate genes localized in the CNV region by combining genes' annotations and patients' phenotype.\n\nObjective #6: FAIRification of hCNV services and datasets\n\nVarious CNV national databases, ELIXIR Core Data Resources, and ELIXIR Deposition Databases are currently being developed by ELIXIR hCNV partners. In order to allow interoperability (including discovery), the FAIR principles (Findable, Accessible, Interoperable, Reusable)37,38 will be applied to those systems to demonstrate the feasibility and utility of distributed CNV databases. This will respect databases' ownerships and national regulations' compliance while allowing searching for similar patients across the network. To respect and follow these principles, we will ensure that data are:\n\n(i) Findable:\n\nThe data should contain globally unique, resolvable and persistent identifiers.\n\nInclude machine-readable descriptions to support structured search and filtering.\n\n(ii) Accessible:\n\nMetadata has to be accessible beyond the lifetime of the digital resource (e.g. using BioSchemas).\n\nClearly defined regarding the condition for access and security protocols for sharing and accessing data.\n\n(iii) Interoperable:\n\nUsage of a specific file format\n\nExtensible machine interpretable formats for data and metadata (e.g. YAML files, JSON-LD)\n\nUse vocabularies (ontologies) and link with other robust resources\n\nIntegration with FAIR resources\n\n(iv) Reusable:\n\nProvide licensing, provenance and description on community-standards.\n\nTask #6.1 will use the French BANCCO database (http://bancco.fr) developed at Aix Marseille University and the CIBERER (Spanish network for research in rare diseases) database developed at the Fundación Progreso y Salud of Sevillas prototypes to demonstrate the benefits of using the FAIR data principles for CNV in diagnostic and research contexts.\n\nTask #6.2 will extend the FAIRification to other non-specific CNV databases such as the European Variation Archive (EVA), RD-CONNECT, arrayMap39 and Database of Genomics Variants Archive (DGVa).\n\nObjective #7: dissemination\n\nThe global adoption of tools and guidelines is strongly linked to the ability to communicate, produce training materials, and train actors as well as patients and the general public.\n\nTask #7.1 will set-up Jamborees to gather experts' point of view on the various objectives and related tasks and developments.\n\nTask #7.2 will set-up regular hackathons to ensure smooth developments and benchmarks by various ELIXIR Nodes.\n\nTask #7.3 will promote the ELIXIR hCNV Community through participation at international meetings, such as GA4GH Plenary. A contact has already been established with the Human Genome Variation Society (HGVS) (see below).\n\nAlignment with ELIXIR Platforms\n\nThe ELIXIR hCNV Community’s objectives have many links to the activities of the ELIXIR Platforms (Figure 1).\n\nLeft: ELIXIR Platforms; Right: ELIXIR hCNV Community objectives.\n\nData Platform\n\nThe aims of this Platform have been described as to drive the use, re-use, and value of life science data. It aims to do this by providing users with robust, long-term sustainable data resources within a coordinated, scalable and connected data ecosystem. Thus, the ELIXIR hCNV Community that will collect high quality genetic and phenotypic data will establish strong links with the ELIXIR Data Platform by providing Deposition Databases for CNV; and by benefiting from literature data integration and scalable curation provided by the Platform.\n\nTools and Compute Platforms\n\nAs described in Objectives #1 and #5, various reference tools will be validated and/or created by the ELIXIR hCNV Community. Their promotion will be facilitated by inclusion in the \"Tools and services registry\" bio.tools (https://bio.tools)40. The activity related to benchmarking will be carried out within the \"scientific benchmark and technical monitoring\" infrastructure (OpenEbench) (https://openebench.bsc.es) and the ELIXIR Compute Platform. In addition, tools will be made interoperable and shall be included as Galaxy workflows41 and available to the community through the \"Cloud and Computing Resources\" from the ELIXIR Compute Platform.\n\nInteroperability Platform\n\nKey to ELIXIR hCNV Community Objective #3 is the development of recommendations to use standardized format(s) to report CNVs. Additionally, Objective #4 will work to extend and harmonize the use of ontologies across the ELIXIR hCNV Community. Both of these objectives align with the “Interoperability with a Purpose at Source” task of the ELIXIR Interoperability Platform Programme for 2019-23.\n\nMore generally, in Objective #6, interoperability will be a major component of the hCNV Community. CNV FAIR databases will therefore strongly interact with the ELIXIR Interoperability Platform at multiple levels.\n\nTraining Platform\n\nFinally, the global recognition of the ELIXIR hCNV Community's achievements will only be made possible through training, capacity building, and dissemination. A strong link has already been established with this ELIXIR Platform for training coordination and additional collaborations including capacity building will be established as systems, databases and tools are released.\n\nAlignment with other ELIXIR Communities\n\nCNVs are genetic mutations found in all organisms. The ELIXIR hCNV Community will naturally strongly interact with the \"Federated Human Data\" and \"Rare Diseases\" ELIXIR Communities (Figure 2). Moreover, the hCNV community will benefit from the \"Federated Human Data\" Community thanks to its experience in human data secure access (GDPR) and ethical aspects (ELSI).\n\nLeft: vertical interaction with all of the current ELIXIR Human Data Communities (Federated Human Data and Rare Diseases). Right: It is predicted that the ELIXIR hCNV Community will interact with other ELIXIR Communities, such as Marine Metagenomics and Plant Sciences; relationships to other ELIXIR Communities will be investigated and developed over time.\n\nGoing beyond these, interactions could be established with the ELIXIR Marine Metagenomics and Plant Sciences Communities in the longer term (Figure 2). In fact, the hCNV detection tools might allow the handling of CNV in plants and marine organisms. Its feasibility will be discussed with these Communities to allow optimal interactions.\n\nIn addition, the creation of pipelines and tools will link to the ELIXIR’s Galaxy Community through the implementation of tools and training materials in their platform.\n\nAlignment with the ELIXIR Industry Programme\n\nDue to the known involvement in human disease, the topic hCNV is of considerable interest to the scientific industry, for example in the field of personalized medicine. It is therefore of critical importance to ensure that the services we provide are of sufficient quality and fit for purpose for adoption in industry as well as in academia. To facilitate knowledge exchange, we will continue to encourage the participation of industrial partners in the ELIXIR hCNV Community in the role of Community Partner(s). This novel form of engagement is anchored in the ELIXIR Scientific Programme 2019–23 and is part of a comprehensive initiative to embed ELIXIR into the wider ecosystem. Furthermore, we propose to use the ELIXIR Industry Staff Exchange program to allow members of the ELIXIR hCNV Community to work with industry partners on projects of mutual interest in the form of short-term staff exchanges.\n\nIntegration at a global level\n\nAs already described, the use of ontologies, nomenclatures and data exchange formats should be viewed in a more global context. In fact, this dimension has already been addressed by the ELIXIR hCNV Community's partners who have identified international projects and organizations with which relationships will be established. Thus, the GA4GH organization will interact and benefit from the hCNV community at various levels. The International Rare Disease Research Consortium (IRDiRC) will not only benefit from the Community but will also be able to promote its achievements through its members and the \"IRDiRC Recognized Resources\" labeling program. HGVS has also been approached to participate to their scientific meetings to disseminate the community's achievements.\n\n\nConclusions\n\nWe believe that this white paper demonstrates the global need for ELIXIR to establish the human Copy Number Variation Community (hCNV) as it responds to a major challenge of NGS data interpretation in the era of whole genome sequencing both for research and in clinical settings. To do so, we have identified seven objectives ranging from CNV detection to data interpretation and sharing, for which various tasks have been described. The interactions with ELIXIR Platforms and Communities, and worldwide integration in the complex landscape of societies, initiatives and projects, has been addressed to avoid duplication of efforts and ensure fruitful collaborations.\n\nFinally, the growing interest for CNV detection and interpretation in human diseases will ensure the global recognition and expansion of the community and will trigger many interactions both for other ELIXIR Communities (such as Marine Metagenomics and Plant Sciences), and industry.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nTjio JH, Levan A: The Chromosome Number of Man. Hereditas. 1956; 42(1–2): 1–6. Publisher Full Text\n\nJacobs PA: An Opportune Life: 50 Years in Human Cytogenetics. Annu Rev Genomics Hum Genet. 2014; 15(1): 29–46. PubMed Abstract \| Publisher Full Text\n\nNowell C: The minute chromosome (Ph1) in chronic granulocytic leukemia. Blut Z Für Gesamte Blutforsch. 1962; 8(2): 65–6. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "74635", "date": "22 Mar 2021", "name": "Antonio Rausell", "expertise": [ "Reviewer Expertise Clinical bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the white paper \"The ELIXIR Human Copy Number Variations Community: building bioinformatics infrastructure for research\", Salgado and co-authors present the working plan of the ELIXIR human CNV community. The manuscript summarises the strategy adopted in order to address current challenges in CNV detection and clinical interpretation. First, the introduction reviews the clinical relevance of structural variants in the human genome as well as the evolution of the technologies used for their identification. Second, a total of 7 main objectives are described, which are further detailed into specific tasks. Here, authors propose the development of enhanced CNV detection protocols, reference datasets, computational methods and analyses pipelines, standards for CNV reporting and data exchange and dissemination actions. The manuscript is clearly written and provides a complete and fair overview of the state-of-the-art in the field and of the challenges ahead. Thus, it not only represents a sound and ambitious action plan but, importantly, an insightful reference for current research in human genetics. I congratulate the authors for such effort. I provide below minor comments and suggestions:\nFor non-familiar readers, I would suggest to add a short paragraph introducing ELIXIR and their missions, and to place the human CNV community in such a broader context.\n\nThe manuscript would benefit from a table summarising the database and software resources previously developed or adopted by the hCNV community that served as a reference or starting baseline for the proposed actions.\n\nI missed some mention to recent long-read sequencing technologies. A discussion about the benefits that they may bring to the human CNV field and whether the hCNV community would consider future actions in this direction would be welcome.\n\nAlso, additional discussion would be welcome as for how the hCNV community effort may indeed boost machine-learning developments oriented to the clinical assessment of CNVs detected from short-read sequencing data. In this direction, the establishment of federated learning strategies across Elixir nodes and associated partners would represent a big step forward. While the manuscript mentions the interaction with the \"Federated Human Data\" and \"Rare Diseases\" ELIXIR Communities, the importance of those aspects would deserve more attention.\n\nThe use of the term \"cytogenetic\" as opposed to \"genomic\" alterations can lead to confusion. I would suggest to rather use structural variants versus single-nucleotide variants and indels.\n\nI would suggest rephrasing of the following two sentences: \"beyond a limited number of cancer-associated gene loci.\" [...] \"It is thus possible to use the technology for genome-scale analysis through to region specific analysis\".\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "88724", "date": "20 Jul 2021", "name": "Andrew Lonie", "expertise": [ "Reviewer Expertise Bioinformatics", "bioinformatics infrastructure", "genomics", "genetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 this is an excellent proposal and I only have minor grammatical suggestions. The science case is strong and is described well; the approach is well thought through and well articulated. This would be a very worthwhile ELIXIR community.\nMinor suggestions:\nMeeting: “The Elixir Human Cnv Community” CNV should be properly capitalised here, as should 'ELIXIR'. And in the previous paragraph, \"The Elixir Human Cnv Community” is already introduced and acronymed as 'hCNV Community'.\nThroughout the document, sometimes 'ELIXIR hCNV Community' is used, sometimes 'hCNV Community;, sometimes hCNV. Would be best to standardise.\nObjective #1: It would be good to discuss how pipelines will be made available for the entire community. It's a complex problem, in practice.\nObjective #2: It would be good to discuss how the reference datasets will be made available in a FAIR way to the global community.\nObjective #3: \"Task #3.2 will develop recommendations to use a standardized format to report CNVs. If a few alternative formats are frequently used, it will provide bioinformatics resources to convert data into the common data exchange format.\" - this sentence doesn't make sense.\nObjective #5: \"The hCNV Community will develop innovative tools to: annotate CNV; facilitate their interpretation through a combinatorial approach; and help to pinpoint key genes in regions of interest. The following tasks have been identified and will most likely evolve as more data become available and as technologies evolve:\"\n\nIt should be,\n\"The hCNV Community will develop innovative tools to annotate CNVs, facilitate their interpretation through a combinatorial approach, and help to pinpoint key genes in regions of interest.\nThe following tasks have been identified and will most likely evolve as more data become available and as technologies evolve:\"\n\"Task #5.1 will define CNV annotations including: type; genotype; genes and transcripts; expression level; exons; regulatory elements; breakpoints/ fusion fragments for WGS only\" - remove the space after \"breakpoints/\".\n\"Task #5.3 will develop specific bioinformatics tools to select candidate genes localized in the CNV region by combining genes' annotations and patients' phenotype.\" - this reads a bit strangely. Candidate genes for what? I assume specific rare diseases?\nObjective #6: \"This will respect databases' ownerships and national regulations' compliance\" - there should be no trailing apostrophe after regulations; the compliance does not belong to the regulations.\nNothing much said about FAIRness of all the other artefacts that will be produced (workflows, standards)...\n\"Alignment with other ELIXIR Communities CNVs are genetic mutations...\"\nMaybe this is better: \"Alignment with other ELIXIR Communities CNVs are genetic variations...\"\n\"Conclusions\": Again, would be good to standardise on a particular nomenclature of hCNV / hCNV Community / ELIXIR HCNV Community / human Copy number Variation Community and stick with it from the start.\nIn summary, this is a very worthwhile community and my comments are generally minor and by way of expression clarifications.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1229
https://f1000research.com/articles/9-1226/v1	12 Oct 20	{ "type": "Research Article", "title": "Anti-hypertensive vasodilatory action of Gynura procumbens mediated by kaempferol 3-O-rutinoside", "authors": [ "Syahirah Shahlehi", "Aziemah Azizi", "Asrin Tengah", "Siti Nornadhirah Amdani", "Mark I. R. Petalcorin", "Syahirah Shahlehi", "Aziemah Azizi", "Asrin Tengah", "Siti Nornadhirah Amdani" ], "abstract": "Introduction: Gynura procumbens (GP), otherwise known as longevity spinach or “Sambung Nyawa” in Malay, is an evergreen herb found in Africa and Southeast Asian countries (including Brunei) used traditionally to treat various diseases such as fever, diabetes and hypertension. We examined GP’s vasodilatory action to determine its possible role via the cholinergic-mediated pathway. Methods: GP leaves were prepared by filtration and evaporation to obtain the aqueous (AEGP) and methanol (MEGP) extracts followed by screening for phytochemical constituents. The total phenol, total flavonoid and flavonol contents were determined using the corresponding Folin–Ciocalteau, and aluminium colorimetric methods and the presence of kaempferol 3-O-rutinoside in the extracts was detected using HPLC analysis. Organ bath studies were conducted to determine the vasodilatory activity using intact and denuded isolated rat aortic rings by exposure to either increasing concentration of extracts (0.25, 0.5, 1.0, and 2.0 mg/mL) or 10 µg/mL kaempferol 3-O-rutinoside in the presence or absence of acetylcholine (ACh) after pre-contraction by noradrenaline (NA). Results: MEGP contained more phytochemical constituents and higher content of total flavonoid and total flavonol but less phenolic content than AEGP. Furthermore, MEGP yielded a 20% elevated amount of kaempferol 3-O-rutinoside than AEGP. Both extracts significantly amplified ACh-endothelium dependent vasodilation and mediated relaxation at 1 mg/mL in endothelium-intact and endothelium-denuded aortic rings with MEGP as a more effective vasodilator than AEGP. Overall, these results imply the involvement of extracts in potentiating cholinergic pathway, which might be mediated by kaempferol, as shown by its vasorelaxation effects in endothelium-intact and –denuded aorta. Conclusions: The present findings demonstrate that the vasodilatory activities of the two Gynura procumbens extracts, AEGP and MEGP, in thoracic aorta rings isolated from rats are potentially mediated via a cholinergic pathway through the action of a flavonoid particularly kaempferol 3-O-rutinoside.", "keywords": [ "Gynura procumbens", "Hypertension", "Vasodilation", "Cholinergic pathway", "Kaempferol", "Acetylcholine", "Muscarinic receptors" ], "content": "Introduction\n\nHypertension persists as high arterial pressure with systolic and diastolic blood pressure (BP) exceeding 140 and 90 mmHg, respectively1 contributing to cardiovascular diseases (CVDs), which are the leading cause of death globally. An estimated 17.9 million people die annually from CVDs, representing 31% of all global deaths2. More than 90% of hypertension is termed as essential or primary hypertension with unknown causes but influenced by environmental, genetic and behavioural factors3,4 including high salt intake, smoking, age, gender, obesity, diabetes, sedentary lifestyles, chronic alcohol consumption and family history of hypertension4,5. The remaining 10% of the cases fall under secondary hypertension caused by potentially reversible biochemical or mechanical pathology associated with renal or adrenal diseases6,7.\n\nA continuous effort has been made to search for a safer and more effective method to control hypertension worldwide. Herbal medicines have been used since the beginning of human civilisation to control hypertension and other cardiovascular disorders including congestive heart failure, atherosclerosis, cerebral and venous insufficiency, arrhythmia and ischemic heart diseases8. Traditional herbs have become significant sources for drug research in pharmaceutical settings as alternatives to conventional synthetic drugs such as thiazide and captopril for hypertension9. Gynura procumbens (longevity spinach or “Sambung Nyawa”) is an evergreen herb that is used traditionally in Africa and Southeast Asia including Brunei, Indonesia, Thailand and Malaysia to treat various diseases such as hypertension, rashes, kidney disease, fever and diabetes10,11. Belonging to the Asteraceae family, this plant is small in size (around 1-3 m in height) with oval-shaped green leaves, fleshy stems and purple tint12, and can thrive in partial, shaded or full sun as long as the soil is moist. The leaves of this plant are traditionally consumed as salad, juice or tea for health benefits.\n\nVarious studies have demonstrated the antihypertensive activity of GP both in-vitro and in-vivo8–10,13–16, acting to lower the blood pressure by stimulating vasodilation besides heart stabilisation and diuretic effects. However, its mechanism of inducing vasodilation is not well understood. Activation of the parasympathetic division of autonomic nervous system induces relaxation of the blood vessel17 via the cholinergic pathway by releasing acetylcholine (ACh) from the nerve terminal in blood vessel walls, which in turn stimulates nitric oxide (NO) production that triggers dilation of vascular smooth muscle. The identity of GP’s active ingredient for vasodilation is likely to be the flavonoids, which may be responsible for its antihypertensive effect10,14,18,19. Kaempferol is a flavonoid that is present in the sub-fraction of the ethanolic extract of GP20,21, and kaempferol 3-O-rutinoside is one of the various types of kaempferol that has been identified in GP14. In this present study, we investigated GP’s vasodilatory action and, then, determined its possible activation of the cholinergic pathway mediated by kaempferol 3-O-rutinoside.\n\n\nMethods\n\nAll chemicals used for methanol extraction, phytochemical content determination including total phenolic, flavonoid, flavonol and high liquid performance liquid chromatography (HPLC) were obtained from Merck, Germany except for Dragendorff’s reagent, mercuric chloride, potassium iodide, iodine, quercetin and kaempferol 3-O-rutinoside, which were purchased from Sigma Aldrich, USA. All reagents for organ bath studies including those to prepare Kreb’s Henseleit solution (Kreb’s solution), sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), glucose, sodium hydrogen carbonate (NaHCO3) and calcium chloride (CaCl2) were purchased either from Sigma Aldrich or Merck.\n\nKreb’s solution was prepared by dissolving compounds in distilled water and adding calcium chloride last with continuous stirring to eliminate the formation of a salt precipitate. For the organ bath study, drugs used were dissolved following the manufacturer’s instructions and GP extracts were dissolved in Kreb’s solution. For HPLC experiment, kaempferol 3-O-rutinoside was dissolved in methanol.\n\nGP was purchased from Rimba Horticulture Centre, Brunei Darussalam. Leaf samples were then verified by Dr Johan Willem Frederick Slik (Associate Professor in Phylodiversity, Faculty of Science, Universiti Brunei Darussalam). The herbal specimen (dried leaves) was deposited into Universiti Brunei Darussalam Herbarium (UBDH) with a sample number 17H8182.\n\nFresh leaves of GP were cleaned, weighed, and oven-dried at 60°C for three days. The dried leaves were then crushed using a blender (Waring 800s, USA) to produce fine powder and filter-sieved in dry form using a mechanical sieve (0.3 mm mesh hole size).\n\nAqueous extract of Gynura procumbens (AEGP) was prepared as described previously22. Briefly, the powdered leaves were macerated twice in distilled water (1:10 ratio of solute to solvent) and kept at 60°C water bath for three days. After filtration, the filtrate was centrifuged at 3000 rpm for 20 min, and the supernatant was recovered, and frozen at -80°C overnight. The sample was then lyophilised by freeze-drying for three days to yield a brown powder of AEGP23 that was stored at 4°C until further use.\n\nMEGP was prepared as described previously24 with slight modifications, using ultrasonic extraction (UE) with 95% methanol in 300 W ultrasonic bath. Briefly, the powdered leaves were placed in a beaker and added with methanol at a 1:20 (solute: solvent) ratio before subjected with ultrasound at 25°C for 30 min. After extraction, the sample was filtered using filter paper, and the powder residues were added again with methanol of 1:20 (solute: solvent) ratio in another cycle of ultrasound. The filtrate obtained was evaporated with a rotary evaporator (Yamato RE301, Japan) at 63°C, and left to dry at 60°C in a drying cabinet for five days yielding a dark greenish-brown sticky solid MEGP that was stored at 4°C until further use. The percentage of yield was calculated using the following equation25:\n\n% Yield = Mass of plant crude extract/ Mass of powder plant in thimble x 100%.\n\nThe extracts were tested using the standard procedures for phytochemical constituents, including alkaloids, glycosides26, flavonoids, saponins, steroids, triterpenoids27 and tannins28. For stock extract solution preparation, AEGP and MEGP (1 g each) were completely dissolved in 100 mL of water and methanol, respectively. The stock solutions obtained were used for phytochemical screening whenever necessary as some of the methods required the solid form of the extracts. Qualitative results were expressed as (+) for the presence and (-) for the absence of phytochemicals. Each test was done by visual observation of colour change or formation of a precipitate after the addition of specific reagents. Hence, the experiments were only carried out once.\n\nAround 15 mg of each extract was separately stirred in 6 mL of 1% HCl at room temperature water bath for 5 min and then filtered using Whatman filter paper (grade 42, 55 mm). The filtrates were divided into two equal parts and used for Dragendorff’s and Mayer’s tests. The orange-red precipitate formation indicated the presence of alkaloids using Dragendorff’s test upon addition of 1 mL of Dragendorff’s reagent (potassium bismuth iodide solution) to the first extract filtrate. Using Mayer’s test, the presence of alkaloids was indicated by the cream-coloured precipitate or turbid solution formation upon addition of 1 mL of Mayer’s reagent (potassium mercuric iodide solution) to the second filtrate.\n\nThe presence of flavonoids was detected by the formation of an intense yellow colour solution that turned into colourless upon addition of few drops of 1 M HCl to a mixture of 1 mL of each extract and 0.2 mL of 1 M NaOH.\n\nUsing Borntrager’s test, the presence of anthraquinone glycosides was detected by the formation of rose-pink to red colour ammoniacal layer upon addition of diluted (10%) ammonia to the lower layer of chloroform that was recovered from boiling and filtering a mixture of 1 mL of each extract, 1 mL of 5% sulphuric acid (H2SO4) and 2 mL chloroform.\n\nThe presence of saponins was detected by the formation of a topmost foamy layer upon thoroughly mixing by hand a dilution of 1 mL of each extract in 20 mL of distilled water for 15 min.\n\nThe presence of steroids was detected by the colour change of the upper layer into red and sulphuric acid (H2SO4) layer, and into fluorescent yellow upon addition of 10 mL of chloroform and an equal volume of concentrated H2SO4 into 1 mg of each extract.\n\nThe presence of tannins was detected by the development of precipitate upon addition of 1% potassium dichromate solution to an equal volume of each extract.\n\nThe formation of reddish-violet colour detected the presence of triterpenoids upon addition of 2 mL of chloroform, 1 mL of acetic anhydride and 1 mL of concentrated H2SO4 to 5 mg of each dry extract.\n\nTotal phenolic content. The total phenolic content of each plant extract was determined using Folin–Ciocalteau assay10,22,26. Briefly, 20 μL of extracts, 100 μL of 2 N Folin–Ciocalteu phenol reagent and 1.58 mL of distilled water were mixed thoroughly. After 8 min, 300 μL of Na2CO3 was added into the mixture and allowed to react at room temperature for two hours before measuring the absorbance at 765 nm using a spectrophotometer. A standard calibration curve of gallic acid (0-4 mg/mL) was prepared, and results were expressed as mg of gallic acid equivalent (GAE) per g of extract. Experiments were replicated four times.\n\nTotal flavonoid content. Total flavonoid content of each extract was determined using aluminium colourimetric method10,22,26 using quercetin (0-200 μg/mL) as a standard. Briefly, 0.5 mL of extract and 0.5 mL of the standard were placed in separate test tubes with 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M potassium acetate, 1.5 mL of 80% methanol and 2.8 mL of distilled water, and mixed thoroughly. A blank was prepared in the same manner where 0.5 mL of distilled water was used instead of the sample or standard, and the aluminium chloride replaced by distilled water. All tubes were incubated at room temperature for 30 min, and the absorbance was measured at 415 nm using the spectrophotometer. Experiments were carried out four times. Results were expressed as mg of quercetin equivalent (QE) per gram of extract.\n\nTotal flavonol content. Total flavonol content of each extract was analysed using aluminium chloride colourimetric method as described previously26. Briefly, quercetin was used to make a standard calibration curve ranging from 0 to 100 μg/mL. The test tube containing 1 mL of extract or 1 mL of standard solution was added with 1 mL of 2% aluminium chloride and 3 mL of 5% sodium acetate and then mixed well. The mixture was then centrifuged at 3000 rpm for 20 min to get a clear solution. The absorbance of the standard and sample was measured at 440 nm using the spectrophotometer. Experiments were replicated four times. Results were expressed as mg of quercetin equivalent (QE) per gram of extract.\n\nHPLC analysis was performed as previously described29 using Agilent 1200 series HPLC system (Agilent LabX, USA) equipped with an autosampler and coupled to a variable UV wavelength detector. Liquid chromatography was performed on a reversed-phase C18 column (internal diameter 4.6 mm, height 250 mm, and particle size 5 mm). The mobile phase consisted of two solvents ([A] 5% CH3COOH, acetic acid and [B] CH3CN, acetonitrile) using the following elution program: 0–10 min 70% A isocratic, 10–20 min gradient to 40% A and then 20–30 min 40% A isocratic again with a flow rate of 1.0 mL/min at 30 ºC to detect peaks at 367 nm. Standard calibration was done using kaempferol 3-O-rutinoside at a concentration ranging from 0 µg/mL to 100 µg/mL. After the calibration curve was generated, both extracts with or without spiking with kaempferol 3-O-rutinoside (100 µg/mL) were run using the same elution program. Experiments were carried out in triplicates.\n\nA total of 25 adult male Sprague Dawley (SD) rats at age of 16–20 weeks, weighing from 250 to 300 g, were obtained from Universiti Brunei Darussalam animal house. The animals were housed in standard environmental conditions (24°C, 60–70% humidity) on a 12 hour-light-dark cycle feeding. The animals were given standard rat pellets and tap water ad libitum.\n\nThe study was conducted following the ethical guidelines of the Laboratory Animal Care (NIH publication #85-23, revised in 1985) and approved by the University Research Ethics Committee (UREC) of PAPRSB Institute of Health Sciences, Universiti Brunei Darussalam.\n\nAfter sacrificing the rats by cervical dislocation, their descending thoracic aorta was removed and placed in a petri dish on ice containing Kreb’s buffer (118.2 mM of NaCl, 4.7 mM of KCl, 2.5 mM of CaCl2, 25 mM of NaHCO3, 1.2 mM of MgSO4, 1.2 mM of KH2PO4, and 11.7 mM of glucose). The rat aorta was dissected, cleared of fats including connective tissues, and cut into rings of 3 mm long. The denuded endothelium was prepared by inserting fine forceps into the lumen of the aorta with gentle rotation. The aortic rings were mounted using two triangular-shaped platinum wire holders in the vessel lumen and suspended horizontally in an organ bath with 10 mL of Kreb’s buffer, thermoregulated at 37°C, and bubbled with carbogen to a resting tension of 1.0 g (Figure 1A). One of the holders was fixed to the bottom of the chambers, and the other was connected to a force-displacement transducer (Grass® Force Displacement Transducer FT03) for measuring isometric tension. The rings were equilibrated at 1.0 g resting tension for 20 min, with bathing solution replacement every 10 min, and tension readjustment to 1.0 g if necessary. The aortic rings were tested for viability using 40 mM KCl before conducting experiments (Figure 1B). Isometric contraction and relaxation were recorded using a Force Displacement Transducer. The transducer signals were displayed, stored and analysed on a computer using AD Instruments model 4/25T with Power Lab Software version 7. The responses (in grams) were recorded using LabChart Reader 7.\n\n(A) Schematic set-up of the organ bath. The aortic rings were mounted using two-triangle-shaped platinum wire holders, suspended horizontally in an organ bath containing 10 mL of the above solution, thermoregulated at 37°C, and bubbled with carbogen with a resting tension of 1 g. Isometric contraction and relaxation were recorded using Force Displacement Transducer. Transducer signals were displayed and stored on a computer. (B) A summarised protocol of one of the organ bath experiments. Before each experiment, the aortic rings were stimulated with 40 mM KCl to produce a contraction, which represents a reference response, ensuring the viability of the rings just before the start of the experiment. Then the buffer solution was replaced for three times, and baseline was stabilised before adding 50 nM of NA for 10 min to induce stable contractions, and 100 μM of ACh was added for 50 min afterwards to produce the first curve. Then, the aortic rings were washed with Kreb’s buffer three times, allowed to stabilise, then added again with 100 μM ACh for 50 min in pre-contracted rat-aorta before adding extracts cumulatively to obtain the second curve.\n\nThe following experiments were conducted using both endothelium-intact and endothelium-denuded aorta, except for the protocol on the addition of noradrenaline (NA) with the extracts or kaempferol, whose effects were previously determined and ruled out as NA-independent regardless of the tissue preparation.\n\nThe aortic rings were pre-contracted by adding 50 nM of NA to induce transient and stable contraction for 60 min to generate the first response curve. Then, the aortic rings were washed with Kreb’s buffer three times, allowing the baseline to stabilise for 10 min before pre-contracted again with 50 nM of NA. After the tonic response or contraction became stable for 20 min, increasing concentrations of AEGP or MEGP (0.25, 0.5, 1 and 2 mg/mL) were added cumulatively at 10 min interval to obtain the second response curve.\n\nThe aortic rings were pre-contracted with 50 nM of NA for 10 min, followed by addition of 100 µM of ACh for 50 min to obtain the first response curve. Then, the aortic rings were washed with Kreb’s buffer three times, allowing the baseline signal to stabilise for 10 min. The aortic rings were pre-contracted again with 50 nM of NA for 10 min followed by addition of 100 μM of ACh for another 10 min before adding AEGP or MEGP cumulatively in increasing concentrations (0.25, 0.5, 1 and 2 mg/mL) every 10 min to obtain the second response curve.\n\nThe aortic rings were pre-contracted with 50 nM of NA for 30 min to obtain the first response curve. Then, the aortic rings were washed with Kreb’s buffer three times, allowing the basal tension signal to stabilise for 10 min. The rings were pre-contracted again with 50 nM of NA for 10 min, followed by addition of 10 μg/mL kaempferol 3-O-rutinoside for 20 min to generate the second response curve. This experiment was to rule out the role of kaempferol in enhancing the ACh-mediated vasodilation.\n\nThe aortic rings were pre-contracted with 50 nM of NA for 10 min, followed by addition of 100 μM of ACh for 20 min to obtain the first response curve. Then, the aortic rings were washed with Kreb’s buffer three times, allowing the basal tension signal to stabilise for 10 min. The rings were pre-contracted again with 50 nM of NA followed by addition of 100 μM of ACh before adding 10 μg/mL kaempferol at 10 min interval to generate the second response curve.\n\nCalculations and statistical analysis were performed using GraphPad Prism (Version 6.0) software. Contractile responses were expressed in grams of tension (g). The vasorelaxation due to ACh, the addition of extracts and kaempferol 3-O-rutinoside, was expressed as a percentage relaxation in response to contraction caused by NA. All results were expressed as mean ± standard error of the mean (SEM) per experimental group with n representing the number of aortae used in each experiment. Data were analysed using two-way analysis of variance (ANOVA), which was followed by Dunnett’s test for significant differences between the control and the experimental treatments, except for the data of kaempferol 3-O-rutinoside with the addition of Ach, which was analysed using unpaired Student’s t-test. The line graph for determining the potent vasodilator between the two extracts was constructed using linear regression. P-values of less than 0.05 (P<0.05) were considered statistically significant.\n\n\nResults\n\nThe percentage yield extracted from powdered leaves of GP using aqueous extraction (AEGP) or methanol extraction (MEGP) is shown in Table 1. After drying the extracted fluid, MEGP gave a higher percentage yield of 8.7%, producing a dark greenish-brown sticky solid material, while AEGP yielded 5.5%, generating a light brown fluffy material. The phytochemical analysis is presented in Table 2, which shows that flavonoids and triterpenoids were detected in both AEGP and MEGP while saponins, steroids and tannins were only seen in MEGP. On the other hand, alkaloids were only detected in AEGP but not MEGP. Glycosides were absent in all extracts. Table 3 presents the total phenolic, flavonoid and flavonol contents in both types of GP extracts stated as mg GAE/g extract for total phenolic content and mg QE/g extract for total flavonoid and flavonol contents. AEGP has higher total phenolic content of 28.23±4.68 mg GAE/g extract, which is twice as that of MEGP (13.82 ± 2.42 mg GAE/g extract). In contrast, MEGP has four times more total flavonoid (10.37 ± 0.07 mg QE/g extract) and two times more total flavonol (4.72 ± 0.04 mg QE/g extract) content than AEGP (2.52 ± 0.21 and 2.35 ± 0.03 mg QE/g extract, respectively). The total phenolic content of GP extracts was based on the standard gallic acid curve obtained at y= 0.4332x+0.0339 (r2= 0.993), while the total flavonoid and flavonol contents were calculated from a standard quercetin curve using the linear regression equations y=0.0029x+0.0458 (r2=0.995) and y=0.0045x+0.039 (r2=0.996), respectively.\n\n+: weak presence, ++: moderate presence, +++: strong presence, and -: Absent.\n\nValues are expressed as mean of mg of GAE or QE per gram of extract ±S.E.M; n=4.\n\nGAE: Gallic acid equivalent, QE: Quercetin equivalent, G. procumbens: Gynura procumbens, SEM: Standard error of the Mean.\n\nThe results of HPLC screening using a reverse-phase C-18 column with a gradient system of acetic acid-acetonitrile combination as the mobile phase for both extracts revealed that when using the spiking technique with the standard reference compound of 100 µg/mL kaempferol 3-O-rutinoside, the height of peaks for both extracts was explicitly increased with the retention time observed at 1.4 min after injection. The amount of kaempferol 3-O-rutinoside calculated based on the difference of the height of peaks at 1.4 min between the standard reference and the extracts were 57.86 µg/mL and 78.35 µg/mL for AEGP and MEGP, respectively. Figure 2 shows the actual HPLC chromatograms for AEGP and MEGP, with and without spiking of 100 µg/mL kaempferol 3-O-rutinoside as the standard reference.\n\n(A) AEGP alone with no spiking. (B) AEGP with spiking using 100 µg/mL of kaempferol-3-O-rutinoside standard reference stock. (C) MEGP alone with no spiking. (D) MEGP with spiking using 100 µg/mL of kaempferol-3-O-rutinoside stock. Detection of kaempferol 3-O-rutinoside was performed at 367 nm; heights of peaks were increased specifically at 1.4 min retention time when spiked with kaempferol 3-O-rutinoside (B and D) as compared with those without spiking (A and C).\n\nBoth AEGP and MEGP extracts were cumulatively added at 10 min interval in increasing concentrations (0.25, 0.5, 1 and 2 mg/mL). Addition of these extracts started at 20 min after the aortic rings were pre-contracted with 50 nM of NA. At the lowest concentration, both AEGP and MEGP produced a minimum relaxation of 12 ± 4.46% and 18 ± 3.22%, respectively (Figure 3). At the highest concentration, both AEGP and MEGP produced a maximum relaxation of 95 ± 4.53% and 112 ± 6.07%, respectively (Figure 3). In the pattern of relaxation induced by both extracts, the percentage relaxation of NA-induced contraction in rat aorta was significantly higher when exposed to increasing concentration of GP extracts, especially from 0.5 mg/mL to 2 mg/mL for both extracts, as compared with that of the control NA alone (Figure 3).\n\nData presented as mean ± S.E.M. The n values for NA and both extracts are 4. P<0.05 for 0.5 mg/mL AEGP, P<0.05 for 0.5 mg/mL MEGP and *** P<0.05 for both 1 mg/mL and 2 mg/mL of AEGP and MEGP as compared with that of the control, NA alone.\n\nIn endothelium-intact rat aorta, both AEGP and MEGP extracts were cumulatively added at 10 min interval in increasing concentrations (0.25, 0.5, 1 and 2 mg/mL) at 10 min interval, starting at 20 min after adding 100 μM of ACh. Addition of these extracts started at 30 min following addition of 100 μM of ACh. At the lowest concentration, both AEGP and MEGP produced the minimum relaxation of 61 ± 13.80% and 72 ± 18.40%, respectively (Figure 4A). At the highest concentration, both AEGP and MEGP produced the maximum relaxation of 87 ± 4.30% and 112 ± 12.3%, respectively (Figure 4A). Relaxation induced by both extracts was significantly different from ACh control alone, especially at 0.5 mg/mL for AEGP and 1 mg/mL for both AEGP and MEGP (Figure 4A).\n\n(A) Endothelium-intact rat aorta (B) Endothelium-denuded rat aorta. Data presented as mean ± S.E.M. The n values for ACh and both extracts are 3 for endothelium-intact rat aorta whereas n values for ACh and both extracts are 6 and 5, respectively, in endothelium-denuded aorta. P<0.05 for 0.5 mg/mL AEGP and 1 mg/mL both AEGP and MEGP as compared with the non-treated control, ACh alone in endothelium-intact rat aorta whereas P<0.05 for 0.5 mg/mL MEGP and 1 mg/mL AEGP as compared with non-treated control, ACh alone, and P<0.05 for 1 mg/mL and 2 mg/mL MEGP vs. control in endothelium-denuded rat aorta.\n\nIn endothelium-denuded rat aorta, both AEGP and MEGP extracts were separately added in increasing and cumulative concentrations (0.25, 0.5, 1 and 2 mg/mL) at 10-min intervals. In both cases, the addition of these extracts started at 30 min following addition of 100 μM of ACh. At the lowest concentration, both AEGP and MEGP produced the minimum relaxation of 46 ± 15.70% and 42 ± 13.10%, respectively (Figure 4B). At the highest concentration, both AEGP and MEGP produced the maximum relaxation of 79 ± 5.60% and 114 ± 8.60%, respectively (Figure 4B). Overall, there was a pattern of relaxation induced by both extracts, where AEGP significantly enhanced relaxation at 1 mg/mL, while MEGP enhanced relaxation at a wider range of 0.5 mg/mL to 2 mg/mL (Figure 4B).\n\nExposure of endothelium-intact rat aorta to either extract when added cumulatively produced a similar vasodilatory effect as a reduction in the percentage of NA peak response. Exposure to increasing concentration of AEGP up to 1 mg/mL reduced the contraction of rat aorta from 39 ± 13.9% to 0 ± 17.1%, and MEGP from 40 ± 18.4% to 5 ± 14.0%. ACh alone without the addition of extracts still showed a 23 ± 4.90% contraction. However, at 2 mg/mL, both extracts had a differential effect, in which MEGP caused further reduction from 5 ± 14.0% to 0 ± 12.3% contraction, whereas AEGP produced a rise of contraction from 0 ± 17.1% to 18 ± 10.2%.\n\nExposure of endothelium-denuded rat aorta to either extract also produced a similar vasodilatory effect, in which the percentage contraction of NA peak response at concentrations reaching up to 1 mg/mL was reduced from 65 ± 9.35% to 0 ± 6.60% for AEGP and 72 ± 13.1% to 21 ± 9.88% for MEGP, respectively. In comparison, ACh alone without the addition of any extract was only able to reduce the contraction from 80 ± 9.31% to 50 ± 7.58%. However, at 2 mg/mL both extracts had a different effect in which MEGP caused a further reduction of contraction from 21 ± 9.88% to 0 ± 8.64%, whereas AEGP produced a rise of contraction from 0 ± 6.60% to 8 ± 5.61%. Overall, Figure 5 demonstrates the vasodilatory action of both GP extracts, expressed as the normalised data for the effect of both AEGP and MEGP observed as a reduction in the contraction of endothelium-intact and denuded aortic rings that were pre-contracted with 50 nM of NA.\n\n(A) Effect of both AEGP and MEGP in endothelium-intact aortic rings. (B) Effect of both AEGP and MEGP in endothelium-denuded aortic rings. Each extract (AEGP or MEGP) was cumulatively added in increasing concentration of 0.25, 0.5, 1, and 2 mg/mL. The effect of control (ACh alone) was measured at 30, 40, 50 and 60 min, corresponding to the time at which the respective concentration of extracts was added cumulatively. Data presented as mean ± SEM (n=3 for control and both AEGP and MEGP in endothelium-intact rat aortic rings; and, n=6 for control, and n=5 for both AEGP and MEGP, respectively, in endothelium-denuded rat aortic rings).\n\nTo elucidate the role of kaempferol-3-O-rutinoside in inducing relaxation, we first determined the vasodilatory effect of kaempferol without the application of ACh in the pre-contracted aorta rings. Kaempferol 3-O-rutinoside was added for 20 min, which started at 10 min after pre-contraction with 50 nM of NA. Addition of 10 µg/mL of kaempferol 3-O-rutinoside caused rapid relaxation (Figure 7), which was significantly increased from 9.8 ± 1.56% to 84.9 ±18.7% at 20 min and from 17.5 ± 5.24% to 94.6 ± 5.59% at 30 min, respectively, as illustrated in Figure 6.\n\nData presented as mean ± SEM The n values for NA alone and NA with kaempferol 3-O-rutinoside are 5 and 3, respectively. P<0.05 for the addition of kaempferol 3-O-rutinoside at 20 and 30 min vs control, NA alone.\n\nThe pre-contracted aortic rings with intact and denuded endothelium were supplemented with 10 µg/mL of kaempferol 3-O-rutinoside at 20 min after addition of 100 µM of ACh to determine the involvement of kaempferol 3-O-rutinoside in potentiating the cholinergic pathway. Addition of kaempferol 3-O-rutinoside significantly enhanced the vascular relaxation evoked by ACh in rat aorta with and without the endothelium. Kaempferol 3-O-rutinoside elicited a maximum relaxation of 76.0 ± 4.66% and 70.7 ± 0.73% with or without endothelium, respectively. In comparison, the control produced relaxation of 41.7 ± 6.47% and 19.9 ± 6.69%, with and without endothelium, respectively (Figure 7).\n\n(A) Endothelium-intact rat aorta (B) Endothelium-denuded rat aorta. Data presented as mean ± SEM The n values for ACh alone and kaempferol 3 O-rutinoside are 6 and 4, respectively in endothelium-intact rat aorta. In contrast, the n values for ACh alone and kaempferol 3 O-rutinoside are 5 and 3, respectively in endothelium-denuded rat aorta. P<0.05 for the addition of kaempferol 3-O-rutinoside at 30 min vs control, ACh alone in both endothelium-intact and –denuded rat aorta.\n\nRaw data for each experiment performed in this study are available, see Underlying data30.\n\n\nDiscussion\n\nVarious studies have been carried out to identify the antihypertensive ability of GP extracts8–10,13–16. The presence of bioactive compounds in GP is believed to contribute to its beneficial properties such as flavonoids, phenolic acids, alkaloids, terpenoids, glycosides and sterols12. In this present study, the antihypertensive property of both crude extracts of AEGP and MEGP was investigated in vitro to determine whether they were able to potentiate the cholinergic mediated vasodilation. Limited studies showed the possible contribution of the cholinergic pathway in enhancing the relaxation effect of GP extracts. Successful extractions with water or methanol from the dried and powdered leaves of GP were achieved, producing the fluffy brown powder and the sticky dark greenish-brown substance as yields for AEGP and MEGP, respectively. The characteristics of AEGP in our study were similar to those reported by Lee et al.23 and Kamaruzaman and Mat Noor31. There was neither an established guideline nor any known protocols for methanol-based extraction for GP plant leaves. However, the characteristics of the dry methanol extracts, yielding a dark brown sticky solid was demonstrated by Korwar et al.32, similar to our findings. The percentage yields for both extracts were different, which might be due to the polarity of the solvents used33. Given an equal initial amount of fresh weight for the plant parts, the aqueous solvent yielded less extract than those extracted with the methanol solvent, suggesting that the plant GP possesses more methanol-soluble components33. Our finding is in concordance with a study done by Njume et al.34 and Ngo et al.35, where absolute methanol or a mixture of 50% (v/v) water with other solvents such as alcohol or acetone was recommended as the solvent of choice to produce high levels of extractable ingredients.\n\nOur results highlighted the observation that both extracts, which were obtained using different solvents, contained phytochemicals, such as flavonoids and triterpenoids. In addition, MEGP responded positively for the saponins, steroids and tannins, while AEGP was detected positive for alkaloids. However, both extracts did not give a positive test for glycosides. The efficiency of methanol in extracting phytochemicals has been reported in the previous studies34,36,37. Our results were consistent with those of others, confirming that methanol is an appropriate solvent for extracting bioactive compounds from GP, giving higher yields than its aqueous counterpart.\n\nPhenolic compounds account for the majority of pharmacological actions. For several decades, phenolic compounds are regarded as having a powerful free radical scavenging activity38,39. As many aspects of CVDs are associated with high oxidative stress, the consumption of polyphenol-rich food that combats oxidative stress can help lower the risk of chronic diseases, including cardiovascular disorders40–42. In this present study, the total phenolic content of AEGP is higher than MEGP. However, the latter was shown to contain more total flavonoids and total flavonol content. The results obtained for the flavonoids are in agreement with the previous report on GP by Afandi et al.22, showing that MEGP contains higher total flavonoid content than AEGP, but the amount of phenolic content was found lowest in AEGP among all the other extract preparations. This variation could be due to differences in the process of extractions and the solvents used. Various factors could contribute to the efficiency of extraction, such as the type and concentration of solvents, pH, time, temperature and solid-liquid ratios43. Besides, the different types and amount of phenolic and non-phenolic compounds extracted are dependent on the extraction solvent used44. The results showing that AEGP contained a lower percentage of total flavonoid and total flavonol compounds than MEGP might be due to flavonoids having low solubility in water20.\n\nThe presence of flavonoids in both extracts suggests they have a considerable role in various pharmacological effects, especially the hypotensive action, which might explain the BP- lowering observation and claims in the traditional practice of consuming the GP leaves to treat hypertension19. Experimental evidence has shown that the flavonoids in GP extracts is responsible for an antihypertensive effect10,14. Kaempferol, which is one of the significant flavonoids dominantly present in the sub-fraction of the ethanolic extract of GP14,20,21, was also detected in both extracts in our study. Kaempferol 3-O-rutinoside was detected in a higher amount from MEGP than AEGP. After quantifying this active ingredient, we decided to test its vasodilatory activity in the organ bath system using rat aorta to elucidate its hypotensive contribution. We first added the extracts after NA-induced contraction of aortic rings to confirm the vasodilatory action of both extracts. Our results highlighted the vascular relaxation effect indicating the antihypertensive property of this plant and confirming reports from previous studies that GP extracts might reduce blood pressure by vasodilation8–10,13,14,45. Next, we investigated whether there is a possible contribution of the cholinergic-mediated pathway in the vasodilatory effect of the extracts. Our present finding demonstrated that the vasorelaxation activity of the extracts further enhanced the ACh-mediated vasodilation in the endothelium-intact aorta, primarily upon exposure to 0.5 mg/mL AEGP and 1 mg/mL of both AEGP and MEGP. These results support the findings of Kaur et al.8, which showed that ACh administration to GP-treated rats in vivo significantly reduced the mean arterial pressure (MAP) and diastolic pressure (DP) of spontaneously hypertensive rat (SHR), and that of Kim et al.13, which detected an increased of NO levels in the serum of Gynura procumbens water extract (GPWE)-treated SHR.\n\nOur data further revealed that the extracts significantly exerted their relaxation effect in the endothelium-denuded aorta, suggesting that the vasodilatory action of AEGP and MEGP extracts is also mediated directly through the vascular smooth muscles. The vascular smooth muscle cells are located within the media of the vascular bed, directly modulating the vascular tone primarily via the changes in sarcoplasmic free Ca2+ concentration46. Various signalling mechanisms could trigger the response of the smooth muscles through an ACh endothelium-independent pathway such as the adrenergic pathway. In the adrenergic pathway, α1 and α2 adrenoceptors mediate contraction of the smooth muscles by coupling to Gq and activating phospholipase C (PLC), thereby increasing the concentration of Inositol 1,4,5-triphosphate (IP3), which in turn, causes the rise in intracellular Ca2+ concentration47. A previous study showed that GP extracts could mediate relaxation via α1receptors in-vivo8. PE (an α adrenoceptor agonist) and methoxamine (a selective α1 adrenoceptor agonist) induced a significant decrease in pressor response of GPWE-treated rats8. From our findings, we speculate that the GP extracts might also mediate relaxation via adrenergic pathway by activating α and β adrenoceptors. A more favored explanation for this action, however, is based on the evidence that we obtained so far, which pointed towards the cholinergic pathway. It is known that ACh fails to induce relaxation when the endothelium is denuded and removed48,49, suggesting that vasorelaxation of smooth muscle is inhibited when the formation of NO is impaired due to activation of M2 and M3 receptors, hence causing contraction instead50,51. Thus, we focused more on investigating the cholinergic endothelium-independent pathway via stimulation of M2 and M3 muscarinic receptors that inhibit the contraction. Overall, our follow-up study elucidated and confirmed that the underlying mechanism of action for both extracts was mediated via the cholinergic pathway, with a significant contribution from kaempferol.\n\nInvestigation on the vasodilators between these two extracts was carried out in both endothelium-intact and endothelium-denuded rat aorta. Results have shown that upon exposure at the same concentration of 2 mg/mL, MEGP produced a more potent vasodilatory effect than that of AEGP. Although at a lower concentration, both extracts responded in a dose-dependent manner, AEGP seemed to reverse the effect into slight contraction upon reaching the concentration of 2 mg/mL, unlike MEGP, which continued to cause further relaxation. Drug effects are caused by the interaction of the drug molecules and the receptor. At low concentration of the drug, many binding sites are still available; however, further increasing the drug concentration causes more drug-receptor complex formation and a substantial increase in drug effect52 until all of the receptor binding sites are occupied. Further increasing the drug concentration causes receptors to become saturated, leading to no or little effect of the drug52. Thus, the slight reverse effect of contraction by AEGP at 2 mg/mL indicates the possible saturation of all the receptors binding sites for the relaxation effect of the blood vessels, limiting any further vasodilation. Thus, in using AEGP, a possibly saturated concentration exceeding 2 mg/mL would not cause any relaxation effect but a contraction of the blood vessels instead.\n\nFlavonoids rich food, primarily via consumption of botanicals, is often associated with a reduction of cardiovascular diseases53–55. Kaempferol 3-O-rutinoside was detected and quantified as one of the active ingredients in GP20. Thus, we decided to test its hypotensive property in an organ bath system using rat aorta. We first carried out the experiments to confirm the relaxant effect of kaempferol 3-O-rutinoside in rat aortic rings, resulting in vasodilatory activity after NA-induced contraction. This result is in agreement with those of previous studies that demonstrated the vasodilatory effects of kaempferol in different vascular beds55–58. Similar findings have also been reported on the vasorelaxant properties of other flavonoids such as quercetin, myricetin, rutin and apigenin57,59. Kaempferol, however, is considered as the most effective among all flavonoids in potentiating vasorelaxation, thus might have more significant potential in preventing and treating CVDs57. We assessed the involvement of kaempferol 3-O-rutinoside in potentiating ACh-dependent endothelium-mediated vasodilation, and ACh-independent mediated contraction. Our results showed that kaempferol elicited the relaxation response produced by ACh in both endothelium-intact and endothelium-denuded rat aorta. Our observation suggests that kaempferol enhanced the relaxation in rat aortic rings possibly via endothelium-dependent and endothelium-independent cholinergic pathway, following previous reports57,58, which is possibly mediated by kaempferol 3-O-rutinoside, as illustrated in Figure 8.\n\nThe figure is a simplified representation showing the main component associated with acetylcholine mediated endothelium vasodilation in the isolated rat aorta (IRA) model and showing the involvement of both extracts in inducing relaxation in vascular endothelium or vascular smooth muscle. In the presence of endothelium, both extracts (AEGP/MEGP) mediate vasodilation possibly by acting on the muscarinic ACh receptor M3by producing NO. When the endothelium is disrupted, the extracts mediate relaxation on vascular smooth muscle possibly via endothelium-independent cholinergic pathway by acting on the muscarinic ACh receptor M2 and the muscarinic ACh receptor M3or via adrenergic pathway by acting on α and β adrenoceptors. The vasorelaxant effect produced by the extracts is mediated by kaempferol, particularly, kaempferol 3-O-rutinoside. ACh; Acetylcholine M2: Muscarinic acetylcholine receptor M2; M3: Muscarinic acetylcholine receptor M3; α: alpha adrenoceptors; β: beta-adrenoceptors; NO: Nitric oxide; GC: Guanylyl cyclase; cGMP: cyclic guanosine monophosphate; AEGP: Aqueous extract of G. procumbens; MEGP: Methanol extract of G. procumbens.\n\n\nSummary and conclusion\n\nThe result of this study shows that crude extracts of AEGP and MEGP possessed different characteristics and physical appearance, as well as the percentage yield. MEGP was a dark greenish-brown sticky solid whereas AEGP was a light brown fluffy material. The percentage yield of MEGP was higher than AEGP. The result of this study also reveals the presence of essential phytoconstituents in both AEGP and MEGP, and MEGP was found to contain more active constituents than AEGP. Besides, MEGP also contained higher total flavonoid and flavonol contents than AEGP, although the amount of phenolic content was lower in MEGP. HPLC study has demonstrated the presence of kaempferol 3-O-rutinoside, where it is higher in MEGP than AEGP. Treatment with the pure active ingredient kaempferol 3-O-rutinoside relaxed both tissue preparations. Overall, these results imply the possible involvement of extracts in the cholinergic pathway, which is possibly mediated by kaempferol 3-O-rutinoside, as shown by its vasorelaxation effects in endothelium-intact and endothelium-denuded aorta. The present finding also demonstrates the vasodilatory effect of the two extracts, in thoracic aorta tissues isolated from rats, possibly via the contribution of the cholinergic mediated pathway. It is also concluded that the main active vasodilatory component in both extracts causing such hypotensive effect is the flavonoid particularly kaempferol 3-O-rutinoside. However, further study is necessary to elucidate its mechanism of action.\n\n\nData availability\n\nOpen Science Framework: Anti-hypertensive Vasodilatory Action of Gynura procumbens. https://doi.org/10.17605/OSF.IO/W6XJV30\n\nThis project contains “Anti-hypertensive Vasodilatory Action of Gynura procumbens.xlsx”, which includes raw data for all experiments performed in this study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International licence (CC-BY 4.0).", "appendix": "References\n\nChobanian AV, Bakris GL, Black HR, et al.: Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003; 42(6): 1206–1252. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Cardiovascular diseases (CVDs). 2019. Reference Source\n\nOparil S, Zaman MA, Calhoun DA: Pathogenesis of Hypertension. Annals of Internal Medicine. 2003; 139: 761–776. 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Reference Source\n\nWang CZ, Mehendale S, Calway T, et al.: Botanical Flavonoids on Coronary Heart Disease. Am J Chin Med. 2012; 39(4): 661–671. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalvamani S, Gunasekaran B, Shaharuddin NA, et al.: Antiartherosclerotic Effects of Plant Flavonoids. Biomed Res Int. 2014; 2014: 480258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChandradevan M, Simoh S, Mediani A, et al.: UHPLC-ESI-Orbitrap-MS Analysis of Biologically Active Extracts from Gynura procumbens (Lour.) Merr. and Cleome gynandra L. Leaves. Evid Based Complement Alternat Med. 2020; 2020: 3238561. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPérez-Vizcaíno F, Ibarra M, Cogolludo AL, et al.: Endothelium-Independent Vasodilator Effects of the Flavonoid Quercetin and Its Methylated Metabolites in Rat Conductance and Resistance Arteries. J Pharmacol Exp Ther. 2002; 302(1): 66–72. PubMed Abstract \| Publisher Full Text\n\nXu YC, Leung SWS, Leung GPH, et al.: Kaempferol enhances endothelium-dependent relaxation in the porcine coronary artery through activation of large-conductance Ca(2+) -activated K(+) channels. Br J Pharmacol. 2015; 172(12): 3003–3014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahobiya A, Singh TU, Rungsung S, et al.: Kaempferol-induces vasorelaxation via endothelium-independent pathways in rat isolated pulmonary artery. Pharmacol Rep. 2018; 70(5): 863–874. PubMed Abstract \| Publisher Full Text\n\nOzü OY, Ertug PU, Karabulut E, et al.: Dose-dependent differential mechanism of quercetin-induced vasodilatations in isolated perfused rat mesenteric vascular bed. International Journal of Pharmacology. 2016; 12(4): 379–386. Publisher Full Text" }	[ { "id": "72826", "date": "13 Oct 2020", "name": "Adi Idris", "expertise": [ "Reviewer Expertise Cell biology and drug delivery" ], "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\nShahlehi et al. reports the vasodilatory effects of the active flavonoid component of Gynura procumbens, kaempferol 3-O-rutinoside, on rat aortic rings. Though the findings are interesting, it is difficult to see the novelty in this work compared to those done by others. Authors are advised to highlight the novelty of their work. Some other comments that authors are recommended to address are listed below.\nBackground:\nAuthors are asked to comment on reference made to Abrika et al 2013, where they looked at the effect of GP flavonoid fraction on rat arterial contraction as I am contending lack of novelty based on this.\n\nResults:\nTables 1 and 3 are unnecessary. Information could easily be stated in text without the need of a table.\n\nFigure 5 is the critical figure in this manuscript. However, error bars are not visible. Is there a reason why there is only n=3 for each treatment arm in figure A and n>5 in figure B? Authors are also asked to keep Y-axis scale consistent between both figures.\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": [] }, { "id": "72829", "date": "22 Oct 2020", "name": "Yun Shin Sew", "expertise": [ "Reviewer Expertise Molecular and safety analysis of food and herb products" ], "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 by Shahlehi et al. reported interesting findings about the vasodilatory effects of two different plant extracts of Gynura procumbens. The authors have shown the one extracted using methanol solvent, MEGP contains higher amount of flavonoids particularly kaempferol 3-O-rutinoside than the water solvent, AEGP. Following that, the authors showed MEGP works better for in vitro vasodilatory performance than AEGP. The authors also claim a probable role of kaempferol 3-O-rutinoside in both extracts as the main causal factor via experimental observations. There are several comments on this article as the followings:\nThe authors should state the volume of individual AEGP and MEGP used for HPLC analysis in the material and method section.\n\nTable 1 should be removed as those are simple information which could be just included in the text.\n\nThere are discrepancies in the usage of asterisk mark for statistical significance level (referring to Figure 3, 4 and 7) which might confuse the readers. Single asterisk often indicate P<0.05 and double asterisk for P<0.01, need to standardize the usage of asterisk(s) in all figures.\n\nIt was a good experimental deign in such as a way that the relaxant effect of kaempferol 3-O-rutinoside in rat aortic rings was investigated. However the amount of kaempferol 3-O-rutinoside tested was pretty low (10ug/mL). Could this be tested with higher amount of kaempferol 3-O-rutinoside since as high as 57.86 ug/mL and 78.35 ug/mL of this compound was found in AEGP and MEGP extract, respectively? Given that higher concentration of extract showed adverse effect of vasodilatory action in Figure 5, will any adverse effect happen when higher kaempferol 3-O-rutinoside is being used (mimicking the concentration of the compound found in each extract)?\n\nJust an additional suggestion for the future works of this study. Though the methanol extract of Gynura procumbens performs better than the one extracted using water (as shown in the present study), methanol extracts of herbs are often considered to be more toxic than water extracts. Therefore, it is necessary to investigate the safety level of this methanol extract. A follow-up in vivo study using rat model should be conducted in future.\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?\nPartly\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/9-1226
https://f1000research.com/articles/9-298/v1	27 Apr 20	{ "type": "Software Tool Article", "title": "RamaNet: Computational de novo helical protein backbone design using a long short-term memory generative adversarial neural network", "authors": [ "Sari Sabban", "Mikhail Markovsky", "Mikhail Markovsky" ], "abstract": "The ability to perform de novo protein design will allow researchers to expand the variety of available proteins. By designing synthetic structures computationally, they can utilise more structures than those available in the Protein Data Bank, design structures that are not found in nature, or direct the design of proteins to acquire a specific desired structure. While some researchers attempt to design proteins from first physical and thermodynamic principals, we decided to attempt to test whether it is possible to perform de novo helical protein design ofjust the backbone statistically using machine learning by building a model that uses a long short-term memory (LSTM) generative adversarial network (GAN) architecture. The LSTM-based GAN model used only theφandψangles of each residue from an augmented dataset of only helical protein structures. Though the network’s generated backbone structures were not perfect, they were idealised and evaluated post generation where the non-ideal structures were filtered out and the adequate structures kept. The results were successful in developing a logical, rigid, compact,helical protein backbone topology. This paper is a proof of concept that shows it is possible to generate a novel helical backbone topology using an LSTM-GAN architecture using only theφandψangles as features. The next step is to attempt to use these backbone topologies and sequence design them to form complete protein structures.", "keywords": [ "Generative Adversarial Network", "De novo Protein Design", "Machine Learning", "Neural Network", "Deep Learning", "Helical Proteins", "Protein backbone", "Long Short-Term Memory" ], "content": "Introduction\n\nThe concept of amino acid sequences folding into globular protein molecules allows for proteins’ large functional diversity, mediating all the functional aspects of living organisms, thus winning themselves attention from biochemists for decades. The fusion of machine learning with computational biology is accelerating research in both fields and bringing humanity closer to the setup of performing most biological research quickly, cheaply, and safely in silico, while only translating the very crucial aspects of it. Having access to a large database of protein crystal structures has led to the use of machine learning to design proteins computationally.\n\nDe novo protein design (i.e. from the beginning) is very well explained in this review1. Proteins fold into a specific shape depending on the sequence of their amino acids, and of course shape dictates function. The driving forces that allow proteins to fold are the hydrogen bond interactions within the backbone and between the side chains, the Van der Waals forces, and principally the interaction of hydrophobic side chains within the core. The space of all possible sequences for all protein sizes is extremely large (as an example there are 20200 possibilities for a 200-residue protein). Thus, is it not surprising that natural proteins exist in clusters close to each other, which is logical since proteins would evolve away from a central functional protein to fold correctly and acquire new folds and functions, rather than go through the tedious ordeal of finding a totally new protein structure within the space of all possibilities. Thus, even though the Protein Data Bank adds about 10,000 new structures to its repository every year, most of these new structures are not unique folds.\n\nThe relationship between the sequence of a protein and its specific structure is understood, but we still lack a unified absolute solution to calculate one from the other. Hence why some research groups generated man-made protein designs by altering already existing natural proteins2, since randomly finding a functionally folded protein from the space of all possible protein sequences is more or less statistically impossible. On the other hand, other researchers have attempted de novo protein design by designing a topology from assembling short sequence peptide fragments taken from natural protein crystal structures3,4, these fragments are calculated statistically depending on the secondary structures they are found in. Sometimes this fragment system is combined with first physical principals to model the loops between secondary structures to achieve a desired three-dimensional topology5. Others have used parametric equations to study and specify the desired protein geometry6–11. These solutions employ an energy function, such as REF2015, that uses some fundamental physical theories, statistical mechanical models, and observations of protein structures to approximate the potential energy of a protein12. Knowing the protein potential energy allows us to guide our search for the structure of a protein given its sequence (the structure resides at the global energy minima of that protein sequence) thus attempting to connect the sequence of a protein with its structure. The critical tool is the energy function, the higher its accuracy the higher our confidence in knowing the computed structure is the real natural structure. Thus, using the energy function to perform structure prediction (going from a known sequence to find the unknown three-dimensional structure) can also be used to perform fixed-backbone design (going from a known three-dimensional structure to find the sequence that folds it). This is where this paper comes in. Where as in de novo design neither backbone nor sequence is known, knowing one results in finding the other using the same energy function1, and a good starting point is to design the backbone.\n\nOther researchers have used machine learning for protein sequence design, employing the constraints (Cα-Cα distances) as the input features for the network and using a sliding window to read a sequence of residues, getting their types and constraints then predicting the next one giving the output prediction as an amino acid sequence13, this architecture reported an accuracy of 38.3% and performs what is called sequence design: designing a sequence for a backbone, so when the protein is synthesised it folds to that backbone. In fact, in the 5 paper the protocol first generates an all-valine backbone, then sequence designs that backbone. In this paper, we want to computationally generate a backbone so it can be sequence designed using other protocols such as RosettaDesign14,15 or the protocol from 13.\n\nThe protein’s backbone can be folded using the ϕ and ψ angles, which are the angles between Cα and N for ϕ and Cα and C for ψ that primarily move in an amino acid’s backbone (not the side chains), and thus can be used as features to control the topology of a backbone. They were even one of the features used to fold proteins and predict their structures in AlphaFold16.\n\nBut the question is: how do we decide the ideal angles for a helix, the length of each helix, the number of helices, as well as the lengths and angles of the loops between the helices that will also result is a compact folded protein backbone. These numerous values can be solved statistically using neural networks. Especially that we want to use the structures in the PDB to forward design (rather then discover) new protein folds that are not resulted from evolution.\n\nThe deep neural network architecture that we chose was a long short-term memory (LSTM) based generative adversarial network (GAN)17. The LSTM is usually used in natural language and data sequence processing, but in our model the LSTM was incorporated into a GAN. The model was constructed from two networks that worked against each other, the first was a generator network that was made up of a stack of LSTM layers, followed by fully connected layers, followed by a mixture density network (MDN) and worked by using random noise numbers as input to build the values for the ϕ and ψ angles. The other network was a discriminator that was made up of a stack of LSTM layers followed by fully connected layers and worked to studying the dataset and determining whether the output from the generator was a truly logical structure or not (fake or real)18.\n\nOur effort in this paper was to use machine learning to learn the general fold of natural proteins, and using this generalising statistical concept to design novel protein backbone topologies, thus only getting the three dimensional backbone structure so it can be used in sequence design using other protocols. Our research at this moment was a proof of concept and only concerned with getting a new and unique folded ideal helical protein backbone rather than a protein with a specific sequence, nor a function, nor a specific predetermined structure. Our system resulted in random yet compact helical backbone topologies only.\n\n\nMethods\n\nThe following steps were used to generate the augmented training dataset, along with details of the neural network architecture and how the output was optimised then evaluated.\n\nThe entire PDB database was downloaded on 28th June 2018 (~150,000 structures), and each entry was divided into its constituent chains resulting in individual separate structures (i.e: each PDB file had only a single chain). Each structure was analysed and chosen only if it contained the following criteria: contained only polypeptides, had a size between 80 and 150 amino acids without any breaks in the chain (a continuous polypeptide), a sum of residues that made up helices and sheets were larger than the sum of amino acids that made up loops, and the final structure having an Rg (radius of gyration) value of less than 15 88 Å. The chosen structures were then further filtered by a human to ensure only the desired structure concepts were selected, removing the structures that slipped through the initial computational filter. Furthermore, a diversity of structure folds were achieved rather than numerous repeats of the same fold (the haemoglobin fold was quite abundant). In previous attempts, a mixture of different structure classes were used, where some structures were only helices, some were only sheets, and the remaining were a mix of the two. However, that proved challenging in optimising the network, and as such a dataset made up of only helical protein structures was chosen for this initial proof of concept. The final dataset had 607 ideal helical structures. These structures were then cleaned (non-amino acid atoms were removed) in preparation to push them through the Rosetta version 3 modelling software that only takes in polypeptide molecules.\n\nThese 607 structures were augmented using the Rosetta FastRelax protocol19. This protocol performs multiple cycles of packing and minimisation. In other words, the protocol performs small slight random angle moves on the backbone and side chains in an attempt to find the lowest-scoring variant, but the random backbone angle moves is what we were after. Its originally intended function was to move a structure slightly to find the conformation of the backbone and side chains that corresponds to the lowest energy state as per the REF15 energy function. Since the protocol performs random moves, a structure relaxed on two separate occasions will result in two molecules that look very similar with similar minimum energy scores, but technically have different ϕ and ψ angle values. This is the concept we used to augment our structures, and each structure was relaxed 500 times to give a final dataset size of 303,500 structures.\n\nUsing only the ϕ and ψ angle values from a crystal structure it was possible to re-fold a structure back to its correct native fold, thus these angles were the only relevant features required to correctly fold a structure, Figure 1A details the range of angles in the un-augmented data. Each amino acid’s ϕ and ψ angle values were extracted and tabulates as in Table 1. This was the dataset used to train the neural network.\n\n7A: Ramachandran plot of the dataset showing the ϕ and ψ angles of each amino acid for each structure. This is the unaugmented data of structures that are only made of helices. Green represents the angles on amino acids in loops, while red represents the angles of amino acids in helices. Some orange can be seen where the DSSP algorithm classified the amino acids as sheets (though there were none). One point to note; the angles here are represented between the range −180° to 180° as is conventional, while in the actual dataset the range was from 0° to 360°. 7B: The network’s output ϕ and ψ angles for 25 structures after the relaxation step. The green dots represent the angles of amino acids within loops, and red within helices clustering around the same location as Figure 1A within the fourth quadrant as is desired for an α-helix that has ideal angles around (−60°,−45°). These structures culminated to all 25 structures in Figure 4, and had an angle range for the helices (−127.4°<ϕ<−44.7°, −71.3°<ψ<30.6°) not including the outliers. The purple dots represent the helices in the control structures, and the black dots the loops.\n\nThe model in Figure 2 was built using the SenseGen model as a template18 and consisted of two networks: a generator G network and a discriminator D network. The G network was constructed from an LSTM layer with 64 nodes, followed by two dense fully connected MLPs with 32 nodes for the first layer and 12 nodes for the second one, both employed a sigmoid activation function:\n\nsigmoid(x)=11+e−x\n\nWhich was followed by an MDN layer employing an MDN activation function:\n\np(y\|x)=∑c=1Cαc(x)D(y\|λ1,c(x),λ2,c(x),...)\n\nc: the index of the corresponding mixture component. α: the mixing parameter. 𝓓: the corresponding distribution to be mixed. λ: the parameters of the distribution 𝓓, as we denote 𝓓 to be a Gaussian distribution, λ1 corresponds to the conditional mean and λ2 to the conditional standard deviation. The training was done using the Adam optimiser, for each parameter ωj:\n\nvt=ρvt−1+(1−ρ)∗gt2Δωt=−ηvt+∈*gtωt+1=ωt+Δωt η:\n\nη: initial learning rate. vt: exponential average of squares of gradients. gt: gradient at time t along ωj. The Adam optimiser had an MDN activation through time loss function defined to increase the likelihood of generating the next time step value. The loss defined as the root mean squared difference between the sequence of inputs and the sequence of predictions:\n\nloss=∑t=1T(xt−yt)2\n\nyt: output. xt: next step sample xt+1 = yt. The D network was constructed from an LSTM layer with 64 nodes, followed by a dense fully connected MLP layer with 32 nodes, and that was followed by a single dense MLP unit layer employing a sigmoid activation function, so that the output of this network was a prediction; the probability of the data being real (indicated by the integer 1) or fake (indicated by the integer 0). The network employed the cross-entropy loss function:\n\nCE=−∑iCtilog(si)\n\nWhere ti and si are the groundtruth and the neural network score for each class i in C. In a binary classification problem, such as the discriminator network output where C′= 2, the Cross Entropy Loss can be defined as:\n\nCE=−∑i=1C'=2tilog(si)=−t1log(s1)−(1−t1)log(1−s1)\n\nWhere it is assumed that there are two classes: C1 and C2. t1 [0,1] and s1 are the groundtruth and the score for C1, while t2 = 1 – t1 and s2 = 1 – s1 are the groundtruth and the score for C2. The G network used random noise as a starting seed, this noise was generated by taking a single randomly distributed number between [0, 1) as the first predicted values, these values were then reshaped to the same shape of the last item of the predicted value resulting in a final shape of (batch_size, step_number, 1). The network predicted the main parameters of the new value (µ, σ, π) several times (according to the number_of_mixtures value) and selected the single mixture randomly but according to the π value. It then predicted the next value according to the normal distribution using the µ and σ values. It added the final value to the prediction chain and then returned to step 2 until the predefined sequence length was obtained. The initial random number was stripped from the returned sequence. Once the networks were constructed, the dataset was normalised and the training was done as follows for each adversarial epoch:\n\n1. Sample minibatch from dataset (Xtrue).\n\n2. Sample minibatch from G network (XG).\n\n3. Train the D network on the training set (Xtrue, XG).\n\n4. Sample minibatch from dataset (Xtrue).\n\n5. Sample minibatch from G network (XG).\n\n6. Train the G network on the training set (Xtrue).\n\nThe full protocol showing the structure of the neural network model and its output. The model employing an LSTM within the generative network and another one within the discriminator network, these two networks work adversarially against each other. The network’s output is the generated ϕ and ψ angles which were applied to a primary structure (a fixed 150 valine length straight structure generated by PyRosetta) that resulted in the development of the secondary structures but not a final compact structure structure due to suboptimal loop structures as a result of their variability in the dataset. To overcome this, the structure was relaxed to bring bring the secondary structure helices together. This did result in more compact structures but was not always ideal, thus a filter was used to filter our non-ideal structures and keep an ideal structure when generated.\n\nThe neural network had the following parameters: the G learning rate was 0.001 while the D learning rate was 0.0003, a drop out rate of 50% was used along with a batch size of 4 over 18,000 epochs.\n\nThe output of the neural network was always a 150 ϕ and ψ angle value combination for a structure with 150 amino acids. A straight chain of 150 valines was computationally constructed using PyRosetta and used as a primary structure. Each amino acid in the primary structure had its angles changed according to the ϕ and ψ angle values, which ended up folding that primary structure resulting in secondary structures of helices and loops between them. The COOH end was trimmed, if it was a loop, until it reached the first amino acid that comprised a helix, thus variable structure sizes where generated. The structure ended up with helices and loops yet still with an open conformation. The generated structure was therefore relaxed using PyRosetta version 4 FastRelax protocol to idealise the helices and compact the structure. Furthermore, not every prediction from the neural network resulted in an ideal structure even after the relax step, therefore we employed a filter to filter out structures we deemed not ideal. The filter discards structures that were less than 80 amino acids, has more residues in loops than in helices, has less than 20% residues making up its core, and has a maximum distance between Cα1 and any other Cα greater than 88 Å (the largest value in the dataset). PyRosetta was used20 since it was easier to integrated the code with the neural network’s python script and combine the Rosetta engine with Biopython21 and DSSP22,23.\n\nAs a control we used the de novo protein design protocol using the Rosetta Script from the 5 paper’s supplementary material. The protocol was modified slightly to accommodate new updates in the Rosetta software suite, but maintained the talaris2014 energy function as in the original paper, and we used the protocol to design helical proteins. These proteins had to pass through several filters including a talaris2014 score of less the -150, a packing threshold of more than 0.50, and a secondary structure threshold of more than 0.90. We attempted to design proteins with 3, 4, and 5 helices to compare the backbone quality our neural network output to the output of the 5 paper.\n\nThis setup used the following packages: python 3.6.9, PyRosett 4, Rosetta 3, Tensorflow 1.13.1, BioPython 1.76, and DSSP 3.01 and was run on GNU/Linux Ubuntu 19.10. Further information on running the setup can be found on this GitHub repository which includes an extensive README file. To train the neural network a 3GB GPU and 12GB of RAM is recommended, while to run the trained neural network and generate a backbone structure an Intel i7-2620M 2.70GHz CPU and 3GB of RAM is recommended.\n\nDetailed in Figure 2, executing the trained network will generate a set of random numbers that are pushed through the Generator network which (using the weights) will modify the values to become values in accordance with the ϕ and ψ angle topologies observed in the training dataset. A simple straight chain of 150 valines is then computationally constructed and the ϕ and ψ angles are applied to each amino acid in order, resulting in the appearance of helical secondary structures. Any tailing loops at the COOH end will be cut out since it interferes with the next step. This structure is then relaxed using the FastRelax protocol which moves the ϕ and ψ angles randomly in its attempt to find the lowest scoring configuration compacting the structure in the process. A filter is applied to determine whether the final structure is ideal or not, its parameters are detailed in the Methods section. If the structure passes the filter the script exists, otherwise it repeats the whole process.\n\n\nResults\n\nThe dataset was named PS_Helix_500 due to the fact that the features used were the ϕ and ψ angles, only strictly helical protein structures were used, and each structure was augmented 500 times.\n\nThe neural network was trained on the dataset for 18,000 epochs (further training collapsed the network, i.e. all outputs were exactly the same structure) with a generally sloping down mean loss as shown in Figure 3 indicating that the G network got better at generating data that the D network classified as real rather than fake. The network was used to generate the ϕ and ψ angles for 25 structures. In other words, random numbers were generated then pushed through the G network where they were modified (using the network’s trained weights) to become the ϕ and ψ angles for 150 residues. This is the angle profile. Using PyRosetta, a simple straight 150-valine chain was constructed and used as a primary structure. The generated ϕ and ψ angles were applied to this primary structure (each amino acid’s angles were changed according to the angle profile) resulting in a folded structure clearly showing helical secondary structures with loops between them. The last tailing loop at the COOH end was truncated, resulting in structures with variable sizes. The loop angles were within the area of the angles found in the dataset, but because they were generated independent of the thermodynamic stability of the structure, the structure did not come together into compact a topology. To push the structure into a thermodynamic energy minima it was relaxed using the PyRosetta FastRelax function which compacted the backbone topology while still having valines as a temporary placeholder sequence. This was repeated 25 times to result in the 25 structures in Figure 4. For comparison five control structures were generated using the de novo design protocol by the previous paper5. Figure 1B shows the Ramachandran plot of the 25 generated structures, where red are the amino acids within helices having angles clustering around the same location as Figure 1A in the fourth quadrant, as is desired for an α-helix, which has ideal angles around (−60°, −45°), our results had an angle range for the helices (−127.4°<ϕ<−44.7°, −71.3°<ψ<30.6°) not including the outliers, the five control structures show angles within the same region (purple for helices and black for loops). The structure generation setup was not perfect at achieving an ideal structure every time, so a filter was deployed to filter out suboptimal structures by choosing a structure that had more residues within helices than within loops, was not smaller than 80 residues, and had more than 20% residues comprising its core. Due to the random nature of the structure generation the efficiency of the network is variable, while generating the 25 structure in Figure 4 the fasted structure was generated after just 4 failed structures, while the slowest structure was generated after 3834 failed structures giving a success rate for the network between 25.0% at its best and 0.025% at its worst, and this took between ~1 minute and ~6 hours to generate a single structure with the desired characteristics utilising just 1 core on a 2011 MacBook Pro with with 4 core Intel i7-2620M 2.70GHz CPU, 3GB RAM, and 120GB SSD. For comparison the protocol by 5 took ~60 minutes for each of the control structures on the same machine. The protocol is summarised in Figure 2, and the results are compiled in Figure 4 showing all 25 structures and the 5 controls.\n\nThe mean loss of the whole network over epoch, for 18,000 epochs, showing a general downward trend. This indicates that in subsequent epochs the G network gets better at generating structures that the D network correctly classifies as real logical structures.\n\nThis figure shows all 25 structures that were generated using the neural network displayed using PyMOL24. It can be seen that all structures have compact helical structures of variable topologies. The 5 control structures at the bottom were generated using the protocol from the 5 paper showing better helices but similar compactness.\n\nThese 25 structures had on average 84.7% of their amino acids comprising their helices, along with and an average of 29.9% of their amino acids comprising their cores, Table 2 shows the Rosetta packing statistic for all 25 structures, along with the five controls, the top7 protein, and the five structures from the 5 paper all showing similar packing values.\n\nThe controls were the structures generated using the modified protocol from 5. For additional comparison the top7 (PDB ID: 1QYS)25 protein packing stat is measured along with the five structures from the protocol paper by 5.\n\n\nDiscussion\n\nIn this paper we outlined how a neural network architecture can design a compact helical protein backbone. We attempted to test our network to generate structures that included sheets, but that failed, mainly due to the wide variation in the loop angles that did not compact a structure to bring the strands together, which sheets rely on more to develop compared to helices.\n\nWe demonstrated that the ϕ and ψ angles were adequate features to design a protein backbone topology only without a sequence. Although we understand the distribution of angles in the Ramachandran plot (the distribution of helix and sheet angles) the neural network’s power comes in the form of combining several dimensional spaces to take a better decision. Given its observation of natural proteins, it can calculate the ideal combination of angles that results in deciding the number and lengths of helices, the loops between them, that will still result in a compactly folded protein backbone.\n\nThe reason we concentrated our efforts on generating a backbone only is because once a backbone is developed it can be sequence-designed using other protocols, such as RosettaDesign14,15 or the protocol from 13.\n\nThough our network had a wide variation of success rates, from high to low, that was due to the random nature of the setup, which was our target to begin with (to randomly generate backbone topologies rather than directly design a specific pre-determined topology). Generating multiple structures and auto filtering the suboptimal ones provided an adequate setup, this achieved our goal of de novo helical protein backbone design within a reasonable time (1–6 hours) on readily available machines.\n\nAs a control we used a slightly modified de novo design protocol from 5, which also performs backbone design (resulting in an all-valine backbone topology) followed by sequence design of that backbone. It has numerous advantages such as generating better helices, but the user must still pre-determine the topology to be generated (decide the number of helices, their lengths and locations, and the lengths of the loops between them), while this neural network automatically takes that decision and randomly generates different backbones, which can be very useful for database generation (see below).\n\nThe neural network is available at this GitHub repository which includes an extensive README file, a video that explains how to use the script, as well as the dataset used in this paper and the weights files generated from training the neural network.\n\nFor future work we are currently working on an improved model that uses further dataset dimensions and features that will allow the design of sheets.\n\nThere are many benefits to generating numerous random protein structures computationally. One benefit can be in computational vaccine development, where a large diversity of protein structures as scaffolds is required for a successful graft of a desired motif26–28. Using this setup, combined with sequence design, a database of protein structures can be generated that provides more variety of scaffolds than what is available in the protein databank, especially that this neural network is designed to produce compact backbones between 80 and 150 amino acids, which is within the range of effective computational folding simulations such as AbinitioRelax structure prediction simulation29.\n\n\nData availability\n\nThe entire Protein Data Bank was downloaded using this command:\n\nAfter which it was processed and filter as described in the Methods section.\n\n\nSoftware availability\n\nSource code available from: https://github.com/sarisabban/RamaNet.\n\nArchived Source code at time of publication: https://doi.org/10.5281/zenodo.3755343 [?]30.\n\nLicense: MIT License.", "appendix": "Acknowledgements\n\nThe corresponding author would like to thank the High Performance Computing Center at King Abdulaziz University for making available the Aziz high performance computer where the corresponding author was able to augment the dataset and perform the Abinitio folding simulations.\n\n\nReferences\n\nHuang PS, Boyken SE, Baker D: The coming of age of de novo protein design. Nature. 2016; 537(7620): 320–7. PubMed Abstract \| Publisher Full Text\n\nDougherty MJ, Arnold FH: Directed evolution: new parts and optimized function. Curr Opin Biotechnol. 2009; 20(4): 486–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang PS, Ban YE, Richter F, et al.: Rosettaremodel: a generalized framework for flexible backbone protein design. PLoS One. 2011; 6(8): e24109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuhlman B, Dantas G, Ireton GC, et al.: Design of a novel globular protein fold with atomic-level accuracy. Science. 2003; 302(5649): 1364–8. PubMed Abstract \| Publisher Full Text\n\nKoga N, Tatsumi-Koga R, Liu G, et al.: Principles for designing ideal protein structures. Nature. 2012; 491(7423): 222–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrigoryan G, Degrado WF: Probing designability via a generalized model of helical bundle geometry. J Mol Biol. 2011; 405(4): 1079–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarbury PB, Plecs JJ, Tidor B, et al.: High-resolution protein design with backbone freedom. Science. 1998; 282(5393): 1462–7. PubMed Abstract \| Publisher Full Text\n\nHuang PS, Oberdorfer G, Xu C, et al.: High thermodynamic stabilityof parametrically designed helical bundles. Science. 2014; 346(6208): 481–485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoh NH, Wang T, Bhate MP, et al.: De novo design of a transmembrane zn2+-transporting four-helix bundle. Science. 2014; 346(6216): 1520–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRegan L, DeGrado WF: Characterization of a helical protein designed from first principles. Science. 1988; 241(4868): 976–8. PubMed Abstract \| Publisher Full Text\n\nThomson AR, Wood CW, Burton AJ, et al.: Computational design of water-soluble α-helical barrels. Science. 2014; 346(6208): 485–8. PubMed Abstract \| Publisher Full Text\n\nAlford RF, Leaver-Fay A, Jeliazkov JR, et al.: The rosetta all-atom energy function for macromolecular modeling and design. J Chem Theory Comput. 2017; 13(6): 3031–3048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, Cao H, Zhang JZH, et al.: Computational protein design with deep learning neural networks. Sci Rep. 2018; 8(1): 6349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuhlman B, Dantas G, Ireton GC, et al.: Design of a novel globular protein fold with atomic-level accuracy. Science. 2003; 302(5649): 1364–8. PubMed Abstract \| Publisher Full Text\n\nMurphy GS, Mills JL, Miley MJ, et al.: Increasing sequence diversity with flexible backbone protein design: the complete redesign of a protein hydrophobic core. Structure. 2012; 20(6): 1086–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenior AW, Evans R: Improved protein structure prediction using potentials from deep learning. Nature. 2020; 577(7792): 706–710. PubMed Abstract \| Publisher Full Text\n\nRadford A, Metz L, Chintala S: Unsupervised representation learning with deep convolutional generative adversarial networks. arXiv. 2015. Reference Source\n\nAlzantot M, Chakraborty S, Srivastava M: Sensegen: A deep learning architecture for synthetic sensor data generation. In: 2017 IEEE International Conference on Pervasive Computing and Communications Workshops (PerCom Workshops) IEEE. 2017; 188–193. Publisher Full Text\n\nTyka MD, Keedy DA, André I, et al.: Alternate states of proteins revealed by detailed energy landscape mapping. J Mol Biol. 2011; 405(2): 607–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudhury S, Lyskov S, Gray JJ: Pyrosetta: a script-based interface for implementing molecular modeling algorithms using rosetta. Bioinformatics. 2010; 26(5): 689–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCock PJ, Antao T, Chang JT, et al.: Biopython: freely available python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009; 25(11): 1422–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoosten RP, te Beek TA, Krieger E, et al.: A series of pdb related databases for everyday needs. Nucleic Acids Res. 2011; 39(Database issue): D411–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTouw WG, Baakman C, Black J, et al.: A series of pdb-related databanks for everyday needs. Nucleic Acids Res. 2015; 43(Database issue): D364–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe PyMOL Molecular Graphics System. 2015; Version 1.8. Reference Source\n\nKuhlman B, Dantas G, Ireton GC, et al.: Design of a novel globular protein fold with atomic-level accuracy. Science. 2003; 302(5649): 1364–1368. PubMed Abstract \| Publisher Full Text\n\nCorreia BE, Bates JT, Loomis RJ, et al.: Proof of principle for epitope-focused vaccine design. Nature. 2014; 507(7491): 201–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzoitei ML, Ban YE, Julien JP, et al.: Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope. J Mol Biol. 2012; 415(1): 175–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzoitei ML, Correia BE, Ban YE, et al.: Computation-guided backbone grafting of a discontinuous motif onto a protein scaffold. Science. 2011; 334(6054): 373–376. PubMed Abstract \| Publisher Full Text\n\nRohl CA, Strauss CE, Misura KM, et al.: Protein structure prediction using Rosetta. Methods Enzymol. 2004; 383: 66–93. PubMed Abstract \| Publisher Full Text\n\nSari S, Mikhail M: sarisabban/RamaNet: First Release (Version v1.0). Zenodo. 2000. http://www.doi.org/10.5281/zenodo.3755343" }	[ { "id": "64120", "date": "02 Jul 2020", "name": "Matias Valdenegro-Toro", "expertise": [ "Reviewer Expertise Machine Learning" ], "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 is about a generative model for protein folding.\nThe main idea is to use a LSTM Generative Adversarial Network, trained on a dataset of real protein backbone angles, in order to generate novel protein designs. This makes sense, as GANs are regularly used as generative models to learn the probability distribution from samples, from where new samples can be generated. These methods are generally applied to images, but it is possible to apply them to other modalities such as sequences, text, etc. I find that this is a novel application that is a bit outside of the common uses of GANs.\nOverall the paper looks good in terms of writing and technical content. I am not an expert in proteins, so I will comment on the Machine Learning parts, which are my expertise.\nThe authors propose the use of an LSTM-GAN, but they do not justify why they use it, in particular the LSTM architecture of a GAN, which is not that common. GANs are mostly used as generative models for images, and there are GAN versions for sequential data. My question is then, what is the motivation to use an LSTM specifically? From what I understand, the dimensionality of the problem is fixed, and protein samples do not have a varying number of folds or angles, in which case a sequential model such an RNN or LSTM does not seem to be necessary. A simple multi-layer perceptron (MLP) could work equally well, it could be interesting if the authors tried this or performed an architecture search to justify their choice.\nThe neural network architectures for the discriminator and generator look fine, it would be nice if the authors mention how they reached these architecture, and if any grid/random search was performed.\nThe choice of activation function seems odd, since the sigmoid activation used in hidden layers can lead to vanishing gradient problems, while the current standard are ReLU activations and its many variations. The choice of the MDN also looks odd, since a GAN generator network generally outputs values in the output range directly, while the MDN outputs a probability distribution from where a sample needs to be drawn. I believe both choices have to be justified appropriately.\nThe authors use the Adam optimizer, which is standard and a good choice for a starting problem. There is a sentence in the paper which I find problematic: \"The Adam optimiser had an MDN activation through time loss function defined to increase the likelihood of generating the next time step value.\" The problem here is that conceptually, an optimizer computes a gradient descent step, an optimizer does not have an activation or loss associated with it, so I am not sure what the authors mean here. The authors mention a L2 loss (mean squared difference), but this is not the loss that is used to train a GAN, even for continuous values.\nThe authors mention that for training a GAN, the binary cross-entropy loss is used, but this is similar and not exactly the loss that is used to train GANs. The correct loss actually consists of two loss functions, one for the generator, and another for the discriminator, and both losses use the output of the D and G networks together. A full description of the losses and its variations can be found in Section 3.2 of [1], and I believe the authors should describe the actual loss they used very carefully, including the interaction of the D and G networks into the loss, as it is the core of the method used to generate protein angles in their paper.\nThe description of the algorithm used to train a GAN looks okay, except for steps 3 and 6 since as I mentioned before, the loss used for training a GAN used both the D and G networks, so this should be explicitly mentioned. Step 4 and 5 are not strictly necessary since a GAN can train the D and G networks on the same batch.\nI briefly analyzed the code linked in the paper, and I am not convinced that the authors are using a Generative Adversarial Network (GAN). The network implemented in the code looks more like an auto-regressive model, and I do not see adversarial training in it. Additional evidence is that in Figure 3 only one loss function is being shown, while as I mentioned before, a GAN uses two loss functions and generally both are plotted in order to visualize training. I believe this is a very important point in order to make the article scientifically sound, but not the only one.\nI think the authors could also discuss what other learning-based methods they tried. Other methods such as Variational Autoencoders (VAEs) come to mind, which are also able to work as generative models.\nI believe that many references are missing from this paper, in particular to the ML community. I have already mentioned the GAN tutorial [1], and additionally I would suggest the Adam optimizer paper [2], and the original Mixture Density Networks paper [3]. Please always make sure that you are citing the techniques you are using from another field.\n[1]: https://arxiv.org/abs/1701.00160 [2]: https://arxiv.org/abs/1412.6980 [3]: http://publications.aston.ac.uk/id/eprint/373/\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5884", "date": "11 Sep 2020", "name": "Sari Sabban", "role": "Author Response", "response": "After close inspection of the SenseGen neural network code (which was adopted from the referenced paper) it is clear that it is an LSTM network and not a GAN, thus the paper was changed to reflect this detail. The point of confusion was that the SenseGen neural network authors used a generator and a discriminator networks, but at the very end of their article they mentioned that they were not connected thus not trained adversarially.To fix this, Fig 2 was changed along with all sections to describe an LSTM rather than a GAN architecture. The output results remain the same since the network was neither changed not re-trained. The paper title was also updated. This should address the comments about the GAN architecture.We did train an MLP-GAN (results which were not as good as the LSTM), as per the recommendation these results were added as a comparison at the end of the results section (fig 5)." } ] } ]	1	https://f1000research.com/articles/9-298
https://f1000research.com/articles/9-1218/v1	09 Oct 20	{ "type": "Research Article", "title": "The role of ozonated Aloe vera oil in full-thickness skin defects: Macrophage count and epithelization length parameter", "authors": [ "Ukio Salferius Tamba", "Muhamad Thohar Arifin", "Muhamad Nur", "Muflihatul Muniroh", "Neni Susilanigsih", "Ukio Salferius Tamba", "Muhamad Nur", "Muflihatul Muniroh", "Neni Susilanigsih" ], "abstract": "Background: Aloe vera has been reported to enhance keratinocyte proliferation and migration, and thereby accelerate wound healing. Ozone therapy is an alternative medicine with disinfecting properties and strong oxidative stress induction capacity. This study aimed to evaluate the effect of Aloe vera oil provided with different dosages of ozone in accelerating the healing rate comparing two-phases; inflammation (day 3) and proliferation (day 7) of full-thickness defect wounds in Sprague Dawley rats as an adjuvant treatment based on macrophage count and new epithelialization length.\n\nMethods: We conducted a study using a post-test only control group design, where a total of 50 Sprague Dawley rats were randomized into ten groups. Two control groups were administered Aloe vera oil (P1, P6), while gentamicin ointment was used in the other control group (P2, P7). Ozonated Aloe vera oil dose was used: 600 mg/ml (P3,P8), 1200 mg/ml (P4,P9), and 1800 mg/ml (P5,P10). Groups P1, P2, P3, P4, P5 were terminated on the 3rd day, while P6, P7, P8, P9, P10 were evaluated on the 7th day. Macrophage counts were assessed using a 100x magnification microscope, through hematoxylin-eosin (HE) staining, and paraffin block with Masson trichrome staining was used to measure the new epithelialization length. Results: There were significant differences in the macrophage count on day three between the group-administered gentamicin (P2) and 1800 mg/ml ozonated Aloe vera oil (P5) (79.96;131.96, p<0,05). The new epithelialization length significantly increased in the group provided with wound treatment up to day 7, using 1800 mg/ml ozonated Aloe vera oil (P10), compared to non-ozonated Aloe vera (P6) and the gentamicin therapy (P7) (1160.88 µm; 1367.64; 2451.40 μm, p<0,05). Conclusions: The application of ozonated Aloe vera oil to full-thickness skin defects in Sprague Dawley rats resulted in a higher macrophage count and longer new epithelialization length than in controls.", "keywords": [ "Aloe vera oil", "Ozone Therapy", "Full-thickness skin defect", "macrophage count", "epithelization" ], "content": "Introduction\n\nSoft tissue wound refers to the damage or loss of tissue continuity, possibly caused by internal factors such as drugs, changes in circulation and metabolic processes, infection, and oxygen transportation failures. External factors include extreme temperatures, injuries, radiation, and the presence of chemical substances1. The body's physiological response, in terms of wound healing, results from various interrelated processes, including coagulation, inflammation, deposition, extracellular matrix differentiation, fibroplasia, epithelialization, contraction, and remodeling1. Furthermore, the remediation course is mediated by various cells, cytokines, matrices, and growth factors, through three phases - inflammation, tissue proliferation, and remodeling2. The process of acute cutaneous wound healing consists of three stages. The first stage is the inflammatory phase, which occurs following the injury and lasts until day-3. The second stage is the proliferation phase that generally takes place from day -7 until day-14, and the third stage is the remodeling phase that can last for several months or even years. The result of a previous study explained that only at specific doses did ozonated oil show a significant increase in the initial phase of wound healing3.\n\nMacrophages play critical roles in all phases of adult wound healing, which are inflammation, proliferation, and remodeling. As wounds heal, the local macrophage population transitions from predominantly pro-inflammatory (M1-like phenotypes) to anti-inflammatory (M2-like phenotypes). Thus, local macrophages retain pro-inflammatory characteristics4.\n\nAloe vera is a thorny plant that grows in tropical and subtropical climates. It has been used for millennia to improve cutaneous wound healing, with this now being known to be due to its ability to increase the proliferation and migration of fibroblasts and human primary epidermal keratinocytes (HPEKs) flow cytometry analysis revealed that cell surface expressions of β1-, α6-, β4-integrin, and E-cadherin increased in HPEKs treated with Aloe vera gel. These increases may contribute to cell migration and wound healing5. Many active ingredients, including vitamins, enzymes, minerals, sugars, lignin, salicylic acids, and amino acids, have been identified from these species. However, therapeutic effects have not correlated well with each component. Recently, a study exploring the biological effects of a single element from Aloe species was conducted. Choi et al. reported that a 5.5 kDa glycoprotein isolated from Aloe vera gel by activity-guided sequential fractionation was found to enhance keratinocyte proliferation and migration, and thereby accelerate wound healing5. However, it is currently believed that biological activities associated with Aloe arise from the synergistic actions of multiple compounds rather than from a single component5.\n\nOzone therapy is an alternative medicine with disinfecting properties and strong oxidative stress induction capacity. These characteristics have been implicated in the stimulation of protective mechanisms for cells and organs by increasing the efficacy of endogenous oxygen-free radicals scavenging6. Moreover, ozone therapy inactivates bacteria and damages cell walls through the oxidation of phospholipids and lipoproteins, and also inhibits fungal growth, damages viral capsids, and interferes with the bacterial reproduction cycle7. This therapy could be applied as an adjuvant or alternative to standard treatments in patients with various types of wounds, particularly when used topical6. Previous studies have highlighted the benefits of ozonated sesame oil in wound healing7.\n\nTherefore, this study aimed to evaluate the effect of Aloe vera oil provided with different dosages of ozone in accelerating the healing rate comparing two-phases; inflammation ( day 3) and proliferation (day 7) of full-thickness defect wounds in Sprague Dawley rats as an adjuvant treatment based on macrophage count and new epithelialization length.\n\n\nMethods\n\nThis was an experimental study using a post-test only control group design. The outcome data were recorded after the duration of the treatment. A total of 50 adult male Sprague Dawley rats were distributed randomly into ten groups, with five in each. A total of ten groups were created, where the controls, P1 (received Aloe vera oil) and P2 (received gentamicin), respectively, for three days. Furthermore, P3, P4, and P5 was administered ozonated Aloe vera Oil at a dose of 600, 1200, and 1800 mg/ml of ozone, respectively for three days. Therefore, P6 (received Aloe vera oil) and P7 (received gentamicin), respectively, for seven days, while P8 P9 and P10 received ozonated Aloe vera Oil at a dose of 600, 1200, and 1800 mg/ml ozone, respectively for seven days. A full-thickness defect measuring 1 cm in diameter was created on the back of the rat. The rats were sacrificed in each group on the third (P1, P2, P3, P4, P5) (inflammation stage of wound healing ) and seventh day (P6, P7, P8, P9, P10) (proliferation stage wound healing), respectively, to obtain the macrophage count and length of new epithelialization (Figure 1).\n\nThe 50 adult male Sprague Dawley rats from a local laboratory-grade rat breeder, with a pure-breeding status, weighing 250 ± 50 grams and lodged in individual stainless-steel laboratory-grade rat cages at room temperature of 28.0 ± 2.0°C, which was monitored with a digital thermometer. The rats were acclimated in the laboratory for one week. Twelve hours light was provided with from 06.00–18.00 and darkness from 18.00–06.00 with allowed access food (BioFeed pellets, manufactured by Karunia Kasih Abadi, Klaten, Indonesia) and water supply (clean drinkable water). These conditions were to ensure the elimination of stress, and feeding was conducted ad libitum. Each cage was inhabited by two to three rats according to the cage size. We did not mix rats from different groups in one cage. We routinely assessed the clinical and behavioral status of the rat every day during routine checks to prevent any clinical or behavioral changes in the laboratory rat. No rats were found dead, having decreased appetite/growth, or exhibited behavioral changes during the study period.\n\nA previous study6 involved the application of ozonated sesame oil (8 microliters) to a wound measuring 3.5 mm in diameter, we employed the same method. The doses used in this investigation were 50.6, 101.3, and 205.5 mg/ml of ozonated Aloe vera oil; hence the amount of ozone provided to each 3.5 mm wound was 0.405 mg, 0.81 mg, and 1.62 mg, respectively. Furthermore, the ozonated oil (Cap) capacity was determined using the formula: Cap = C x FRate x T, where Concentration (C) was set at 45 mg/L, and FRate(Flow rate) at 1.5 liters/minute, while Timeincludes 30, 60 and 120 minutes. The manufacture and testing of ozonated Aloe vera were carried out at the Plasma Research Center (PRC), Diponegoro University, Indonesia. The tools used for making ozonated oil were ozone generators (ozone generator manufactured in-house by Plasma Research Center, Diponegoro University) and magnetic stirrers. The ozone outlet is connected to an anti-oxidation hose with a diffuser, which served to increase the effectiveness of ozone absorption in the oil. Chemical characterizations (acidity value (AV), iodine value (IV); and peroxide value (PV)) of ozonated Aloe Vera oil samples have also been performed. As for PV evaluation, it was determined using both iodometric titrations according with the official monograph of the European Pharmacopoeia as well as placing the sample at reflux for 60 minutes18. Physical characterization has been performed by viscosity measurements (Viscomate VM-10AL, CBC Europe) at both the temperatures of 22 and 35_0.2 1C. A total of 40 ml Aloe vera oil was used in this experiment. Previous studies have used three ozone concentration – low, medium and high – which we replicated6. In this experiment the ozone concentration used were 600mg/ml as the low PV concentration; 1200 mg/ml for medium, and 1800 mg/ml as the high PV concentration. Table 1 shows the peroxide value (PV) concentration in every ozone dose.\n\nFull-thickness wounds of one-centimeter diameter were made on the back of the rat (Figure 2). The procedure involved anesthetizing the Rats with a mixture of Ketamine (100 mg/mL; Ketamin Cas Number: 6740-88-1 Ikapharmindo, Indonesia) -Xylazine (20 mg/mL; Xyla Cat. No.: IX2 Interchemie werken \"De Adelaar\" BV Local Distributor: PT Tekad Mandiri Citra). A Mixture dose of 80 mg/kg Ketamine; 10 mg/kg Xylazine via the intraperitoneal route, followed by the careful placement of the rat in the left lateral decubitus position. The hair on the dorsum area was shaved, and povidone-iodine was used to clean the skin, followed by ethanol 70%. The wound area was marked, and the lesion was made by cutting the skin using a scalpel to the base of the panniculus carnosus. The fatty and connective tissues were cleaned, and the site irrigated with normal saline (if necessary), before wrapping with a band-aid. Furthermore, the non-adhesive cottonoid was applied to covering the wound. The rats were then placed in a clean and warm cage with adequate lighting to recover from the anesthesia.\n\nA: Image of the wound creation process on the back of rat; B: Example of full-thickness defect wound (round shape in this study); C: Placement of non-adhesive segment of Hansaplast plaster on wound model; D: Elastomull bandage was used to wrap the thorax and dorsum area of rat, and tightness was adjusted to ensure proper breathing (adequate chest expansion) and movement of extremities before returning to the cage.\n\nEight microliters of either untreated Aloe vera oil or Ozonated aloe vera were applied twice a day (every 12 hours) on the wounds, and 1 cm gentamycin ointment was used on the control group.\n\nSurgical excision of the wounded area was performed at days 3 and 7 in each group. Skin tissue was immersion fixed in 10% NBF (neutral-buffered formalin) (Medici, medical labware, Jakarta Indonesia. Cat. NBF-19L) for 24 hours at room temperature. Sections (4 mm) were deparaffinized in xylene (Xylene Cath No 1.08661.2500 MERCK Merck Millipore Germany) and rehydrated in alcohol (Dipanax, Cath No 20501021606, Tangerang, Indonesia) gradients. Hematoxylin and eosin (H&E) staining (Hematoxylin Solution, Harris Modified, MFCD00078111 Merck Germany; Eosin Y Solution, Product No HT110132Sigma-Aldrich USA) were performed for histology9. The macrophage count was assessed microscopically, using a 100x magnification binocular microscope. The length of new epithelialization was examined by mason staining to the paraffin block10, followed by the measurement of epithelial tissue thickness with a microscope, under 40x magnifications. The results were then documented/photographed with optics (Miconas, Indonesia).\n\nThis research was approved and declared ethically feasible by the Ethics Commission of the Public Health Faculty, Diponegoro University Semarang, with ethical clearance number 132/EC/H/KEPK/FK-UNDIP/X/2019. All efforts were made to alleviate harm to animals by administering anesthesia to all of the study animals before full-thickness wound model creation, keeping the animals in a well-maintained cage, and ensuring graceful termination of animals before we took tissue samples for a histopathology examination.\n\nData analysis was performed using SPSS 21 version. All data were analyzed using descriptive statistics. Normality tests were performed using the Shapiro wilk test. Homogeneity testing and one-way ANOVA were performed to compare the control with the treatment groups. Post hoc Games-Howell was used to determine the difference between each variable. This process was conducted to investigate the presence of significant differences, and p-value <0.05 was considered statistically significant.\n\n\nResults\n\nTable 2 shows the differences in the mean macrophage count of treatment groups 1-10, and the highest mean value was recorded on day-3, in P5(administered 1800 mg of ozonated Aloe vera oil) as 131.96. Meanwhile, the lowest (79.96) was measured on day-3, in P2, with gentamicin treatment (Figure 3). The result of the data normality test shows the absence of normal data distribution (p=0.028 or p<0.05). Hence, data transformation was performed to generate normally distributed data (with a p-value range of 0.065 to 0.884).\n\n* Normal (p > 0.05); £ Shapiro-wilk\n\nAll normally distributed data were tested using the one-way ANOVA to ascertain the presence of significant differences in mean macrophage count between the ten groups. Table 3 shows that the data obtained were not homogenous, at p=0.028, or p<0.05. However, one-way ANOVA test resulted in a p-value of <0,001, indicating the presence of a significant difference between groups. Therefore, a Post hoc Games-Howell test was then conducted to observe the variation in the mean macrophage count of two groups. Table 4 shows the results of the Post hoc Games-Howell test, and a significant difference (p<0,05) was observed between the group provided with gentamicin (P2) and 1800 mg/ml of ozonated Aloe vera oil (P5) on day-3, at 79.96 and 131.96, respectively. Also, there was substantial variation between the groups administered 1200 mg/ml (P4) and 1800 mg/ml (P5) of ozonated Aloe vera oil sacrificed on day 3, with those euthanized Aloe vera oil on day 7 (P6), at 168.92, 131.96 and 69.08, respectively.\n\n* Significant (p < 0.05); ** Homogenous (p > 0.05)\n\n* Significant (p < 0.05)\n\nTable 5 shows the variation in the mean length of new epithelialization between the ten treatment groups, with the highest value of 2451.40, recorded in group P10 provided with 1800 mg/ml of ozonated Aloe vera oil, on day-7. Meanwhile, the least (799.49) was reported on day-3, in group P3 administered 600 mg/ml of ozonated Aloe vera oil (Figure 4). The data normality test result shows the normal distribution in the ten treatment groups, with p-value ranging from 0.083 to 0.970. Therefore, all normally distributed data were tested using the one-way ANOVA to investigate any significant differences in mean macrophage count between the ten groups. Based on the normality test, Table 6 demonstrated non-normal distributed data, with p=0.013 or p<0.05. However, one-way ANOVA test showed a p-value of <0.001, indicating the absence of any significant differences between treatment groups. Therefore, a Post hoc Games-Howell test was conducted to evaluate the variation in the mean length of new epithelialization between two groups.\n\n* Normal (p > 0.05); £ Shapiro-wilk\n\n* Significant (p < 0.05)\n\nThe result of the Post hoc Games-Howell test (Table 7) shows a substantial increase in the length of new epithelialization, particularly in groups treated using 1800 mg/ml of ozonated Aloe vera oil (P10) for seven days (2451.40 μm), compared to those administered Aloe vera oil therapy (P6) and gentamicin therapy (P7), at 1160.88 µm and 1367.64 µm, respectively. Furthermore, ozonated Aloe vera oil therapy proved effective in each group of Sprague Dawley rats with full-thickness skin defect, based on the increased epithelialization.\n\n* Significant (p < 0.05).\n\nThis process occurred during the wound healing process with gentamicin (P7), with no significant difference (1367.64 µm), compared to P6 treated with Aloe vera oil (1160.88 µm), P8 with 600 mg/ml of ozonated Aloe vera oil (2074.48 µm), and P9 with 1200 mg/ml of ozonated Aloe vera oil (1984.09). Therefore, the administration of ozonated Aloe vera oil at specific doses has a significant positive effect on the healing response of full-thickness defect wounds in Sprague Dawley rats, leading to faster new epithelialization length development.\n\n\nDiscussion\n\nThe process of acute cutaneous wound healing generally consists of three stages, including the inflammatory phase experienced immediately after the injury, which extends to day-7. The second stage is proliferation, observed to day-14, and the third is the remodeling phase, known to last for several months or even years6. Previous studies have demonstrated a significant increase in the initial phase of wound healing in response to moderate doses of ozonated oil (1500 mg/dl)6. The current study showed the effectiveness of 1800 mg/ml ozonated Aloe vera oil in promoting the wound healing response of full-thickness defects in Sprague Dawley.\n\nAccording to Valacchi et al.6, ozone is an efficient bactericidal, anti-fungi, and anti-viral agent, hence the propensity to reduce infection and improve skin unification during wound healing. The effect of ozonated oil on wound closure rate depends on the peroxide content6, as ozone possesses powerful oxidative characteristics in both gas and liquid phases, therefore producing microbicidal effects needed to damage the cell wall and cytoplasmic membrane of bacteria and fungi. This cascade of events leads to increased cell permeability, consequently facilitating the entry of ozone molecules11.\n\nThe use of ozonated oil also prevents superinfections and stimulates tissue reconstruction through increased cell proliferation and the formation of new vascularization6. Also, an increase in oxygen tension at the wound site elevates the tendency of granulation tissue formation, which has implications on the wound closure rate4. The augmented expression of transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF), known to play critical roles in the wound healing process, has also been achieved in clinical and experimental wound healing research with ozone therapy12.\n\nThe results of data analysis showed differences in the mean macrophage count between groups treated with 600, 1200, and 1800 mg/ml of ozonated Aloe vera oil and the control, administered Aloe vera oil or gentamicin alone. The evaluation parameter was higher in groups provided with 1200 mg/ml and 1800 mg/ml of ozonated Aloe vera oil for three days compared to those given gentamicin for three days and Aloe vera oil for seven days. This outcome was probably due to the increased oxidative role of ozone during the early phase of the inflammatory process13.\n\nOzone has been widely recognized as an anti-viral, bactericidal, and anti-fungal agent, and used in clinical therapy for its beneficial wound healing effects. This element is not able to penetrate the cell but is able to bind unsaturated fatty acids, and consequently form reactive oxygen species (ROS)6.\n\nOne form of ROS compound is hydrogen peroxide, known as a non-radical oxidant and implicated as an ozone mediator in several biological processes, including wound therapy14. Besides, ROS plays a crucial role in these processes by decreasing blood flow and forming a thrombus.\n\nROS are known to assist in the transportation of neutrophils through the blood vessels, therefore serving as a protection against bacteria by signaling the immunocytes, including monocytes, to fight pathogenic germs15. Furthermore, ROS stimulates endothelial cells to form new blood vessels, form an extracellular matrix (including collagen synthesis), and keratinocyte proliferation15, although excessive production leads to oxidative stress. This condition causes adverse effects on the wound healing process, hence the need for antioxidants to achieve a dynamic balance15.\n\nThe mean value of total macrophage produced on day 4 – 7 was lower than day 1 – 3. According to previous research, this variation was affiliated with the presence of vitamins, enzymes, proteins, carbohydrates, minerals (calcium, sodium, magnesium, zinc, and iron), and amino acids in Aloe vera, which play a major role in treating inflammation16. Also, various anti-inflammatory agents, including salicylic acid, Indomethacin, Manosa-6-phosphate, B sitosterol, as well as components of Lignin, Saponin, and Anthaquinone consisting of Aloin, Barbaloin, Anhtranol, Anthracene, Aloetic acid, Aloe-emodin, have been identified as essential ingredients of antibiotics and pain killer drugs. Meanwhile, Aloe vera is also rich in vitamins, polysaccharides, and other components with health benefits. The anti-inflammatory constituents can stimulate the immune system, leading to cell regeneration, and the consequent acceleration of healing in the inflammatory phase16.\n\nBased on the results of new epithelialization length measurement, ozone induces a notable effect on the wound healing process. Furthermore, the administration of 1800 mg/ml ozonated Aloe vera oil for seven days had a significant positive mean difference compared to the other groups.\n\nVarious studies have been conducted to observe the role of topical Aloe vera on the wound healing process. Oryan et al.17 examined a 2×2 cm wound on the back of rats treated with Aloe vera gel, and the parameters evaluated include wound surface, size shrinkage, and epithelialization. Teplicki et al.8 demonstrated increased healing in terms of proliferation and migration of fibroblasts and keratinocytes after treatment. Aloe vera shows effectiveness through increased cell migration, keratinocyte function, and epithelialization repair8.\n\nThe current study results indicate the fastest wound healing rate in the group treated with the highest dose of ozonated Aloe vera oil (1800 mg/ml). This was followed by others, in the order of 600 mg/ml and 1200 mg/ml, the positive control administered gentamicin, and then Aloe vera oil, respectively. Therefore, further research is required to examine the effect of ozonated Aloe vera oil to ascertain the most effective dose for wound healing.\n\nThis study has several limitations, including the lack of uniformity in the hygiene level of each rat, which possibly influenced the wound healing process. Also, the wound tissue sampling was not conducted identically, therefore making it impossible to determine the exact phase of wound healing encountered on day seven, as the process in Sprague Dawley rats is perhaps different from humans.\n\n\nConclusion\n\nBased on the results and discussion, ozonated Aloe vera oil applied to full-thickness skin defect in Sprague Dawley rats resulted in higher macrophage count and longer new epithelialization length than the control group.\n\nHowever, uniformity in the hygiene level of each rat is important in order to accelerate wound healing processes and minimize infection. Also, there is a need for further research to investigate the exact component in Aloe vera oil responsible for the treatment progression. Furthermore, subsequent studies are also to determine the effective dose to facilitate implementation is not only experimental animals, but also humans, to observe the antioxidant effect of ozonated Aloe vera oil.\n\n\nData availability\n\nOpen Science Framework: The Role of Ozonated Aloe vera Oil in Full-Thickness Skin Defect: Macrophage Count And Epithelization Length Parameter, https://doi.org/10.17605/OSF.IO/7A82V18\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThe authors would like to express our gratitude to the Center Plasma Research (CPR), Diponegoro University, Semarang.\n\n\nReferences\n\nRobson MC: Wound healing: biologic features and approaches to maximum healing trajectories. Curr Probl Surg. 2001; 38(2): 72–141. PubMed Abstract \| Publisher Full Text\n\nLi J, Chen J, Kirsner R: Pathophysiology of acute wound healing. Clin Dermatol. 2007; 25(1): 9–18. PubMed Abstract \| Publisher Full Text\n\nTravagli V, Zanardi I, Valacchi G, et al.: Ozone and ozonated oils in skin diseases: a review Mediators Inflamm. 2010; 2010: 610418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrzyszczyk P, Schloss R, Palmer A, et al.: The Role of Macrophages in Acute and Chronic Wound Healing and Interventions to Promote Pro-wound Healing Phenotypes. Front Physiol. 2018; 9: 419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoriyama M, Moriyama H, Uda J, et al.: Beneficial Effects of the Genus Aloe on Wound Healing, Cell Proliferation, and Differentiation of Epidermal Keratinocytes. PLoS One. 2016; 11(10): e0164799. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValacchi G, Lim Y, Belmonte G, et al.: Ozonated sesame oil enhances cutaneous wound healing in SKH1 mice. Wound Repair Regen. 2011; 19(1): 107–15. PubMed Abstract \| Publisher Full Text\n\nLim Y, Phung AD, Corbacho AM, et al.: Modulation of cutaneous wound healing by ozone: Differences between young and aged mice. Toxicol Lett. 2006; 160(2): 127–34. PubMed Abstract \| Publisher Full Text\n\nTeplicki E, Ma Q, Castillo DE, et al.: The Effects of Aloe vera on Wound Healing in Cell Proliferation, Migration, and Viability. Wounds. 2018; 30(9): 263–268. PubMed Abstract\n\nFeldman AT, Wolfe D: Tissue Processing and Hematoxylin and Eosin Staining. In: Day C. (eds) Histopathology. Methods in Molecular Biology (Methods and Protocols). Humana Press, New York, NY. 2014; 1180. PubMed Abstract \| Publisher Full Text\n\nSuvik A, Effendy AW: The use of modified Masson's trichrome staining in collagen evaluation in wound healing study. Mal J Vet Res. 2012; 3(1): 39–47. Reference Source\n\nNagayoshi M, Kitamura C, Fukuizumi T: Antimicrobial effect of ozonated water on bacteria invading dentinal tubules. J Endod. 2004; 30(11): 778–781. PubMed Abstract \| Publisher Full Text\n\nSaini R: Ozone therapy in dentistry: A strategic review. J Nat Sci Biol Med. 2011; 2(2): 151–153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDanusantoso H: Peran radikal bebas terhadap beberapa penyakit paru. Jurnal Kedokteran Trisakti. 2003; 22(1): 31–36. Reference Source\n\nBocci VA: Scientific and medical aspects of ozone therapy. State of the art. Arch Med Res. 2006; 37(4): 425–35. PubMed Abstract \| Publisher Full Text\n\nDunnill C, Patton T, Brennan J, et al.: Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process. Int Wound J. 2017; 14(1): 89–96. PubMed Abstract \| Publisher Full Text\n\nCarolia N, Sukohar A: Pengaruh Pemberian Ekstrak Lidah Buaya Konsentrasi 25%, 50%, 75%, dan 100% terhadap Jumlah Makrofag pada Radang Mukosa Mulut Tikus Putih Jantan Galur Sprague Dawley. Jurnal Kedokteran Universitas Lampung. 2016; 1: 243–246. Reference Source\n\nOryan A, Mohammadalipour A, Moshiri A, et al.: Topical Application of Aloe vera Accelerated Wound Healing, Modeling, and Remodeling: An Experimental Study. Ann Plast Surg. 2016; 77(1): 37–46. PubMed Abstract \| Publisher Full Text\n\nMuhamad TA: The Role of Ozonated Aloe vera Oil in Full-Thickness Skin Defect: Macrophage Count And Epithelization Length Parameter. 2020. http://www.doi.org/10.17605/OSF.IO/7A82V" }	[ { "id": "83934", "date": "03 Jun 2021", "name": "Yos Adi Prakoso", "expertise": [ "Reviewer Expertise Skin immunology", "skin wound healing", "modified herbal therapy", "wound infection expert", "histopathology" ], "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\nDear Authors\nI'm sorry to say that this paper cannot be accepted for indexing due to a lot of errors were observed from the methods and analysis.\nYou were not citing the appropriate references that fell within your scope of the manuscript.\n\nYou have limited references.\n\nThe parameters and analysis are not sufficient. For example: how can you count the macrophages within the skin tissue without using immunohistochemistry? I thought that it is impossible to be conducted.\n\nThe quality of your histological images is low and you make a wrong assessment of your interpretation. This study parameter should be recalibrated by the pathologist or expert of skin pathologist due to the mismatch of staining with the assessed parameters.\n\nThe data of normality and homogeneity is not applicable to be embedded within the text.\n\nYour discussion is not enough to present the present result.\n\nI hope you can fix the problems.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "86729", "date": "08 Jul 2021", "name": "Huzwah Khaza'ai", "expertise": [ "Reviewer Expertise Wound healing", "antioxidant", "vitamin E", "diabetic and alzheimer's disease" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study was to investigate the effects of ozonated Aloe vera oil on full-thickness wounds. However, the study is not comprehensive enough to achieve an understanding of the mechanism of healing processes by ozonated Aloe vera oil. Theoretically, the healing process is involved in 4 stages which are homeostasis, inflammation, proliferation, and remodelling. The study only focuses on stages 2 and 3 looking at the macrophages and epithelialization. Due to the poor resolution of histological data the counting of macrophages is ambiguous.\nAre days 3 and 7 really representing stages 2 and 3 of wound healing, which normally can only be observed in a continuous study from day 0 until day 21?\n\nThe peroxide value/total antioxidant status should also be measured in wound tissues.\n\nHow is the dosage of Aloe vera oil determined?\n\nThe Conclusion does not focus on the findings. The reviewer has no clue if ozonated oil or Aloe vera is involved in wound healing.\n\nThe author needs to do more comprehensive work before this work can be accepted for indexing. The author also must clearly determine the problem statement.\nThe study is still in its early stages and therefore I would Not Approve this paper due to the many reasons as stated above.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\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? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1218
https://f1000research.com/articles/9-1216/v1	09 Oct 20	{ "type": "Method Article", "title": "Non-invasive somatotransgenic bioimaging in living animals", "authors": [ "Juliette M. Delhove", "Rajvinder Karda", "Lorna M. FitzPatrick", "Suzanne M.K. Buckley", "Simon N. Waddington", "Tristan R. McKay", "Rajvinder Karda", "Lorna M. FitzPatrick", "Suzanne M.K. Buckley", "Simon N. Waddington", "Tristan R. McKay" ], "abstract": "Bioluminescence imaging enables noninvasive quantification of luciferase reporter gene expression in transgenic tissues of living rodents. Luciferase transgene expression can be regulated by endogenous gene promoters after targeted knock-in of the reporter gene, usually within the first intron of the gene. Even using CRISPR/Cas9 mediated genome editing this can be a time consuming and costly process. The generation of germline transgenic (GLT) rodents by targeted genomic integration of a gene expression cassette in embryonic stem (ES) cells is commonplace but results in the wastage of large numbers of animals during colony generation, back-crossing and maintenance. Using a synthetic/truncated promoter-driven luciferase gene to study promoter activity in a given tissue or organ of a GLT also often results in unwanted background luciferase activity during whole-body bioluminescent imaging as every cell contains the reporter. We have developed somatotransgenic bioimaging; a method to generate tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in rodents that substantially reduces the number of animals required for experimentation. Bespoke designed TFARs are delivered to newborn pups using viral vectors targeted to specific organs by tissue-tropic pseudotypes. Retention and proliferation of TFARs is facilitated by stem/progenitor cell transduction and immune tolerance to luciferase due to the naïve neonatal immune system. We have successfully applied both lentiviral and adeno-associated virus (AAV) vectors in longitudinal rodent studies, targeting TFARs to the liver and brain during normal development and in well-established disease models. Development of somatotransgenic animals has broad applicability to non-invasively determine mechanistic insights into homeostatic and disease states and assess toxicology and efficacy testing. Somatotransgenic bioimaging technology is superior to current whole-body, light-emitting transgenic models as it reduces the numbers of animals used by generating only the required number of animals. It is also a refinement over current technologies given the ability to use conscious, unrestrained animals.", "keywords": [ "somatotransgenic", "luciferase", "germline transgenic", "biosensor", "lentivirus", "AAV" ], "content": "\n\nRapid generation of a light-emitting transgenic; can be used in conjunction with whole-body transgenic animals to increase the amount of data obtained per animal and allows for within animal comparisons to be made making the data more reliable and sensitive.\n\nReduced number of animals (a cohort of approximately 16 animals for multiple time points across the entire experiment compared to 16 animals PER time point); more data from same number of animals as serial measures can be performed; refined techniques to allow for conscious imaging; reduced anesthesia events which decreases animal distress leading to improved welfare of the animals.\n\nTime (weeks to generate and validate models compared to months to generate a whole-body transgenic and subsequently validate the model); less space required for transgenic colony; speed of experiments; cost benefit; easy to implement as long as a lab is already biosafety Class 2 compliant.\n\nDisease modelling (eg fibrosis, hypoxic ischemia, inflammation); monitoring of developmental pathways; mechanistic interrogation of molecular signaling pathways in embryonic and pluripotent stem cells (NRF2, NF-κB, PI3K, Wnt, TGFβ, BMP, Notch).\n\nEfficacy of drugs on candidate pathways.\n\n\nIntroduction\n\nIn the past, the study of gene expression during normal development or induced by disease in rodent models have been assessed largely by end-point assays involving the sacrifice of large cohorts of animals or restricted to invasive serial blood or urine sampling. Whole body imaging has been a game changer in this context and bioluminescence imaging (BLI) has become one of the most prevalent non-invasive modalities in academic research. Luciferase enzymes, derived from many different invertebrate species, catalyze their substrates in a reaction that emits photons of light in a dose responsive and quantifiable manner1. Bioluminescent light, resulting from the activity of luciferase genes optimized for expression in mammalian cells, can be used as a quantifiable surrogate for gene promoter activation in vivo and detected using highly sensitive charge-coupled device (CCD) cameras2. Germline, light-producing transgenic (LPT) mice, where a luciferase transgene is inserted in the first intron of an endogenous gene, can be subjected to BLI to quantify gene activity in living animals3. Genome editing technologies have expedited the generation of whole body luciferase reporter transgenics4, and have massively increased the usage of LPT rodents, but critically do not reduce animal wastage in transgenic colony formation. In fact, the numbers of mice used in biomedical research has increased substantially since CRISPR/Cas9 genome editing has been applied to the production of transgenic mice with the time taken for mouse models to be created taking months instead of years to generate. The UK Home Office statistics notes that the number of procedures involving genetically altered animals rose by 36% between 2007 and 2016. Furthermore, it was reported that in 2016, 226,000 animals were used to create new lines of genetically altered animals5.\n\nOne of the greatest challenges facing users of germline LPT mice is that the luciferase reporter is present in every cell of the mouse. Researchers studying gene activity in a particular tissue or organ must be able to differentiate between target bioluminescence and the surrounding noise or targeting of the signal from a different organ entirely6. For many visceral organs this is practically impossible. We have developed an alternative approach by targeting conditionally activated luciferase reporter cassettes using viral vectors (lentivirus and adeno-associated virus) administered directly to target tissues in the newborn rodent to produce tissue-restricted somatotransgenic animals2. Viral transduction is further targeted by the use of pseudotyping with alternative viral envelopes known to have a specific cellular or tissue tropism7. Resultant somatotransgenic rodents have tissue-restricted expression of a luciferase gene conditionally activated by a minimally defined promoter or a synthetic promoter. To date, we have designed and validated in vitro over 20 synthetic transcription factor activated reporter (TFAR) constructs based on a minimal promoter sequence activated by serial transcription factor binding motifs2. These reporter elements have been selected to interrogate some of the most common signaling pathways known to be aberrant in neurological diseases, cancer, fibrosis and drug toxicity, the details of which are highlighted in Table 1. Any small rodent can be subjected to somatotransgenic bioimaging (SB) and there is no colony breeding wastage.\n\nnuclear factor kappa-light-chain-enhancer of activated B cells (NFκB); lipopolysaccharide (LPS); bone morphogenetic protein 2a (BMP2a); T-cell factor/lymphoid enhancer-binding factor (TCF/LEF); lithium chloride (LiCl2); activator protein 1 (AP-1); phorbol myristate acetate (PMA); Leukemia inhibitory factor (LIF); hepatic nuclear factor 4 beta (HNF4-β); hydrogen peroxide (H2O2); 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD); nuclear factor of activated T-cells (NFAT); Notch intracellular domain (NICD); liver x receptor (LXR); transcription factor EB (TFEB).\n\nFurthermore, SB can be layered on either gene knockout, chemically/biologically induced or naturally occurring rodent disease models2. The combined flexibility and reliability of SB means that the numbers of animals required for experiments is substantially reduced. As an example, a literature review of over 20 comparable studies of liver disease revealed that the common analytical methodologies were serial blood, bile and urine sampling involving repeated anaesthesia as well as periodic sacrifice for histological analysis. These involved large cohorts of between 3-12 animals being sacrificed at up to 10 timepoints meaning as many as 120 animals are used in each experiment. Typically, our experiments with somatotransgenic animals and longitudinal imaging contains 6-8 experimental and 6-8 control animals per experiment. This translates into a 7-10 fold reduction in the number of animals used as compared to the current standard methodologies of analysis.\n\nIt is established that repeated anesthesia is not only distressing to rodents but also disrupts physiological and pathological processes, notably in the brain8. BLI is routinely carried out on anesthetized mice in order to define a photonic capture region of interest on a static target. Since SB is based on complete capture from a tissue-restricted luciferase reporter gene, rodents can remain conscious and unrestrained during imaging. Our comparisons of SB in brains and livers showed no difference between conscious or unconscious bioimaging9. This represents a significant refinement to animal husbandry, as well as increases the frequency with which rodents can be subjected to bioimaging. Thus meaning more data can be obtained from the same number of animals. SB can be used by any researcher in academia or industry with access to an appropriate in vivo imaging system, such as the IVIS® imaging platform, to detect BLI and assess changes in gene expression resulting from disease onset or drug intervention.\n\nHere we present methodologies describing the development of the viral reporter vectors, the production of TFAR-containing virus, and methods for in vitro validation. Technical details of our in vivo validations for 2 disease models of inflammation, bile duct ligation and a model of hyperoxia, are detailed and experimental data presented.\n\n\nMaterials\n\nMluI (New England Biolabs; R0198S)\n\nXhoI (New England Biolabs; R0146S)\n\nBamHI (New England Biolabs; R0136S)\n\nEcoRI (New England Biolabs; R0101S)\n\npENTR-1A® (Invitrogen; A10462)\n\ncPPT forward sequencing primer - (GTGCAGGGGAAAGAATAGTAG)(Sigma-Aldrich)\n\nGateway™ LR Clonase™ II enzyme mix (Invitrogen; 11791020)\n\nDH5α chemically competent cells (Invitrogen; 18258012)\n\nOne Shot™ Stbl3™ competent cells (Invitrogen; C737303)\n\nampicillin antibiotic (Sigma-Aldrich; A9393)\n\nLB agar (Sigma-Aldrich; L3027)\n\nLB broth powder (Sigma-Aldrich; L3147)\n\nagarose gel powder (Sigma-Aldrich; A4718)\n\nHEK293T cell line (VWR; MSPP-CRL3216)\n\nDulbecco’s Modified Eagle’s Media (DMEM) (Sigma-Aldrich; D6429)\n\nFetal calf serum (FCS)(Invitrogen; 10082147)\n\nPenicillin/Streptomycin (Pen/Strep) (Sigma-Aldrich; P4333)\n\nPhosphate buffered saline (PBS) (Sigma-Aldrich; D8662)\n\nTrypsin EDTA dissociation media (Sigma-Aldrich; T4299)\n\nOpti-MEM I reduced serum medium (Invitrogen; 31985070)\n\npMD2.G (VSV-G envelope) (Addgene; #12259)\n\npCMVd8.74 (gag-pol, tat, rev) (Addgene; #22036)\n\nPsPAX2 (Addgene #12260)\n\nRETRO-TEK HIV-1 p24 antigen ELISA (Zeptometrix; 0801200)\n\nKAPA SYBR FAST qPCR kit (Roche; KK4600)\n\nbranched PEI (MW ~25,000) (lentivirus production) (Sigma Aldrich; #408727)\n\n0.45 µM PVDF filter (Sigma-Aldrich; P1938)\n\npHGT1 Ad5 helper plasmid (MTA Harvard Medical School, USA)\n\nBenzonase (Sigma-Aldrich; E8263)\n\nAAV pro 293T cells (Takara; 632273)\n\nSodium chloride (Sigma-Aldrich; S9888)\n\nPolyethyleneimine (PEI) (Polysciences; 24765)\n\n0.45 µm filter membrane (Millipore; SCHVU05RE)\n\nCell lifter (Corning; 3008)\n\n0.45 µm syringe filters (Sartorius; 17598)\n\n0.22 µm centrifuge tube filter (Costar; 8160)\n\n10 ml syringe (Terumo; SS+10ES1)\n\n50 ml syringe (BD Plastipak; 300865)\n\nDisposable needles (BD Microlance; 301155)\n\nBenzonase nuclease (Sigma; E1014-25KU)\n\nAmicon Ultra-15 centrifugal filter units (Millipore; UFC910024)\n\nSlide-A-lyzer dialysis cassette 10,000 MWCO (Thermo Scientific; 66810)\n\n5 ml FACS tubes (Falcon; 352053)\n\nSodium deoxycholate (Sigma; D6750-100G)\n\nGlycine (Sigma; G8898-500G)\n\nPOROS™ CaptureSelect™ AAV Resin (Thermo Scientific; A36739)\n\nÄKTAprime plus (High performance liquid chromatography - HPLC system (GE Healthcare; 11001313)\n\nDPBS (1X) (Gibco; 14190-094)\n\nPBS tablets (Sigma; P4417-100TAB)\n\nLuna Universal Probe qPCR MasterMix (NEB; M3004S).\n\n96-well PCR plate 0.1 mL format.\n\nMicroAmp® Optical Adhesive Film (Applied Biosystems).\n\nHeLa cell line (Sigma; 93021013)\n\nHuh7 cell line (JCRB Cell Bank; JCRB0403)\n\nHepG2 cell line (Sigma; 85011430)\n\nRPMI-1640 media (Sigma-Aldrich; R8758)\n\nultra-pure LPS-EB (InvivoGen; tlrl-3pelps)\n\nActivin A (PeproTech; AF-120-14E)\n\nBMP2a (PeproTech; AF-120-02)\n\nCobalt Chloride (Sigma-Aldrich; 60818)\n\nLithium Chloride (LiCl2) (Sigma-Aldrich; L9650)\n\nEstradiol (Sigma-Aldrich; E2758)\n\nNutlin-3a (Sigma-Aldrich; SML0580)\n\nPhorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich; P8139)\n\nLY294002 (StemCell Technologies; #72152)\n\nhuman recombinant LIF (StemCell Technologies; #78055)\n\nPurmorphamine (Sigma-Aldrich; SML0868)\n\nHydrogen peroxide (H2O2) (Sigma-Aldrich; 216763)\n\n2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Sigma-Aldrich; NIST1614)\n\nIonomycin (Sigma-Aldrich; I0634)\n\nLuciferase assay lysis buffer (0.65% NP40 (Sigma-Aldrich; NP40), 10 mM Trizma® base (Sigma-Aldrich; T1699) (pH 8.0), 1 mM Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich; E9884)(pH 8.0), 150 mM NaCl(Sigma-Aldrich; S9888))\n\nluciferase assay buffer (25 mM Trizma® base (Sigma-Aldrich; T1699) pH 7.8, 1 mM 1,4-Dithiothreitol (DTT) (Sigma-Aldrich; 10197777001), 1 mM EDTA (Sigma-Aldrich; E9884), 1% Triton™ X-100 (Sigma-Aldrich;), 8 mM MgCl2(Sigma-Aldrich; M8266), 3 ml glycerol (Sigma-Aldrich; G5516), 1.25 mM rATP (Promega; E6011), 0.5% BSA (Sigma-Aldrich; A2153))\n\nLuminometer such as the GloMax (Promega; E9032)\n\nBio-rad protein assay (Bio-rad; #5000006)\n\nCD1 mice (Charles River Laboratories)\n\n4% isofluorane (Abbott Laboratories)\n\n33-gauge Hamilton needle (Fisher Scientific, Loughborough)\n\nbupivacaine hydrochloride (Advanz Pharma)\n\nD-luciferin, potassium salt (Gold Biotechnologies; LUCK)\n\ncooled charged-coupled device camera (IVIS machine) (Perkin Elmer)\n\nLiving Image Software (v4.1) (Perkin Elmer)\n\n\nMethods\n\nLentivirus cloning. A simple two-step cloning process was developed to generate lentiviral TFAR vectors using Gateway® cloning. We first generated the parental lentiviral vector by cloning the Gateway® (GW) Destination recombination site into a blunted unique MluI restriction site of a 2nd generation HIV-1 based lentiviral backbone upstream of a bicistronic 3xFLAG-FLuc-F2A-eGFP reporter gene cassette to produce pLNT-GW-Luc-eGFP. We then generated a shuttle vector containing the adenovirus E1A minimal promoter (MP) sequence cloned into a unique XhoI restriction site in the Gateway® shuttle vector, pENTR-1A. This shuttle plasmid (pENTR-MP) was used as a sub-cloning vector to introduce de novo synthesized regulatory sequences (Aldevron, North Dakota, USA) upstream of the MP (existing TFARs listed in Table 1) into BamHI/EcoRI restriction sites. Vectors were confirmed by Sanger sequencing using a cPPT forward primer (GTGCAGGGGAAAGAATAGTAG) then recombined into pLNT-GW-Luc-eGFP using Gateway® cloning (Figure 1A).\n\nResponse elements are cloned into the pENTR vector. A recombination reaction is performed to shuttle the response element into the lentiviral or AAV backbone upstream of a 3xFLAG-FLuc-2A-eGFP bicistronic reporter.\n\nAAV cloning. The AAV8-Gateway®-Luc-T2A-eGFP was completely de novo synthesized (Aldevron). This AAV plasmid consisted of a Gateway® sequence, placed upstream of the Luc-2A-eGFP reporter cassette. AAV TFARs were generated by Gateway® cloning as with the lentiviral vectors (Figure 1B).\n\nBoth lentivirus and AAV vectors were generated by transfection of human embryonic kidney, HEK293T, producer cells with accessory plasmids as previously detailed2. Lentiviral titer was generated based on the p24 ELISA assay whereas AAV was titered by quantitation of capsid protein by viral genome copy numbers per ml10,11.\n\nAll newly generated TFARs were comprehensively evaluated for predicted responses to known activators/inhibitors on multiple cell lines, including HEK293T, HeLa, Huh7, HepG2, and primary human fibroblasts, prior to being employed in vivo2. The reporters, in vitro agonists, concentrations, and duration of activation are outlined in Table 1.\n\nSpecifically, for the HIF response element (HRE) construct that can be used to determine the induction of hypoxia, HEK293T and HeLa cells were transduced with LNT-HRE-Luc-eGFP at a multiplicity of infection (MOI) of 10. Three days post-transduction, transduced cells were replated in two sets of triplicates, one for HRE activation and one for basal levels of luciferase activity. The following day one triplicate set of cells were exposed to the agonist, CoCl2 (100 mM/µl), in RPMI-1640 for 12 hours while the non-activated cells were only placed in RPMI-1640. A time course of 12, 24, 48, and 72 hours post agonist activation was performed. At each time point, the cells were lysed in a standard luciferase assay lysis buffer and stored for future luciferase assay analysis.\n\nAll procedures were performed under United Kingdom Home Office Project License 70/8030, approved by the ethical review committee, and followed institutional guidelines at University College London. All methods were performed in accordance with the relevant guidelines and regulations. Male and female outbred wild type CD1 mice were used and were supplied by Charles Rivers Laboratories. Mice were used for adult interventions as close as possible to 6 weeks of age and 25g.\n\nIVC cages were used to house the mice, with a maximum of 5 per cage. Male and female mice were housed separately post weaning. Food and water were routinely monitored by the animal technicians at the facility. The temperature of the room was set at 25°C with light/dark cycles alternating every 12 hrs. Mice were bred onsite and time-mated in order to plan experiments as accurately as possible and avoid wastage of unused pups. A total of 34 mice were used in the studies, described below.\n\nNeonatal intracranial and intravenous injections. Mice either received intracranial or intravenous injections, never both. Skilled technicians can achieve more than 99% success rate for intravenous injections and no failed intracranial injections. No anaesthesia was used as the procedure is very quick and involves a single needle puncture per mouse. For intracranial injections, mice (postnatal day 1 (P1)) received unilateral injections of concentrated lentiviral or AAV vector (5 μl in total) into the cerebral lateral ventricles using a 33-gauge Hamilton needle. The site of injection was located two-fifths of the distance between the eye and the lambda intersection of the skull as described and illustrated by Kim et al.12. During the procedure, mice were held in an upright position, with their heads firmly placed between the handler’s thumb and index finger. The 33-gauge Hamilton needle was held perpendicular to the mouse head and injected at a depth of approximately 3mm. The vector was slowly controlled and injected free handed into the lateral ventricles. At P1, a 33-gauge needle can easily penetrate the skull and there is no reflux of injectate as the ventricular system is sufficiently voluminous to accommodate the extra volume. Biodistribution of vector has been confirmed using this methodology9. For intravenous injections, P1 pups received intravenous injections of lentiviral or AAV vectors into the superficial temporal vein (20 μl) using a 33-gauge Hamilton needle. This procedure was performed free handed over approximately 5 seconds. Pups were returned as soon as possible to the dam and the nest undisturbed as much as possible. Animals were monitored daily for distress or discomfort such as the presence of a dull/ruffled coat, poor posture (hunched), pale or sunken eyes, a change in normal behaviour, reduced food or water intake, dehydration, any discomfort when handled, a reluctance to move, and any significant weight loss. In this cohort, we noted no significant signs of pain or discomfort following vector administration.\n\nValidation and disease modelling induction of somatotransgenic bioimaging\n\nEffects of carrier gas on hypoxia-inducible factor (HIF) induction\n\nAll mice received neonatal intracranial injection of lentivirus vector containing the HIF response element upstream of a luciferase-eGFP bicistronic cassette (LNT-HRE-Luc-eGFP) at P1. After weaning, age-matched mice were randomly allocated to two groups (N=11 for each group based on power calculations) for imaging at 0, 2, 4, 6 and 8 hours. Randomization was achieved by using Microsoft Excel to generate a random column of numbers against mouse numbers and then sorting the list of mice using the SORT function. The groups received isoflurane mixed with either 100% oxygen or air. The group receiving 100% oxygen exhibited consistently lower HIF activation than the group receiving air (21% oxygen); this was significantly different at 6 hours (P=0.0465, repeated measures ANOVA where one factor was with/without 21% oxygen and the repeated measure was time). See data availability section.\n\nThis data suggests that the carrier gas, itself, may influence physiological and pathological processes and supports the concept that conscious bioimaging may be preferable, where possible9.\n\nPartial bile duct ligation in mice\n\nAll mice received neonatal intravenous injection of lentivirus vector containing the truncated GFAP promoter driving a luciferase-eGFP bicistronic cassette (LNT-GFAP-Luc-eGFP) at P1.\n\nMice were anaesthetized with 4% isoflurane at between 40–60 days after birth, shaved and disinfected around the surgical area, and a midline laparotomy (≈ 15 mm) performed just caudal to the sternum using aseptic surgery techniques. The median and left lobes of the liver were exteriorized and kept moist with sterile gauze. The bile duct was ligated with 6.0 silk suture to occlude outflow from the left and median lobes but not occlude bile outflow from the right and caudate lobes as described by Yang and colleagues13. The liver was returned to the abdomen and the wound closed in two stages using 6.0 silk suture; skin was closed using a subcuticular suture with buried knots (N=7). Sham operated mice received laparotomy only (N=5). Mice were recovered in a warmed chamber and monitored to make sure they were not under any unnecessary distress. Clinical record sheets were used to detail observations and post-operative treatments that were given. Pre- and immediate post-operative doses of topical bupivacaine hydrochloride (2 mg/kg) and subcutaneous 50 µl morphine (6.67 mg/ml) was administered for systemic post-operative analgesia14. Following surgery, mice were single housed and provided with moist food to aid consumption of food and prevent significant weight loss. Monitoring sheets recorded age, weight, disease and surgical status. Mice were monitored for approximately 1 hour post-surgery, and subsequently checked and weighed to monitor for overt signs of discomfort and significant weight loss. A loss of weight of more than 15% required humane euthanasia of the animal. In this cohort, the animals were not found to have negative post-operative outcomes, which is a significant improvement over performing full bile duct ligations which often result in pain, jaundice, and death to the animals. No animals within our cohort required euthanasia using partial bile duct ligation.\n\nWhole-body bioluminescence imaging. Mice which underwent partial bile-duct ligations were anesthetized with isoflurane containing 100% oxygen, all other cohorts of mice described herein were imaged while conscious and free-moving. Mice received an intraperitoneal injection of 15 mg/mL of D-luciferin. Mice were imaged after 5 minutes using a cooled charged-coupled device camera (IVIS imaging machine) for between 1 second and 5 minutes. The regions of interest (ROI) were measured using Living Image Software and expressed as photons per second per centimeter squared per steradian (photons/second/cm2/sr). The imaging chambers used to image conscious mice for the intracranial injected mice had the following dimensions; 5 cm × 5 cm × 6 cm. The Perspex box which was used to image the conscious mice which had received an intravenous injection is described in detail in Karda et al.9. Where possible, BLI was carried out in adult reporter rodents on three consecutive days to establish a robust median baseline; subsequent data points were expressed as fold-change over this internal standard for each individual animal.\n\nEuthanasia. At experiment conclusion, mice were culled using rapid cervical dislocation to induce rapid loss of consciousness. To reduce the possibility of operator error, the main concern for animal welfare using this technique, the procedure was performed by adequately trained personnel with a high degree of technical proficiency only. Where possible, cervical dislocation was performed while mice were still under non-recovery isoflurane anaesthetic. Death was assured by decapitation.\n\nIn consultation with a statistician, power calculations are performed based on previous data using the free GPower (v3.0.10) online software. Using the probability of finding an effect set at 80%, the probability of incorrectly rejecting the null hypothesis when it is true set at 0.05 (alpha), and using a two-sided test, a power calculation was performed in order to determine the sample size required for each group under investigation. Using our previous data involving the activation of the Smad2/3 binding element using the potent agonist, activin A, a standard deviation of 1.75 was used. The mean of group 1 (control) was set at 5.13, and the mean of group 2 was set at 7.8 (experimental). Based on these values, the effect size is expected to be large (1.52 fold change). An effect size greater than 0.8 is indicative of a large effect size, indicating that 93% of the control group will be below the mean of the experimental group. The experiment was sufficiently powered with a sample size of eight experimental and eight control animals to detect a change in the total amount of photonic output between two groups with a power of 80%. For in vivo data, repeated measurements following agonist administration were analyzed with repeated measures analysis of variance using GraphPad Prism v8.0.0 with a significance level of P <0.01. Area under curve data for experiments with two groups were analyzed by Student’s one-tailed t-test. For areas under curve derived from two or more groups, one-way analysis of variance (ANOVA) with Newman-Keuls post-hoc multiple comparison test was performed.\n\n\nProtocols\n\nWe have generated a comprehensive library of lentiviral TFAR vectors and are developing a similar library of AAV vectors (Table 1). These TFARs are available to any academic researcher on request. We also have developed a simple two-step protocol for shuttling de novo synthesized transcription factor binding sequences into our parental vectors.\n\nTFAR design, synthesis, and sub-cloning\n\n1. Derive minimal consensus binding sequence for the candidate transcription factor from the literature.\n\n2. Design serial transcription factor binding sequence (TFBS) by interspersing 4-10 binding sequences with 10 random nucleotides using non-coding combinations of the 4 nucleotides.\n\n3. De novo synthesize the resultant sequence flanked by BamHI (5’-) and EcoRI (3’-) restriction sites for directional cloning into pENTR-MP, a Gateway pENTR vector containing a minimal promoter sequence 5’-GGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACTCTCTTCC-3’.\n\n4. Clone TFBS into pENTR-MP using DH5α chemically competent cells to produce pENTR-TFAR.\n\n5. Confirm clones by sequencing using the pENTR forward primer which binds approximately 82 bp from the BamHI cloning site. (ACTGATAGTGACCTGTTCGTTGC).\n\nCloning synthetic promoter into the lentiviral cassette.\n\n1. Perform a single-step LR Clonase recombination reaction between the pENTR-MP vector containing the desired synthetic promoter with the LNT-GW-Luc-eGFP vector as per manufacturer’s standard instructions.\n\n2. Transform 1 μl of the LR recombination reaction into One Shot™ Stbl3™ (Invitrogen) competent cells and grow cells overnight at 30°C on ampicillin-laden (100 μg/ml) LB-agar plates.\n\n3. Select colonies and grow in a shaking incubator (280 rpm) overnight at 30°C in 5 mls of LB broth containing ampicillin (100 μg/ml).\n\n4. Screen and select colonies for positive clones using the BamHI restriction enzyme and gel electrophoresis. Correct clones contain 4 fragments of 10,928, approximately 200-300 bp (depending on response element and minimal promoter cloned in), 81 bp, and 12 bp (not seen on gel). A plasmid that has not undergone recombination and is still the LNT-GW-Luc-eGFP parental backbone that does not contain the biosensing response element contains 5 fragments of the following sizes: 10, 928 bp, 857 bp, 702 bp, 228 bp, 12 bp (not seen on gel). Example gel provided as underlying data15.\n\n5. Confirm clones by sequencing following the recombination reaction into the lentiviral backbone by using the cPPT forward primer (GTGCAGGGGAAAGAATAGTAG).\n\nCloning synthetic promoters into the AAV cassette.\n\n1. As described above for cloning synthetic promoters into the lentiviral cassette, perform LR Clonase recombination reaction between pENTR-MP containing the synthetic promoter of choice with the AAV-GW-Luc-eGFP, as per manufacture’s protocol (see Note 1 for Clonase storage recommendations).\n\n2. Transform 1 μl of the LR recombination reaction into One Shot™ Stbl3™ (Invitrogen) competent cells (see Note 2) and grow cells overnight at 30°C on ampicillin-laden (100 μg/ml) LB-agar plates.\n\n3. Select colonies and grow in a shaking incubator (280 rpm) overnight at 30°C in 5 mls of LB broth containing ampicillin (100 μg/ml).\n\n4. Screen colonies for positive clones using appropriate restriction enzyme depending on the desired synthetic promoter and then perform gel electrophoresis.\n\n\nNotes\n\n1. The LR Clonase enzyme mix is unstable even at -20°C for extended periods. Thus, it is recommended that small aliquots of 5 μl are made and preferably stored at -80°C to reduce the number of freeze/thaw cycles and retain as much enzyme activity.\n\n2. One Shot™ Stbl3™ competent cells have been designed specifically for the propagation of unstable DNA sequence such as those found within the lentiviral backbone which contains direct repeats.\n\nGeneration of high-titer TFAR lentivirus.\n\n1. Seed HEK293T cells (mycoplasma negative – see Note 3) at approximately 2x107 cells per T175 cm2 flask and incubate at 37°C, 5% CO2 overnight to achieve up to 90% confluence.\n\n2. Mix lentiviral plasmids in Opti-MEM™ I reduced serum medium (7.5 mls / T175 flask) in the following quantities (per flask): 50 μg transfer vector containing transgene, 32.5 μg gag-pol packaging vector (pCMVΔR8.74) OR psPAX2 and 17.5 μg VSV-G envelope vector (pMD2.G).\n\n3. In a separate tube, mix 1 μl 10 mM branched PEI in 7.5 mls Opti-MEM™ I reduced serum medium® (per flask). Incubate for 5 minutes.\n\n4. Mix DNA and PEI solutions together in a 1:1 ratio (total of 15 mls per flask) and incubate at room temperature for 20 minutes to allow DNA:PEI complexes to form (see Note 4 for PEI preparation using branched PEI). This is hereon described as transfection media (Opti-MEM, DNA:PEI complex).\n\n5. Remove media from cells in flask and add 15 mls of transfection media.\n\n6. Incubate transfection media on cells in an incubator at 37°C / 5% CO2 for 3 hours.\n\n7. Remove transfection media and replace with 15 mls complete media (DMEM + 10% FCS + 1% Pen/Strep).\n\n8. After 24 hours, remove media and replenish cells with new culture medium.\n\n9. After a further 48 hours, collect the virus-containing medium and filter through a 0.45 μm PVDF filter (see Note 5).\n\n10. Subject virus-containing medium to overnight centrifugation (16-20 hours) at 5000g at 4°C.\n\n11. Repeat harvest and overnight centrifugation for 72-hour post-transfection supernatant.\n\n12. Resuspend the viral pellet in 50 μl Opti-MEM I reduced serum medium® (per T175 flask of cells used) and gently mix every 20 minutes for 1 hour at 4°C and cryopreserve at -80°C.\n\n13. Lentiviral titer is obtained using a RETRO-TEK HIV-1 p24 antigen ELISA as per manufacturer’s protocol.\n\nGeneration of high-titer TFAR AAV.\n\n1. Seed 6–7 × 106 HEK293T cells into 15cm plates so that they are 70-80% confluent and incubate at 37°C, 5% CO2 overnight.\n\n2. Mix 10.5µg of the AAV transgene plasmid, 10.5µg of pDG8 plasmid expressing AAV2 Rep, AAV8 Cap gene and 31.5µg pHGT1 (Ad helper plasmid) to 1.5mls of Opti-MEM reduced serum medium. Add 758 μl of stock transfection agent PEI (2mg/ml) to Opti-MEM (1.5ml/dish), mix and leave at room temperature for 15-20 minutes.\n\n3. Drop-wise, add 3 ml of transfection mix to each dish and gently distribute\n\n4. After 24 hours remove media and replace with 15 mls of DMEM (2% fetal calf serum and 1% Pen/Strep antibiotic)\n\n5. After 48 hours harvest the cells by scrapping the cells off the dish and pooling the media and cells to 50 ml.\n\n6. Spin at 1500 rpm* for 5 minutes and pour supernatant into a receiver bottle and store at -20°C. revolutions per minute (herein, on a centrifuge with size of rotor radius 17.4 cm).\n\n7. Re-suspend pelleted cells in 1 ml TD lysis buffer (140mM NaCl, 5mM KCl, 0.7mM K2HPO4, 3.5mM MgCl2, 25mM Tris. pH7.5), pool and store in -80°C.\n\n8. Freeze thaw pellet 5 times and add 25 ul/ml of 20% sodium deoxycholate.\n\n9. Add 8 ul/40 mls of cells Benzonase and incubate at 37°C for 30 minutes. Spin at 4000rpm for 30 minutes at 18°C and filter using 0.45 um filters. Repeat this twice.\n\n10. For supernatant, aliquot into 50 mls tubes and add 5ul of benzonase per 50mls of supernatant.\n\n11. Add 100 ul of MgSO4 and incubate for 30 minutes at 37°C, after which spin at 4000 rpm for 30 minutes at 4°C. Filter using 0.45 um filters, repeat twice.\n\n12. Run the cell pellet and supernatant through the HPLC machine using the POROS column16.\n\n13. Add virus to dialysis cassette and place cassette into 1 L of PBS. Keep spinning overnight at room temperature on a slow rotating rotor.\n\n14. Prime the membrane of the Amico Ultra tube by adding 5 mls PBS and centrifuge for 5minutes at 4000 rpm.\n\n15. Remove PBS and place virus onto membrane. Spin at 4000 rpm for 5 minutes\n\n16. Wash membrane with vector collected and add additional 100 ul PBS. Place vector into a 2 ml centrifugal tube with 0.22 um filter. Spin for 3 minutes at 13,000K on a benchtop centrifuge.\n\n17. Remove filter and aliquot vector. Store at -80°C.\n\n18. AAV vector was quantified using qPCR as per previously published protocol16, but briefly, samples were treated with DNase to release DNA. Serial dilutions were prepared to be used as standards. Both standards and samples were tested in triplicate. Cycling parameters were: 95°C for 10 minutes, followed by 40-45 cycles of 95°C for 15 seconds and 60°C for 45 seconds. Vector titers were calculated using the following formula: Titre (vc/mL) = C/5 × 1000 × D × 10 × 2 (See note 6).\n\n\nNotes\n\n3. Mycoplasma infections have the capacity to reduce lentiviral titers. Therefore, perform a mycoplasma test using PCR with the following primers: Forward (5’- gggagcaaacaggattagataccct - 3’) and Reverse (5’- tgcaccatctgtcactctgttaacctc -3’) with the following cycling parameters: 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds repeated for 30 cycles. A 1.5 - 2% gel will yield a 300 bp fragment on an agarose gel. Alternatively use a fluorescence-based method using a kit such as MycoAlert™ Mycoplasma Detection kit (Lonza).\n\n4. To make up 10 mM branched PEI (MW ~25,000), in a fume hood, add 10 ml water to 10 ml PEI and vortex. Add 12 N HCL, 1 ml at a time, and vortex (this is approximately 10 ml HCL) then top up to 41.2 ml with water. Make sure that this final solution is at pH 7.0. Vortex and store in small aliquots at -80°C.\n\n5. Both PES and PVDF filters are suitable but PVDF has been shown to be lower protein binding.\n\n6. Where C is the copy number measured from the reaction, 5 is the volume of sample used, 1000 is the conversion factor from µl to ml, D is the dilution factor, 10 is the initial dilution of the AAV from the digestion (5 µl to 50 µl), and 2 takes into account the complementary strand that is not targeted by the primers within the reaction.\n\nIn vitro validation of HRE reporter constructs.\n\n1. Plate cell lines at ~60% confluence and transduce with virus at an MOI of 10.\n\n2. Amplify transduced cells for at least 72 hours before replating to allow the virus to integrate and express basal levels of the transgenes.\n\n3. Add 250 µM CoCl2 in RPMI-1640 media to activate HRE activity for 12 hours followed by addition of 500 µl of RPMI-1640 media (see note 6).\n\n4. At 24 hours post-activation, replace with complete media.\n\n5. For luciferase activity, lyse approximately 5x105 cells in 300 μl luciferase lysis buffer (0.65% NP40, 10 mM Trizma® base (pH 8.0), 1 mM EDTA (pH 8.0), 150 mM NaCl)\n\n6. Clarify the soluble fraction of the supernatant by centrifugation at 13,000xg for 1 minute.\n\n7. Add 20 μl cell extract to 20 μl assay buffer (25 mM Trizma® base pH 7.8, 1 mM DTT, 1 mM EDTA, 1% Triton™ X-100, 8 mM MgCl2, 3 ml glycerol, 1.25 mM rATP, 0.5% BSA).\n\n8. Inject D-luciferin substrate (Gold Biotechnology) into each well at a final concentration of 1.5 mM.\n\n9. Measure luminescence output using a suitable microplate reader with luminescent capabilities.\n\n10. Perform a protein assay to normalise relative photonic light units to total protein.\n\n11. Only if a statistically significant response to agonist or antagonist was achieved, using appropriate statistical evaluation, would the TFAR be deemed suitable for in vivo experiments.\n\n\nNotes\n\n6. It is important to note that histidine inhibits CoCl2, and DMEM contains high concentrations of histidine compared to other media. It is therefore imperative to use RPMI-1640 for HRE induction using this agonist.\n\n1. Administer concentrated lentiviral or AAV vector (5 μl total) by unilateral injection into the cerebral lateral ventricles using a 33-gauge Hamilton needle (see note 7).\n\n2. Perform intravenous injections of lentiviral or AAV vectors (20 μl total) into the superficial temporal vein using a 33-gauge Hamilton needle.\n\n1. As previously described9, anesthetize mice (see Note 8) with 4% isofluorane in 100% O2 (see Note 9). Inject 300 μl D-luciferin solution at a concentration of 15 mg/ml (a dose of approximately 150 mg/kg) into the intraperitoneal cavity (see Note 10).\n\n2. Image the unconscious mice in the warmed lightproof detection chamber of the IVIS in vivo imaging system (see Note 11). Commence imaging 5 minutes after D-luciferin administration (t=0) (see Note 12). An overlay of the two images is generated using Living Image software to create a pseudo-colored image to depict luminescent intensities over each animal.\n\n3. Define regions of interest (ROIs) manually using a standard area for each organ.\n\n4. Prior to agonist-mediated activation or surgical induction of disease, each of the animals is administered with D-luciferin and imaged three times within 72 hours in order to ascertain a robust median baseline measurement of bioluminescent imaging which can subsequently be used to express all future data points as a fold-change over this baseline value.\n\n5. The type of statistical test depends upon the nature of the biosensor and the kinetics of the response. Two possible approaches are; A) for each animal in the two experimental groups, obtain the area under the curve using the parallelogram method. Compare using a Student’s t-test if data is normally distributed. If mathematical transformations were not sufficient to satisfy parametric assumptions we suggest using a non-parametric test e.g. Mann-Whitney U-test. B) compare two or more experimental groups over time using analysis of variance (ANOVA) with repeated measures. If ANOVA shows a significant difference between groups, perform a post-hoc test (e.g. Tukey, Bonferroni or Sidak) to test which time points might be significantly different (see Note 13).\n\n\nNotes\n\n7. The 33-gauge Hamilton needle should be kept moist at the tip by placing a wet paper towel around the needle. This helps reducing the friction against the new-born mouse skin.\n\n8. Mice can be injected without anesthesia. However, anesthesia reduces mobility and improves injection accuracy. Inhalation or injectable anesthetics are avoided as the nose cone impairs access to the injection sites (especially the superficial temporal vein).\n\n9. Mice may also be anesthetized using air or air and a nitrous oxide mix. The choice of carrier gas may affect the chosen biosensor as well as firefly luciferase activity (since this is an oxygen-dependent reaction).\n\n10. When administering the D-luciferin via intraperitoneal injection, make sure that the bladder or other internal organs are not penetrated by the needle. This can be achieved by “tenting” the skin for injection. Similarly, ensure penetration into the peritoneal cavity by watching for, and avoiding the formation of a subcutaneous bleb.\n\n11. To achieve optimal luciferase expression from the mice, it is best to use white furred mice as the black-furred mice prevent the bioluminescence from penetrating through.\n\n12. Waiting 5 minutes after D-luciferin administration permits time for entry of the D-luciferin into the bloodstream. It is worth performing a preliminary experiment to determine the kinetics for optimal bioluminescence for different cell and tissue targets. In addition, alternative routes of D-luciferin administration (e.g. intranasal) may be used.\n\n13. It is good practice to perform a preliminary experiment to gauge the kinetics of the biosensor response and to identify timeframes of these responses in order to refine subsequent statistical tests. The statistical test and the time points of analysis should be decided before the experiment is performed, not afterwards.\n\n\nResults\n\nLentiviral delivery of vectors containing transcription factor binding elements upstream of a bicistronic reporter cassette offer a unique method with which to interrogate biological pathways. HRE is used as a biological sensor of hypoxia, important in cancer and ischemia models of disease. Here we present data validating our biosensing constructs in vitro (Figure 1) prior to in vivo (Figure 215) validation and experimentation.\n\nHEK293T (A) and HeLa (B) cells were transduced with pLNT-HRE-Luc-eGFP and either activated (n=3) with 250 µM CoCl2 (red bars) or not activated (n=3) (grey bars) in triplicate for each of the time points (T = 12, 24, 48, and 72 hours). Data was analyzed using a two-way ANOVA with repeated measures with Bonferroni post-hoc test to correct for multiple testing. Bars represent mean ± SD with adjusted p values indicated above each timepoint (=<0.05, =<0.01, =<0.001, **=<0.0001).\n\nIn vitro validation of the pLNT-HRE-Luc-eGFP construct. Prior to using the LNT-HRE-Luc-eGFP vector to interrogate signaling profiles in disease models in vivo, it was important to first validate this construct and establish its ability to be induced by agonists in vitro. CoCl2 is a chemical inducer of the hypoxia-inducible factor that mimics hypoxia. In the presence of CoCl2, a highly significant increase in luciferase-mediated bioluminescence fold-change (Δ bioluminescence) over non-activated cells was observed in vitro (Figure 215) prior to in vivo (Figure 315) validation and experimentation.\n\nLNT-HRE-Luc-eGFP transduced animals were imaged at 0,2,4,6 and 8 hours in either 100% oxygen (n=11) (red) or air (n=11) (black) carrier gas. An increase in oxygen results in lower hypoxia-induced luciferase expression with data expressed as a fold change in bioluminescence (Δ bioluminescence) from time point 0. Δ Bioluminescence was plotted per individual animal (A) and as mean±SD (B). Statistical significance of > 0.05 is indicated by (Sidak’s multiple comparison test, Graphpad Prism 8.0).\n\nResponse to oxygen content of carrier gasses during anesthesia. Following validation of the hypoxia response element in vitro, we chose to use it to ask whether in vivo HIF activity is modulated by the anesthetic carrier gas. 100% oxygen is frequently used as a carrier gas to ensure sufficient tissue oxygenation during anesthesia however, theoretically, it may downregulate HIF activation as this response element responds inversely to hypoxia, or the lack of oxygen. Mice underwent anesthesia with isoflurane using either air or 100% oxygen as the carrier gas and HRE signaling monitored at a series of time points using bioluminescence imaging. A change in bioluminescence (Δ bioluminescence) compared to time point 0 was noted over time with oxygen expectedly decreasing the expression of the HIF response element (Figure 3A-B15). This indicates the importance of selecting the correct carrier gas for anaesthetic induction when interrogating HIF-responsive signaling using luciferase expression and imaging.\n\nSomatotransgenic bioimaging to assess GFAP activation in a model of cholestatic liver injury. The partial BDL model is an established model of cholestatic liver disease17. Currently, analysis of liver disease consists of terminal endpoint analysis of groups of animals at serial time points, and/or serial bleedings for analysis of serological biomarkers of liver function, particularly liver enzymes and total serum bile acids. We have previously studied signal transduction pathways of inflammation and WNT-signaling in this model14. Here, we asked whether expression of GFAP, a marker of activated hepatic stellate cells that are a major cell type involved in liver fibrosis, was altered following induction of liver injury. P1 mice received LNT-GFAP-Luc-eGFP, a lentiviral vector containing the luciferase-eGFP transgene driven by the bioresponsive glial fibrillary acidic protein (GFAP) response element. At 45 days of age mice were randomly allocated to two groups, one of which received partial bile duct ligation; controls received sham surgery without ligation of the bile duct (Figure 4A–B15). There was a consistent increase in bioluminescence 3 days after induction of the model versus sham controls. One particular mouse showed a remarkable, sustained increase in luciferase output following induction. This data illustrates the strength of continual non-invasive measurements, in that it is possible to identify consistent outliers, which would not be possible with terminal end-point analysis, due to a lack of data on expression kinetics.\n\nLNT-GFAP-Luc-eGFP-transduced animals were subject to partial bile duct ligation (n=7)(red) as a model of cholestatic liver injury. Another group of animals underwent sham surgery only (n=5)(black). Fold-change in bioluminescence (Δ bioluminescence) was calculated before and after model induction and plotted as individual animals (A) and means ± SD (B).\n\n\nDiscussion\n\nThe development of the described methodologies has a broad scope of applicability for monitoring of disease progression, induction, and therapeutic intervention. The generation of biosensing, reporter animals that can undergo longitudinal assessment of transcription factor activity has significant implications for the number of animals currently being utilized within both academic and industry research and development. This novel platform of somatotransgenic bioimaging is also capable of generating longitudinal datasets from small cohorts of animals. F0 cohorts based on appropriate power calculations can be administered with TFAR vector and assayed almost immediately, depending on the experiment, after drug administration or disease state induction. In many instances, animals can be reused for further follow-up studies without any due stress or requirement for serial anesthesia. This approach results in a massive reduction in animals required for a predicted outcome and a clear decrease in procedures stressful to animals.\n\n\nData availability\n\nOpen Science Framework: Non-invasive somatotransgenic bioimaging in living animals. https://doi.org/10.17605/OSF.IO/XF7EZ15\n\nThis project contains the following underlying data:\n\n1. 293T – HRE – CoCl2 – Luciferase assay calculations.csv - (in vitro luciferase assay using the LNT-HRE-Luc-eGFP and cobalt chloride agonist in HEK293T cells).\n\n2. HeLa – HRE – CoCl2 – Luciferase assay calculations.csv - (in vitro luciferase assay using the LNT-HRE-Luc-eGFP and cobalt chloride agonist in HeLa cells).\n\n3. BDL NC3Rs raw data.csv - (In vivo raw data for GFAP activation using bile duct ligations (BDL)).\n\n4. HRE NC3Rs raw data.csv - (In vivo raw data for HRE activation using the model of hypoxia).\n\n5. pLNT-GW-Luc-eGFP.dna – (plasmid map of the lentiviral construct containing the Gateway cassette upstream of luciferase and eGFP).\n\n6. pLNT-HRE-Luc-eGFP.dna – (plasmid map of the lentiviral construct containing the HIF response element (HRE) upstream of luciferase and eGFP following recombination).\n\n7. Gel image of LNT-GW-Luc-eGFP before recombination.tif – (BamHI digest of LNT-GW-Luc-eGFP prior to recombination reaction).\n\n8. BamHI digest of pLNT-HRE, WNT, PI3, HNF4, Gli-Luc-eGFP clones.tif – (BamHI digest of LNT-HRE, WNT, PI3, HNF4, Gli-Luc-eGFP response elements following recombination reaction).\n\n9. pLNT-HRE-Luc-eGFP sequencing to confirm HRE.abi – (sequencing of LNT-HRE-JDG and alignment to theoretical sequence of LNT-HRE-JDG).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWilson T, Hastings JW: Bioluminescence. Annu Rev Cell Dev Biol. 1998; 14: 197–230. PubMed Abstract \| Publisher Full Text\n\nBuckley SMK, Delhove JMKM, Perocheau DP, et al.: In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters. Sci Rep. 2015; 5: 11842. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDodd KW, Burns TC, Wiesner SM, et al.: Transgenic mice expressing luciferase under a 4.5 kb tyrosine hydroxylase promoter. Cureus. 2011; 3(8): e34. Publisher Full Text\n\nRojas-Fernandez A, Herhaus L, Macartney T, et al.: Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9. Sci Rep. 2015; 5: 9811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOffice UH: Annual statistics of scientific procedures on living animals Great Britain 2015. 2013.Reference Source\n\nZhang N, Ahsan MH, Purchio AF, et al.: Serum amyloid A-luciferase transgenic mice: response to sepsis, acute arthritis, and contact hypersensitivity and the effects of proteasome inhibition. J Immunol. 2005; 174(12): 8125–8134. PubMed Abstract \| Publisher Full Text\n\nKarda R, Delhove JM, Buckley SM, et al.: Generation of Light-Emitting Somatic-Transgenic Mice for Disease Modelling of Hypoxic Ischaemic Encephalopathy. Reprod Sci. 2015; 22: 174A–174A.\n\nWu L, Zhao H, Weng H, et al.: Lasting effects of general anesthetics on the brain in the young and elderly: \"mixed picture\" of neurotoxicity, neuroprotection and cognitive impairment. J Anesth. 2019; 33(2): 321–335. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarda R, Perocheau DP, Suff N, et al.: Continual conscious bioluminescent imaging in freely moving somatotransgenic mice. Sci Rep. 2017; 7(1): 6374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFagone P, Wright JF, Nathwani AC, et al.: Systemic errors in quantitative polymerase chain reaction titration of self-complementary adeno-associated viral vectors and improved alternative methods. Hum Gene Ther Methods. 2012; 23(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZeltner N, Kohlbrenner E, Clément N, et al.: Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors. Gene Ther. 2010; 17(7): 872–879. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim JY, Ash RT, Ceballos-Diaz C, et al.: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo. Eur J Neurosci. 2013; 37(8): 1203–1220. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang M, Ramachandran A, Yan HM, et al.: Osteopontin is an initial mediator of inflammation and liver injury during obstructive cholestasis after bile duct ligation in mice. Toxicol Lett. 2014; 224(2): 186–195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelhove JMKM, Buckley SMK, Perocheau DP, et al.: Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice. Sci Rep. 2017; 7: 41874. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelhove J: Non-invasive somatotransgenic bioimaging in living animals. 2020. http://www.doi.org/10.17605/OSF.IO/XF7EZ\n\nDiaz JA, Geard A, FitzPatrick LM, et al.: Continual Conscious Bioluminescent Imaging in Freely Moving Mice. Methods Mol Biol. 2020; 2081: 161–175. PubMed Abstract \| Publisher Full Text\n\nHeinrich S, Georgiev P, Weber A, et al.: Partial bile duct ligation in mice: a novel model of acute cholestasis. Surgery. 2011; 149(3): 445–451. PubMed Abstract \| Publisher Full Text" }	[ { "id": "72772", "date": "03 Nov 2020", "name": "Tingting Xu", "expertise": [ "Reviewer Expertise bioimaging", "bioreporter" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the somatotransgenic bioimaging technology in living mice using transcription factor activated luciferase reporter (TFAR) delivered by viral vectors. Specifically, the manuscript describes in detail: 1) design and cloning of transcription factor-minimal promoters, 2) design and production of lentiviral and adeno-associated viral delivery vectors, 3) imaging of HIF response element in response to different anesthesia carrier gas, and 4) imaging of GFAP activity in liver injury. Overall, the somatrotransgenic bioimaging technology is advantageous over the traditional transgenic model by significantly reducing the number of animals needed. This manuscript also demonstrates imaging in conscious animals which further reduces animal stressed associated with anesthesia.\nSome details of the method/protocol is missing:\n\nPage 8, left column, under 'Whole-body bioluminescence imaging. How many mg D-luciferin per g of body weight?\n\nPage 10, left column, bullet point 18. What serial dilutions were used as standards?\n\nPage 11, right column, bullet points 12 and 13. Please provide more details about how to perform these preliminary experiments.\nData still needed to support the conclusion:\n\nIn vitro validation data of GFAP reporter\n\nActual animal images of Figure 3 and Figure 4. This is an animal bioimaging manuscript after all. Need the animal photos.\n\nPage 13, figure 4: Any statistics performed to compare the Sham and BDL groups?\nOther formatting issues:\nPage 12, left column, top 3 lines: Double check the figure numbers. They don't match.\n\nPage 11, left column, subtitles 'neonatal intracranial and intravenous injections' and 'continued monitoring of TFAR activity in anesthetised or conscious mice' : double check their formatting. As of now they appear to be part of notes, but they should be the subheadings for protocols.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "73989", "date": "16 Nov 2020", "name": "Antonius Plagge", "expertise": [ "Reviewer Expertise neurobiology", "lentivirus vectors", "bioluminescence", "preclinical imaging", "genetically modified mouse models", "epigenetics", "genomic imprinting", "neuroendocrinology" ], "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\nDelhove et al. describe Lenti- and Adenovirus vector-based methods to generate and analyse somatotransgenic mice that express firefly luciferase as a reporter gene to monitor via bioluminescene the response of transfected cells or tissues to various stimuli. They generated a Gateway system to easily add genetic promoter/response elements to a bicistronic cassette of Luc and eGFP. They describe a range of tested artificial promoter response elements (TFARs), which contain multiple copies of specific transcription factor binding sites and are useful to monitor certain physiological or developmental processes like hypoxia. Virus particles containing such reporter constructs are injected into neonatal mice, leading to transfection of endogenous cells in the surrounding tissue, which then permanently express luciferase and GFP. The authors demonstrate their techniques with the hypoxia induced response element (HRE) and a GFAP response element in in vitro and in vivo experiments. This protocol is supported by several publications from this author group over the last ~5 years, in which they continuously developed and refined their methods. In my view, this approach of somatotransgenic luciferase reporter gene mice, which cannot be maintained by breeding and need to be generated newly for each experiment, can be useful in some experimental set-ups, but cannot fully replace germline transgenic or knock-in mice that express luciferase from cell-type specific promoters. The manuscript is overall well written and detailed, thus allowing replication of the methods by other groups. The authors mentioned that they would distribute their plasmid constructs upon request, but I was wondering whether they are considering to deposit them with Addgene for easier and more general availability?\n\nThe injection of virus particles into neonatal mice (intracranial, iv) requires training and well developed skills. The authors didn’t really comment on potential side effects from such procedures. For example, what are the chances that mice develop permanent brain damage following intracranial virus injections? What is the rate of losses of neonatal mice following injections? Such details should be discussed in the paper.\nThe virus injections might label various cell types in the tissue or region surrounding the injection site. The response of those cell types to the challenge provided, e.g. hypoxia, might differ and this might therefore not be recognised with this approach. Can this be discussed in more detail?\n\nThe authors should add references to table 1 TFARS, where validated by publications. The last factor cassette TFEB / autophagy seems to be limited to cell culture? Please clarify.\nNote 12 on page 11 – subcutaneous luciferin injection should be added as a recommended (Perkin Elmer) alternative injection route.\nPage 12 top: “Here we present data validating our biosensing constructs in vitro (Figure 1) prior to in vivo (Figure 2) validation and experimentation.” Should read Figures 2 and 3\nFigure 3 legend: please clarify whether these were intracranial injections.\nThe GFAP promoter/response element used in Figure 4 does not seem to be listed in table 1? Please clarify and provide more detail.\nPlease clarify for all relevant figures whether ΔBioluminescence is based on difference in radiance (photons/sec/cm2/sr) or on difference in Flux (photons/sec).\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre the 3Rs implications of the work described accurately? Yes\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "96779", "date": "13 Dec 2021", "name": "Felicity Gavins", "expertise": [ "Reviewer Expertise Inflammation and immune cell trafficking", "pharmacology and preclinical imaging. Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Delhove et al., focusses on trying to address some of the issues that surround the use of bioluminescence imaging e.g. the wastage of large numbers of animals, unwanted background luciferase activity during bioluminescent imaging, and the time consuming and costly process of genome editing for luciferase transgene expression. To overcome these issues, the authors developed tissue-restricted transcription factor activated luciferase reporter (TFAR) cassettes in animals (a method termed ‘somatotransgenic bioimaging’).\nOverall, this is a nicely written and interesting methodology article. The fact that the authors have been developing and fine tuning their methodology over the last few years, makes the protocol one which is likely to be taken up by other researchers wanting to perform bioluminescence imaging or more specifically, somatotransgenic bioimaging, to address their scientific questions. That being said, it would have been nice to see a more open and easily accessible way for researchers to be able to have access to the TFARs, rather than solely gaining access on request. In addition, it would have been helpful to have more information regarding the impact and uptake of this type of methodology, especially in the context of the 3Rs in other areas of research, not just in the context of liver disease models.\nThe 3Rs benefit seems somewhat inflated with respect to the numbers of animals that could be reduced if somatotransgenic bioimaging were performed. The authors discuss that in their longitudinal imaging experiments they use 6-8 somatotransgenic experimental and 6-8 control animals per experiment, which translates into 7-10 fold reduction in the number of animals used in liver disease models as compared to the current standard methodologies of analysis. However, while there is no doubt that the use of somatotransgenic bioimaging may reduce animal use, the reduction proposed may be higher than that suggested by the authors because e.g. the periodic sacrifice for histological analysis may still be needed, especially by other groups.\nTable 1 is useful, but references for each response element would be helpful. In addition, the response elements used in the figures do not seem to be present in the table. These should be added.\nThe conscious imaging element that the authors proposed is interesting and physiologically relevant. However, is the imaging only possible using the specific custom-made “periscopic chamber to permit simultaneous collection of light emission from both ventral and dorsal surfaces” (Karda et al., 2017)?\nUnder the paragraph entitled ‘Whole-body bioluminescence imaging’, it is not clear as to whether the ip injection of D-luciferin was administered per gram weight of the animal.\nThe notes provided in the methods section are helpful. However, as this is a method article of a novel methodology, more information should be provided. This may also increase uptake of this specific method e.g. can the authors provide data obtained for the alternative routes of administration of the D-lucferin,\nThe authors mention that it is best to use “white-furred mice as the black-furred mice prevent the bioluminescence from penetrating through”. Given the number of transgenic animals that are on C57Bl/6 background, this statement would seem rather confusing. Would the authors provide more information as to how researchers should deal with these issues e.g. shaving of the mice.\nProviding kinetic experiments of D-luciferin for different cell and tissue targets would be a helpful addition.\nFigure 1 seems to be missing, or the figures are incorrectly labelled?\nFigure 2. Baseline (t=0h) levels should be provided.\nFigure legends need more information so that the reader does not need to refer back to the article e.g. Figure 3 ”transduced animals were imaged….”. Where were they imaged? More details needed regarding the TFAR.\nNo imaging pictures have been provided. These should be included.\nFigure 4 is lacking statistics. If nothing was significant, this should be made clear in the figure legend and article text.\n\nAre a suitable application and appropriate end-users identified? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre the 3Rs implications of the work described accurately? Partly\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1216
https://f1000research.com/articles/9-971/v1	11 Aug 20	{ "type": "Research Article", "title": "Occupational structure of bearers of Jewish rabbinical, occupational and generic surnames", "authors": [ "Alexander Jonathan Vidgop", "Nelly Norton", "Nechama Rosenberg", "Malka Haguel-Spitzberg", "Itzhak Fouxon", "Alexander Jonathan Vidgop", "Nelly Norton", "Nechama Rosenberg", "Malka Haguel-Spitzberg" ], "abstract": "We study choice of profession in three groups of Russian-speaking Jewish families with different occupational distributions of the ancestors. This study continues exploration of the persistence of social status of families over centuries that was initiated in recent years. It was found previously that in some cases professions remain associated with the same surnames for many generations. Here the studied groups are defined by a class of the surname of individuals composing them. The class serves as a label that indicates a professional bias of the ancestors of the individual. One group are the bearers of the class of surnames which were used by rabbinical dynasties. The other group is constituted by occupational surnames, mostly connected to crafts. Finally, the last group are generic Jewish names defined as surnames belonging to neither of the above groups. We use the database that consists of 858 and 1057 of the first two groups, respectively, and 7471 generic Jewish surnames. The statistics of the database are those of individuals drawn at random from the considered groups. We determine shares of members of the groups working in a given type of occupations together with the confidence interval. The occupational type’s definition agrees with International Standard Classification of Occupations. It is demonstrated that there is a statistically significant difference in the occupational structure of the three groups that holds beyond the uncertainty allowed by 95% confidence interval. We quantify the difference with a numerical measure of the overlap of professional preferences of different groups. We conclude that in our study the occupational bias of different population groups is preserved at least for two centuries that passed since the considered surnames appeared.", "keywords": [ "Occupational Structure", "Statistics", "Intergenerational Mobility" ], "content": "Introduction\n\nRecently usage of surnames in studies of intergenerational mobility, such as investigations of temporal changes of representations of different surnames in various social groups, has developed into an established tool of research (see e.g. Clark, 2015; Clark, 2012; Clark et al., 2015; Güell et al., 2015 and references therein; Santavirta & Stuhler, 2019 for a recent review). In a typical study, the frequency of occurrence of certain surnames in different professional or elite or other population groups is considered. This frequency is compared with the frequency of the surname’s occurrence in the general population. If it is found that the surname occurs in some group significantly more than in the general population, then the surname is overrepresented in the group. Conversely, if the surname occurs less, then it is underrepresented.\n\nProbably the main result of the aforementioned extensive studies is that the over- or under-representations do not change over long periods of time, much longer than would be implied by the conventional mobility measures. Those measures average over society, thus hiding the underlying low mobility rates for a given surname. These studies have been performed for different countries and cultures (see Clark, 2015 and references therein).\n\nIn this work, we perform a study for a population of Russian-speaking Jews who have not been so far considered in this type of study. We investigate occupational distribution of three different groups in the population that are defined by different biases in the occupational distribution of their ancestors. The size of 9315 individuals of the studied pool of data allows us to derive rigorous statistics of the groups (we use here rounding, explained below). We demonstrate that the distributions are different beyond the uncertainty allowed by the confidence interval. This finding shows that biases in occupational distributions can be preserved for at least two centuries that passed, since all Jews have had an inherited surname. This provides yet another demonstration of comparatively slow surnames’ mobility, defined as social mobility of individuals with a given surname, (Clark, 2015). Our results also provide the occupational distribution of the Russian speaking Jewry of the twentieth century, a result that has its own interest.\n\nThe objectives of our study are quite similar to those of Clark (2012), where the statistics of several groups of surnames in Sweden are considered. Clark (2012) considered noble surnames, names that were once given to nobility; Latinized surnames were adopted by the educated class and certain other groups. Similarly, we consider rabbinical surnames (counterpart of nobility), surnames of craftsmen (hereafter called occupational), and others that fall outside of these categories (generic).\n\nRabbinical surnames are those whose first bearer was a rabbi. Rabbis constituted the elite of their time, the most respected class of the Jewish population, and they can be considered as a kind of the Jewish nobility. The rabbi of the surname’s origin could be a prominent figure living many centuries ago, for example as in the case of Luria, Shapiro or Halperin. Family names of these dynasties were often taken as a sign of distinction. However, other rabbinical families are “only” two hundred years old, for example Rabinovich. For these clans, in the beginning of their history, the profession of the rabbi was often passed from father to son for a number of generations. Occupational surnames derive from the name of the craft of the first bearer of the name. Craftsmen had the knowledge of their craft and present a rough counterpart to the educated class considered by Clark (2012), for example Schuster (shoemaker), Mednik (tinker) and Portnoi (tailor). In these families the professions were also often passed between generations. Finally, generic surnames consist mostly of surnames whose origin has nothing to do with the professions of their founder.\n\n\nMethods\n\nOur data was acquired over four years (November 2015 - February 2020) from individuals who were part of an educational family history program that was implemented by the Am haZikaron Institute for Science and Heritage of the Jewish People in Tel Aviv, Israel.\n\nThe individuals were Russian-speaking Jewish family members residing in the Former Soviet Union (FSU). They voluntarily provided genealogical data for the program via online forms that were sent to them before their arrival in Tel Aviv (see section Data collection). The forms were presented in the native language of the individual (Russian) and informed the individuals that their data could be used for future academic purposes. Completion of the form was taken as consent to allow their data to be used for this academic study (some participants chose not to complete the form). This study did not seek ethical approval as it was deemed low risk, none of the participants were considered vulnerable, the participants consented for their data to be used in future academic research, and all participants were over 18 years of age.\n\nData from an individual was not selected for inclusion in this study if it was intentionally distorted. Data verification was accomplished during a meeting in Tel Aviv between the authors.\n\nInformation was obtained on professions of a participant’s family members for the last four generations. This resulted in a collection of data on individuals who were born throughout the 20th century. The educational programs at Am haZikaron are open to all, and so there was no known statistical bias toward any particular professions. Similarly, there is no known correlation between the starting letter of the name and the profession. Hence, to the best of our judgement, the obtained data, arranged alphabetically, is a random list of Jewish individuals and their professions. The randomness holds up to small clusters of individuals who belong to the same family and have some correlations. These correlations are yet to be studied and are not the focus of our study. It will be seen later that randomness is consistent with the statistics.\n\nIn the research by Clark et al (Clark, 2015; Clark, 2012; Clark et al., 2015), the major source of information were professional directories that list all individuals in a particular professional area. In contrast, our data is a random pool of the population that necessitates a different methodology for analysis. We partitioned the population into the three groups of surnames (rabbinical, occupational, generic), which could be biased with respect to their occupational distribution due to the bias in their ancestry. We checked if the bias persists through time and found that there is a statistically significant difference in the professional preferences of the three groups.\n\nWe collected the data over four years until the pool included a statistically significant amount of surnames where the confidence interval allowed to reasonably fix the share of each profession in the total population. We obtained data on 858 (57.8.% men) bearers of rabbinical and 1057 (59.7% men) bearers of occupational surnames. The other 7471 (57.6% men) individuals had a generic surname, which was neither rabbinical not occupational. Men are slightly overrepresented since the maiden names of the female family members were sometimes unknown to the participants. This slight difference in gender composition of the groups may cause some professional bias; however, this is negligible compared with the magnitude of the groups’ differences (shown below).\n\nThe studied population included different birth cohorts. The earliest birth date for an individual in the data was 1858 and the latest 2001. For adequate comparison of the groups, we must have roughly the same share of each group born in each of the considered generations. Therefore, we divided the historical period spanned by our data into four periods (1858-1894, 1895-1930, 1931-1966, 1967-2001; Table 1).\n\nFrom Table 1, it is seen that the birth date distributions of the groups are very similar so that the comparison of occupations is reasonable. The difference of shares of different generations holds for many reasons: each participant was asked to fill the data for two parents, four grandparents and eight great grandparents, where the data on the older generations was often forgotten, while the younger generation could still be studying or have no profession yet. However, the precise form of the birth date distribution is irrelevant for our comparative study, for only birth date distributions of different groups are similar.\n\nThe birthplaces were scattered all over the territory of the FSU. Jewish families have a long tradition of studying and those who would want to acquire education would typically receive such an opportunity. In other words, with a good approximation, an individual born into a Jewish family of the FSU would have an equal opportunity for getting that or another profession irrespective of birthplace. Therefore, we disregarded the geographical factor in our study.\n\nNo detectable bias toward some profession due to a different number of reported family members was observed. This number was never too large, and rarely reached five individuals (other ancestors were not Jewish and not considered by our study).\n\nThe data on professions was self-reported in the native (Russian) language of the participant and was not standardized. We processed the data to a standard of occupations according to the methodology below.\n\nGrouping the data by profession. We separated the data into the three groups described (rabbinical, occupational, generic). We then grouped the professions into 23 narrower professional activities. These were defined either by their significant presence in the data, e.g. bookkeepers who constituted about 5% of all individuals, or by a unique character of the profession, for example interpreter/linguist. The groupings of professions, when performed, did not contradict the International Standard Classification of Occupations. The 23 categories of professions were as follows:\n\n1. Engineer – by far the largest fraction of the studied population\n\n2. Physician\n\n3. Teacher\n\n4. Bookkeeper\n\n5. Worker\n\n6. Creative profession\n\n7. Economist\n\n8. Head/chief officer\n\n9. Nurse\n\n10. Researcher\n\n11. Clerical worker\n\n12. Armed forces\n\n13. Programmer\n\n14. Salesmen\n\n15. Businessman\n\n16. Legal professional\n\n17. Driver\n\n18. Interpreter, linguist\n\n19. Literary worker\n\n20. Pharmacist\n\n21. Librarian\n\n22. Psychologist\n\n23. Rarely occurring profession (other)\n\nWe calculated the number of individuals having each one of the above professions for each of the three studied groups of surnames. The main target of our study is the share of each profession (Pi) in each of the considered three groups of surnames. Thus, if N is the total number of members of Russian-speaking Jewish families with a generic surname then PiN is the total number of members of these families with the i-th profession. For instance, P21N would be the total number of librarians. The data on the full group consisting of millions of people (as defined by the fraction of Russian-speaking Jews whose names are neither rabbinical nor occupational where we count not only our contemporaries but all those who lived in the twentieth century) are unavailable. Thus, we have the standard problem of constraining Pi from the incomplete information on the studied groups that is at our disposal. This is done by the statistical analysis relying on the observation that with good approximation our data constitute a random pool of the considered population groups.\n\nA typical result of counting the professions is presented in Table 2 where the example of generic surnames is used. The total pool of data consisted of 7471 individuals. Due to presence of correlated clusters of individuals in the data, we found it instructive to use coarse-grained variables Xi(k) for the statistical calculations. These variables separate the data into blocks of hundreds. For the pool size of 100, the statistical properties are seen already. Frequencies of different professions in each block of 100 are similar with some fluctuations. The usage of the variables Xi(k) is necessary for the statistical considerations explained in detail below, otherwise they give an idea how the described laws apply in practice when the sample sizes are moderate. The statistics of Xi(k) answers the question – if we took a pool of 100 representatives of one of the groups what would be the typical occurrences of each profession?\n\nThe first column provides the profession. The entries of the first row provide the considered range of numbers of people in the list considered in the corresponding column. For instance, the second column describes occupational distribution of 100 individuals with numbers from 1 to 100, the third column describes 100 individuals with numbers from 101 to 200 and so forth. Thus X7(300) is the number of individuals with the profession of economist in the range from 201 to 300. This number is located at the intersection of eighth row and fourth column, X7(300)=2 – our list contains two economists in the portion of the list defined by 201–300 range.\n\nThus we took out of our pool the first 100 individuals and determined the numbers Xi(100) of individuals with the i-th profession. Then we took 100 more individuals and determined the numbers Xi(200) of individuals with the i-th profession in the range of 101–200. Continuing in this way, we determined Xi(k) determined by the columns in Table 2. We used the pool size up to 1000 because this size allows comparison with the groups of rabbinical and occupational surnames where the total pool was about 1000. Thus, we used the pool of 7471 as the control size that allows to test how well the pool of 1000 individuals represents the whole considered group. Average over a limited pool size k gives an approximation pi(k) for Pi defined above. Up to fluctuations pi(k) monotonously approach Pi on increasing k and we can hope that a reasonable approximation to Pi can be obtained already from the largest k available from our data. Indeed, we demonstrate quantitatively that distributions derived from the pools of 1000 and 7471 individuals are rather similar. Thus, making the reasonable assumption that pool size of 7471 represents the full group accurately, which is proved by the calculation of confidence intervals, we conclude that the occupational distribution of the generic surnames can be derived quite accurately (quantified below) from the distribution of 1000 individuals. Assuming that the groups of bearers of rabbinical and occupational surnames are similar statistically we can then conclude that the distributions for these groups, derived from the study of about 1000 individuals, provide a good characterization of the full groups. Despite that, we rely in our conclusions on the rigorously defined confidence intervals; qualitatively it is probable that the means obtained from studies of 858 rabbinical and 1057 occupational surnames provide accurate idea of the full groups.\n\nDistance between the distributions. There was a need for quantitative comparison of distributions Pi of the three considered groups (Figure 1). Thus, considering the finite pool sizes’ approximations to Pi in Figure 1 (in %), it is seen that they are different; however how different? To make the comparison, we calculated the distance between the distributions. There is no unique conventional definition of the distance between probability distributions. We used the Hellinger distance (see for example. Yang & Le Cam, 2000), whose definition can be understood by observing that since the distributions are normalized, ∑i=123Pi=1, then what needs the comparison are the shapes of the distributions. For instance, for all three distributions in Figure 1, the heights of all bars sum to 100 and only the shapes distinguish the distributions. The shapes can be compared by considering the overlap, which is conveniently defined by the Bhattacharyya coefficient (Bhattacharyya, 1943):\n\n\n\nwhere k and p are indices of the considered two groups (in this equation and below); when there is a need to indicate to which group Pi refers, we use the notation Pir, Pio, Pig, for the Pi of rabbinical, occupational and generic surnames, respectively. The Bhattacharyya coefficient is the scalar product (type of overlap) of two vectors in 23-dimensional space with components Pik and Pip. The square root is introduced in the definition because Pik are unit vectors in the 23-dimensional space, which allows definition of “the shape of the distribution” as the direction of these unit vectors. The coefficient changes between zero, holding for non-overlapping distributions, and one, holding for identical distributions. The Hellinger distance between the distributions is then defined as:\n\n\n\nThus, H(k, p) is proportional to the Euclidean distance between two vectors Pik and Pip in the 23 dimensional spaces. It provides a good definition of the distance because the Euclidean distance does.\n\nConfidence interval under the random sampling assumption\n\nOur sample consists of 858 bearers of rabbinical surnames, 1057 bearers of occupational surnames and 7471 bearers of generic surnames (we often use in the calculations the rounded number of 7400). These samples are quite large; however, the population means that can be derived from them still contain quite a large uncertainty. Here we describe the derivation of the confidence interval, i.e. the interval within which the population means are contained with high probability (95% probability in our study). The derivation in this section is done assuming that our list of individuals is a random sample of the studied population groups. This is a good assumption up to the presence of sequences of 3–5 individuals with the same surname who belong to the same close family. These individuals can be assumed to have a certain correlation of professions, which violates the assumption of random sampling. The consistency of the assumption of random sampling despite these correlations will be demonstrated in the next section.\n\nThe material in the rest of this section is mostly well-known. We consider a total population of X individuals, of whom Xi have a property “i”. In the application of interest in this work, this property is a certain profession; however, the nature of this property is irrelevant for general considerations. We make a random sampling of the population, i.e. we pick an individual at random. Then, by definition of random sampling, the individual with property “i” is picked with probability pi = Xi/X. If we continue the random sampling, then the probability distribution of the number Xi(N) of individuals with the property “i” in randomly picked N individuals is given by the binomial distribution with the success probability pi. Here we assume that N is much smaller than both Xi and X so that the random sampling occurs in approximately identical conditions. The average and variance of Xi(N) are given by the well-known formulas of binomial distribution\n\n\n\nwhere here and below the angular brackets stand for averaging. Large N binomial distribution can be approximated by Gaussian, implying that the distribution of Xi(N) is Gaussian and is determined uniquely by the mean and the variance above. We find that the distribution of x≡(Xi(N)- pi N)/[pi N(1- pi)]1/2 is the standard normal distribution with zero mean and unit variance. For this distribution, it is well-known that the probability that x will fall between -1.96 and 1.96 is approximately 0.95. This probability equals the probability that Xi(N)/N falls between pi-[pi(1- pi)/N]1/2 1.96 and pi+[pi(1- pi)/N]1/2 1.96 that is designated by P(pi-1.96pi(1-pi)/N≤Xi(N)/N≤pi+1.96pi(1-pi)/N) and obeys\n\n\n\nOur data provides Xi(N)/N, from which we want to find the confidence interval of pi that is the interval to which pi belongs with 95% certainty. If the observation provides the value Xobs for Xi(N) then, with 95% probability, the unknown quantity pi obeys\n\n\n\nThis inequality is equivalent to (Xobs - pi N)2 ≤1.962 pi N(1- pi), which gives\n\n\n\nWe find\n\n\n\nwhere we defined\n\n\n\nWe have neglected terms of order 1/N and introducing (pi)obs≡ Xobs/N that\n\n\n\nwith 95% probability. This has the same form as Equation (2) above because pi and (pi)obs coincide in the leading order in N>>1. For not so large sample, however, there is a difference, the property which is often not mentioned in the discussions.\n\nIn the rest of this work we keep using the definition of the confidence interval by 95% certainty. This and the factor of 1.96 above are somewhat arbitrary. If we used 90% confidence interval instead, then 1.645 would be present in the formula above instead of 1.96. This would result in more differences between the studied groups; however, we prefer to stick to the more conservative estimate of the differences.\n\nData consistency with random sampling. We have already reported that the assumption of perfectly random sampling is not accurate. However, our data is still composed of independent information units where the unit is defined by the information provided by one participant. This unit is the information on the close family of the participant. Hence for a large number of participants, considering with very good approximation the information that they provide as independent, the data is still the sum of independent identically distributed random variables. The variable is the number of people with profession “i” in one reported family. Therefore, by the central limit theorem (see e.g. Gnedenko & Kolmogorov, 1968), the distribution of the number of representatives of each profession is still Gaussian, as in the case of random sampling. Having the Gaussian distribution, we can then evaluate the confidence interval and obtain the appropriate generalization of the results of the previous section. Below we quantify these considerations and provide the changes that are in order.\n\nTable 3 provides a typical excerpt from our list of individuals and their professions. It is seen that some surnames are included multiple times. In Table 3, individuals with the same surname do not necessarily belong to the same family, as seen from the places of origin. In some cases (not shown), the bearers of the same names did belong to the same family and had similar professions. For instance in the list of 858 individuals with rabbinical surnames, we found two Berlins who were architects, two Wahls who were salesmen, two Halperins who were accountants, two Hellers who were teachers, three Hellers who were engineers, four Hellers who were economists, two Ginzburgs who were accountants and two who worked in delivery, two Gordovers who were engineers, two Gordons who were workers, and nine Horowitzs who were engineers. These correlations might cause the statistics to behave differently from the situation where each individual is picked at random from the studied group. This issue must be considered quantitatively. We observed previously that the number of reported people from the same family rarely exceeded five and typically was much less. For quantitative treatment of the effect of these correlations of limited range (below five), we consider the statistics of Xi(N) – the number of individuals with profession “i” in the list with total number of individuals N. We introduce the random variable xi(p). This variable equals 1 if the individual in place “p” of the list has profession “i” and 0 otherwise. Then\n\n\n\nSome surnames are different spellings of the same name. These spellings were created in the course of family migrations during centuries because spellings of the same name in official documents differed in different countries, for example due to spelling mistakes in the records.\n\nWe assume that the list is ordered so that individuals with possible correlations of professions are grouped together; the list can be thought of as obtained in this way. We pick at random individuals from the considered group (bearers of rabbinical or occupational or generic surname) and then we pick their close family of random size as determined by the statistics of family sizes (which is of no interest here). This implies that xi(p) in the equation above have finite correlation range so that the variance < xi(p) xi(p+k)>- < xi(p) > < xi(p+k)> is non-zero for some positive integer k. k is bounded from above by kmax, which is formally given by the maximal family size, which is fifteen (individual, parents, great and great great-parents); however, this is five in reality as we have already explained. For large N>>kmax the distribution of Xi(N) is still Gaussian as in the case of random sampling. This can be seen as the result of application of the central limit theorem to the sum of independent random variables where each variable is the sum of xi(p) over one family, i.e.\n\n\n\nHere we introduced the random variable (yfamily)l , which counts the number of representatives of profession “i” in the l-th family where l is the index of the family. Thus the statistics of (yfamily)l could be obtained by partitioning the considered group of population (rabbinical, occupational or generic) into families, where the family is defined as information unit in our data and not otherwise, with the unit’s definition given in the beginning of this section. Considering (yfamily)l with different l as independent, we conclude from the central limit theorem that Xi(N) is approximately Gaussian random variable, which is uniquely characterized by its mean and variance (a finer consideration can be done using the version of the central limit theorem for random variables with fast decaying correlations, see Gnedenko and Kolmogorov). These are given by\n\n\n\nwhere we used directly the definition Xi(N)= ∑Np=1 xi(p). Here <xi(p)>= pi, where pi were defined in the previous section. We can consider the last sum as sum of diagonal and off-diagonal elements. The sum over the diagonal elements is readily found by using that xi(p)=1 with probability pi and xi(p)=0 with probability 1-pi. Thus <x2i(p)>=pi and\n\n\n\nwhich is the same answer as for the random sampling. It is the sum of the off-diagonal terms ∑p≠k (<xi(p) xi(k)>- <xi(p) > <xi(k)>), a non-trivial quantity, which characterizes the correlations of professions within one family. Thus <xi(p) xi(p+1)> is the probability that two consecutive individuals from the list have identical profession “i”. This probability is larger than <xi(p)> <xi(p+1)>, which would hold if professions of the individuals were independent. The finite difference <xi(p) xi(p+1)>- <xi(p)> <xi(p+1)> is caused by the finite probability that individuals p and p+1 belong to the same family and thus have positively correlated professions. It seems inevitable that within-family correlations are positive so that\n\n\n\nThe left-hand side of the above equation is identically zero for k> kmax (see above). It is then readily seen from the definition in Equation (3) above that the variance obeys\n\n\n\nwhere the constant ci is independent of N. The question is how different ci is from the random sampling value pi(1-pi) because of the described correlations.\n\nWe face the problem of estimating the normalized dispersion ci for our unknown sampling statistics. This can be done quite accurately in the case of bearers of generic surnames where we have data on more than 7400 individuals. This is accomplished by partitioning the list into 74 hundreds and considering the corresponding 74 numbers [Xi(100)]k with k running from 1 to 74 as independent realizations of Xi(100). Here the independence of [Xi(100)]k with different k holds due to 100>>kmax, which implies that the number of correlated professions in different hundreds is negligibly small in comparison with the total numbers. Indeed, we have\n\n\n\nwhere <xi(p)(xi(k)> differs from <xi(p)><xi(k)> only for indices p and k in narrow vicinity of p=k=100, which can be neglected. We find the independent variables law <[Xi(100)]k[Xi(100)]k+1>=<[Xi(100)]k><[Xi(100)]k+1> (similar consideration can be done for higher order correlations). This independence is the reason why we group the data in blocks of 100 (of course the block size is not defined uniquely). We observe that Yi≡(1/74) ∑74k=1([Xi(100)] k)2 is a sum of independent Gaussian variables and hence it is Gaussian itself. The average of Yi is <(Xi(100))2> and its dispersion is\n\n\n\nwhere we used independence of [Xi(100)] k with different k and that Gaussianity of [Xi(100)] k implies <([Xi(100)] k)4>=3 <([Xi(100)] k)2>2=3 <(Xi(100)])2>2. Thus the distribution of (Yi/<(Xi(100))2>-1) √37 is the standard normal distribution with zero mean and unit variance. We find that the value of Yi obtained from our data limits <(Xi(100))2> within the interval given by\n\n\n\nwith the confidence level of 95% (see above). The random sampling would give <(Xi(N))2>= pi N+N(N-1) pi2 with N=100 as seen readily from the properties of the binomial distribution. The comparison between the dispersion determined from our data and the dispersion predicted by the random sampling assumption is provided in Table 4.\n\nThe agreement is found to be much narrower than allowed by the confidence interval provided in the fourth and fifth columns.\n\nWe find that the observed dispersion agrees with the prediction of the random sampling assumption with accuracy which is well beyond what could be hoped for, as is clear from the confidence interval. Here we used for pi the values obtained from averaging over the sample of more than 7400 individuals, where we neglect the error using the large sample size. The observed agreement over as many as 23 categories is completely consistent with the assumption that the data are equivalent to random sampling of the group of bearers of generic surnames. In other words, the correlations between the profession of different individuals, which are present in the data, are negligible. Since these correlations do not seem to be different for other groups (bearers of rabbinical and artisanal surnames) then we will assume in the Results below that our data provides the random sampling of all the considered population groups. We have also derived dispersion for other groups and saw that the assumption works well (these comparisons are not provided since for these groups the sample of about 1000 individuals is too small for reaching rigorous conclusions).\n\n\nResults\n\nThe results for the occupational distribution of the generic surnames derived from data presented in Table 2 are given in Table 5. For this surnames’ class we have a rather large pool, which allows us to obtain a distribution with high accuracy, as presented in the third column, which gives the mean together with the confidence interval. It is seen that the means are fixed rather sharply and for many categories the range of values around the mean, allowed by the confidence interval, is narrow. Yet sharper results hold for the total pool of 9315 individuals consisting of all the surnames, i.e. the generic, rabbinical and occupational surnames together (here 9315 is found using the data on only 7400 out of 7471 individuals with generic surname). The distribution, presented in the fourth column, provides us with rather detailed information on the occupations of the Russian-speaking Jews that seemingly was not considered previously. Finally, the second column presents the same distribution, however obtained by restricting the pool of generic surnames to the first 1000 individuals. This distribution is provided for comparison with rabbinical and occupational surnames in Table 6 where the total available pool is about 1000 in both cases.\n\nThe third column provides population means together with the confidence interval as derived from the full pool of 7400 individuals. For comparison with other surnames’ classes, we present in the second column also the distribution that would be derived by using only the first 1000 names.\n\nThe second column provides the distribution of the generic surnames (considered previously), the third gives the distribution for the joined classes of generic and rabbinical surnames and the fourth for generic and occupational surnames.\n\nWe observe that the distributions of bearers of generic surnames and of the total considered population are rather similar. In fact, within the confidence interval, the distributions agree (we observe however quite significant difference in the predicted means in the rows marked with blue color). The Bhattacharyya coefficient of these distributions is 0.9998 and the Hellinger distance between the distributions is 0.014 (the calculation demands the full and not rounded numbers). The coefficient is very close to one and the distance is very small; however, the interpretation of these numbers is not obvious. We need a scale to tell which distance is large and which small because the vectors representing the distributions belong to a high-dimensional space. For comparison, the coefficient and the distance for the distributions of the generic surnames in the second and third columns of the table are 0.9975 and 0.05 respectively. These numbers are also very close to one or small despite the difference of the distributions being quite appreciable (the difficulty in introducing measures of similarity in high dimensional spaces is sometimes known as “the dimensionality curse”; see e.g. Aggarwal et al., 2001).\n\nThus, we compare the distributions of generic and all surnames directly (Table 5). The distributions, as given by the mean values, are very similar. We marked in blue the only three rows for which the distributions differ appreciably where the maximal difference is 12%. The distributions’ difference is insignificant both because the share of the rabbinical and occupational surnames in the total population is not that large (~20%), and the difference of the distributions of the generic, rabbinical and occupational surnames is not very large. Yet the difference exists, and it is statistically significant as we will demonstrate.\n\nThe lack of the appreciable difference between the total distribution and the generic distribution has the origin that is similar to that of measured high mobility rates in different countries. These measurements do not contradict low mobility rates measured by surname, as explained in Clark, 2015. Different surnames are over- or under-represented in different social groups for long periods of time; however, when society’s average is taken for deriving the mobility rate of society, the over- and under-representation average out producing overall high mobility rates. Similarly, the deviations of the rabbinical and occupational surnames’ distributions from that of the generic surnames often occur in opposite directions, so that after averaging the difference disappears. This point is illustrated in Table 6. We see that for the largest absolute deviations, marked in red, the deviations are opposite, including the row corresponding to head/chief officer. Indeed, for this row, the deviations of the generic and all surnames are strongest.\n\nFurthermore, we see from Table 5 that population means predicted from the study of 1000 and 7400 individuals are consistent within the confidence interval. Moreover, the average values coincide with high accuracy – for 13 occupational categories the difference is <20% (these rows are left unmarked). For the rest of the categories, marked in red, the difference is larger; however it is never dramatic – the statistics derived from the study of 1000 individuals gives a very good idea of the much more precise statistics derived from 7400 individuals.\n\nFinally, Table 7 presents the full data for the occupational distributions of the rabbinical, occupational and generic surnames. We saw on the example of the generic surnames (Table 5) that the means obtained from the pools of about 1000 individuals provide good orientation for the actual Pi. Therefore, we provide in separate columns the means of the three groups. It is seen that the differences are significant. For many categories, these differences continue beyond those allowed by the confidence intervals which are provided in the corresponding columns. Thus, we marked in red the rows where occupational distributions differ with 95% probability. We marked with blue the categories where the difference can be claimed with a slightly smaller probability of about 90%. The three Bhattacharyya coefficients and Hellinger distances measuring the similarity and difference of the considered three distributions are given respectively by\n\nBC(r,g)=0.988, H(r,g)=0.1095, BC(o,g)=0.9942, H(o,g)=0.0763, BC(o,r)=0.9818, H(o,r)=0.1348,\n\nShares Pi(in per cent) of the i-th profession are provided for each group together with their 95% confidence intervals. The sizes of the respective pools are provided in the first row. Red indicates Pi that are different beyond the statistical error; blue indicates those that are different with high probability, e.g. would differ if we used 90% confidence interval.\n\nwith obvious notations (e.g. H(r,g) is the distance between rabbinical and generic surnames’ distributions). We see that occupational and rabbinical surnames’ distributions are the most different pair whereas occupational and generic surnames’ distributions are the least different pair. This does not correspond to the differences in Table 7 (colored red/blue) where the largest number of differences is between the generic and rabbinical surnames. The reason is that the pools of rabbinical and occupational surnames are smaller, which results in a larger uncertainty due to finite confidence intervals. In contrast, the overlap coefficients and the distances above are derived from the mean values only and do not reflect the magnitude of the confidence intervals.\n\nWe recall that the overlap and distance for the distributions of 1000 and 7400 generic surnames are 0.9975 and 0.05, respectively. We see, by comparison with the equation above, that these distributions are closer than distributions of different groups which is necessary for consistency. Moreover, assuming that a similar difference between distributions of 1000 and 7400 would exist for rabbinical and occupational surnames (as would be found if we had a larger pool of data), we see that the numbers are consistent with the assumption that distributions of rabbinical and occupational surnames differ from the generic surnames’ distribution and from each other.\n\n\nDiscussion and Conclusions\n\nAn individual’s choice of profession is determined by a multitude of genetic and environmental factors that are largely unknown. However, undoubtedly the family into which the individual is born is one of the main factors of influence. Family differences could persist for many generations via choice of partners. Indeed, marriages occur between individuals having similar social and genetic backgrounds who can preserve the differences in their offspring (see Güell et al., 2015; Clark, 2015). This reproduction mechanism (which is not a literal transmission of profession from generation to generation, that was never present in our data, but rather a transmission of certain statistical preferences in occupational choices) is, however, imperfect. The differences caused by family origin gradually dissolve with time and their complete disappearance has been observed to take centuries (Clark, 2015). In this work, we continued this direction of studies by comparison of occupational differences of the three groups of Russian-speaking Jewish families.\n\nWe observed that having a surname at which origin was a rabbi, a craftsman or neither of the above categories would create a difference of occupational preferences of the individual. For instance, some fraction of individuals having a rabbinical surname are actually the descendants of the rabbi who was at the name’s origin (and not unrelated individuals with the same surname). This results in a difference of occupational preferences of members of this group from the average preferences of the population. Since these names originated from nine to two hundred years ago then the differences in the preferences could be negligibly small today. However, previous studies, such as those reviewed by Clark (2015), indicate that the differences can still be appreciable. Our study confirmed that in fact the occupational preferences of bearers of rabbinical, occupational and generic surnames differ beyond statistical uncertainty. We remark that the studied groups themselves typically do not identify themselves as different. It can be firmly stated that most of the Russian-speaking bearers of the rabbinical surnames are either completely unaware of their name’s origin or see in it little meaning to their lives. Similar facts hold for the other groups. This differs from some cases, e.g. the case of Swedish nobility (Clark, 2015).\n\nCan we understand the differences provided in Table 7? Most of the differences are readily explained by low mobility, making the assumption that bearers of the names correlate appreciably with the origin of the studied surnames. Thus, the proportion of engineers among the bearers of rabbinical surnames is higher than in the other two groups. This profession demands much study and abilities for complex mind constructions that is evidently present in the profession of Jewish rabbi. The largest difference between all the three groups is in the fraction of members of the groups who are workers. The fraction for rabbinical surnames is between 0.117 and 0.163; however for generic surnames it is enclosed between 0.177 and 0.195 and for occupational surnames it is between 0.208 and 0.264. The corresponding means of occupational and rabbinical surnames differ by 1.69 times. This is a significant difference, indicating the initial inclinations of bearers of rabbinical surnames for non-worker type of activity, and conversely inclinations of craftsmen toward that kind of activity, persisted for at least two hundred years of history. These conclusions and numbers are in complete accord with those provided by Clark (2015). We find that bearers of rabbinical surnames pick the profession of heads/chief officers more than twice as often than the bearers of generic surnames. This is reasonable since rabbis led their communities. The difference from occupational surnames is somewhat smaller. Another significant difference between the groups is that bearers of both occupational and rabbinical surnames have almost identical preferences for clerical work, which are less than those of generic surnames. Inclination for clerical work would not be anticipated from a rabbi or a craftsman.\n\nWe also calculated the occupational distribution of all the surnames, i.e. general Russian-speaking Jewish families, which is of its own interest. We demonstrated that the distribution is very similar to that of bearers of generic surnames. This indicates that, despite differences between generic, rabbinical and occupational surnames being pronounced, they average out in the distribution of the general population. This is both because the deviations of the occupational preferences of bearers of rabbinical and occupational surnames from those of generic surnames are often opposite and because those groups are not as many. Thus, in our pool, rabbinical and occupational surnames constitute 20.5% (1915 individuals) of the total, where 9.2% are rabbinical surnames.\n\nThe above difference between rabbinical, occupational and generic surnames finds reasonable explanation in the personal features of rabbis and craftsmen. Similarly, we could explain the bearers of occupational surnames preference for the profession of programmer. What came as a less intuitive result is that the share of researchers among the bearers of the rabbinical surnames is larger than the average; however not that large. Further studies are needed since the difference with our data can be claimed with less than 90% probability. The bearers of the rabbinical surnames were found to have preferences for creative professions that are larger, however not much larger than the average. The bearers of occupational professions have lower preference for these professions.\n\nProbably the most surprising of our findings is that bearers of rabbinical surnames have almost twice lower preference for legal professions than the rest of the population. It seems that since the profession of the rabbi demands the ability to learn and apply the religious law, flexibility of mind, and ability to defend sometimes opposite viewpoints, then it must be the opposite. Indeed, we checked the names of famous Russian Jewish lawyers and discovered that their surnames are overwhelmingly generic (the most famous Jewish Russian lawyer living today has an occupational surname of Reznik, which means “ritual slaughterer” ). We do not have a good explanation for this observation; however, it seems to be confirmed by the lists of prominent people of the profession in question.\n\nFinally, the proportion of drivers among the bearers of rabbinical surnames is less than in the general Jewish population, which appears reasonable. The mean fractions of literary workers and psychologists can differ by more than four times; however, the statistical error in these rather rare groups of the population is quite large and further studies are needed.\n\nHere, we demonstrated the difference between the groups. The next step would be finding the actual mobility rates that characterize how fast the difference between the groups disappears. Thus, in our data we could consider the occupational distributions of each one of the four generations and compare them, where the generation is defined by an appropriate temporal period (see above and Clark, 2015). The pools that we have at our disposal are however too small for reaching definite conclusions. Therefore, the calculation of the intergenerational mobility is left for future work.\n\n\nData availability\n\nThe dataset described here cannot be shared openly due to the identifiable nature of the data (surnames, occupations, birth dates, birthplaces). Any researchers wishing to access the underlying data can contact the corresponding author (itzhak8@gmail.com). Data will be shared under the following conditions: researchers will need to declare that they are currently undertaking similar research, that the data will not be shared with anyone other than the researcher who requested it, and it will be exclusively used for academic purposes.", "appendix": "References\n\nAggarwal CC, Hinneburg A, Keim DA: On the surprising behavior of distance metrics in high dimensional space. In: International conference on database theory. Springer, Berlin, Heidelberg. 2001; 420–434. Publisher Full Text\n\nBhattacharyya A: On a measure of divergence between two statistical populations defined by their probability distributions. Bull Calcutta Math Soc. 1943; 35: 99–109. Reference Source\n\nClark G: The son also rises: Surnames and the history of social mobility. Princeton University Press. 2015; 49. Reference Source\n\nClark G: What is the true rate of social mobility in Sweden? A surname analysis, 1700–2012. Manuscript, Univ. California, Davis. 2012. Reference Source\n\nClark G, Cummins N, Hao Y, et al.: Surnames: A new source for the history of social mobility. Explor Econ Hist. 2015; 55: 3–24. Publisher Full Text\n\nGnedenko BV, Kolmogorov AN: Limit distributions for sums of independent random variables. Addison-Wesley, Massachusetts. 1968. Reference Source\n\nGüell M, Rodríguez Mora JV, Telmer CI: The informational content of surnames, the evolution of intergenerational mobility, and assortative mating. Rev Econ Stud. 2015; 82(2): 693–735. Publisher Full Text\n\nSantavirta T, Stuhler J: Name-Based Estimators of Intergenerational Mobility: Evidence from Finnish Veterans. 2019. Reference Source\n\nYang GL, Le Cam LM: Asymptotics in Statistics: Some Basic Concepts. Berlin: Springer. 2000. Publisher Full Text" }	[ { "id": "69224", "date": "28 Aug 2020", "name": "Gregory Clark", "expertise": [ "Reviewer Expertise social mobility" ], "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 seeks to detect whether there is occupational persistence among Russian Jewish immigrants to Israel and their ancestors with the occupational types implied by their surnames - rabbinical, craft occupations, or nondescript. The authors find that the occupational pattern of the inheritors of rabbinical surnames does differ still from the inheritors of nondescript surnames, while the craft surname inheritors show less difference.\nA strength of the article is the database the authors have collected by surveying Russian Jewish immigrants to Israel of up to 4 generations of occupations in each family. This database has occupational information on more than 9,000 linked individuals.\nThe article has limitations however. First the focus on occupational structure makes the problem one of high dimensions statistically, with limitations on the statistical significance of results, and the results hard to evaluate. It is hard, for example, to compare what is happening with this group compared to other populations. Simpler questions could be addressed with this data which would yield statistically much stronger results. For example, we can assign a status score to each occupation by looking at average earnings by occupation now in Israel. Then we could calculate this average status score across each of 4 generations. We could then estimate how quickly across generations status was regressing to the mean in this population. And occupational persistence may be just a variety of status persistence if occupations vary by status.\nI am most familiar with English data, and here the intergenerational persistence of status is much stronger than the persistence of occupational type. Priests have sons who are almost equally likely to be army officers, lawyers, doctors, civil servants, engineers, and university researchers.\nA second limitation of the article is that while the nondescript surnames constitute about 80% of the sample, for some reason only a subset of these cases are used in much of the analysis. It was never clearly explained why this was the case. If the authors have the data then I do not see why this limitation would be imposed.\nA third limitation is that while the data covers 4 generations, in the analysis it was all lumped into one pool, losing all the temporal information. I think this was because it was necessary because of the high dimension statistical problem to aggregate the data to obtain results significant at conventional significance levels. But why introduce this feature of the data if no use is made of it?\nA fourth limitation is that the statement that these surnames were at least 200 years old in origin was never justified. How do the authors know that?\nA suggestion for further work would be to look at other surname types. In 19th century Russia the surname ending ..ski was elite, while the endings ..ov, ..in, and ..ev were lower class. Do any of these immigrants have such endings and do they show any distinctiveness?\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? Partly\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?\nPartly\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": [ { "c_id": "5991", "date": "08 Oct 2020", "name": "Itzhak Fouxon", "role": "Author Response", "response": "Review by Prof. Gregory Clark This article seeks to detect whether there is occupational persistence among Russian Jewish immigrants to Israel and their ancestors with the occupational types implied by their surnames - rabbinical, craft occupations, or nondescript. The authors find that the occupational pattern of the inheritors of rabbinical surnames does differ still from the inheritors of nondescript surnames, while the craft surname inheritors show less difference. Response: the description is accurate with sole reservation that the participants were visitors of Israel and not immigrants. The misunderstanding probably stems from reading \"The individuals were Russian-speaking Jewish family members residing in the Former Soviet Union (FSU). They voluntarily provided genealogical data for the program via online forms that were sent to them before their arrival in Tel Aviv (see section Data collection)....\" which does allow for misunderstanding. We introduced the sentence \"Upon the completion of the educational program, the participants returned to their home countries.\" for clarification. Review: A strength of the article is the database the authors have collected by surveying Russian Jewish immigrants to Israel of up to 4 generations of occupations in each family. This database has occupational information on more than 9,000 linked individuals. Response: We thank the reviewer for kind opinion of our efforts in data collection. Review: The article has limitations however. First the focus on occupational structure makes the problem one of high dimensions statistically, with limitations on the statistical significance of results, and the results hard to evaluate. Response: It is true that having a high dimensional statistics made our task much harder. We needed to collect data on a large number of participants which took us four years. We stopped the data collection when we saw that the results are already statistically significant. We had the purpose in this paper to show that with 95% probability the statistics of different groups are different. We believe that this observation deserves to be reported. There is much more to be learnt from the data as the referee remarks below. Review: It is hard, for example, to compare what is happening with this group compared to other populations. Response: We did in fact check that we can make a comparison with non-Jewish participants of the program (who belong to Jewish families by marriage). However doing all this work in one paper would be too much (this work took a lot of time) and we left the detailed study for future work. It is true that for comparison with yet other populations would demand a different approach. Review: Simpler questions could be addressed with this data which would yield statistically much stronger results. For example, we can assign a status score to each occupation by looking at average earnings by occupation now in Israel. Then we could calculate this average status score across each of 4 generations. We could then estimate how quickly across generations status was regressing to the mean in this population. And occupational persistence may be just a variety of status persistence if occupations vary by status. Response: We thank the referee for an excellent idea for future work. In fact, we struggled to get statistically significant results for separate generations, we simply would need a much larger database. Using the status score would allow us to get these results, it seems. We could estimate regression to the mean and mobility. This would make our work a contribution to the study of mobility, which is a traditional object of much interest. At the same time, our data gives detailed occupational structure of these generations as a whole i.e. of Russian Jewry during the twentieth century. This object does not give us details of generational distribution, however gives us details on occupational distribution, albeit coarse-grained over four generations. The occupational distribution has its own interest, as we hope the referee would agree. Thus we could quantify if members of rabbinical families are strongly overrepresented in research work and study other questions. Our opinion is that both studies are valuable and \"much stronger results\" still does not mean that our work does not present interest. Review: I am most familiar with English data, and here the intergenerational persistence of status is much stronger than the persistence of occupational type. Priests have sons who are almost equally likely to be army officers, lawyers, doctors, civil servants, engineers, and university researchers. Response: We are aware of the outstanding research by the referee in this direction. If the conclusions of our study can be applied to English data then they tell that spreading of professions of descendants of priests will produce a distribution different from that of descendants of craftsmen. Review: A second limitation of the article is that while the nondescript surnames constitute about 80% of the sample, for some reason only a subset of these cases are used in much of the analysis. It was never clearly explained why this was the case. If the authors have the data then I do not see why this limitation would be imposed. Response: We did use the full data e.g. table 7, providing the total distributions, states that data on 7471 individuals with nondescript surnames was used. However in the course of the discussion we compare the distributions for 7471 and 1000 individuals with nondescript surnames in order to show the reader what the difference could be for rabbinical and occupational surnames where we have data on 1000 however not on 7471 individuals. For stressing this point we introduced in the caption of Table 7 the sentence \"Confidence intervals of generic surnames are significantly narrower than in other groups thanks to much larger pool of available data.\" Review: A third limitation is that while the data covers 4 generations, in the analysis it was all lumped into one pool, losing all the temporal information. I think this was because it was necessary because of the high dimension statistical problem to aggregate the data to obtain results significant at conventional significance levels. Response: It is the true reason. We include in the revised version an explicit statement in the sentence:\"We did not perform separate study of different cohorts since the data available for them would not be statistically significant.\" Review: But why introduce this feature of the data if no use is made of it? Response: Our main target in the paper is showing the groups' difference. This could occur because different generations could be present differently in these groups. For instance by pure accident we could have much more representatives of early generations for rabbinical surnames than for occupational ones. Then a possible reason for the difference could be time dependence of the average occupational distribution in the society. Thus we write that \"For adequate comparison of the groups, we must have roughly the same share of each group born in each of the considered generations.\" The data processing would not change if we omitted the feature, however the implications would change should we have found a strong difference of generations present in the groups. Review: A fourth limitation is that the statement that these surnames were at least 200 years old in origin was never justified. How do the authors know that? Response: Surnames' adoption by Jews was a process that took many centuries. However the official laws ordered universal adoption of hereditary family names everywhere Jews, considered in this study resided, at about 1800. Strictly speaking, inaccuracy of law enforcement in Russia, introduced some exceptions to this rule where some Jews could avoid the census and the names' adoption for some time. However these exceptions are statistically negligible. We introduced the sentence \"Formation of Jewish surnames with few exceptions finished by the beginning of the 19th century, see e. g. Beider, 2008.\" that provides a reference with exposition of the history of the surnames' adoption by Jews. We thank the reviewer for this comment, this had to be told. Review: A suggestion for further work would be to look at other surname types. In 19th century Russia the surname ending ..ski was elite, while the endings ..ov, ..in, and ..ev were lower class. Do any of these immigrants have such endings and do they show any distinctiveness? Response: These endings are atypical endings for the Jewish surnames and rather belong to Russian surnames. We'd be glad to make this study however we do not have these data at the moment. Review: I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Response: We cordially thank the Referee for in-depth study of the paper and constructive critics and comments. We hope that our revisions in the text and responses above resolve the significant reservations by the referee and the present version of the paper can be approved." } ] }, { "id": "69225", "date": "18 Sep 2020", "name": "Jan Stuhler", "expertise": [ "Reviewer Expertise Economics", "labor economics", "migration", "intergenerational mobility" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 recent development in inter-generational research is the use of names to overcome data limitations. While surnames are a rather indirect measure of family links, they are informative about distant ancestors. Compared to conventional studies based on direct family links, name-based studies can therefore capture socioeconomic differences over much longer time intervals. Studies such as Clark (2015) or Barone and Mocetti (2020)1 show that status differences between surnames can persist over many centuries, which suggests that the traditional evidence based on direct parent-child links understates the extent to which advantages are transmitted from one generation to the next. Recent multi-generational studies provide some support for this view, but point to somewhat lower persistence than the surname-based studies (Santavirta and Stuhler, 2019).\nThis new study by Vigdop, Norton, Rosenberg, Haguel-Spitzberg and Fouxon contribute to this literature, but adopt the surname-based approach with a twist: Rather than studying surnames per se, they compare three groups of surnames defined by their professional “bias” in the past, distinguishing names associated with rabbinical dynasties, occupational as well as generic names. This categorization exploits the distinguished position of rabbis in the Jewish population, and is comparable to the consideration of noble names in Clark (2012). The authors show that the occupational distributions of Russian-speaking Jewish families in these three groups are different, even though that in most cases, the names were inherited for many generations.\nThe argument is transparently and convincingly laid out, and the main finding seems well supported by the data. I however have a number of minor comments on 1) the sample and data collection specific to this study, 2) more general methodological issues, and 3) possible extensions of this study in future research.\nSample and data collection:\n\nOne wonders how representatives the sample will be, given that it was collected related to a particular educational program implemented at one particular institution. It would be useful to compare the distribution of occupations and individual characteristics to external evidence from data sources that ought to be representative, and that distinguish immigrants from the former Soviet Union. The Israeli Labor Force Survey might be such source (as for example used in Cohen-Goldner and Paserman, 20112).\nA related issue is that respondents were are asked about their distant ancestors, such that the occupational variable might be affected by “recall bias”. It is also imaginable that such recall errors are systematic (e.g., if family folklore tends to overstate the importance of a family’s ancestors), and that this tendency varies between the groups considered in the study (e.g., if families are aware of the rabbinical origin of their name and interpret their family history accordingly). Such recall bias would tend to be more pronounced for distant ancestors. The inclusion of distant ancestors is not necessary for the analysis, and it would be useful to know if the main findings hold even when restricting attention to the subsample of the responds and more immediate ancestors.\nMore general methodological issues:\nOne of the main criticism against the name-based approach is that it measures status persistence on the group (surname) level rather than individual-level persistence. The group-level perspective becomes problematic if the group definitions correlate with third factors that itself have implications for socioeconomic status and intergenerational mobility. In particular, surnames may differ systematically with race or location. It would therefore be interesting to test whether families in the three groups came from systematically different regions in the Former Soviet Union (e.g., by coding regions in terms of their geographic location, their population density, or other regional characteristics).\nAt first, the splitting of the total sample into blocks of hundreds seems a little odd. The best predictor of the population mean is the sample mean (e.g., the last column in Table 2), so it is unusual to split the sample into subsamples to do separate analysis within each of these subsamples (e.g., the other columns in Table 2). Of course, the purpose of this procedure is to illustrate how sensitive the results are to sample size, given that we have much fewer rabbinical and occupational than general surnames. However, it does not become sufficiently clear why (i) we cannot just estimate standard errors for the occupational shares, and the difference in occupational shares between surname groups, and (ii) in which way splitting the total sample into separate blocks can be informative about the random sampling assumption. To motivate that early on would be useful, because the discussion of this issue in the current paper version takes much space and becomes fairly technical.\nIn the final part of the manuscript it becomes clear why sampling uncertainty is such an important issue – the Bhattacharyya coefficient and the Hellinger distance do not account for sampling uncertainty, so tends to indicate that occupational distributions are more distant when the sample size is small. This caveat could be mentioned earlier on. As such, the Bhattacharyya and Hellinger measures cannot fully serve their intended purpose. The authors address this issue by assessing the sample fractions and corresponding confidence intervals for each of the professions in Table 7. These “case studies” illustrate that the difference in occupational distributions is indeed systematic, and not just a statistical artifact from sampling error. However, this evidence is not integrated with the summary measures, and for future research it would be useful to study whether we can find a measure of occupational distance that already accounts for sampling error.\nMinor presentational issues: In the abstract, it could be briefly noted what database you are referring to (e.g., that the data is self-collected). In Table 7, it could be clarified if you formally test the hypothesis that the difference in population means between the groups is zero, or simply compare whether the confidence intervals are overlapping.\nPossible extensions and future research:\nA potential fruitful avenue for future research would be the application of occupational prestige scores that rank occupations according to their socioeconomic status. Of course, these type of rankings are not without issues – the standing of an occupation may differ between countries, or vary over time. See Yuchtman and Fishelson (1972)3, for a consideration of these issues in the Israelian context. However, such issues would be less problematic if our main focus is on the comparison between groups, and errors in the rankings affect the groups similarly. By applying occupational scores, we transform the categorical into ordered data, which would allow for a number of interesting extensions in future research. Most importantly, it would allow us to change focus from group differences in the occupational structure as such (in any direction) to systematic differences in socioeconomic status, and therefore intergenerational mobility.\nOnce occupational scores are matched to the data, we can also apply the type of name-based estimators that are increasingly used in the intergenerational literature (for an overview, see Santavirta and Stuhler, 2020). For example, we can study to what extent surnames or the rabbinical/occupational/general categorization can predict socioeconomic success, using the approach by Güell et al. 2015. The other widely used approach is the so-called grouping estimator, to measure how quickly the difference between groups regress to the mean. While the estimator depends on sample size, it performs better in the type of data structure that we face here, in which complete lineages are sampled. In particular, it would be interesting to understand if the apparent status differences between rabbinical, occupational and general surnames have remained stable, or reduced over the generations covered by your sample.\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5992", "date": "08 Oct 2020", "name": "Itzhak Fouxon", "role": "Author Response", "response": "Review by Prof. Jan StuhlerA recent development in inter-generational research is the use of names to overcome data limitations. While surnames are a rather indirect measure of family links, they are informative about distant ancestors. Compared to conventional studies based on direct family links, name-based studies can therefore capture socioeconomic differences over much longer time intervals. Studies such as Clark (2015) or Barone and Mocetti (2020)1 show that status differences between surnames can persist over many centuries, which suggests that the traditional evidence based on direct parent-child links understates the extent to which advantages are transmitted from one generation to the next. Recent multi-generational studies provide some support for this view, but point to somewhat lower persistence than the surname-based studies (Santavirta and Stuhler, 2019).This new study by Vigdop, Norton, Rosenberg, Haguel-Spitzberg and Fouxon contribute to this literature, but adopt the surname-based approach with a twist: Rather than studying surnames per se, they compare three groups of surnames defined by their professional “bias” in the past, distinguishing names associated with rabbinical dynasties, occupational as well as generic names. This categorization exploits the distinguished position of rabbis in the Jewish population, and is comparable to the consideration of noble names in Clark (2012). The authors show that the occupational distributions of Russian-speaking Jewish families in these three groups are different, even though that in most cases, the names were inherited for many generations.The argument is transparently and convincingly laid out, and the main finding seems well supported by the data. I however have a number of minor comments on 1) the sample and data collection specific to this study, 2) more general methodological issues, and 3) possible extensions of this study in future research.Response: We thank Prof. Jan Stuhler for kind opinion of our work. We hope that we could incorporate the minor comments in the revised version, please see below.Review: Sample and data collection:One wonders how representatives the sample will be, given that it was collected related to a particular educational program implemented at one particular institution. It would be useful to compare the distribution of occupations and individual characteristics to external evidence from data sources that ought to be representative, and that distinguish immigrants from the former Soviet Union. The Israeli Labor Force Survey might be such source (as for example used in Cohen-Goldner and Paserman, 20112).Response: The participation in our program was obligatory for all Russian-speaking participants of a larger program which was held for many years and in which over all about one million young Jews took part. This larger program is extremely inclusive and the only bias that it could introduce is that all participants had some degree of affiliation with the Jewish people. Hence, strictly speaking, our findings are confined to this subgroup of carriers of rabbinical, occupational and generic surnames. The Labor Force Survey obviously would have a similar bias and we do not see how we could perform a study beyond the above limitation. It seems to us that this bias is not so restrictive since it roughly corresponds to the original statement that the study was performed for Russian-speaking Jewish families. We have introduced in the text an explicit statement \"Our program was obligatory for participants of a larger, very inclusive program so that to the best of our knowledge the only bias in the sample was some degree of affiliation with the Jewish people.\" We hope that this resolves the issue. Review: A related issue is that respondents were are asked about their distant ancestors, such that the occupational variable might be affected by “recall bias”. It is also imaginable that such recall errors are systematic (e.g., if family folklore tends to overstate the importance of a family’s ancestors), and that this tendency varies between the groups considered in the study (e.g., if families are aware of the rabbinical origin of their name and interpret their family history accordingly). Such recall bias would tend to be more pronounced for distant ancestors. The inclusion of distant ancestors is not necessary for the analysis, and it would be useful to know if the main findings hold even when restricting attention to the subsample of the responds and more immediate ancestors.Response: We agree that this could be an issue. Unfortunately cutting the data would result in statistically insignificant sample. We believe though that recall bias is not high and the mistake in reporting the profession of grandparents can be neglected (The reported data on the ggparents boiled down to parents' data on their gparents. The participants could tell that they do not know as they did occasionally). Moreover the recall bias, in this case, could act in the direction of erasing differences between the groups rather than amplifying them. Indeed, the Soviet totalitarian regime made any professions of ancestors other than peasants and workers very problematic. Cutting the heads which were above the crowd was more than usual. For instance wood merchants would be reported as wood experts, the profession of rabbi would be suppressed as much as possible and so forth. Hence the recall bias almost certainly acted in the opposite direction of erasing all differences between the USSR citizens. Review: More general methodological issues:One of the main criticism against the name-based approach is that it measures status persistence on the group (surname) level rather than individual-level persistence. The group-level perspective becomes problematic if the group definitions correlate with third factors that itself have implications for socioeconomic status and intergenerational mobility. In particular, surnames may differ systematically with race or location. It would therefore be interesting to test whether families in the three groups came from systematically different regions in the Former Soviet Union (e.g., by coding regions in terms of their geographic location, their population density, or other regional characteristics).Response: we thought of this factor to which we devoted this paragraph that probably went overlooked: \"The birthplaces were scattered all over the territory of the FSU. Jewish families have a long tradition of studying and those who would want to acquire education would typically receive such an opportunity. In other words, with a good approximation, an individual born into a Jewish family of the FSU would have an equal opportunity for getting that or another profession irrespective of birthplace. Therefore, we disregarded the geographical factor in our study.\"Review: At first, the splitting of the total sample into blocks of hundreds seems a little odd. The best predictor of the population mean is the sample mean (e.g., the last column in Table 2), so it is unusual to split the sample into subsamples to do separate analysis within each of these subsamples (e.g., the other columns in Table 2). Of course, the purpose of this procedure is to illustrate how sensitive the results are to sample size, given that we have much fewer rabbinical and occupational than general surnames. However, it does not become sufficiently clear why (i) we cannot just estimate standard errors for the occupational shares, and the difference in occupational shares between surname groups, and (ii) in which way splitting the total sample into separate blocks can be informative about the random sampling assumption. To motivate that early on would be useful, because the discussion of this issue in the current paper version takes much space and becomes fairly technical.Response: We introduced in the revised version the early stage explanation: \"In contrast with individual data which is not randomly sampled, the blocks can be be considered as a result of random sampling where a hundred was taken from the population and then, independently, another hundred and so forth, see below. Moreover separation into blocks demonstrates what we can anticipate to see if we pick 100 members from the considered population: \" We hope that this early explanation helps the reader. Review: In the final part of the manuscript it becomes clear why sampling uncertainty is such an important issue – the Bhattacharyya coefficient and the Hellinger distance do not account for sampling uncertainty, so tends to indicate that occupational distributions are more distant when the sample size is small. This caveat could be mentioned earlier on. As such, the Bhattacharyya and Hellinger measures cannot fully serve their intended purpose. The authors address this issue by assessing the sample fractions and corresponding confidence intervals for each of the professions in Table 7. These “case studies” illustrate that the difference in occupational distributions is indeed systematic, and not just a statistical artifact from sampling error. However, this evidence is not integrated with the summary measures, and for future research it would be useful to study whether we can find a measure of occupational distance that already accounts for sampling error.Response: We introduced in the revised version the early stage explanation: \"We will see below that on their own the distances do not allow the distributions’ comparison, however in conjunction with statistical analysis they become useful.\" The problem of introducing a distance accounting for sampling error seems to us very difficult indeed. Review: Minor presentational issues: In the abstract, it could be briefly noted what database you are referring to (e.g., that the data is self-collected). In Table 7, it could be clarified if you formally test the hypothesis that the difference in population means between the groups is zero, or simply compare whether the confidence intervals are overlapping.Response: We introduced in the abstract \"self-collected\". We introduced in the table's caption the sentence \"The table tests the hypothesis that the occupational distributions of the groups are identical by checking the overlap of the confidence intervals.\" Review: Possible extensions and future research:A potential fruitful avenue for future research would be the application of occupational prestige scores that rank occupations according to their socioeconomic status. Of course, these type of rankings are not without issues – the standing of an occupation may differ between countries, or vary over time. See Yuchtman and Fishelson (1972)3, for a consideration of these issues in the Israelian context. However, such issues would be less problematic if our main focus is on the comparison between groups, and errors in the rankings affect the groups similarly. By applying occupational scores, we transform the categorical into ordered data, which would allow for a number of interesting extensions in future research. Most importantly, it would allow us to change focus from group differences in the occupational structure as such (in any direction) to systematic differences in socioeconomic status, and therefore intergenerational mobility.Once occupational scores are matched to the data, we can also apply the type of name-based estimators that are increasingly used in the intergenerational literature (for an overview, see Santavirta and Stuhler, 2020). For example, we can study to what extent surnames or the rabbinical/occupational/general categorization can predict socioeconomic success, using the approach by Güell et al. 2015. The other widely used approach is the so-called grouping estimator, to measure how quickly the difference between groups regress to the mean. While the estimator depends on sample size, it performs better in the type of data structure that we face here, in which complete lineages are sampled. In particular, it would be interesting to understand if the apparent status differences between rabbinical, occupational and general surnames have remained stable, or reduced over the generations covered by your sample.Response: We agree with the referee that these are good directions for future research. We would like to thank Prof. Stuhler for devoting time to our work and providing thoughtful comments." } ] } ]	1	https://f1000research.com/articles/9-971
https://f1000research.com/articles/9-1103/v1	08 Sep 20	{ "type": "Research Article", "title": "Taxono-genomics description of Olsenella lakotia SW165T sp. nov., a new anaerobic bacterium isolated from cecum of feral chicken", "authors": [ "Supapit Wongkuna", "Sudeep Ghimire", "Tavan Janvilisri", "Kinchel Doerner", "Surang Chankhamhaengdecha", "Joy Scaria", "Supapit Wongkuna", "Sudeep Ghimire", "Tavan Janvilisri", "Kinchel Doerner" ], "abstract": "Background: The microbial community residing in the animal gastrointestinal tract play a crucial role in host health. Because of the high complexity of gut microbes, many microbes remain unclassified. Deciphering the role of each bacteria in health and diseases is only possible after its culture, identification, and characterization. During the culturomics study of feral chicken cecal sample, we cultured a possible novel strain SW165T. Methods: For the possible novel strain SW165T, phenotypic characterization was performed using colony morphology, Gram staining, growth in different temperature and pH and motility. Biochemical assays included carbon source utilization, enzymatic activity, cellular fatty acids and short chain fatty acid production. 16S rRNA sequencing and whole genome sequencing and comparison was performed for genetic analysis. Results: This strain was isolated from cecal content of feral chickens in Brookings, South Dakota, USA. Phylogenetic analyses based on 16S rRNA gene sequence revealed that the closest valid neighbor was Olsenella profusa DSM 13989T (96.33% similarity) within the family Atopobiaceae. Cells were Gram-strain-positive and obligately anaerobic bacilli in chains. The optimum temperature and pH for the growth of the microorganism were 37-45oC and pH 6.0-7.5 respectively. This strain produced acetic acid as the primary fermentation product. Major fatty acids were C12:0, C14:0, C14:0 DMA and summed feature 1 (C13:1 at 12-13 and C14:0 aldehyde). Strain SW165T exhibited a genome size of 2.43 Mbp with a G+C content of 67.59 mol%, which is the second highest G+C content among members of the genus Olsenella. The digital DNA-DNA hybridization and OrthoANI values between SW165T and DSM 13989T were only 17.6 ± 5.3 and 74.35%, respectively. Conclusion: Based on the phenotypic, biochemical, and genomic analyses, we propose the new species of the genus Olsenella, and name it Olsenella lakotia SW165T sp. nov., (=DSM 107283 =CCOS 1887) as the type strain.", "keywords": [ "Olsenella lakotia SW165T sp. nov.", "culturomics", "chicken gut microbiota", "taxono-genomics" ], "content": "Introduction\n\nThe chicken gut harbors highly diverse microbes1. The gut microbes are known for their nutritional benefits by producing short chain fatty acids, enzymes, amino acids along with their ability to resist pathogens, immunity development and maintain homeostasis2. Even though culture independent methods have highlighted the functional capability of gut microbes, validation of these functions requires their cultivation, identification, and characterization. Most of the intestinal bacteria have been never isolated in the laboratory3,4 thus hindering the understanding of their ecological and functional roles in the gut. Recently, “culturomics” strategy drives discovery of previously uncultured species based on modified culture conditions, such as media, temperature, pH and atmosphere and rapid identifying methods; matrix-assisted desorption ionization- time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing5–7. We employed culturomics to isolate bacteria from cecum of feral chickens. Based on bacterial isolation and identification, strain SW165T was found as a new species within the genus Olsenella.\n\nThe members of the genus Olsenella are strictly anaerobic, Gram-positive, non-motile, non-spore-forming bacilli or cocci. This genus was first named by Dewhirst et al. 20018 and amended by Zhi et al 20099 and Kraatz et al 201110. This genus has recently been reclassified as a member of the family Atopobiaceae under order Coriobacteriales, class Coriobacteria, and phylum Actinobacteria11. The genus Olsenella consists of nine published species; O. uli12, O. profusa8, O. umbonata10, O. scatoligenes13, O. urininfantis14, O. congonensis15, O. provencensis16, O. phocaeensis16, and O. mediterranea16. The members of this genus are strictly anaerobic, Gram positive, non-motile, non-spore forming rod shaped with G+C content of DNA 62–64%8,13,17. The main habitats of the Olsenella are the oral cavity and gastrointestinal tract of humans18–21, animals and various anaerobic environmental sites22–24. In chicken cecum, many members of genus Olsenella have been reported in the chicken microbiome in metagenomic-based studies15,25. However, only O. uli was isolated from chicken gut26.\n\nThe taxono-genomics approach uses combination of phenotypic and genotypic characterization to describe new bacteria27,28. Phenotypic investigation includes morphological, physiological, and biochemical assays. Genome-based and 16S-based analysis are used in genotypic characterization. In this study, strain SW165T was described using taxono-genomics and compared to its closely related phylogenetic neighbors. Following analysis, we found that strain SW165T belongs to a novel species for which the name Olsenella lakotia SW165T sp. nov. is proposed.\n\n\nMethods\n\nThe cecal content of feral chickens was collected in Brookings, South Dakota, USA. For cultivation, the samples were transferred into an anaerobic workstation (Coy Laboratory) containing 85% nitrogen, 10% hydrogen and 5 % carbon dioxide and plated on a modified Brain Heart Infusion (BHI-M) agar containing 37 g/L of BHI, 5 g/L of yeast extract, 1 ml of 1 mg/ml menadione, 0.3 g of L-cysteine, 1 ml of 0.25 mg/L of resazurin, 1 ml of 0.5 mg/ml hemin, 10 ml of vitamin and mineral mixture, 1.7 ml of 30 mM acetic acid, 2 ml of 8 mM propionic acid, 2 ml of 4 mM butyric acid, 100 µl of 1 mM isovaleric acid, and 1% of pectin and inulin. After 3 days of incubation at 37°C under anaerobic conditions, single colony of strain SW165 was identified by MALDI-TOF mass spectrometry using a Microflex spectrometer (Bruker Daltonics, Bremen, Germany). The strain was maintained in BHI-M medium and stored with 10% (v/v) Dimethyl Sulfoxide (DMSO) at -80°C.\n\nFor morphological characterization, the strain SW165T was anaerobically cultivated in BHI-M medium, pH 6.8-7.2, at 37o C. Colony morphologies were examined after 2–3 days of incubation on BHI-M agar plates. Gram-staining was performed using a Gram-Staining kit set (Difco), according to the manufacturer’s instructions. Cell morphologies were examined by scanning electron microscopy (SEM) of cultures during exponential growth. Aerotolerance was examined by incubating cultures for 2 days separately under aerobic and anaerobic conditions. Growth of strain SW165T at 4, 20, 30, 37, 40 and 55°C was determined. For determining the range of pH for growth of SW165T, the pH of the medium was adjusted to pH 4.0–9.0 with sterile anaerobic stock solutions of 0.1 M HCl and 0.1 M NaOH. Motility of this microorganism was determined using motility medium with triphenyltetrazolium chloride (TTC)29. The growth was indicated by the presence of red color, reduced form of TTC after it is absorbed into bacterial cell wall.\n\nBiochemical tests to determine standard taxonomic characteristics for strain SW165T were performed in triplicate. The utilization of various substrates as sole carbon and energy sources and enzyme activities were performed using the AN MicroPlate (BIOLOG) and API ZYM (bioMérieux) according to the manufacturer's instructions. Reference strain, DSM 13989T purchased from the DSMZ culture collection and isolated strains SW165T were simultaneously cultured in BHI-M medium at 37°C for 24 h under anaerobic condition before cell biomass were harvested for cellular fatty acid analysis. The fatty acids were extracted, purified, methylated and analyzed using gas chromatography (Agilent 7890A) based on the instruction from Microbial Identification System (MIDI)30. Metabolic end-products such as short-chain fatty acids of strain SW165T and DSM 13989T grown in BHI-M were determined using a gas chromatography. The cultures were deproteinized with 25% metaphosphoric acid before supernatant collection. The supernatant was analyzed for the presence of acetic acid, butyric acid, isovaleric acid and propionic acid using GC (Themo ScientificTM TRACETM 1310 GC equipped with a TraceGOLDTM TG-WaxMS A GC column.).\n\nGenomic DNA of the strain SW165T was extracted using a DNeasy Blood & Tissue kit (Qiagen), according to the manufacturer’s instructions. 16S rRNA gene sequence was amplified using universal primer set 27F (5’- AGAGTTTGATCMTGGCTCAG-3’; Lane et al., 1991) and 1492R (5’- ACCTTGTTACGACTT- 3’; Stackebrandt et al., 1993)31,32, and sequenced using a Sanger DNA sequencer (ABI 3730XL; Applied Biosystems). The 16S rRNA gene sequence of SW165 was then compared with closely related strains from the GenBank (www.ncbi.nlm.nih.gov/genbank/) and EZBioCloud databases (www.ezbiocloud.net/eztaxon)33. Alignment and phylogenetic analysis were conducted using MEGA7 software34. Multiple sequence alignments were generated using the CLUSTAL-W35. Reconstruction of phylogenetic trees was carried out using the maximum-likelihood (ML)36, maximum-parsimony (MP)37, and neighbor-joining (NJ)38 methods. The distance matrices were generated according to Kimura's two-parameter model. Bootstrap resampling analysis of 1000 replicates was performed to estimate the confidence of tree topologies.\n\nThe whole genome sequencing of strain SW165T was performed using Illumina MiSeq sequencer using 2x 300 paired end V3 chemistry. The reads were assembled using Unicycler that builds an initial assembly graph from short reads using the de novo assembler SPAdes 3.11.139. The quality assessment for the assemblies was performed using QUAST5.0.240. Genome annotation was performed using Rapid Annotation using Subsystem Technology (RAST) server41. The digital DNA-DNA hybridization (dDDH) was performed using Genome-to-Genome Distance Calculator (GGDC) web server (http://ggdc.dsmz.de) to estimate the genomic similarity between strain SW165T and the closest phylogenetic neighbor. Average nucleotide identity (ANI) between strain SW165T and the closely related strains was also calculated using the OrthoANI software42. Distribution of functional categories of strain SW165T was compared to Olsenella species and was presented in a heatmap generated using Explicet version 2.10.543.\n\n\nResults\n\nStrain SW165T was isolated from cecal contents of feral chicken in anaerobic chamber (Coy Laboratory Product, MI, USA). Colonies of SW165T on BHI-M agar were 0.2–0.5 cm in diameter, appeared white, smooth, and umbonate with entire circular edges when grown at 37°C anaerobically after 48 hours of incubation. After cultivation, the colonies of this strain were subjected to identification by MALDI-TOF using a Microflex spectrometry (Bruker Daltonics, Bremen, Germany). MALDI-TOF did not identify the strain as the scores obtained were < 1.70. Thus, full length 16S rRNA gene was sequenced using Sanger sequencing method. The 16S rRNA of the strain SW165T showed 96.33% identity with O. profusa DSM 13989T (GenBank accession no. AF292374), the validly closest species within phylogenetical nomenclature (Figure 1). The current cut off for species delineation from its nearest neighbor based on 16S rRNA is 98.7%44. As the identity of 16S rRNA of strain SW165T was lower than threshold, it was considered as a representative of putatively novel species within the genus Olsenella in the family Atopobiaceae. Phylogenetically, the strain was found to cluster together with other members of genus Olsenella, as shown in Figure 1, validating that SW165T belongs to genus Olsenella taxonomically.\n\nTree shows phylogenetic position of Olsenella lakotia DSM 107283T and closely related species in the family Atopobiaceae. GenBank accession numbers of the 16S rRNA gene sequences are given in parentheses. Black circles indicate that the corresponding branches were also recovered both by maximum-likelihood and maximum parsimony methods. Bootstrap values (based on 1000 replications) greater than or equal to 70% are shown in percentages at each node. Bar, 0.01 substitutions per nucleotide position.\n\nPhenotypic growth of strain SW165T was observed on modified BHI-M agar after 2–3 days of incubation at temperature between 37°C and 45°C and pH between 6.0–7.0. The optimum temperature and pH for the growth were at 45°C and pH 7.0, respectively. Strain SW165T grew only under anaerobic conditions, suggesting obligate anaerobic nature. Bacterial cells were Gram-stain-positive bacilli (0.5–2.0 µm), growing in pairs or as short chains and were non-motile (Figure 2).\n\nCells were anaerobically cultured for 24 hours at 37o C in BHI-M medium. Bar, 2 μm; uncropped/unedited image.\n\nTo further analyze the biochemical properties of the strain, we performed the carbon source utilization assay using BIOLOG AN microplate and compared it to closely related taxa. Strain SW165T consumed various carbon sources for the growth, which differed from related strains in the utilization of D-fructose, L-fucose, D-galactose, maltose, D-melibiose and D-raffinose, and in the non-utilization of dulcitol. Based on enzymatic activity test, the strain produced several enzymes, including alkaline phosphatase, leucine arylamidase, cysteine arylamidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, and β-glucosidase. Interestingly, alkaline phosphatase α-galactosidase, β-galactosidase and β-glucuronidase are not reported from its closest neighbors (Table 1). Furthermore, the dominant cellular fatty acids of the strain SW165T were saturated, including C 12 : 0 (25.5%) and C 14 : 0 (22.83%). Moreover, other dominant fatty acids were C 14 : 0 DMA (15.61%) and summed feature 1 [C13:1 and/or C14:0 aldehyde; 13.94%]. However, there were distinct quantities of some fatty acids between SW165T and the relative strains (Table 2). The major short chain fatty acids produced by SW165 when cultivated in BHI-M were acetic acid (3.74 mM) followed by propionic acid (0.53 mM).\n\nColumn headers show Strains designated in the following numbers: 1 - SW165T; 2 - O. profusa DSM 13989T; 3 - O. uli DSM 7084T; 4 - O. umbonata DSM 22620T; 5 - O. scatoligenes DSM 28304T. Results for metabolic end products of SW165 are from this study with cells that were cultured for 3 days at 37°C in BHI-M. +, positive; -, negative; w, weak; ND, not determined.\n\n†Data from Kraatz et al. (2011)10\n\n‡Data from et al. (2015)13\n\nA/a, acetic acid; L, lactic acid; f, formic acid. Capital letters indicate major end products.\n\nStrains: 1 - SW165T; 2 - O. profusa DSM 13989T; 3 - O. uli DSM 7084T; 4- O. umbonata DSM 22620T; 5- O. scatoligenes DSM 28304T. Values are percentages of total fatty acids detected. Fatty acids with contents of less than 1% in all strains are not shown; ND, Not detected.\n\n‡Data from et al. (2015)13\n\n*Summed features are fatty acids that could not be separated using the MIDI System. Summed feature 1 contains C13 : 1 and/or C14 : 0 aldehyde. Summed feature 13 contains C15 : 0 anteiso and/or C14 : 0 2-OH.\n\nWe examined the genome of the strain SW165T to investigate its differentiation from the neighbors. The genome size of strain SW165T was 2,427,227 bp with 67.59 mol% G+C content. The draft genome was assembled into 33 contigs with 2,228 protein-coding sequences and 52 RNAs (Table 3) and is visualized as Figure 3. The genomes sizes for the Olsenella species were comparable to one another except for O. urininfantis whose was only 1.75 Mbps. However, the G+C contents strain SW165T and O. mediterranea were the highest but comparable to other neighboring Olsenella species (Table 3). The genome of SW165T possessed a total of 1, 230 genes with putative function and 998 genes as hypothetical proteins. Among 1,230 genes, 823 were classified as features in subsystem, following functional categories (COGs). Majority of categories included amino acid and derivatives (172 genes), carbohydrates (163 genes), and protein metabolism (132 genes) (Extended data: Supplemental Table 145).\n\nFrom outside to the center: coding sequences on the forward strand (CDS +), coding sequences on the reverse strand (CDS -), tRNAs, rRNAs, GC content, and GD skew.\n\nFurthermore, we compared the genome of SW165T to its neighbor using OrthoANI as shown in Figure 4. The genome of SW165 was only 73.41% identical to its nearest neighbor O. profusa DSM 13989T. Also, the OrthoANI values for SW165T and closely related strains ranged from 65.40 to 74.18 % (Figure 4) indicating that the genome of SW165T is unique compared to its neighbors. The proposed cut off for OrthoANI for the species delineation is 95-96% identity46,47. In addition, dDDH between SW165T and the closest neighbor, O. profusa DSM 13989T was only 17.6 ± 5.3. These values were lower than threshold of ANI and dDDH for delineating prokaryotic species, suggesting that these strains are distinct species. Also, the gene distribution into COGs was comparable in all eight compared Olsenella genomes (Figure 5). Hence, the phenotypic and genetic discrepancy of the SW165T with its close neighbor apparently supports that strain SW165T represents a new species of the genus Olsenella.\n\nHeatmap represents OrthoANI values generated using OAT software between O. lakotia and related taxa with valid taxonomy.\n\nThe functional features were predicted based on the clusters of orthologous groups. Heatmap was generated from genome annotation of individual species by RAST using Explicet software.\n\n\nDiscussion\n\nCulturomics of the gut microbiota has evolved as a strong tool to increase the isolation of diverse previously uncultured bacteria from the gut5,48. The cultivation of the gut microbiota enables to improve the health through an enhanced understanding of their roles in the gut ecosystem and finally to the host. Thus, using the culturomics strategy, we were able to isolate previously uncultured bacterium SW165T from cecal content of feral chicken and finally characterize and describe it using taxono-genomics as a novel microorganism.\n\nThe novelty of a prokaryotic organism is universally determined by the comparison of 16S rRNA gene sequence homology49. The threshold values are used at distinct taxonomic levels46. In this context, the newly discovered bacterium was initially validated using the full-length 16S rRNA gene sequences, which were thereafter used for taxonomic classification. Phylogenetic analysis of 16S rRNA gene showed that the novel strain SW165T clustered with closely related taxa in the genus Olsenella within the family Atopobiaceae (Figure 1). This genus composes of nine species, most of which are members of gut microbiota of humans and animals. However, O. uli is only a species that have been isolated from chicken gut26. Remarkable, this study revealed a new member of Olsenella from gut microbiota of chicken.\n\nPhenotypic analyses are performed to differentiate closely valid bacteria. Based on phenotypic tests, strains SW165T appeared several distinct properties compared to other members of the genus Olsenella (Table 1 and Table 2). The obvious distinct phenotypic features were observed in biochemical tests including enzymatic activity and carbon source utilization, thereby they might be important parameters for discriminating closely related species. These differences suggested the novelty of this microorganism belonging to Olsenella.\n\nIn addition to 16S based comparison, whole genome can be used for distinguishing, distinct bacteria. Recently, digital DNA-DNA hybridizations (dDDH) becomes a key measurement in delineation of prokaryotic species. It is an in-silico genome-to-genome comparison inferring whole genome distance to mimic DDH50. Besides, Average Nucleotides Identification (ANI) is another particular tool that confirm the taxonomic delineation. It measures the overall similarity between two genome sequences51. Recent publication of novel bacteria trend to perform genome-based analysis to support the results of 16S rRNA gene-based analysis. The strengths of genome-based analyses include comparison of all nucleotides in prokaryotic taxonomy and functional prediction52. Based on genomic evidence, strain SW165T showed low similarity in terms of OrthoANI with Olsenella species of the family Atopobiaceae (Figure 4). Further, genome features and distribution of predicted functional categories of strain SW165T was corresponded to all other Olsenella species (Figure 5 and Table 3). Thus, we proposed the strain SW165T as a new species Olsenella lakotia SW165T sp. nov., within the family Atopobiaceae.\n\nO. lakotia sp. nov. (la.ko’tia N.L n. referring to native American tribe). Cells are strictly anaerobic, Gram-positive streptobacillus and non-motile. The average size of each cell is 0.5–2.0 µm. Colonies are visible on BHI-M agar after 2 days and are approximately 0.2–0.5 cm in diameter, cream-white, smooth, slightly umbonate with entire circular margin. The microorganism exhibits optimal growth in BHI-M medium at 45°C and pH 7.0. The strain utilizes arbutin, cellobiose, dextrin, D-fructose, L-fucose, D-galactose, α-D-glucose, maltose, D-mannose, D-melibiose, D-raffinose, salicin, sucrose and turanose as a carbon source. Positive enzymatic reactions are obtained for alkaline phosphatase, leucine arylamidase, cysteine arylamidase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase and β-glucosidase. The volatile fatty acid produced by this strain is acetic acid. The primary cellular fatty acids are C12 : 0, C14 : 0, C14 : 0 DMA and summed feature 1. The genome is 2,427,227 bp and its G+C is 67.59 mol%.\n\nThe type strain SW165T (=DSM 107283 =CCOS 1887) was isolated from the cecum of feral chicken was deposited in the DSMZ and CCOS collections under accession numbers DSM 107283 and CCOS 1887 (Extended data: Supplemental Data 153), respectively. The 16S rRNA and genome sequence are available in GenBank under accession numbers MK963074 and BioProject PRJNA545153, respectively.\n\n\nData availability\n\nOlsenella sp. strain SW165 16S ribosomal RNA gene, partial sequence, Accession number MK963074: https://www.ncbi.nlm.nih.gov/nuccore/MK963074\n\nOlsenella lakotia SW165 Genome sequencing and assembly, Accession number PRJNA545153: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA545153\n\nFigshare: Extended data; Supplemental Table 1 (Functional categories (COGs) from genome of strain SW165T), https://doi.org/10.6084/m9.figshare.12793544.v145.\n\nFigshare: Supplemental Data 1 (DSMZ and CCOS accession numbers of strain SW165T), https://doi.org/10.6084/m9.figshare.12793610.v153.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nSW gratefully acknowledges Science Achievement Scholarship of Thailand (SAST) for providing fellowship. 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Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12793610.v1" }	[ { "id": "71020", "date": "11 Sep 2020", "name": "Yung-Fu Chang", "expertise": [ "Reviewer Expertise microbial pathogenesis", "molecular diagnosis", "vaccine development", "genomics", "and proteomics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 “Taxono-genomics description of Olsenella lakotia SW165T sp. nov., a new anaerobic bacterium isolated from the cecum of feral chicken’’ was submitted by Scaria et al. The authors have characterized a new bacterium strain isolated from chicken. It is a well-written manuscript and the data is very solid to support their conclusion. I have enclosed the manuscript with a minor revision which you can see here.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\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": [ { "c_id": "5939", "date": "21 Sep 2020", "name": "Surang Chankhamhaengdecha", "role": "Author Response", "response": "Thank you for reading and correcting our manuscript. We really appreciate you approval." } ] }, { "id": "71021", "date": "24 Sep 2020", "name": "Mohamed N. Seleem", "expertise": [ "Reviewer Expertise Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes the isolation and characterization of a proposed new species “lakotia” within the genus Olsonella. The isolate has been characterized using the combination of genome sequencing and phenotypic assays. The phenotypic characterization includes nutrient utilization using Biolog plates, enzyme profiling using API zyme, and cellular membrane fatty acid profiling using FAME. At the phenotypic level, the proposed new species show clear differences with Olsonella umbonata, the closest taxonomic neighbor. At the genomic level, the proposed new species is only 74% similar to other species in the genus Olsonella. Therefore, the proposed species meets the genomic and phenotypic requirements of being designated as a new species. However, some minor changes could improve the clarity of the manuscript:\nTable 1. Tittle of the table reads “ Characteristics of O. lakotia and closely related strains” Authors should instead give the phenotype reported here. Therefore, please revise the title to improve the clarity; eg. Phenotypic properties of O. Lakota... Likewise, in the table, column header should be “Phenotpic properties” instead of “characteristic”. Please give species names in the column headers instead of numbers.\n\nTable 3. Authors have given bioproject number instead of genbank accession number for O. lakotia SW165. The genbank accession number was anot accessible from the Bioproect page in NCBI. Please remove the bioproject number and instead give Genbank SRA accession number here.\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?\nNot applicable\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/9-1103
https://f1000research.com/articles/9-240/v1	06 Apr 20	{ "type": "Software Tool Article", "title": "QUARTIC: QUick pArallel algoRithms for high-Throughput sequencIng data proCessing", "authors": [ "Frédéric Jarlier", "Nicolas Joly", "Nicolas Fedy", "Thomas Magalhaes", "Leonor Sirotti", "Paul Paganiban", "Firmin Martin", "Michael McManus", "Philippe Hupé", "Nicolas Joly", "Nicolas Fedy", "Thomas Magalhaes", "Leonor Sirotti", "Paul Paganiban", "Firmin Martin", "Michael McManus" ], "abstract": "Life science has entered the so-called ’big data era’ where biologists, clinicians and bioinformaticians are overwhelmed with unprecedented amount of data. High-throughput sequencing has revolutionized genomics and offers new insights to decipher the genome structure. However, using these data for daily clinical practice care and diagnosis purposes is challenging as the data are bigger and bigger. Therefore, we implemented software using Message Passing Interface such that the alignment and sorting of sequencing reads can easily scale on high-performance computing architecture. Our implementation makes it possible to reduce the time to delivery to few minutes, even on large whole-genome data using several hundreds of cores.", "keywords": [ "High-Throughput Sequencing", "Alignment", "Sorting", "High-Performance Computing", "MPI" ], "content": "Introduction\n\nSince the first next generation sequencing technology was released in 2005 (Kchouk et al., 2017), considerable progress has been made in terms of sequencing quality, diversity of protocols and throughput of the machines. As of today, the most recent generation of sequencers can easily produce terabytes of data each days and we expect this exponential growth of the sequencing to continue. This data tsunami raises many challenges, from data management to data analysis, requiring an efficient high-performance computing architecture (Lightbody et al., 2019). Indeed, the throughput capacity of the sequencers tends to overwhelm the capacity of common computer architectures and the data analysis workflow to handle such amount of data in a reasonable time. As we have entered the era of genomic medicine, delivering the results to the clinicians within a short delay to guide the therapeutic decision is a challenge of the utmost importance in daily clinical practice. Several national initiatives worldwide such as France, USA, UK or Australia (Stark et al., 2019) promote the use of genomics into healthcare. There is no doubt that exascale informatic architecture and software are required to tackle the challenges raised by the genomic medicine.\n\nA typical bioinformatics workflow to analyze high-throughput sequencing (HTS) data consists of a set of systematic steps of pre-processing to i) align (or map) the sequencing reads on a reference genome and ii) to sort the alignments according to their coordinates on the genome. Those steps are fundamental for the efficiency and relevance of the downstream analysis to decipher genomic alterations such as mutations or structural variations. Traditional tools for aligning the reads are BWA-MEM (Li & Durbin, 2010) and SOAP2 (Li et al., 2009a), and the sorting is usually performed with Samtools (Li et al., 2009b), Sambamba (Tarasov et al., 2015), Picard tools and GATK (McKenna et al., 2010). Most of the time, these steps are very time consuming (up to several days for whole genome analysis) as they suffer from bottlenecks at the CPU, IO and memory levels. Therefore, removing these bottlenecks would make it possible to reduce the time-to-delivery of the results such that they could be available within a reasonable delay when very large data are produced by the sequencers.\n\nIn order to tackle the aforementioned challenges to align and sort the sequencing data, we developed software based on the Message Passing Interface (MPI) communication protocol (Gropp et al., 1996) that makes it possible to fully benefit of parallel architecture of supercomputers Hupé & Jarlier (2020a; 2020b). MPI can therefore reduce the different bottlenecks by capitalizing on the low-latency network fabrics generally available on modern supercomputers. This allows an efficient distribution of the workload over the available resources of the supercomputers thus providing the expected scalability.\n\n\nMethods\n\nFor the alignment, the software consists of a MPI wrapper to the BWA-MEM software such that the original code remains unchanged. The wrapper uses parallel IO and shared memory (Figure 1). It first parses the input FASTQ files in order to define chunks of equal size. The different chunks are actually represented as two offsets (start and end) from the original FASTQ file thus reducing the amount of information to store. The reference genome is loaded into the shared memory on each computer node. Then, each chunk is processed in parallel threads by the original BWA-MEM algorithm. The results is finally written thanks to a shared file pointer in a unique SAM file. For a human genome, the size of the reference genome file in the shared memory takes around 5 GB and the total memory used by a BWA-MEM thread is around 300 MB. The original BWA-MEM processes a chunk of reads at a time that contains the same number of nucleotides which is also the case with our parallel implementation in order to allow the reproducibility with the original algorithm. We name our code alignment mpiBWA (Hupé & Jarlier, 2020a).\n\nEach core is in charge of aligning a chunk of the FASTQ file. The reference genome is stored in shared memory of each node of the computing cluster. Once aligned, the results are written into a SAM file. To ensure scalability, the read and write operation require a parallel filesystem.\n\nFor the sorting, the software implements a parallel version of the bitonic sort proposed by Batcher (1968) for sorting any sequence of elements of size n = 2k being of power of 2. Its complexity is 𝒪 (n log2n) that is higher than 𝒪 (n log n) from the popular merge sort algorithm. However, the bitonic sort is very suitable for parallel implementation since it always compares elements in predefined sorting network that is independent of the input data. To understand how the algorithm works, we first defined in what follows some concepts (see Grama et al. (2003) for details).\n\nThe algorithm relies on a bitonic sequence that is a sequence of values 〈a0, a1, … , an−1〉 with the property that 1) there is an index i, 0 ≤ i ≤ n−1 such that 〈a0, a1, … , ai〉 is monotonically increasing and 〈ai+1, … , an−1〉 is monotonically decreasing, or 2) there exists a cyclic shift of indices so that the condition 1) is satisfied. From any bitonic sequence s = 〈a0, a1, … , an−1〉, the bitonic split operation consists in transforming the input sequence s into these two subsequences:\n\n\n\nBatcher (1968) proved that both s1 and s2 are bitonic sequences and the elements of s1 are smaller than the elements of s2. Thus, a recursive bitonic split from a bitonic sequence of size n = 2k , until the sequence obtained are of size one allows the sorting of the input bitonic sequence in k splits as shown in Figure 2. The procedure of sorting a bitonic sequence from bitonic split operation is called bitonic merge. For each split of a bitonic merge, n/2 comparisons are performed during which the two numbers are exchanged if not in the right order using a compare-exchange operation. A bitonic merge procedure of a sequence of size n is noted BMn⊕ if the comparisons sort the number in monotonically increasing order or BMn⊖ for monotonically decreasing order.\n\nEach horizontal line represents an input and each arrow represents a two-input/two-output comparator which performs compare-exchange operation. Depending on its direction, the outputs are sorted in increasing (decreasing) order with an downwards (upwards) arrow. First, the comparator network transforms an input sequence of 16 unordered numbers into a bitonic sequence alternating BM2⊕/BM2⊖, BM4⊕/BM4⊖and BM8⊕/BM8⊖ bitonic merges. Then, the obtained bitonic sequence is sorted with a recursive bitonic merge procedure BM16⊕ (adapted from Grama et al. (2003)).\n\nSorting any sequence of unordered number thus requires to convert the input sequence into a bitonic sequence. This is obtained using a bitonic sorting network (see Figure 2) that sorts n = 2k numbers using k − 1 bitonic sorting stages, where the i-th stage is composed of n/2i alternating increasing BM2i⊕ and decreasing BM2j⊖ bitonic merges. The final stage being BMn⊕ to obtain the sorted sequence.\n\nIt is straightforward to generalize the bitonic sort algorithm on parallel architecture as shown in Figure 3. Each processor is in charge of an even number m = n/p elements (where p is the number of processors that is a power of 2) that are first sorted using a merge sort algorithm. Then, each comparison over the bitonic sorting network is replaced by a pair of processors with performs a compare-split operation. During the compare-split operation, the two sorted sequences from each processor are merged into one monotonic sorted list, then bisected into two (lower and higher) sequences. After the compare-split, one processor will keep the lower m elements from sequence and the other processor will keep the higher m elements according to the direction of the arrow in the step (see Kim et al. (2001) & Grama et al. (2003) for details). This parallelization allows the sorting of n elements in 𝒪 (log2n) time using a bitonic sorting network.\n\n(a) Each horizontal line represents an input of elements attached to a processor. Each processor first locally sorts its list of 4 elements, then, the parallel sort performs three steps of compare-split operations over the bitonic sorting network. (b) During the compare-split operation, each pair of processors first exchanges their information: each processor sends its block to the other process, then it merges the two sorted blocks into one list and stores only the appropriate half of the merged block to be used in the next step (adapted from Kim et al. (2001) & Grama et al. (2003)).\n\nThe workflow for the sorting is described in Figure 4. The SAM file is read and split into p blocks that are dispatched across the p processors. Each block is parsed such that for each line of the SAM file, we extract the genomic coordinates of each read, its sequence (and all other information contained in the SAM file) and the line is indexed according to its offset in the SAM memory buffer of a processor. Then, the genomic coordinates are sorted with the bitonic sort as shown in Figure 3. During the sorting, a vector with five values follows the bitonic sorting network including the genomic coordinate c, the rank ri of the processor that parsed the block of the input SAM file, the offset oi of the line from the input SAM file, the rank rd of the processor that will write the block of the sorted data in the destination SAM file and the offset od of the line in the sorted destination SAM file. Obviously, the values rd and od are known when the bitonic sort is completed. It is important to highlight that the parallel computation is performed by different processors that are located on different compute nodes. This means that a block of data that has been read by a given processor is not accessible by another processor. Moreover, to optimize the writing of the sorted destination SAM file, it is essential to write the data in contiguous block. Thus, it is necessary that a processor in charge of the writing owns locally the data from a block of contiguous offsets od in the sorted destination file. This implies that all the data have to be shuffled across all the processors that have to exchange data in an all-to-all communication step. To optimize the communication between the processors during the shuffle, we have implemented a Bruck algorithm (Bruck et al., 1997) of time complexity 𝒪 (log2 p). The Bruck algorithm is performed twice as shown in Figure 4 and can be seen as a joint procedure between two tables (the elements of the table being located on different processors). During the first Bruck phase, the rd and od are sent back to the corresponding processor from where the data originated, and then, during the second Bruck phase, all the data (i.e. the appropriate lines of the SAM file) are sent to the processor that has been assigned to write the data. During the second phase the original data is copied into the memory buffer and then exchanged.\n\nThe SAM file is read and split over the 4 processors P0 to P3 that sort the data with the bitonic sort. Then, a first Bruck phase sent the rd and od values back to processor from where the data originated. The data from the SAM file are sent by a second Bruck phase to the processor that has been assigned to write the contiguous blocks (during this step, the original data is copied into the memory buffer and then exchanged). Finally, the data can be written on a parallel filesystem. c: genomic coordinate. ri: rank of the processor that parsed the block of the input SAM file. oi: offset of the line from the input SAM file. rd: rank of the processor that will write the block of the sorted data in the destination SAM file. od: offset of the line in the sorted destination SAM file.\n\nNote that when the SAM file contains several chromosomes, each chromosome is sorted successively. Therefore, the upper memory bound depends on the size of the whole SAM file plus internal structures plus the size of the biggest chromosome. In this case, the total amount of memory used is around 1.5 the size of the original SAM file if the file contains read on all the chromosome of the human genome. If the SAM file contains only one chromosome, then the total amount of memory required is 2.5 the size of the original SAM file. To be efficient, the sorting requires a number of cores being a power of 2. We name our code for sorting mpiSORT (Hupé & Jarlier, 2020b).\n\nWe benchmarked mpiBWA and mpiSORT on the HG001 NA12878 sample from GIAB (Zook et al., 2014). This sample is a whole genome with 300X depth of coverage composed of 2.13 billions 2x250 pair-ended reads. The BAM file has been downloaded from:\n\nurl: ftp://ftp-trace.ncbi.nlm.nih.gov/ReferenceSamples/giab/data/NA12878/\n\nfolder: NIST_NA12878_HG001_HiSeq_300x/NHGRI_Illumina300X_novoalign_bams/\n\nThe BAM file has been converted into FASTQ files using bedtools bamtofastq. In order to test the scalability of our software with respect to the size of the data, we downsampled the original 300X sample to obtain FASTQ files corresponding to depths of coverage ranging from 28X to 300X with a geometric progression with a common ratio of 1.6. The sequences have been aligned on the human genome GRCh38.\n\nWe ran the benchmark on a computing cluster equipped with Intel Xeon® Gold 6148 CPU @ 2.40GHz (Skylake architecture). The nodes are interconnected with Intel® Omni Path Architecture (OPA) of 100 Gbps speed. The parallel file system is BeegFS with two servers. We compile the programs with GCC 8.3 and use Open MPI 3.1.4. Jobs have been submitted using slurm scheduler.\n\nThe code has been written in C programming language using MPI directives. mpiBWA encapsulates the original BWA-MEM version 7.15. Two implementations for mpiBWA exist: the first outputs one SAM file with all the chromosomes, the second (named mpiBWAByChr) outputs one SAM file per chromosome. Moreover, mpiBWA comes along with mpiBWAIdx, which is responsible for creating a binary image of the reference genome. This image is subsequently loaded in the shared memory by mpiBWA. Then, every mpiBWA process on the same computing node will share the same genome reference in order to save memory usage. mpiBWAIdx does not need MPI to run.\n\nAs our software rely on the MPI standard, mpirun must be available to run the program. Several MPI implementations exist such as mpich, open-mpi or Intel® MPI Library.\n\n\nUse cases\n\nThe Figure 5 shows the scalability of both mpiBWA (with the mpiBWAByChr implementation) and mpiSORT with varying sample sizes (from 28X to 300X) using computation distributed over 128 cores. The output files from mpiBWAByChr have been used as input by mpiSORT. Both algorithms efficiently scale as the input data are bigger, the walltime to process the data being proportional to their input size. We assessed how much time was spent on pure computation versus IO (i.e. reading the input files and writing the output files): mpiBWA spent more than 97% of its walltime in computation while mpiSORT spent between 50% to 60% in IO. The walltime to analyze the biggest sample of 300x is 9h hours for the alignment and less than one hour for the sorting.\n\nThe FASTQ files are first aligned with mpiBWAByChr, SAM files are generated as outputs and then sorted with mpiSORT and -u option. 128 cores have been used over 16 and four nodes with mpiBWA and mpiSORT, respectively. The black dots represent the theoretical values of the walltime with respect to the 300X depth of coverage being set as the reference.\n\nThe first step consist in building the binary image of the reference genome that will be used in the shared memory by mpiBWA:\n\n\n\nThis creates the file myReferenceGenome.fa.map.\n\nImportantly, mpiBWAIdx requires the following files that are generated by BWA index:\n\n\n\nThe .map file needs only to be generated once. Then, mpiBWA is executed with the mpirun program, for example:\n\n\n\nThis command launches two processes MPI with eight BWA threads each (16 cores will be used). If you want to split the results of the alignment by chromosome, use mpiBWAByChr, for example:\n\n\n\nThe total memory used during the alignment if approximately the size of the .map file plus the size of the SAM chunks loaded by each BWA tasks. A BWA thread takes around 300 MB of memory.\n\nFrom the results obtained from mpiBWAByChr, each SAM files can be sorted as follows:\n\n\n\nThis command launches four processes MPI.\n\nAs mpiSORT requires all the input SAM file to be loaded into the memory, the program is memory bounded. Therefore, a lot of attention has to be paid to define how many cores are needed to process the data. For example, let’s assume that the computing cluster consists of nodes with 190 GB of RAM memory with 40 cores each (thus 4.75 GB per core is available but this value can be rounded to the lower limit of 4.5 GB to leave some free memory on the node). To choose the number of cores, the following rule has to be applied: the total memory for sorting a SAM file that contains only one chromosome is around 2.5 times the size of the SAM file. For instance, sorting a chr1.sam file of size 209 GB with 4.5 GB per core requires 128 cores, for a of size 110 GB it requires 64 cores. We remind that the bitonic sort algorithm requires a number of cores that is a power of 2, this is the reason why the number of cores has to be set to the upper bound to the closest power of 2.\n\n\nConclusion\n\nIn this paper, we described parallel algorithms to process sequencing data that fully benefit from high-performance architecture (Hupé & Jarlier (2020a; 2020b)) that differ from other implementations using embarrassingly parallel approaches (Decap et al., 2015; Kawalia et al., 2015; Puckelwartz et al., 2014). Our implementation is based on bitonic sorting network, Bruck algorithm and IO optimizations with MPI directives. The scalability of the software strongly relies on the underlying hardware architecture, such as the low latency interconnection and distributed file system. Interestingly, our implementation allows the generation of aligned read files for each chromosome that can be further sorted in parallel thus reducing the time of downstream analysis whenever an analysis per chromosome is possible. The performance we obtained showed that MPI is very relevant for the field of genomics. The tools we developed pave the way towards the use of whole genome sequencing in daily clinics in order to meet the deadline and deliver results to the clinician in real-time for precision medicine as the time to delivery can be reduced to several minutes if the code is distributed over several hundreds of cores.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSource code for mpiBWA available at: https://github.com/bioinfo-pf-curie/mpiBWA.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3727143 (Hupé & Jarlier (2020a)).\n\nSource code for mpiSORT available at: https://github.com/bioinfo-pf-curie/mpiSORT.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3727145 (Hupé & Jarlier (2020b)).\n\nAll documentation is available at: https://github.com/bioinfo-pf-curie/QUARTIC.\n\nLicense: CeCILL Version 2.1.\n\n\nAuthor contributions\n\nF.J. and N.J. developed and tested mpiBWA. F.J., N.F., L.S., T.M, P.P. and M.F. developed and tested mpiSORT. M.M. provided technical expertise and access to computing cluster facilies to benchmark the code. F.J. coordinated the developments. F.J. and P.H. wrote the manuscript. P.H. supervised the study.", "appendix": "Acknowledgments\n\nWe are very grateful to the national infrastructure France Génomique for providing us with an access to their computing cluster and we especially thank Claude Scarpelli as the head of its informatics department. We thank François Prud’homme and Sébastien Astruc from the ICT department at Institut Curie. We also thanks Intel Corporation collaborators for fruitfull discussions and help.\n\n\nReferences\n\nKchouk M, Gibrat JF, Elloumi M: Generations of sequencing technologies: from first to next generation. Biology and Medicine. 2017; 9(3). Reference Source\n\nLightbody G, Haberland V, Browne F, et al.: Review of applications of high-throughput sequencing in personalized medicine: barriers and facilitators of future progress in research and clinical application. Brief Bioinform. 2019; 20(5): 1795–811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStark Z, Dolman L, Manolio TA, et al.: Integrating genomics into healthcare: A global responsibility. Am J Hum Genet. 2019; 104(1): 13–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Durbin R: Fast and accurate long-read alignment with burrows-wheeler transform. Bioinformatics. 2010; 26(5): 589–595. 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. 2009a; 25(16): 2078–2079. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi R, Yu C, Li Y, et al.: Soap2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009b; 25(15): 1966–1967. PubMed Abstract \| Publisher Full Text\n\nTarasov A, Vilella AJ, Cuppen E, et al.: Sambamba: fast processing of NGS alignment formats. Bioinformatics. 2015; 31(12): 2032–2034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKenna A, Hanna M, Banks E, et al.: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010; 20(9): 1297–1303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGroppa W, Lusk E, Doss N, et al.: A high-performance, portable implementation of the MPI message passing interface standard. Parallel Computing. 1996; 22(6): 789–828. Publisher Full Text\n\nHupé P, Jarlier F: bioinfo-pf-curie/mpibwa: version-1.0. 2020a. http://www.doi.org/10.5281/zenodo.3727143\n\nHupé P, Jarlier F: bioinfo-pf-curie/mpisort: version-1.0. 2020b. http://www.doi.org/10.5281/zenodo.3727145\n\nBatcher KE: Sorting networks and their applications. In Proceedings of the April 30–May 2,1968, spring joint computer conference. 1968; 307–314. Publisher Full Text\n\nGrama A, Karypis G, Kumar V, et al.: Introduction to Parallel Computing. Addison-Wesley, second edition, 2003. ISBN 0201648652 9780201648652. Reference Source\n\nKim YC, Jeon M, Kim D, et al.: Communication-efficient bitonic sort on a distributed memory parallel computer. In Proceedings. Eighth International Conference on Parallel and Distributed Systems. ICPADS 2001. IEEE. 2001; 165–170. Publisher Full Text\n\nBruck J, Ho CT, Kipnis S, et al.: Efficient algorithms for all-to-all communications in multiport message-passing systems. Parallel and Distributed Systems, IEEE Transactions on. 1997; 8(11). Publisher Full Text\n\nZook JM, Chapman B, Wang J, et al.: Integrating human sequence data sets provides a resource of benchmark SNP and indel genotype calls. Nat Biotechnol. 2014; 32(3): 246–51. PubMed Abstract \| Publisher Full Text\n\nPuckelwartz MJ, Pesce LL, Nelakuditi V, et al.: Supercomputing for the parallelization of whole genome analysis. Bioinformatics. 2014; 30(11): 1508–1513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDecap D, Reumers J, Herzeel C, et al.: Halvade: scalable sequence analysis with mapreduce. Bioinformatics. 2015; 31(15): 2482–2488. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKawalia A, Motameny S, Wonczak S, et al.: Leveraging the power of high performance computing for next generation sequencing data analysis: tricks and twists from a high throughput exome workflow. PLoS One. 2015; 10(5): e0126321. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "62078", "date": "04 May 2020", "name": "Ramon Amela Milian", "expertise": [ "Reviewer Expertise bioinformatics" ], "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\nJarlier and colleagues introduce their work on parallelizing popular bioinformatics programs, which have become the bottleneck for the current scenario of massive NGS data generation even in clinical settings.\nAuthors describe two MPI implementations for the alignment and sorting steps used as part of the NGS data preprocessing in order to better take advantage of parallel architectures. Algorithms are well described and the documentation is good, making really easy the reproducibility of their results 1. In addition, authors provide software containers (singularity), which can be used in controlled environments including HPC installations.\nDespite the existing benchmarking efforts carried by authors, a scalability study is missing. Ideally, benchmarking should include varying resources allocation so readers can benefit from such comparison. We would like to suggest, as you have done with varying deep coverage sizes, you make the scalability study through a varying number of processors e.g. 4, 8, 16, 32, 64, 128. It would be also interesting to use as a baseline the normal execution of those programs to illustrate the improvement of the MPI implementation regarding the standard approaches. This way, it would be possible to answer the following questions:\nIs the MPI version faster when using a single node?\n\nOtherwise, from which point is better to use the MPI version than the most common software. I.e. in case the MPI is 2 times slower but scales linearly, the execution time would be shorter when giving the MPI implementation at least 2 nodes.\nThis way, the potential users would know better the scenarios in which using this implementation becomes more interesting.\nYou have mentioned there are similar approximations to this problem. However, you just mention them in the conclusions. Wouldn’t it make sense to discuss similarities and differences with other implementations in the text?\nApart from the benchmarking component, it would be interesting to further discuss the scenarios where implementations like this one will be used. It is not clear that clinical settings will have internally very powerful computational facilities for their daily use.\nFinally, we would like to mention some minor points related to the text itself.\nAbstract: Moving from the general problem to the specific solution without mentioning that the NGS data preprocessing (alignment and sorting) represent major bottlenecks to timely results deliver for diagnostic use.\nMethods:\nAlignment. reference genome file > indicate a version.\n\nSorting section. Apparently there is a miswriting of the theoretical computational cost of the algorithm > O (n log2 n) that is higher than O (n log n).\nMinor:\nIntroduction: data each days > data each day.\n\nIntroduction: align and sort the sequencing data, we developed software > align and sort sequencing data, we have developed software.\n\nSorting section: by a pair of processors with performs a compare-split operation > by a pair of processors which performs a compare-split operation.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5610", "date": "23 Jun 2020", "name": "Philippe Hupé", "role": "Author Response", "response": "First, we would like to thank the reviewers. We are very grateful for their time, contribution and very valuable comments that significantly helped to improve the article and the documentations of the tools. You will find below a detailed answer to the different issues that we have addressed in the revised manuscript. Best regards, Frédéric Jarlier and Philippe Hupé Detailed response to the reviewer1's comments > Despite the existing benchmarking efforts carried by authors, a scalability study is missing. Ideally, benchmarking should include varying resources allocation so readers can benefit from such comparison. We would like to suggest, as you have done with varying deep coverage sizes, you make the scalability study through a varying number of processors e.g. 4, 8, 16, 32, 64, 128. Indeed, this benchmark varying the number of cores was clearly missing. Therefore, a scalability study has been added for both mpiBWA and mpiSORT using 8, 16, 32, 64, 128 cores. The results of this benchmark are described in the section \"Use cases\" and in Figure 6. We took this opportunity of this new benchmark to use the last version (1.1) of our MPI implementations (Figure 5have been therefore updated). > It would be also interesting to use as a baseline the normal execution of those programs to illustrate the improvement of the MPI implementation regarding the standard approaches. As suggested, we also compared our MPI implementations with respect to a reference baseline using the traditional tools (bwa and samtools). The walltimes of the reference baseline are presented in the section \"Use cases\" and in Figure 6. > This way, it would be possible to answer the following questions: > > Is the MPI version faster when using a single node? > The results show that the walltimes are similar between mpiBWA and bwa. mpiSORT is much faster than samtools offering a speed-up over 6. > Otherwise, from which point is better to use the MPI version than the most common software. I.e. in case the MPI is 2 times slower but scales linearly, the execution time would be shorter when giving the MPI implementation at least 2 nodes. > The results of the scalability benchmark and the comparison with respect to the traditional tools demonstrate that both mpiBWA and mpiSORT perform efficiently on a single node and on multiple nodes. > This way, the potential users would know better the scenarios in which using this implementation becomes more interesting. > The results of the scalability benchmark show that the MPI implementation can be very versatile. Our MPI implementations can easily address the two typical scenarii: when time-to-delivery matters, the scalability allows the processing of the data very quickly using multiple cores and nodes. when the throughput matters (i.e. number of samples that can be analyzed at the same time), you can process a sample on a single node. We also added a benchmark section in the documentation of the github repository such that the user can reproduce the benchmark to figure out what is the best scenario according to the computing infrastructure that is used. The documentations on the github repository describe how to assess the memory and cpu usage for the traditional tools and the MPI implementations as we did for the scalabitity study. The documentation is available here: https://github.com/bioinfo-pf-curie/mpiSORT/blob/master/docs/README.md#benchmark https://github.com/bioinfo-pf-curie/mpiBWA/blob/master/docs/README.md#benchmark > You have mentioned there are similar approximations to this problem. However, you just mention them in the conclusions. Wouldn’t it make sense to discuss similarities and differences with other implementations in the text? We added in the conclusion that: Indeed, this differs from other implementations using embarrassingly parallel approaches (Puckelwartz et al., 2014; Decap et al., 2015; Kawalia et al.,2015} that rely on the MapReduce paradigm: in this case, input files are first split into small chunks, each one being analyzed by a process, and the different output files are then merged into a single output. With MPI, splitting the input file is not necessary. > Apart from the benchmarking component, it would be interesting to further discuss the scenarios where implementations like this one will be used. It is not clear that clinical settings will have internally very powerful computational facilities for their daily use. > We agree that the access to a powerful and up-to-date computing infrastructure may be a bottleneck. We added in the conclusion of manuscript: This implies that the hospitals or institutions need a powerful state-of-the-art informatic architecture that is either available locally or provided with mutualized resources through national infrastructures that are certified to process healthcare data. > Finally, we would like to mention some minor points related to the text itself. > > Abstract: Moving from the general problem to the specific solution without mentioning that the NGS data preprocessing (alignment and sorting) represent major bottlenecks to timely results deliver for diagnostic use. We agree that the Abstract needed to be improved. It has been substantially modified in the revised version. > Methods: > > Alignment. reference genome file > indicate a version. > We added in the text \"The reference genome of your choice\" as the user can use any genome. > Sorting section. Apparently there is a miswriting of the theoretical computational cost of the algorithm > O (n log2 n) that is higher than O (n log n). The complexity are correct. Its is O (n log2 n) for the bitonic sort, it is higher that other sort algorithms but it can be easily parallelized. > > Minor: > > Introduction: data each days > data each day. > > Introduction: align and sort the sequencing data, we developed software > align and sort sequencing data, we have developed software. > > Sorting section: by a pair of processors with performs a compare-split operation > by a pair of processors which performs a compare-split operation. > The typos have been corrected." } ] } ]	1	https://f1000research.com/articles/9-240
https://f1000research.com/articles/9-307/v1	29 Apr 20	{ "type": "Research Article", "title": "Cooking with biomass fuel and cardiovascular disease: a cross-sectional study among rural villagers in Phitsanulok, Thailand", "authors": [ "Chudchawal Juntarawijit", "Yuwayong Juntarawijit", "Yuwayong Juntarawijit" ], "abstract": "Background: Burning biomass fuel is a major source of indoor air pollution; about 40% of Thai people still use biomass for cooking. There is increasing evidence of the association between biomass smoke exposure and serious health effects including cardiovascular disease. The object of this cross-sectional study was to investigate the association between biomass use for household cooking and cardiovascular outcome, including coronary heart disease, hypertension, high cholesterol, diabetes mellitus, and stroke among rural villagers in Phitsanulok, Thailand. Methods: Data from 1078 households were collected using a face-to-face interview questionnaire. Results: After being adjusted for gender, age, cigarette smoke, secondhand smoke, and exposure to other sources of air pollution, it was found that the family members of cooks using biomass fuel were at risk of coronary heart disease (CHD; OR=4.35; 95%CI 0.10–18.97), high blood pressure (HBP; OR=1.61; 95%CI 1.10–2.35), high cholesterol (HC; OR=2.74; 95%CI 1.66–4.53), and diabetes (OR= 1.88; 95%CI 1.03–3.46). Compared to LPG use, using wood was associated with stroke (OR=7.64; 95%CI 1.18–49.61), and using charcoal was associated with HC (OR=1.52; 95%CI 1.04–2.24). Compared to never user, household cooks who sometimes use charcoal had an increased risk of HBP (OR=2.04; 95%CI 1.32–3.15), HC (OR=2.61; 95%CI 1.63–4.18), and diabetes (OR=2.09; 95%CI 1.17–3.73); and cooks who often use charcoal had an elevated risk of stroke (OR=3.17; 95%CI 1.04–9.71), and HC (OR=1.52; 95%CI 1.02–2.27) to their family members. Conclusions: The study results were consistent with those found in studies from other parts of the world, and supports that exposure to biomass smoke increase cardiovascular diseases. The issue should receive more attention, and promotion of clean fuel use is a prominent action.", "keywords": [ "Biomass fuel", "cardiovascular diseases", "household air pollution", "kitchen smoke", "cooking fume" ], "content": "Introduction\n\nCooking smoke is a major source of household air pollution, which affects billions of people around the world, especially in developing countries. Globally, nearly 3 billion people still use solid fuels (wood, charcoal, crop residues, and dung) for cooking and heating1. Smoke from wood burning contains a large number of pollutants, including particulate matter, carbon monoxide, nitrogen dioxide, formaldehyde, and a number of highly toxic organic compounds, such as benzene, 1, 3 butadiene, benzo[a]pyrene and other toxic polycyclic aromatic hydrocarbons2.\n\nThe use of solid fuel for cooking and/or household energy sources increases respiratory and non-respiratory illnesses in both adults and children. Those effects that are well established are acute respiratory infections, chronic obstructive pulmonary disease (COPD), lung cancer, asthma, tuberculosis, and cataracts3,4. In children, biomass use is related to mortality, and acute lower respiratory tract infections, and some other non-respiratory illness, such as poor lung function, low birthweight, nutritional deficiency, and impairment of learning ability5,6.\n\nThough with limited evidence, recent studies linked biomass smoke exposure and cardiovascular diseases (CVD), e.g. coronary heart disease (CHD), hypertension or high blood pressure (HBP), diabetes, and stroke7–10. In laboratory studies, chronic exposure to biomass smoke increased the thickness and plaque of blood vessels11. In epidemiological studies, Peruvians who live in high altitude environments and use biomass fuel had an elevated prevalence of HBP12. A study among villager women in Bangladesh reported an association between elevated cumulative exposure to biomass smoke and the prevalence of HBP13. A similar result was found in a study in Shanghai Putuo, which found using solid fuel increases the risk of HBP, CHD, and diabetes14; and a study in Shanxi, China reported an increased risk of HBP, CHD, stroke, diabetes, and dyslipidemia15 A recent study by Yu et al.16 also linked solid fuel use to cardiovascular mortality.\n\nOn a global scale, CVD is the number one cause of death and is responsible for about 18 million deaths annually17. In Thailand, CVD accounts for 23% of the national mortality18. Currently, there is no study on the effect of biomass smoke on CVD in Thailand. It was reported that about 40% of Thai households still use biomass, mainly charcoal, wood, and agriculture residue, for cooking19. The objective of this study is to investigate a possible association between biomass use for cooking and cardiovascular diseases, including CHD, HBP, HC, diabetes, and stroke. The study uses data from a cross-sectional survey among rural villagers in Phitsanulok, Thailand. The result could be used for disease prevention and control, and to support the global literature.\n\n\nMethods\n\nThis is cross-sectional study. Participants are rural villagers living in Phitsanulok Province, Thailand. Phitsanulok is a midsize province located about 400 km north of Bangkok. There are 866,891 people in the area of 9 districts. Most of the people are rice farmers20.\n\nParticipants were randomly selected using multistage sampling. Out of the 9 districts in Phitsanulok province, 5 were randomly selected. In each district, one sub-district and a local health-promoting hospital were approached. In each sub-district with support from the local health-promoting hospital, a total of 1,150 households were approached and 1,134 (98.6%) people agreed to participate in the study. In each household, only one participant who was responsible for household cooking and aged over 20 years was selected. After data cleanup, 56 (4.9%) items of data were missing important information, such as age, gender, cooking practice. The final data from 1,078 people were used for statistical analysis.\n\nThe minimum sample size was calculated to be 1,034, using unmatched cross-sectional study with the following assumptions: two-sided significance level = 95%; power of detection = 80%; percent unexposed with outcome = 5%; and odds ratio = 2.0.\n\nData was collected using a face-to-face interview questionnaire, which was administered by 15 village health volunteers (provided as Extended data in English21). The interviews took place in the house of participants. The data was collected during the period of May–June 2017. Health volunteers were all trained on how to properly carry out the interview and use the questionnaire. The questionnaire was designed to collect information on demographic data, fuel use for cooking, and other cooking practices. In addition to general demographic data, participants were also asked a history of tobacco use (ever, never), and working in factory environments using “yes” or “no” questions. Ever smoker referred to those who smoke more than 100 cigarettes in their lifetime. Data on pesticide use was also measured by “yes” or “no” questions: “Have you ever spray or mix pesticide?”. For cooking fuel data, we asked about the types of fuel they used for cooking food (wood, charcoal, LPG, electricity), and the frequency of using each types of fuel. Data collected on cooking practices were types of cooking oil, the frequency of tears while cooking (TWC) (never, sometimes, often), kitchen location (inside a house, outside a house, both inside and outside a house), and the characteristics of kitchen ventilation (good or poor ventilation).\n\nThe presence of cardiovascular disease was determined by the participant response to the question: “Have you ever been diagnosed with the following diseases (coronary heart disease (CHD), high blood pressure (HBP), high cholesterol (HC), diabetes mellitus, stroke) by a medical doctor?”. For diseases among their family members, we asked “Did you have a family member with the following diseases?”.\n\nThe content validity of the questions was tested by three experts, and the Index of Item Objective Congruence (IOC) was between 0.7–1.0. The questionnaire was also tested for question sequencing and understanding using a group of 30 people with a similar background to the intended participants.\n\nDemographic and prevalence of cardiovascular disease were descriptively analyzed. Comparison between groups were analyzed using chi-square test for categorical variables, and independent t-test for continuous variables. The association between cardiovascular disease was analyzed using logistic regression with odds ratios (OR) and 95 percent confidence interval (CI) adjusted for gender (male, female), age (continuous data), cigarette smoking (ever, never), living smoker (yes, no), working with smokers (yes, no), and exposure to air pollution (yes, no). All statistical analyses were performed using IBM SPSS version 19 and OpenEpi (online version 3.01). Statistical significance was set at a p-value of less than 0.05.\n\nThe study was approved by the Ethical Committee of Naresuan University (COA No. 485/2016), and written informed consent from the respondents was obtained before the interviews were conducted.\n\n\nResults\n\nMost of the respondents were women (84.2%) with a mean age of 53.04 ± 12.93 yr. The highest education levels were primary school or high school. Most were farmers (36.0%) and 20.2% were causal workers on farms. About 10% were smokers and 33% lived with a smoker. Additional information on the demographic data is shown in Table 1 and in Underlying data22.\n\nAbout 70% of the respondents reported using biomass for cooking (Table 2). However, when asked for fuel types that they usually use for cooking, 64.5% reported LPG and 32.3% charcoal. Among those who use charcoal, 38.6% use it often. About half have a kitchen located inside a house with good ventilation. Almost all reported having TWC either sometimes or often. Most of them cook every day.\n\n* P–value of chi square test for difference between biomass user and not use group, 2–trail\n\n** Significantly difference, p <0.05\n\nThe study found HBP, HC, and diabetes to be the most common cardiovascular outcomes (Table 3). Compared to non-user group, biomass users had a significantly higher prevalence of HBP, and HC, and their family members also had more incidence of HBP, HC, diabetes, and heart disease.\n\na disease of respondent (R)\n\nb disease of respondent’s family member (F)\n\n* P–value of chi square test for difference between diseases prevalence among biomass use and LPG use\n\n** Significant at P < 0.05, 2–tail\n\nFurther analysis using logistic regression and control variables, revealed that compared to gas users, biomass users had family members with elevated CHD, HBP, HC, and diabetes (Table 4). Among different types of fuel, household cooks using wood had a significant elevated risk of CHD (OR=7.64, 95%CI 1.18-49.61), and their family members had an elevated risk of HC (OR=1.52, 95%CI 1.04-2.24). Comparing frequency of charcoal use, those who use charcoal sometimes or often are more likely to have CHD, HBP, HC, and diabetes as compared to those who never use charcoal. The family members of charcoal users also had a significant increase of HC and stroke. When using TWC as an indicator for smoke exposure, it was found that those who always had TWC had significantly increased risk of stroke (OR=2.16; 95%CI 1.08-4.32), and those with sometimes TWC had a CHD risk (OR=2.64; 95%CI 1.02-6.81). Regarding kitchen location, the family members of cooks having kitchens both inside and outside a house had an elevated risk of stroke (OR=4.60; 95%CI 1.14-18.54).\n\n*Logistic regression, adjusted for gender (male, female), age (continuous), cigarette smoking (ever, never), living with smoker (yes, no), working with smoker (yes, no), pesticide use (yes, no), working in a factory (yes, no)\n\n\nDiscussion\n\nThis study presented an association between cardiovascular diseases and exposure to smoke from biomass, mainly charcoal, which is relatively cleaner when compared to wood, coal, or dung, a biomass which were often found in the literature. The study also showed that biomass use not only affects household cooks but also their family members. It was found that biomass users have a higher prevalence of HBP and HC, and their family members had a higher prevalence of HBP, HC, diabetes, and CHD (Table 3). Further analysis using logistic regression and control for potential confounder showed a significant OR of biomass use and CHD(F), HBP(F), HC(F), and diabetes(F) (Table 4). Compared to LPG, wood use also had a strong association with stroke (OR=7.64; 95%CI 1.18–49.61). Among charcoal users, those who use it sometimes or often had an elevated risk of CHD, HBP, HC, and diabetes for themselves, and risk of HC and stroke for their family members. The results are consistent with the literature. Previous research found biomass smoke contains a lot of pollutants, especially fine particulates, and carbon monoxide which are known to cause cardiovascular effects2. In laboratory studies, biomass smoke exposure was associated with endothelial inflammation23.\n\nFor hypertension, we found both cooks and their family members have a higher prevalence of HBP (Table 3). Further analysis indicated an elevated risk of HBP (OR= 1.61; 95%CI 1.10–2.35) among family members of cooks using biomass for cooking (Table 4). As compared to those who never use it, cooks who sometimes use charcoal have twice the risk of HBP (OR=2.04; 95%CI 1.32–3.15) and those who use charcoal over twenty years have 1.38 times the risk of HBP (OR=1.38; 95%CI 1.01–1.89). In the literature, there is increasing evidence to link biomass smoke and HBP24,25. A study in Peru found that biomass users had an increased risk of both prehypertension (OR=5.0; 95%CI 2.6–9.9), and hypertension (OR=3.5; 95%CI 1.7–7.0)12. In Bangladesh, it was found that among rural women, each additional year of biomass smoke exposure could increase the risk of HBP by 61% (OR=1.61; 95%CI 1.16–2.22)26. In Bangladesh, it was found that among rural women, one additional year of biomass smoke exposure to increase risk of HBP by 61% (OR=1.61; 95%CI 1.16–2.22)13. Recent studies in Honduras also linked PM2.5 and black carbon exposure and HBP among women using traditional and improved stoves25.\n\nThe current study also found a higher prevalence of HC among cooks and their family members using biomass fuel (Table 3) with a significant OR of 2.74 (95%CI 1.66–4.53) for family members (Table 4). The result showed a difference in the risk of HC among those who use wood, charcoal, and LPG. This risk also varied particularly according to the frequency of charcoal use. Compared to nonusers, an elevated risk of HC was found among cooks who sometimes use charcoal (OR=2.04; 95%CI 1.32–3.15), and among those who use charcoal over 20 years (OR=1.73; 95%CI 1.22–2.44). Among cooks, every year of using charcoal will increase risk of HC by about 1% (OR=1.010; 95%CI 1.002–1.017). Risk of HC was also increased among family members of cooks who often use charcoal (OR=1.52; 95%CI 1.02–2.27). Though the evidence was limited, other studies have found an association between cholesterol and COPD, a disease often found among biomass users26. A study in Ghana also found a strong association between wood smoke exposure and several hematological and biochemical indices, including HC (OR=20.44; 95%CI 2.610–160.2)27. The higher OR might be explained by the difference in biomass types, which was found to be wood in other studies, while most of respondents in this study use charcoal which is relatively cleaner.\n\nWe found about 10% of the respondents had type 2 diabetes and the prevalence of the disease was higher among biomass users (Table 3). Logistic regression analysis revealed a significant risk of diabetes among cooks using charcoal sometimes (OR=2.09; 95%CI 1.17–3.73) as compared to the never user group (Table 4). Among family members of cooks, risk of diabetes was elevated by using biomass fuel (OR=1.88; 95%CI 1.03–3.46), and years of using charcoal (OR=1.013; 95%CI 1.001–1.024). Similar results have also been reported by several studies on the effect of particulate matter or traffic-related air pollutants on diabetes28. In addition, experimental studies may provide potential mechanisms, including glucose homeostasis, systemic inflammation, stress in the liver and endoplasmic reticulum, and alterations of mitochondrial and other adipose tissue29. Currently, epidemiological studies on the effect of indoor air pollution on diabetes are rare. A study of women in Honduras reported an association between the prevalence of prediabetes/diabetes and PM2.5 in kitchen biomass cooking stoves30. This was consistent with the results from a previous study from Shanghai Putuo, which also found an elevated risk of several cardiovascular diseases including diabetes (OR=2.48; 95%CI 1.59–3.86) among people using solid fuel at home14.\n\nThose who use biomass for cooking had a risk of CHD 4.35 times (95%CI 0.10–18.97) of LPG users; and those using charcoal sometimes had risk of CHD 4.11 times (95%CI 1.40–12.11) of never user group. These results are consistent with evidence from cigarette smoke and ambient air pollution. In animal studies, biomass fuel smoke caused arteriosclerotic effects in animal blood vessels11. Studies found COPD as a risk factor of CHD31; and our previous study found elevated chronic symptoms, such as chronic cough, dyspnea and runny nose which is a sign of COPD among cooks using biomass fuel for cooking26. Epidemiological studies also reported an association between solid fuel smoke exposure and CHD32. A study in Pakistan found that rural women who currently use solid fuel had an increased risk of acute coronary syndrome (OR=4.8; 95%CI 1.5–14.8)33. This is consistent with a study from Shanghai Putuo, which found solid fuel use in the home is associated with CHD (OR=2.58; 95%CI 1.53–4.32)14, and study from Shanxi, China found an elevated risk of CHD (OR=2.25) among solid fuel users15.\n\nIn this study, respondents who use wood (OR=7.64; 95%CI 1.18–49.61) and charcoal (OR=2.03; 95%CI 0.58–7.09) had an elevated risk of stroke as compared to clean fuel users (Table 4). Among charcoal users, those using charcoal sometimes (OR=1.66; 95%CI 0.44–6.29) and often (OR=2.76; 95%CI 0.56–13.50) seem to have a higher risk of stroke but a significant elevation was found only among the family members of cooks using charcoal often (OR=3.17; 95%CI 1.04–9.71). This was consistent with the literature. The association between household solid fuel use and stoke were also reported in a study from Shanghai Putuo (OR=1.87; 95% CI 1.03–3.38)14, and study from Shanxi, China (OR=1.64)15. In ambient settings, a long-term effect of PM exposure on cardiovascular disease, including stroke, was well established34. It was estimated that for each 10 µg/m3 increment in PM10, risk of overall stroke events will increase by 1.06 times (95%CI 1.02–1.11), and the risk of stoke mortality by 1.08 times (95%CI 0.99–1.18)35.\n\nOne potential drawback of this study was the use of self-reported data of diseases. Without the confirmation of medical records, the survey diseases are subjected to information bias. However, the bias will be distributed equally to all comparison groups, and this tends to underestimate the result. The number of participants included in this study was also rather small to detect the actual association of a rare disease, e.g. stroke. By using cross-sectional design, the study result cannot explain the causal relationship, because it is not known whether exposure or the disease occurred first. However, the problem is minimal for rare diseases.\n\n\nConclusions\n\nThe results from this study support research findings in other part of the world that using biomass for cooking increases the risk of cardiovascular diseases. This study also confirms the negative effects of using charcoal, which is considered to be a relatively cleaner fuel as compared with wood, dung, coal, and other agricultural residues. Concerned organizations should pay more attention to the issue and promote clean fuel usage.\n\n\nData availability\n\nFigshare: Household cooking and cardiovascular diseases, https://doi.org/10.6084/m9.figshare.12117066.v222.\n\nThis project contains the following underlying data:\n\nHousehold cooking and cardiovascular diseases.sav (Collected demographic and cardiovascular diseases data)\n\nData dictionary.docx (Word document containing dictionary for study dataset)\n\nFigshare: Questionnaire-household cooking and cardiovascular disease, https://doi.org/10.6084/m9.figshare.12121887.v221.\n\nThis project contains the following extended data:\n\nQuestionnaire-household cooking and cardiovascular disease.docx (Study questionnaire in English)\n\nQuestionnaire-household cooking and cardiovascular disease-Thai.docx (Study questionnaire in Thai)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to thank the participants in this study. Our appreciation also goes to local health promoting hospitals in Phitsanulok and the village health volunteers for data collection. We would like also to thank Mr. Kevin Mark Roebl of the Division of International Affairs and Language Development, Naresuan University for editing assistance.\n\n\nReferences\n\nWHO: Household Energy and Health Household Energy and Health. Geneva, Switzerland; 2006.\n\nNaeher LP, Brauer M, Lipsett M, et al.: Woodsmoke health effects: a review. Inhal Toxicol. 2007; 19(1): 67–106. PubMed Abstract \| Publisher Full Text\n\nKim KH, Jahan SA, Kabir E: A review of diseases associated with household air pollution due to the use of biomass fuels. J Hazard Mater. Elsevier; 2011; 192(2): 425–31. PubMed Abstract \| Publisher Full Text\n\nFullerton DG, Bruce N, Gordon SB: Indoor air pollution from biomass fuel smoke is a major health concern in the developing world. Trans R Soc Trop Med Hyg. Elsevier; 2008; 102(9): 843–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOwili PO, Muga MA, Pan WC, et al.: Cooking fuel and risk of under-five mortality in 23 Sub-Saharan African countries: a population-based study. Int J Environ Health Res. 2017; 27(3): 191–204. PubMed Abstract \| Publisher Full Text\n\nWHO: Indoor air pollution. 2020. Reference Source\n\nHaber G, Witberg G, Danenberg H: [Air pollution and cardiovascular disease]. Harefuah. 2007; 146(10): 738–43. PubMed Abstract\n\nMortimer K, Gordon SB, Jindal SK, et al.: Household air pollution is a major avoidable risk factor for cardiorespiratory disease. Chest. American College of Chest Physicians; 2012; 142(5): 1308–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajagopalan S, Brook RD: THE INDOOR-OUTDOOR AIR-POLLUTION CONTINUUM AND THE BURDEN OF CARDIOVASCULAR DISEASE: AN OPPORTUNITY FOR IMPROVING GLOBAL HEALTH. Glob Heart. Elsevier; 2012; 7(3): 207–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurroughs Peña MS, Velazquez EJ, Rivera JD, et al.: Biomass fuel smoke exposure was associated with adverse cardiac remodeling and left ventricular dysfunction in Peru. Indoor Air. 2017;27(4): 737–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPainschab MS, Davila-Roman VG, Gilman RH, et al.: Chronic exposure to biomass fuel is associated with increased carotid artery intima-media thickness and a higher prevalence of atherosclerotic plaque. Heart. 2013; 99(14): 984–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurroughs Peña M, Romero KM, Velazquez EJ, et al.: Relationship between daily exposure to biomass fuel smoke and blood pressure in high-altitude Peru. Hypertension. 2015; 65(5): 1134–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarman N, Haque MA, Rahman AKMF, et al.: Association of biomass fuel smoke exposure and hypertension among rural women of Bangladesh: A cross-sectional study. Indian J Public Health. 2019; 63(3): 258–60. PubMed Abstract \| Publisher Full Text\n\nLee MS, Hang JQ, Zhang FY, et al.: In-home solid fuel use and cardiovascular disease: a cross-sectional analysis of the Shanghai Putuo study. Environ Health. 2012; 11(1): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQu W, Yan Z, Qu G, et al.: Household Solid Fuel Use and Cardiovascular Disease in Rural Areas in Shanxi, China. Iran J Public Health. 2015; 44(5): 625–38. PubMed Abstract \| Free Full Text\n\nYu K, Lv J, Qiu G, et al.: Cooking fuels and risk of all-cause and cardiopulmonary mortality in urban China: a prospective cohort study. Lancet Glob Health. 2020; 8(3): e430–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Cardiovascular diseases (CVDs). Fact Sheets. 2017. Reference Source\n\nWorld Health Organization: Noncommunicable Disease (NCD) Country Porofiles, 2018. 2018. Reference Source\n\nNSO National Office of Statitics, UNICEF, Fund UNC, et al.: Thailand Thailand Monitoring the situation of children and women Multiple Indicator Cluster Survey. 2012. Reference Source\n\nWikipedia: Phitsanulok Province. 2020. Reference Source\n\nJuntarawijit C: Questionnaire-household cooking and cardiovascular diseases. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12121887.v2\n\nJuntarawijit C: Household cooking and cardiovascular diseases. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12117066.v2\n\nCaravedo MA, Herrera PM, Mongilardi N, et al.: Chronic exposure to biomass fuel smoke and markers of endothelial inflammation. Indoor Air. 2016; 26(5): 768–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDutta A, Ray MR: Hypertension and respiratory health in biomass smoke-exposed premenopausal Indian women. Air Qual Atmos Heal. 2014; 7(2): 229–38. Publisher Full Text\n\nYoung BN, Clark ML, Rajkumar S, et al.: Exposure to household air pollution from biomass cookstoves and blood pressure among women in rural Honduras: A cross-sectional study. Indoor Air. 2019; 29(1): 130–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuntarawijit Y, Juntarawijit C: Cooking smoke exposure and respiratory symptoms among those responsible for household cooking: A study in Phitsanulok, Thailand. Heliyon. 2019; 5(5): e01706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDadzie EK, Ephraim RKD, Afrifa J, et al.: Persistent exposure to wood smoke is associated with variations in biochemical and hematological indices among regular wood burners in the Cape Coast metropolis, Ghana. Sci African. 2019; 4: e00100. Publisher Full Text\n\nPark SK: Ambient air pollution and type 2 diabetes: Do the metabolic effects of air pollution start early in life? Diabetes. American Diabetes Association Inc; 2017; 66(7): 1755–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajagopalan S, Brook RD: Air pollution and type 2 diabetes: mechanistic insights. Diabetes. American Diabetes Association; 2012; 61(12): 3037–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajkumar S, Clark ML, Young BN, et al.: Exposure to household air pollution from biomass-burning cookstoves and HbA1c and diabetic status among Honduran women. Indoor Air. 2018; 28(5): 768–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMüllerova H, Agusti A, Erqou S, et al.: Cardiovascular comorbidity in COPD: systematic literature review. Chest. 2013; 144(4): 1163–78. PubMed Abstract \| Publisher Full Text\n\nFatmi Z, Coggon D: Coronary heart disease and household air pollution from use of solid fuel: a systematic review. Br Med Bull. 2016; 118(1): 91–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFatmi Z, Coggon D, Kazi A, et al.: Solid fuel use is a major risk factor for acute coronary syndromes among rural women: a matched case control study. Public Health. 2014; 128(1): 77–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee KK, Miller MR, Shah ASV: Air pollution and stroke. J Stroke. Korean Stroke Society; 2018; 20(1): 2–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScheers H, Jacobs L, Casas L, et al.: Long-Term Exposure to Particulate Matter Air Pollution Is a Risk Factor for Stroke: Meta-Analytical Evidence. Stroke. 2015; 46(11): 3058–66. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62897", "date": "19 May 2020", "name": "Thandi Puoane", "expertise": [ "Reviewer Expertise Public Health", "Nutrition Epidemiology", "Cardiovascular risk factors" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 cross-sectional study undertaken to investigate a possible association between biomass use for cooking and cardiovascular diseases, including CHD, HBP, HC, diabetes, and stroke. Biomass users had a significantly higher prevalence of HBP, and HC, and their family members also had more incidence of HBP, HC, diabetes, and heart disease.\nIn the abstract: Change \"object\" to \"objective\" of this study.\n\nThe abbreviation “LPG\" used from the abstract was not defined.\n\nThe conclusion should be improved giving a summary of the findings first then move onto saying “these findings support those of...\"\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "71210", "date": "22 Sep 2020", "name": "Nilima Barman", "expertise": [ "Reviewer Expertise Non communicable disease", "Laboratory medicine", "Public health" ], "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 has emphasized on detrimental effects of biomass cooking fuel on cardiovascular health. The results will help in future policy making regarding cooking fuel. The overall article is well-written but some of the issues need to be clarified as described below.\nIn the abstract:\n\nThe methods should be improved, giving a clear description of cooking fuel.\n\nThe author mentioned 'hypertension' in the objective, but high blood pressure (HBP) in the result section. Terminology should be consistent throughout the manuscript.\n\nIn the conclusion, the author stated, 'the study results were consistent with those found in studies from other parts of the world...'. This statement should be discussed in the discussion, not in the conclusion. The conclusion should be based on the authors' main findings.\n\nIn the introduction:\nPlease clarify the term 'Cooking smoke,' whether it means smoke from cooking or from fuel?\n\nIn the methods:\nThe study design is well articulated. But self-reported cardiovascular diseases may give a vague impression to the readers, although the author mentioned it as a limitation. In a matter of sense, the authors showed more than two-thirds (71.9 %) of participants had primary education who acted as self reporters of disease condition. So, in my opinion, the authors should have a strong justification in favor of including self-reported cardiovascular diseases with authentic scientific references.\n\nAgain, do the authors cross-check self-reported disease conditions with the patient's medical or laboratory reports or drug history? The mentioned high cholesterol (HC) is instead a biochemical abnormality apart from a disease condition.\n\nIn statistical analysis:\nThe regression model needs a precise description. Is it a multivariate or multinominal model?\n\nIn a logistic regression model, the cardiovascular disease condition of family members are also encountered. Are the adjusting confounding variables like age and sex in that regression model in relation of family members’ age and sex, or the respondents’? It should be precisely mentioned in description of regression model.\n\nIn results:\nIt seems confusing between the data on 'Fuel use for cooking' in Table 1 and the total no of biomass and LPG users in Table 2. (In Table 1, LPG users are 695 participants, but in Table 2, it is 322 (by addition of 56 male and 266 female). Same for the biomass fuel. Please clarify it.\n\nThe footnote of Table 2 had a term '2-trail'. Is it trail or tail? Please correct it.\n\nIn Table 4, the significant value is given in bold letters. Please mention it.\n\nIn discussion:\nIn the 4th paragraph, the authors stated that 'We found about 10% of the respondents had type 2 diabetes'. It is hard to believe self-reported evidence of type 2 diabetes. Please clarify this or correct it.\n\nIn conclusion: the same as in abstract.\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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\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": [ { "c_id": "5970", "date": "08 Oct 2020", "name": "Chudchawal Juntarawijit", "role": "Author Response", "response": "Response to reviewerIn the abstract: Comments: The methods should be improved, giving a clear description of cooking fuel. Responses: A short description of cooking fuel was added to the methods section. Because F1000Research has set a maximum limit of 300 words for abstract, no more detailed information could be added.Comments: The author mentioned 'hypertension' in the objective, but high blood pressure (HBP) in the result section. Terminology should be consistent throughout the manuscript. Responses: The term “high blood pressure” was replaced by “hypertension”. Comments: In the conclusion, the author stated, 'the study results were consistent with those found in studies from other parts of the world...'. This statement should be discussed in the discussion, not in the conclusion. The conclusion should be based on the authors' main findings. Responses: Yes, I agree that “the statement should be discussed in the discussion”. However, we believed we had already done that enough to justify the statement, which is our main finding.In the introduction:Comments: Please clarify the term 'Cooking smoke,' whether it means smoke from cooking or from fuel? Responses: The meaning of cooking smoke was clarified and more information was added to the first paragraph in Introduction.In the methods:Comments: The study design is well articulated. But self-reported cardiovascular diseases may give a vague impression to the readers, although the author mentioned it as a limitation. In a matter of sense, the authors showed more than two-thirds (71.9 %) of participants had primary education who acted as self reporters of disease condition. So, in my opinion, the authors should have a strong justification in favor of including self-reported cardiovascular diseases with authentic scientific references.Responses: Yes, I agree that using self-report data is a limitation of this study. However, since the data was collected by a well trained and experienced village health volunteer, the problem was expected to be minimal. The quality of the answer to this question may not depend much on their background education of respondents. In addition, this information bias, if occurred, will equally distribute among groups (case and control). Comments: Again, do the authors cross-check self-reported disease conditions with the patient's medical or laboratory reports or drug history? The mentioned high cholesterol (HC) is instead a biochemical abnormality apart from a disease condition. Responses: Yes, it is good if we can do the cross-check self-reported conditions. However, we did not do that.In statistical analysis:Comments: The regression model needs a precise description. Is it a multivariate or multinominal model? Responses: Thank you for reminding. In this study, we use binary multiple logistic regression.More detail of the model was added to the statistic description.Comments: In a logistic regression model, the cardiovascular disease condition of family members are also encountered. Are the adjusting confounding variables like age and sex in that regression model in relation of family members’ age and sex, or the respondents’? It should be precisely mentioned in description of regression model.Responses: Thank you to raise the issue. It is a good point which could be another limitation of this study. We didn’t have enough information of the family members, so just use the data of the respondents.More information was added in the description of the regression model.In results:Comments: It seems confusing between the data on 'Fuel use for cooking' in Table 1 and the total no of biomass and LPG users in Table 2. (In Table 1, LPG users are 695 participants, but in Table 2, it is 322 (by addition of 56 male and 266 female). Same for the biomass fuel. Please clarify it. Responses: Table 1 showed data on what types of fuel the respondents usually use for cooking, and some of them use more than one fuel types. However, data in Table 2 was from another question which asked whether the respondents use biomass or not, and those who answered “no” was then classed as none biomass user or LPG user (assumed that only few use electric strove).Comments: The footnote of Table 2 had a term '2-trail'. Is it trail or tail? Please correct it. Responses: The error was corrected.Comments: In Table 4, the significant value is given in bold letters. Please mention it. Responses: The statement was added to Table 4 footnote.In discussion:Comments: In the 4th paragraph, the authors stated that 'We found about 10% of the respondents had type 2 diabetes'. It is hard to believe self-reported evidence of type 2 diabetes. Please clarify this or correct it. Responses: Actually, we asked whether the respondents had ever been diagnosed by a medical doctor to have type 2 diabetes. Also the data was collected by village health volunteer, who were well trained as public health staff and know the disease which is very common in Thailand." } ] } ]	1	https://f1000research.com/articles/9-307
https://f1000research.com/articles/9-300/v1	28 Apr 20	{ "type": "Research Article", "title": "Transcutaneous bilirubin level to predict hyperbilirubinemia in preterm neonates", "authors": [ "Dewi Rahmawati", "Mahendra Tri Arif Sampurna", "Risa Etika", "Martono Tri Utomo", "Arend F. Bos", "Dewi Rahmawati", "Risa Etika", "Martono Tri Utomo", "Arend F. Bos" ], "abstract": "Background: Hyperbilirubinemia is common in neonates, with higher prevalence among preterm neonates, which can lead to severe hyperbilirubinemia. Assessment of total serum bilirubin (TSB) and use of a transcutaneous bilirubinometer (TcB) are existing methods to identify and predict hyperbilirubinemia. This study aimed to determine TcB cut-off values during the first day for preterm neonates to predict hyperbilirubinemia at 48 and 72 hours. Methods: A total of 90 neonates born ≤35 weeks were included in the study. They were divided into two groups (Group I: 1000-1500 grams; Group II: 1501-2000 grams). The bilirubin level was measured on the sternum using TcB at the ages of 12, 24, and 72 h. TSB measurements were taken on the third day or if TcB level reached ± 1.24 mg/dL phototherapy threshold and if TcB showed abnormal results (Group I: 5.76-8.24 mg/dL; Group II: 8.76-11.24 mg/dL). Hyperbilirubinemia was defined as TSB ≥7 mg/dL for group I and >10 mg/dL for group II. Results: In total, 38 group I neonates and 48 group II neonates were observed. Almost half of neonates in group I (44.7%) were suffering from hyperbilirubinemia at the age of 48 hours, with 45.8% of group II at the age of 72 hours. To predict hyperbilirubinemia at the age of 48 hours, the best 24-hour-age TcB cut-off values were calculated to be 4.5 mg/dL for group I and 5.8 mg/dL for group II. To predict hyperbilirubinemia at the age of 72 hours, we determined 24-hour-age TcB value of 5.15 mg/dL for group II. Conclusion: TcB values in the early days of life can be used as hyperbilirubinemia predictors on the following days for preterm neonates. Close monitoring should be managed for those with TcB values higher than the calculated cut-off values.", "keywords": [ "transcutaneous bilirubin", "preterm neonates", "predict", "hyperbilirubinemia" ], "content": "Introduction\n\nHyperbilirubinemia is a common condition occurring in neonatal periods1, with a prevalence of around 60% in term neonates and 80% in preterm neonates. Preterm neonates have greater risk of severe hyperbilirubinemia, which can lead to encephalopathy2. This condition is preventable if early detection and prompt treatment can be arranged and managed properly1,3,4.\n\nVisual assessment is not reliable especially in the first 24–48 hours, since only 80% of jaundiced babies can be recognized visually if the bilirubin level reaches > 6 mg/dL5–8. High bilirubin levels can be dangerous, since preterm neonates have a greater risk of low bilirubin kernicterus2.\n\nTotal serum bilirubin (TSB) measurement remains the gold standard for diagnosing hyperbilirubinemia. The drawbacks of this procedure, however, are that it is painful, causes stress to the neonates, has a greater risk of infection, and needs a couple of hours to get the results9–11.\n\nTrancutaneous bilirubinometry (TcB) is a non-invasive procedure to identify hyperbilirubinemia. A number of studies have been conducted to validate TcB to assess whether it can be used safely. These found that TcB has good correlations with TSB. The use of TcB can also reduce the need for blooding sampling by 41–73%10,12,13.\n\nDue to the burdens of an occurrence of hyperbilirubinemia, its early detection and prediction are crucial. TSB or TcB is recommended to predict neonatal hyperbilirubinemia for neonates with >35 weeks of gestation13–15. Some studies using TcB to predict hyperbilirubinemia have already been conducted, but all of them recruited only late preterm and term neonates5,16. For preterm neonates, one study was already conducted using TSB measurement at the age of 6 until 24 hours to predict hyperbilirubinemia in the following hours or days4. As far as the researchers know, there has been no previous study using TcB to predict hyperbilirubinemia for preterm neonates. Therefore, the aim of this study was to use TcB to predict hyperbilirubinemia in preterm neonates to prevent complications since visual assessment is no longer reliable.\n\n\nMethods\n\nThis was a cohort study conducted in the Neonatal Intensive Care Unit (NICU) at Dr Soetomo General Hospital for 5 months (September 2018–January 2019). This study was approved by Dr. Soetomo General Hospital Surabaya Ethics Committee (No. 0586/KEPK/Ix/2018). An informed consent was signed by parents after they understood the information for consent. Study size retrieved in this research used purposive sampling with inclusion and exclusion criteria (a flow diagram is available as Extended data)17,18 during the research period. The sample size that we retrieved was estimated by applying Hulley et al.19 formulation of which confidence interval was at 95%, coefficient correlation at 0.84 and standard deviation of 1.84. Therefore, we applied minimum sample of 20 samples for each group, classified by infants’ body weight in certain ranges. While staying in accord with the minimum sample size, we expanded our samples up to 45 infants for each group with total sample of 90 infants. Yet, we had excluded four data due to missing TSB measurement.\n\nRace and thickness of melanin layer of skin tissue were taken into account as confounding variables, and as variables able to modify and differ the outcomes of others. Therefore, to control for study bias, the subjects addressed for this study were those subjects which had similar ethnic backgrounds, which were Malay Mongoloid.\n\nThe inclusion criteria were: 1) born at ≤35 weeks of gestational age and with a birth weight of <2000 g, and 2) parental consent by signing a form. The exclusion criteria were: 1) being diagnosed as hyperbilirubinemia at the age of 12 hours, 2) having any major congenital anomaly, 3) being discharged from hospital at an age of less than 3 days. Neonates who received phototherapy before the observation was done, missed TSB, or voluntarily resigned from this study were excluded from the study. The subjects recruited were divided into two groups, neonates with birth weights of 1001–1500 g (Group I) and 1501–2000 g (Group II).\n\nThe bilirubin level of each neonate was measured on the sternum by TcB (Dräger® Jaundice Meter 105) at the age of 12 hours, 24 hours, and 72 hours with ±3 hours tolerance (the TcB measurement could be taken within 3 hours before/after the exact time). The TSB measurement was taken for each neonate at the age of 3 days or if the TcB bilirubin level was ≥5.76 mg/dl for group I and TcB ≥8.76 mg/dl for group II and it had to be taken within 6 hours before/after the TcB measurement. The TSB measurement also had to be taken if the TcB measurement showed abnormal results. Hyperbilirubinemia was defined as TSB ≥7 mg/dl for preterm neonates with birth weights of 1000–1500 g and TSB >10 mg/dl for preterm neonates with birth weights of 1501–2000 g.\n\nThe data was analysis by Microsoft Office Excel, IBM SPSS Statistics Version 21. We performed receiver operating characteristic (ROC) curve analysis to determined cut off point of TcB level to predict hyperbilirubinemia at the age of 48 and 72 hours. We calculated the specificity, sensitivity, positive predicted value (PPV), negative predicted value (NPV), and likelihood ratio.\n\n\nResults\n\nThere were 90 preterm neonates recruited for this study, 40 of whom weighed 1000–1500 grams (Group I) and 50 of whom weighed 1501–2000 grams (Group II). Only 38 neonates of group I and 48 neonates of group II were observed until the end of the study. Four neonates were excluded from the study due to missing TSB results.\n\nMaternal and neonatal characteristics are shown in Table 1. For group I, the mean gestational age of group I was 32.29 ± 1.84 weeks, with a mean birth weight of 1273.68 ± 177.34 grams. Meanwhile for group II the mean gestational age was 33.69 ± 1.26 weeks and a mean birth weight of 1792.70 ± 145.86 grams. Based on risk factors of ABO-incompatibility, one subject of group I who suffered hyperbilirubinemia at the age of 48 hours. Meanwhile, two subjects in group II suffered hyperbilirubinemia at the age of 48 hours and another two subjects at the age of 72 hours at the end of observation, the maximum bilirubin level was 15.2 mg/dl for group I and 16.33 mg/dL for group II. Most neonates of group I (44.7%) suffered hyperbilirubinemia at the age of 48 hours, while most neonates of group II (45.8%) at the age of 72 hours (Figure 1). We found the TSB mean in group I at the age of 24, 48, and 72 hours to be 7.9 mg/dL, 9.16 mg/dL, and 9.3 mg/dL respectively, and 11.01 mg/dL, 10.23 mg/dL, and 11.04 mg/dL respectively, in group II.\n\n*Descriptive analysis was used.\n\nMaternal and neonatal rhesus were positive.\n\nA ROC curve was constructed to determine a hyperbilirubinemia threshold based on the data collected. We found that the AUC (area under curve) of the TcB bilirubin level at the age of 12 hours to predict hyperbilirubinemia at the age of 48 hours for group I was 0.804 (p 0.002) with a cut-off point of 2.35 mg/dL (sensitivity 79.20% and specificity 71.40%). For the TcB bilirubin level at the age of 24 hours to predict hyperbilirubinemia at the age of 48 hours, we found an AUC 0.771 (p 0.06), with a cut-off point of 4.50mg/dl (sensitivity 87.50% and specificity 64.26%) (Figure 2a, Figure 2b, Table 2).\n\n(a) Receiver operating characteristic (ROC) curve for TcB at the age of 12 hours to predict hyperbilirubinemia at the age of 48 hours for group I. (b) ROC curve for TcB at the age of 24 hours to predict hyperbilirubinemia at the age of 48 hours for group I. (c) ROC curve for TcB at the age of 12 hours to predict hyperbilirubinemia at the age of 48 hours for group II. (d) ROC curve for TcB at the age of 24 hours to predict hyperbilirubinemia at the age of 48 hours for group II.\n\n†Receiver operative characteristic curve analysis was used. Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value; LR, likelihood ratio.\n\nWe found the AUC of TcB bilirubin levels at the age of 12 hours to predict hyperbilirubinemia at the age of 48 hours for group II was 0.658 (p = 0.083), with a cut-off point of 3.05 mg/dL (sensitivity 66.7% and specificity 66.7%). The AUC of TcB bilirubin level at the age of 24 hours was 0.732 (p = 0.011), with a cut-off point of 5.80 mg/dL (sensitivity 80% and specificity 63.6%). (Figure 2c, Figure 2d and Table 3).\n\n†Receiver operative characteristic curve analysis was used. Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value; LR, likelihood ratio.\n\nThe TcB bilirubin level of group I at the age of 12 hours, 24 hours, and 48 hours to predict hyperbilirubinemia at the age of 72 hours showed a very weak AUC, which were 0.243 (p = 0.386); 0.297 (p = 0.494); 0.500 (p = 1.000) respectively, therefore no cut-off point could be determined.\n\nThe TcB bilirubin level of group II at the age of 12 hours to predict hyperbilirubinemia at the age of 72 hours showed a weak AUC (0.499; p = 0.991) with a cut-off point of 2.65 mg/dL (sensitivity 60% and specificity 46%). At the age of 24 hours, we found TcB AUC 0.751 (p = 0.008), with a cut-off point of 5.15 mg/dL (sensitivity 74.3% and specificity 76.9%). Meanwhile, at the age of 48 hours the TcB AUC was 0.731 (p = 0.015), with a cut-off point 8.65 mg/dL (sensitivity 67.6% and specificity 61%) (Figure 3a–c and Table 4).\n\n(a) Receiver operating characteristic (ROC) curve for TcB at the age of 12 hours to predict hyperbilirubinemia at the age of 72 hours for group II. (b) ROC curve for TcB at the age of 24 hours to predict hyperbilirubinemia at the age of 72 hours for group II. (c) ROC curve for TcB at the age of 48 hours to predict hyperbilirubinemia at the age of 72 hours for group II.\n\n†Receiver operative characteristic curve analysis was used. Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value; LR, likelihood ratio.\n\n\nDiscussion\n\nThis study has determined a TcB cut-off value of 4.5 mg/dL at the age of 24 hours in group I (1000–1500 grams) and 5.8 mg/dL in group II (1501–2000 grams) as predictive of hyperbilirubinemia at the age of 48 hours. To predict hyperbilirubinemia at the age of 72 hours, this study could not determine any TcB cut-off value for group I (1000–1500 gram) as a result of very weak correlation. The TcB cut-off value of 5.15 mg/dL at the age of 24 hours was determined as the best predictor for hyperbilirubinemia at the age of 72 hours in group II (1501–2000 grams). This cut-off level was established with a sensitivity value ranging from 74.3% to 87.5% at 24 hours after birth. Similar studies have already been conducted, but those studies recruited only late preterm neonates. Lavanya et al. found that TcB values measured in the first 24–48 hours of life can predict hyperbilirubinemia at an age of more than 48 hours20. Bansal et al. determined that TcB values of >4.6 mg/dl at the age of 12–24 hours (sensitivity 83.09%; specificity 87.37%; PPV 90.4% and NPV 78.3%) and >7.4 mg/dl at the age of 24–48 hours (sensitivity 93.55%; specificity 82.11%; PPV 81.69% and NPV 95.35%) are predictors for hyperbilirubinemia in the first 48 hours of life5. Other studies conducted used TSB values to predict hyperbilirubinemia in the following days. Mayer recruited preterm neonates weighing 1000–1500 grams and determined a capillary TSB value of 3.55 mg/dl at the age of 12 hours as the best predictor of significant hyperbilirubinemia (sensitivity 94.4%, PPV 98.1%, and NPV 40%)4.\n\nMost neonates recruited in this study suffered hyperbilirubinemia before the age of 72 hours old. This study also showed that smaller babies suffered peak incidence earlier (at 48 hours) than larger babies (at 72 hours). Hyperbilirubinemia is more prevalent in preterm neonates4,5,20) and is usually more severe and has longer duration compared to that in term neonates4. This is caused by increased bilirubin production, decreased bilirubin excretion, increased enterohepatic circulation, lower albumin level and a weak albumin-bilirubin bond11,21. Early detection of hyperbilirubinemia will decrease its mortality and morbidity. The need for reliable methods to predict hyperbilirubinemia is important. The use of a noninvasive procedure, like TcB, in the first 6–24 hours of life is recommended as a marker of bilirubin production22 and it can decrease the need for blood sampling23.\n\nIn group I at the ages of 24, 48, and 72 hours, we found TSB mean values of 7.9, 9.16, and 9.3 mg/dL, respectively; in group II, these values were 11.01, 10.23, and 11.04 mg/dL, respectively. This is similar to previous study which found that TSB mean values in the first 5 days of the lives of preterm neonates were 10–12 mg/dL24. However, this study found there were preterm neonates who reached a TSB value >15 mg/dL in the first 72 hours. However, it is possible for preterm neonates to have high bilirubin levels in the first days of life, which can lead to hyperbilirubinemia complications if not recognized and treated properly. The previous study conducted by Bhutani et al. also indicated that neonates who suffer from hyperbilirubinemia in the following days had higher percentiles on the first day of life25. Therefore, the American Academy of Pediatrics (AAP) recommends routine checks of TSB or TcB along with risk factor assessments in the first days of life14.\n\nTo the knowledge of the researchers, this was the first study conducted to predict hyperbilirubinemia in preterm neonates weighing 1000–2000 grams using TcB. One limitation of the study was that it could not determine TcB cut-off values to predict hyperbilirubinemia at the age of 72 hours for preterm neonates weighing 1000–1500 grams due to a lack of subjects able to complete the study, since most had already developed significant hyperbilirubinemia by this time. This, it is hoped that future similar studies will be able to recruit larger populations.\n\n\nConclusion\n\nTcB values in the early days of life can be used as a predictor of hyperbilirubinemia in the following days for preterm neonates. It is possible for preterm neonates to have high bilirubin levels in the first few days of their lives. Therefore, daily measurement of TcB is important for early identification of hyperbilirubinemia, especially in order to prevent complications in certain more vulnerable preterm neonates. Close monitoring should be arranged for those who have TcB values higher than the cut-off values.\n\n\nData availability\n\nFigshare: Datasheet TcB and TSB - Group 1. https://doi.org/10.6084/m9.figshare.11948490.v126.\n\nThis project contains data gathered for neonates in group 1 (those 1000–1500 grams).\n\nFigshare: TcB Level and TSB-MTA - Group 2. https://doi.org/10.6084/m9.figshare.11948529.v127.\n\nThis project contains data gathered for neonates in group 2 (those 1500–2000 grams).\n\nFigshare: Supplemental File - Flow Chart Study of TcB and TSB. https://doi.org/10.6084/m9.figshare.12017586.v118.\n\nThis project contains a study flow diagram.\n\nFigshare: STROBE checklist for ‘Transcutaneous bilirubin level to predict hyperbilirubinemia in preterm neonates’. https://doi.org/10.6084/m9.figshare.11991672.v217.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank neonatal unit at Dr. Soetomo Academic Teaching Hospital members who also gave contributions within the study progress, as follows:\n\n1. Siti Annisa Dewi Rani, MD and Muhammad Pradhika Mapindra, MD as research assistants whom are employed in our Neonatal Unit for data editing\n\n2. Spencer Lemaich who contributed to proofread the manuscript in English;\n\n3. Head of each Neonatal Unit ward: Mrs. Pamiani, Mrs. Wahyu, and Mrs. Peni for supporting our study and coordinating each of their nursing team to collaborate with us;\n\n4. All our colleagues in Neonatal Unit at Dr. Soetomo Academic Teaching Hospital.\n\n\nReferences\n\nHan S, Yu Z, Liu L, et al.: A Model for Predicting Significant Hyperbilirubinemia in Neonates From China. Pediatrics. 2015; 136(4): e896–905. PubMed Abstract \| Publisher Full Text\n\nWatchko JF, Maisels MJ: Jaundice in low birthweight infants: pathobiology and outcome. Arch Dis Child Fetal Neonatal Ed. 2003; 88(6): F455–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiskin A, Tamir A, Kugelman A, et al.: Is visual assessment of jaundice reliable as a screening tool to detect significant neonatal hyperbilirubinemia? J Pediatr. 2008; 152(6): 782–787.e2. PubMed Abstract \| Publisher Full Text\n\nMayer I, Gursoy T, Hayran M, et al.: Value of twelfth hour bilirubin level in predicting significant hyperbilirubinemia in preterm infants. J Clin Med Res. 2014; 6(3): 190–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBansal R, Agarwal AK, Sharma M: Predictive Value of Transcutaneous Bilirubin Levels in Late Preterm Babies. Int J Contemp Med Res. 2016; 3(6): 1661–3. Reference Source\n\nel-Beshbishi SN, Shattuck KE, Mohammad AA, et al.: Hyperbilirubinemia and transcutaneous bilirubinometry. Clin Chem. 2009; 55(7): 1280–7. PubMed Abstract \| Publisher Full Text\n\nNewman TB, Xiong B, Gonzales VM, et al.: Prediction and prevention of extreme neonatal hyperbilirubinemia in a mature health maintenance organization. Arch Pediatr Adolesc Med. 2000; 154(11): 1140–7. PubMed Abstract \| Publisher Full Text\n\nSgro M, Campbell D, Shah V: Incidence and causes of severe neonatal hyperbilirubinemia in Canada. CMAJ. 2006; 175(6): 587–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanpavat S, Nuchprayoon I: Transcutaneous bilirubin in the pre-term infants. J Med Assoc Thai. 2007; 90(9): 1803–8. PubMed Abstract\n\nJangaard KA, Curtis H, Goldbloom RB: Estimation of bilirubin using BiliChektrade mark, a transcutaneous bilirubin measurement device: Effects of gestational age and use of phototherapy. Paediatr Child Heal. 2006; 11(2): 79–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSajjadian N, Shajari H, Saalehi Z, et al.: Transcutaneous bilirubin measurement in preterm neonates. Acta Med Iran. 2012; 50(11): 765–70. PubMed Abstract\n\nvan Imhoff DE, Hulzebos CV, Bos AF, et al.: Transcutaneous bilirubin measurements in preterm infants: Effects of photo-therapy and treatment thresholds. In: The management of hyperbilirubinemia in preterm infants. 2017; 77–90.\n\nDijk PH, Hulzebos CV: An evidence-based view on hyperbilirubinaemia. Acta Paediatr. 2012; 101(464): 3–10. PubMed Abstract \| Publisher Full Text\n\nAmerican Academy of Pediatrics Subcommittee on Hyperbilirubinemia: Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics. 2004; 114(1): 297–316. PubMed Abstract \| Publisher Full Text\n\nAlizadeh Taheri P, Sadeghi M, Sajjadian N: Severe neonatal hyperbilirubinemia leading to exchange transfusion. Med J Islam Repub Iran. 2014; 28(1): 64. PubMed Abstract \| Free Full Text\n\nBhat RY, Kumar PC: Sixth hour transcutaneous bilirubin predicting significant hyperbilirubinemia in ABO incompatible neonates. World J Pediatr. 2014; 10(2): 182–5. PubMed Abstract \| Publisher Full Text\n\nSampurna MTA, Rahmawati D, Etika R, et al.: STROBE Analysis - TcB and TSB. figshare. Preprint. 2020. http://www.doi.org/10.6084/m9.figshare.11991672.v2\n\nSampurna MTA, Rahmawati D, Etika R, et al.: Supplemental File - Flow Chart Study of TcB and TSB. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12017586.v1\n\nHulley SB, Cumming SR, Browner WS, et al.: Designing Clinical Research. Fourth Edition. 4th Edition. Wolters Kluwer, Lippincott Williams Wilkins. Philadelphia, PA 19103 USA. LIPPINCOTT WILLIAMS & WILKINS, a WOLTERS KLUWER business; 2013. Reference Source\n\nLavanya KR, Jaiswal A, Reddy P, et al.: Predictors of significant jaundice in late preterm infants. Indian Pediatr. 2012; 49(9): 717–20. PubMed Abstract \| Publisher Full Text\n\nLeite MD, Facchini FP: [Evaluation of two guidelines for the management of hyperbilirubinemia in newborn babies weighing less than 2,000 g]. J Pediatr (Rio J). 2004; 80(4): 285–90. PubMed Abstract \| Publisher Full Text\n\nÜnsür MT, Ünsür E, Inan N, et al.: The predictive value of first-day bilirubin levels in the early discharge of newborns. Iran J Neonatol. 2015; 6(3): 1–5. Reference Source\n\nGrabenhenrich J, Grabenhenrich L, Bührer C, et al.: Transcutaneous bilirubin after phototherapy in term and preterm infants. Pediatrics. 2014; 134(5): e1324–9. PubMed Abstract \| Publisher Full Text\n\nKaplan M, Wong R, Sibley E, et al.: Neonatal Jaundice and Liver Disease. In: Martin’s Neonatal-Perinatal Medicine. 2011; 1443–90. Reference Source\n\nBhutani VK, Johnson L, Sivieri E: Predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns. Pediatrics. 1999; 103(1): 6–14. PubMed Abstract \| Publisher Full Text\n\nSampurna MTA, Rahmawati D, Etika R, et al.: Datasheet TcB and TSB - Group 1. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11948490.v1\n\nSampurna MTA, Rahmawati D, Etika R, et al.: TcB Level and TSB-MTA - Group 2. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11948529.v1" }	[ { "id": "62844", "date": "11 May 2020", "name": "Claudio Tiribelli", "expertise": [ "Reviewer Expertise Bilirubin", "Jaundice" ], "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 study investigating in preterm neonates (PTNs) the predictive value of the bilirubin level assessed by transcutaneous technique (TcB) for hyperbilirubinemia (HB). 90 PTNs were and divided into two groups according to the weight (1500 g as diving value). Bilirubin was prospectively measured at different time points (12, 24, 48, and 72 h). Bilirubin level was confirmed by serum bilirubin measurement (TSB) if the TcB was “increased”. ROC curves were used to assess a 24 h TcB value predicting 48 and 72 h HB. Although the data may be of interest, the study suffers several intrinsic weaknesses what must be addressed before being considered further.\nMajor Critiques:\nThe indication when TSB was measured is unclear and confusing. In the abstract it is stated that TSB was assessed before 72 h if TcB showed “abnormal results”. What does this mean? Detailed numerical values must be provided to allow to understand why TSB was performed.\n\nOn what basis the TSB value of ≥ 7 mg/dL in group 1 and > 10 mg/dL HB in group 2 was selected? This needs to be scientifically substantiated. At what time this level was measured?\n\nHow TSB was measured? Were the lab values confirmed by internal calibration? This must be clarified.\n\nFig 1 shows that the time course of HB is different in the 2 groups being the bilirubin peak reached 24 h later in group 2. This difference accounts for the different TcB cutoff values. This needs to be considered and addressed in the discussion.\n\nHow was the correlation between TcB and TSB at 72h? This information is important to assess the reliability of the two techniques (see also point #3).\n\nThe abstract is inaccurate as data reported are different from those indicated in the text (see for example the lack of 48H values).\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5660", "date": "07 Sep 2020", "name": "Mahendra Tri Arif Sampurna", "role": "Author Response", "response": "1). Reviewer's Comment: The indication when TSB was measured is unclear and confusing. In the abstract it is stated that TSB was assessed before 72 h if TcB showed “abnormal results”. What does this mean? Detailed numerical values must be provided to allow to understand why TSB was performed. Author's response: Dear Reviewer, Thank you for your comments. We added in our method à variables section as follows: The bilirubin level of each neonate was measured on the sternum by TcB (Dräger® Jaundice Meter 105) at 12 hours, 24 hours, and 72 hours with ±3 hours tolerance (the TcB measurement could be taken within three hours before/after the exact time). The TSB measurement was taken for each neonate at the age of three days or if the TcB bilirubin level was ≥5.76 (7-1.24) mg/dL for Group I and TcB ≥8.76 (10-1.24) mg/dL for Group II and it had to be taken within six hours before or after the TcB measurement (assumption of TcB standard deviation being ±1.24 mg/dL). The TSB measurement also had to be taken if the TcB measurement showed abnormal results. Hyperbilirubinemia was defined as TSB ≥7 mg/dL for preterm neonates with birth weights of 1000–1500 g and TSB >10 mg/dL for preterm neonates with birth weights of 1501–2000 g as suggested by the Kaplan et al., in Martin’s Neonatal-Perinatal Medicine (2011). 2). Reviewer's Comment: On what basis the TSB value of ≥ 7 mg/dL in group 1 and > 10 mg/dL HB in group 2 was selected? This needs to be scientifically substantiated. At what time this level was measured? Author's response: Dear Reviewer, Thank you for your comments. The cut off 7 and 10 mg/dl are based on the recommendation of Martin Fanaroff (Reference source: Kaplan M, Wong R, Sibley E, et al.: Neonatal Jaundice and Liver Disease. In: Martin’s Neonatal-Perinatal Medicine. 2011; 1443–90.) In this recommendation they use birth weight category regardless postnatal age in hours. 3). Reviewer's comment: How TSB was measured? Were the lab values confirmed by internal calibration? This must be clarified. Author's response: Dear Reviewer, Thank you for your comments. TSB measurements were performed by retrieving peripheral blood samples of each subject. Added in methods à variables section : Total serum bilirubin was measured in the central laboratory using SIEMENS Dimension® with a modified Doumas 22 reference method, which is a modification of the diazo method described by Jendrassik and Grof in 1938 23. Internal calibration was completed daily, with a quality control printout. The Indonesian External Quality Assurance Service performed external quality control. 4). Reviewer's comment: Fig 1 shows that the time course of HB is different in the 2 groups being the bilirubin peak reached 24 h later in group 2. This difference accounts for the different TcB cut-off values. This needs to be considered and addressed in the discussion. Author's comment: Dear Reviewer, Thank you for the comment. We added in the discussion: It shows that that higher birth weight has opportunity to be hyperbilirubinemia later than the lower birthweight. The lower birthweight has lower threshold bilirubin, because they have higher chance to become encephalopathy in lower bilirubin level (high risk infants). The younger gestational age and lower birth weight, lead to a higher prevalence of infants developing hyperbilirubinemia. It is a result of excessive neonatal red cell hepatic and immaturity of the gastrointestinal system. Prematurely delivered infants have a likelihood of slower maturation of hepatic bilirubin uptake and conjugation. 5). Reviewer's comment: How was the correlation between TcB and TSB at 72h? This information is important to assess the reliability of the two techniques (see also point #3) Author's response: Dear Reviewer, Thank you for your comments. We actually did not assess the correlation between TcB and TSB at 72 hours of age. We would like to mention our reasons why we did not perform the assessment as the followings: We were not specifically aiming at performing diagnostic test; We only discovered fewer observed subjects who had reached observation at 72 hours of age thus the sample size at that age was not abundant in number. Most of the infants underwent phototherapy before 72 hours (group 1). Those infants were excluded from the study for further analysis. 6) Reviewer's Comment: The abstract is inaccurate as data reported are different from those indicated in the text (see for example the lack of 48H values). Author's response: Dear Reviewer, Thank you for noticing. We adapted our abstract accordingly We also found that there was an unsuitable data between those mentioned in the study abstract and the ones in figures. However, we would like to inform that the proportional data provided in the study abstract is correct already meanwhile the ones in figures were attained by integrating the data percentages. Therefore, we are going to amend and adjust the data provided in abstract." } ] }, { "id": "62845", "date": "21 May 2020", "name": "Tina M. Slusher", "expertise": [ "Reviewer Expertise Pediatric Global Health", "Neonatal Hyperbilirubinemia", "Pediatric Critical Care" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is an interesting study looking at the correlation between transcutaneous bilirubin (TcB) and serum bilirubin and the predictive value of subsequent clinically significant hyperbilirubinemia in neonates. However, there are some problems with the study that need to be addressed.\n\nThey authors state that TcB has not been used in premature infants with the exception of late preterms. However, in a systematic review by Nager et al. most of the 22 articles they mention include very preterm infants. Perhaps the authors need to mean to say in their population but this needs to be clarified.\n\nEach country does indeed need to develop their own criteria for diagnosis and treatment levels of hyperbilirubinemia based on risk in their environment, treatments available and other specifics related to their own country. However, authors do need to tell us where there cutoffs for each group came from---are the in country normal, standard cutoffs for their hospital or region or how were they selected.\n\nNumbers too small to extrapolate widely to Indonesia or beyond.\n\nRhesus of mothers and infants missing; G6PD status missing if not done state that.\n\nAlthough not a major problem or limitation this article could be improved by having a native English speaker read and make minor edits throughout.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\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?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5661", "date": "07 Sep 2020", "name": "Mahendra Tri Arif Sampurna", "role": "Author Response", "response": "1). Reviewer's comments: This study is an interesting study looking at the correlation between transcutaneous bilirubin (TcB) and serum bilirubin and the predictive value of subsequent clinically significant hyperbilirubinemia in neonates. However, there are some problems with the study that need to be addressed. The authors state that TcB has not been used in premature infants with the exception of late preterms. However, in a systematic review by Nager et al. most of the 22 articles they mention include very preterm infants. Perhaps the authors need to mean to say in their population but this needs to be clarified. Author's response: Dear Reviewer, Thank you for your comments. Yes, indeed you are right, thanks for your suggestion. What we meant here is similar with the study by Mayer (Mayer I, Gursoy T, Hayran M, Ovalı F. Value of twelfth-hour bilirubin level in predicting significant hyperbilirubinemia in preterms Infants. J Clin Med Res. 2014;6(3):190–6) but they used TSB to predict significant hyperbilirubinemia later on. Practice in our hospital so far is using Kramer and few TSB measurement to start phototherapy. So, we give another alternative which is non-invasive, easy to use, and applicable in our setting in Indonesia. We added in our introduction as : In a systematic review by Nagar et al.16, most of 22 articles studied the accuracy of TcB to estimate TSB, and TcB could be used in clinical practice to reduce blood sampling. Some studies have used TcB to predict significant hyperbilirubinemia in subsequent days, but all of them recruited only late preterm and term neonates 5,17 . For preterm neonates, one study was already conducted using TSB measurements at 6- 24 hours to predict hyperbilirubinemia in the following hours or days 4 . As far as the researchers know, there have been no previous studies using TcB to predict subsequent, significant hyperbilirubinemia for older preterm neonates. Therefore, this study aimed to use TcB to predict hyperbilirubinemia in preterm neonates to prevent complications since visual assessment is unreliable. 2) Reviewer's comment: Each country does indeed need to develop their own criteria for diagnosis and treatment levels of hyperbilirubinemia based on risk in their environment, treatments available and other specifics related to their own country. However, authors do need to tell us where there cutoffs for each group came from---are the in country normal, standard cutoffs for their hospital or region or how were they selected Author's response: Thank you for your comments. We added in our method à variables section as follows: The bilirubin level of each neonate was measured on the sternum by TcB (Dräger® Jaundice Meter 105) at 12 hours, 24 hours, and 72 hours with ±3 hours tolerance (the TcB measurement could be taken within three hours before/after the exact time). The TSB measurement was taken for each neonate at the age of three days or if the TcB bilirubin level was ≥5.76 (7-1.24) mg/dL for Group I and TcB ≥8.76 (10-1.24) mg/dL for Group II and it had to be taken within six hours before or after the TcB measurement (assumption of TcB standard deviation being ±1.24 mg/dL). The TSB measurement also had to be taken if the TcB measurement showed abnormal results. Hyperbilirubinemia was defined as TSB ≥7 mg/dL for preterm neonates with birth weights of 1000–1500 g and TSB >10 mg/dL for preterm neonates with birth weights of 1501–2000 g as suggested by the Kaplan et al., in Martin’s Neonatal-Perinatal Medicine (2011).21 3). Reviewer's comment: Numbers too small to extrapolate widely to Indonesia or beyond. Author's response: Dear Reviewer, This is the study limitation and has been addressed in discussion section. We added in the discussion section in the last paragraph: A limitation of the study was that it could not determine TcB cut-off values to predict hyperbilirubinemia at the age of 72 hours for preterm neonates weighing 1000–1500 grams due to a lack of subjects able to complete the study, since most had already developed significant hyperbilirubinemia by this time. 4). Reviewer's comment: Rhesus of mothers and infants missing; G6PD status missing if not done state that. Author's response: Dear Reviewer, Thank you for your comments. We even hardly measured Blood Group ABO and Rhesus of the mother and babies since the babies are not yellow. We have not used it as screening program. G6PD measurement is not affordable for our majority of citizen. We need to give consent first when the bilirubin is not yet decrease and rise again after intensive phototherapy, or there is sign of haemolysis Thank you for your suggestion. Indeed, this is also our limitation that we added in the discussion section on the last paragraph To the knowledge of the researchers, this was the first study conducted to predict significant hyperbilirubinemia in preterm neonates weighing 1000–2000 grams using TcB. A limitation of the study was that it could not determine TcB cut-off values to predict hyperbilirubinemia at the age of 72 hours for preterm neonates weighing 1000–1500 grams due to a lack of subjects able to complete the study, since most had already developed significant hyperbilirubinemia by this time. The mothers’ rhesus blood groups and the babies’ G6PD levels were not obtained. Hopefully, future similar studies will be able to recruit larger populations. 5) Reviewer's comment: Although not a major problem or limitation this article could be improved by having a native English speaker read and make minor edits throughout Author's response: Dear Reviewer, Thank you for your comments We have already proofread this manuscript by Proofreading Service." } ] } ]	1	https://f1000research.com/articles/9-300
https://f1000research.com/articles/9-1207/v1	08 Oct 20	{ "type": "Research Article", "title": "Annotation and curation of human genomic variations: an ELIXIR Implementation Study", "authors": [ "Alessia David", "Valérie Barbié", "Marcella Attimonelli", "Roberto Preste", "Enni Makkonen", "Heidi Marjonen", "Mats Lindstedt", "Kati Kristiansson", "Sarah E. Hunt", "Fiona Cunningham", "Ilkka Lappalainen", "Michael J.E. Sternberg", "Valérie Barbié", "Marcella Attimonelli", "Roberto Preste", "Enni Makkonen", "Heidi Marjonen", "Mats Lindstedt", "Kati Kristiansson", "Sarah E. Hunt", "Fiona Cunningham", "Ilkka Lappalainen", "Michael J.E. Sternberg" ], "abstract": "Background: ELIXIR is an intergovernmental organization, primarily based around European countries, established to host life science resources, including databases, software tools, training material and cloud storage for the scientific community under a single infrastructure. Methods: In 2018, ELIXIR commissioned an international survey on the usage of databases and tools for annotating and curating human genomic variants with the aim of improving ELIXIR resources. The 27-question survey was made available on-line between September and December 2018 to rank the importance and explore the usage and limitations of a wide range of databases and tools for annotating and curating human genomic variants, including resources specific for next generation sequencing, research into mitochondria and protein structure. Results: Eighteen countries participated in the survey and a total of 92 questionnaires were collected and analysed. Most respondents (89%, n=82) were from academia or a research environment. 51% (n=47) of respondents gave answers on behalf of a small research group (<10 people), 33% (n=30) in relation to individual work and 16% (n=15) on behalf of a large group (>10 people). The survey showed that the scientific community considers several resources supported by ELIXIR crucial or very important. Moreover, it showed that the work done by ELIXIR is greatly valued. In particular, most respondents acknowledged the importance of key features and benefits promoted by ELIXIR, such as the verified scientific quality and maintenance of ELIXIR-approved resources. Conclusions ELIXIR is a “one-stop-shop” that helps researchers identify the most suitable, robust and well-maintained bioinformatics resources for delivering their research tasks.", "keywords": [ "ELIXIR", "survey", "database", "bioinformatics tools" ], "content": "Introduction\n\nELIXIR is an intergovernmental organization, primarily centred in Europe, established to host resources, such as databases, software tools, training material and cloud storage for the scientific community, under a single infrastructure. ELIXIR started in 2013 and now includes 22 state members and over 220 research Institutions. By providing a one-stop-shop for the scientific community, it aims to help researchers identify the most suitable bioinformatics resources (and the appropriate training material and workshops) to deliver their research task. Moreover, ELIXIR recognizes and facilitates sharing of data and exchange of expertise between its members, with the goal of agreeing on best practice.\n\nIn 2018, ELIXIR commissioned an international survey on the usage of databases and tools for annotating and curating human genomic variants with the aim of improving ELIXIR resources. Here we present the result of the survey.\n\n\nMethods\n\nA 27-question survey (Extended data) was designed and agreed upon by several ELIXIR members from various European countries. The survey was constructed around six main themes (detailed in “Questionnaire structure”) aimed at exploring the usage of ELIXIR resources and tools. Six ELIXIR nodes/partners informed the construction of the survey. Two researchers/research centres were identified by each of the six ELIXIR nodes to participate in a pilot test. The survey was modified based on the feedback received. The final questionnaire was made available on-line using the Webropol survey website between September and December 2018. Responses were uploaded to a Finland-based university server. Members of the Finland ELIXIR node accessed the responses and stored them in a .csv file for analysis. Information about participation was displayed on the survey and completion of the survey was taken as confirmation of participation.\n\nConsidering the absence of identifying information in data published here, and the non-sensitive nature of the survey, no ethical approval was sought for this study. No information presented here can be used to identify survey participants.\n\nFor each country, an ELIXIR representative was asked to recruit prospective survey respondents from academia, clinical diagnostics, clinical research, industry and the Government within their country. No additional eligibility criteria were set. Participation to the survey was advertised using several means, such as the mailing lists of universities, research centers, private companies and research institutes within each European country. Prospective participants were asked to fill the anonymized on-line questionnaire. A three-month deadline was given for completing the on-line questionnaire. Reminders to complete the survey were published in the regular newsletters at the recruited institutions. This approach did not allow calculation of the response rate.\n\nThe survey collected two types of data:\n\n- quantitative data: this included category ranking metrics, as well as general frequency of use of the tools and databases surveyed. Whenever possible, a list of possible choices was provided for selection, to allow proper survey analysis and interpretation.\n\n- qualitative data: this included participant comments.\n\nThe survey was divided in six sections (the full questionnaire with the list of tools, databases and resources that were surveyed is presented in Extended Data):\n\nSection 1 - Background information\n\nSection 2 - Resources for annotating and curating human genomic variants (9 questions)\n\nSection 3 - Next generation sequencing (3 questions), (If no, skip this section)\n\nSection 4 - Mitochondria (4 questions), (If no, skip this section)\n\nSection 5 - Proteins (5 questions), (If no, skip this section)\n\nSection 6 - ELIXIR (3 questions)\n\nSections 1, 2 and 6 were open to all participants, whereas sections 3 to 5 were only open to participants who worked or had an interest in working in these specific fields. Accordingly, respondents were asked to skip the sections that were not pertinent to their work or that of their group.\n\nThe complete list of tools and databases surveyed is presented in Extended data.\n\nData analysis was performed using RStudio (version 0.98.1062) and R (R version 3.4.4)1. Quantitative data are presented as absolute and relative frequency. Qualitative data were analysed per theme.\n\n\nResults\n\nA total of 92 questionnaires were available for analysis. Data were collected from 18 European countries and the Unites States (Extended data: Figure S1). Finland contributed to the majority of answers (30%, n=28), followed by the United Kingdom (11%, n=10). The large majority of respondents (89%, n=82) identified themselves as belonging to academia or a research environment (Extended data: Table S1). The large majority (51%, n=47) of respondents gave answers to the questionnaire in relation to their work and that of a small group of (<10) people. This was followed by answers given in relation to individual work (33%, n=30) and the work of a large group of (>10) people (16%, n=15). As the survey was sent to colleagues by the organisers, the responders may well not be representative of the wider community. Additionally, most of the responders were from European countries and the responses could be substantially different elsewhere, such as from the USA.\n\nSection 2 of the survey aimed at identifying key data resources and tools used by the scientific community primarily in ELIXIR member states. Respondents were asked to rank 52 resources (listed in Extended data: Table S2). A total of 2055 responses were collected from 88% (82) responders. In total, 22 resources were considered critical/very important by more than 50% of respondents who knew the resources and considered them relevant to their work (Figure 1). Three ELIXIR resources, Ensembl2, the Ensembl Variant Effect Predictor (VEP)3 and UniProt4, were within the top ten. Several additional resources were listed by respondents and are presented in Extended data: Tables S3 and S4.\n\nA total of 78 responses were collected from 50 (54%) respondents on the standards and guidelines used for the interpretation of sequence variants. 66% (n=33) of respondents followed the American College of Medical Genetics (ACMG) standards and guidelines for the interpretation of sequence variants5, 22% (n=11) national guidelines, and 8% (n=4/50) in house guidelines. 4% (n=2) answered “other guidelines” and listed the European Leukemia Net (ELN) guidelines6 and the “Sequence Ontology base classification for variants”7. With regards to the human genome reference assembly version, approximately one third (38%, n=31/81) of respondents used both GRCh37 and GRCh38, 44% (n=36/81) GRCh37 only, 15% (n=12/81) GRCh38 alone, and 2% (n=2/81) GRCh37, GRCh38 and older versions (Figure 2).\n\nFour types of data/data formats used in human genome variation analysis (VCF/BCF, BAM, FASTQ, FASTA) were considered critical/very important by the majority of respondents (Extended data: Table S5). Additional data formats that were listed as critical/very important were: BED (cited 4 times), gVCF (cited twice), PLINK8, IMPUTE9 and Hail MatrixTable. Genome-wide association studies (GWAS) and rare disease studies were listed as the most important topics and analytical operations in human genome variation analysis performed by respondents to this survey (Extended data: Table S6).\n\nOne of the main goals of this survey was to identify user requirements in the use of resources for human genome variation annotation and curation. A total of 777 responses on the importance of basic features of databases and tools were analysed. The majority of respondents (>83%) considered critical/very important a resource’s free/low cost, open license to academia, the ability to assess the quality of its data, good user documentation and its availability in English. An easy-to-use web browser and privacy policy were deemed critical/very important by >77% of respondents (Table 1).\n\nA total of 1173 responses were analysed on the importance of technical features in variation analysis, annotation tools and databases. Good curation of the database was the top scoring technical feature, ranked critical/very important by 92% of respondents. The following five technical features were considered critical/very important by >75% of respondents: i) fit for purpose data used by the resource, ii) the scientific coverage and comprehensiveness of the resource; iii) scalability to high-throughput analysis; iv) availability of datasets for download; and v) ability to analyse large datasets/queries (Table 2).\n\nThe next section of the survey focused on Next Generation Sequencing (NGS). 66 (72%) respondents worked with NGS and ranked 6 sequencing methods listed in Extended data: Table S7. Whole exome, panel DNA, whole genome and RNA sequencing were used by more than 60% of respondents. Methylation sequencing was the least used technique (28% of respondents) but was the technique the majority of respondents would like to use (23% of respondents). Several other techniques were listed among “Other”, such as single-cell RNA sequencing, ribosome profiling, chromatin conformation techniques (3C, 4C, HiC, capture HiC), assay for transposase-accessible chromatin using sequencing (ATAC-SEQ), targeted RNA sequencing, fusion genes on cDNA, microbiome sequencing, GRO-seq.\n\nThe next section of the survey explored the use of resources related to the study of mitochondria. Only 15 (16%) respondents worked with mitochondrial DNA (mtDNA) alone or in combination with nuclear genes involved in mitochondrial functionality. Of these, 7 used NGS alone, 2 NGS and Sanger sequencing and 6 could not specify. Additional methods listed under “Other methods” were whole genome sequencing (WGS), whole exome sequencing (WES), RNA-seq and mtDNA included in WGS or WES.\n\nAmong the mitochondria-dedicated databases, MITOMAP10 was ranked as critical/very important by 65% of respondents, followed by HmtDB (Human Mitochondrial Database)11 by 44% and HmtVar (Human Mitochondrial Variants Database)12 by 33%. With the exception of MITOMAP, MitoCarta13 and HmtDB, more than half of the respondents were unaware of all other databases (Extended data: Table S8).\n\nAmong the six tools for mitochondria-related research that were ranked, MToolBox14, HaploGrep215, MitoTip16 and MitImpact217 were equally reported as critical/very important by 18% of respondents (Extended data: Table S9). The following mitochondrial databases/tools not included in the survey were listed among “Other” and ranked critical/very important: MitoFates18 and an in house built annotation pipeline. mvTool (part of MSeqDR.org) and MitoMaster (part of MITOMAP) were listed among “Other tools” used for mitochondria-related research.\n\nThe next section of the survey explored the use of resources related to the study of protein structures. 21 out of 92 (22.8%) respondents worked with protein structure, whereas 5 (5.4%) did not but would like to. The results of the survey are presented based on 182 answers from these 26 respondents. Databases of experimentally determined protein structure and complexes, such as PBD19 and PDBe20, were considered critical/very important by 65% (n=17/26) of respondents, followed by tools that report on the structural consequences of variants (62%, n=16/26). All 7 tools/databases that were surveyed were ranked critical/very important by over 45% of respondents (Table 3). The following tools/databases were not surveyed but cited by respondents as key resources for structural modelling of variants: Pymol21, Rosetta22, KiNG23, Modeller24, PolyPhen-225, SNP3D26, SNPs&GO27, SAAPdab/SAAPpred28, HOPE29 and Yasara30.\n\nKey limiting factors to the use of tools for modelling the structural consequences of variants were the lack of expertise in the area (54%, n=13/24), difficulties in using the tools (42%, n=10/24), and difficulties in interpreting the results (38%, n=9/24) (Figure 3). Lack of high throughput capability was listed among “Other limitations”. The difficulty to translate protein dynamics into structural models was also cited.\n\nThe last section of the questionnaire covered questions related to ELIXIR as a platform that coordinates the tools and databases surveyed in this study and the benefits that ELIXIR, as a European intergovernmental organization, offers to the scientific community. The majority of respondents (52%, n=42/92) were not aware that the tools and databases surveyed in Q25 are part of ELIXIR (Extended data: Figure S2). However, the long-term sustainability of ELIXIR core resources and the verified scientific quality of datasets were considered critical/very important by >76% (n=68) of participants who answered this question. The international standards for describing and saving data, the verified quality and maintenance of ELIXIR resources and facilities to find the right research tools were also considered critical/very important by the majority of respondents (>60%) (Table 4). Additional challenges that were identified by respondents regarding the annotation and curation of human genetic variants were related to the quality of data: incorrect entries, conflicting annotation, limited access to expert curation and classification of variants obtained in routine diagnostic setting and limited number genotype-phenotype annotations. An important caveat is the ascertainment bias that the survey was primarily sent to colleagues of the authors and this probably is not a representative sample of the community involved in the annotation and curation of human genomic variations.\n\n\nConclusions\n\nThis survey shows that the scientific community in ELIXIR member states considers several resources supported by ELIXIR crucial or very important Moreover, it shows that the work done by ELIXIR is greatly valued. In particular, most respondents acknowledged the importance of key features and benefits promoted by ELIXIR, such as the verified scientific quality and maintenance of ELIXIR-approved resources.\n\n\nData availability\n\nOpen Science Framework: ELIXIR, https://doi.org/10.17605/OSF.IO/SWX4231.\n\nOpen Science Framework: ELIXIR, https://doi.org/10.17605/OSF.IO/SWX4231.\n\nThis project contains the following extended data:\n\n- Copy of the online survey\n\n- Figure S1. Number of responses from the countries that participated in the survey\n\n- Figure S2. Results for Q25: “Before the survey, were you aware that the resources listed below are part of ELIXIR?\n\n- Tables S1. Answers to Q2: Place of work. More than one answer was allowed per participant\n\n- Table S2. List of Databases and Tools surveyed in Q4\n\n- Table S3. Additional Resources listed by Respondents under “Other” in Q4 and considered Critical or Very Important\n\n- Table S4. Other resources listed by Respondents in Q5\n\n- Table S5. The importance of data formats in human genome variations analysis (Q8)\n\n- Table S6. List of the topics and analytical operations in human genome variation analysis considered most important in the Respondents’ work (Q9)\n\n- Table S7. Answers to Q14 (Do you use these sequencing methods?)\n\n- Table S8. Mitochondrial databases (Q18)\n\n- Table S9. Mitochondrial tools (Q18)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nR Core Team: R: A language and environment for statistical computing. Vienna, Austria; 2012.\n\nYates AD, Achuthan P, Akanni W, et al.: Ensembl 2020. Nucleic Acids Res. 2020; 48(D1): D682–D688. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLaren W, Gil L, Hunt SE, et al.: The Ensembl Variant Effect Predictor. Genome Biol. 2016; 17(1): 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019; 47(D1): D506–D515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichards S, Aziz N, Bale S, et al.: Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. 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PubMed Abstract \| Publisher Full Text\n\nDavid A: Elixir. 2020. http://www.doi.org/10.17605/OSF.IO/SWX42" }	[ { "id": "76824", "date": "22 Jan 2021", "name": "Lina Ma", "expertise": [ "Reviewer Expertise Big Data Integration and Analytics", "Non-coding RNA Annotation and Curation" ], "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 systematically analysed the results and statistics of the questionnaire for an international survey on the usage of ELIXIR databases and tools, which are mainly devoted to annotating and curating human genomic variants. This survey would benefit both ELIXIR and researchers in helping them to identify the most suitable and robust bioinformatics resources, and thus make those resources well-maintained. I have two major concerns regarding this work.\nIt is not clear how the 52 resources for Q4 (How important the following resources in your work) in Section 2 (Resources for annotating and curating human genomic variants) were selected. As not all of the resources are related with human genomic variants annotation and curation, the results or the question itself may be misleading. For example, ArrayExpress may be critical/important for gene expression studies, and Europe EMC may be critical/important for literature retrieval. However, they may not be critical/important for variation analysis. This should be addressed in the manuscript to avoid misunderstanding.\n\nA small number of questionnaires are available for some questions. Especially, for sections 4 and 5, there are only about 20 participants. I am afraid some conclusions may not be well-supported by the statistics. MitoCarta, which is considered to be one of the most popular mitochondrial databases according to its high citations and z-index, ranks the first among the 53 related resources collected by Database Commons (https://bigd.big.ac.cn/databasecommons). However, only 24% of participants considered MitoCarta to be critical/important. Therefore, the authors should conclude with caution and discuss this in the manuscript.\n\nAlso, there is one typo in row 7 of ‘Protein structure’. ‘PBD’ should be ‘PDB’.\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? Partly", "responses": [] }, { "id": "145730", "date": "18 Aug 2022", "name": "Osamu Ogasawara", "expertise": [ "Reviewer Expertise Bioinformatics", "high performance computing" ], "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 conducted a systematic survey of the usage of databases and tools for annotating and curating human genomic variants with the aim of improving ELIXIR resources. Eighteen European countries and the United States participated in the survey and a total of 92 questionnaires were collected and analyzed. The survey questions were carefully designed and consisted of a series of questions about what kind of databases and tools users need, and what kind of technical requirements they would like to have in their computing infrastructure. The results of the survey are quite interesting and will be useful to researchers in many fields, not just those working on computational infrastructure and tools. In addition, the questions themselves and the survey results are provided as Extended Data which is very useful for ensuring the reproducibility of the paper's analysis results and for readers to verify any questions they may have.\nMajor comments:\nSome items may be underestimated due to the mixing of questionnaire results from researchers with different project sizes and other different characteristics. A brief discussion of this effect should be added.\nFor example, for Question 11, \"Run locally,\" the text indicates that 28% of the respondents chose \"critical\" as the 8th item from the top of Table 2, however, if this calculation is done (by using Extended Data) only for the large group (>10 people), 40% of the respondents chose \"critical\" as their response, and it comes in the upper part of Table 2. Thus, not all of the respondents to the questions are highly interested, but researchers with certain characteristics may be highly interested in some of the items. (On the other hand, as far as I can tell from my research using Extended Data, most of the questions in Questions 10, 11, and 26 seem to have almost the same ratio of critical, very important, and useful, even if the size of the research project varies. The few exceptions include “Data in large number of global populations available”, “Open interfaces (for example REST)”, and “Response time of key web pages and search functions”. These are all items in Q11, and the large group had a high percentage of \"critical\" responses.\n\nSince the purpose of the questionnaire survey is stated in the Abstract as \"with the aim of improving ELIXIR resources\", it would be better to briefly explain in the conclusion the idea of how to reflect the results of this survey in the management of ELIXIR.\n\nMinor comment:\nIn the Abstract, the authors wrote “Results: Results: Eighteen countries participated in the survey and a total of 92 questionnaires were collected and analysed”, but as shown at the beginning of the Results (page 3) and in Figure S1, 19 countries (Eighteen European countries and the United States) participated.\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? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-1207
https://f1000research.com/articles/9-281/v1	23 Apr 20	{ "type": "Review", "title": "Gene editing in dermatology: Harnessing CRISPR for the treatment of cutaneous disease", "authors": [ "Catherine Baker", "Matthew S. Hayden", "Catherine Baker" ], "abstract": "The discovery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized gene editing research. Through the repurposing of programmable RNA-guided CRISPR-associated (Cas) nucleases, CRISPR-based genome editing systems allow for the precise modification of specific sites in the human genome and inspire novel approaches for the study and treatment of inherited and acquired human diseases. Here, we review how CRISPR technologies have stimulated key advances in dermatologic research. We discuss the role of CRISPR in genome editing for cutaneous disease and highlight studies on the use of CRISPR-Cas technologies for genodermatoses, cutaneous viruses and bacteria, and melanoma. Additionally, we examine key limitations of current CRISPR technologies, including the challenges these limitations pose for the widespread therapeutic application of CRISPR-based therapeutics.", "keywords": [ "CRISPR", "dermatology", "gene editing", "genodermatoses", "viruses", "cutaneous disease" ], "content": "Introduction\n\nGene editing technologies have been transformative in biological research and show immense potential for the study and treatment of inherited and acquired human diseases1. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system uses programmable RNA-guided CRISPR-associated (Cas) nucleases to change, remove, or add genetic material to specific locations within the genome2. In comparison with other site-specific nucleases, such as zinc finger nucleases (ZFNs), meganucleases (MNs), and transcription activator-like effector nucleases (TALENs), CRISPR-Cas nucleases are easier to design and implement through the simple manipulation of a guide RNA sequence.\n\nDermatologic conditions hold particular appeal as targets for CRISPR-Cas therapeutics. There are several well-described, monogenic inherited skin disorders, such as the epidermal blistering disorders, that are considered ideal candidates for genome editing therapeutics. In addition, the skin is an easily accessible organ that allows for extraction and in vitro culture of target cells as well as direct localized administration of CRISPR-Cas therapeutics through topical, grafting or injection methods3. Lastly, for the same reason, the visibility of skin allows for simpler monitoring of the genetically edited cutaneous tissues for both efficacy and potential deleterious effects3.\n\nOngoing research efforts are exploring a variety of CRISPR-Cas approaches to the development of new CRISPR-Cas therapeutics for dermatology. Though most experiments have focused on ex vivo manipulation of diseased primary cell lines, researchers are increasingly developing in vivo and ex vivo techniques with translational potential. We propose that, for a variety of reasons, dermatology is likely to continue to be at the center of the development and clinical application of CRISPR-Cas therapeutics. For example, one of the first human trials involving CRISPR-Cas9 is geared toward treating refractory melanoma, among other neoplasms4. Therefore, in this review we will focus on the current research and potential future applications of therapeutic CRISPR-Cas nucleases in dermatology.\n\n\nMechanisms of genome engineering with CRISPR-Cas\n\nThere are several types of CRISPR-Cas systems (I-III), and numerous subtypes, that have been identified in bacteria and archaea, but the type II CRISPR-Cas9 system is the best studied, particularly in terms of its application to dermatology therapeutics5. The type II CRISPR system provides bacteria with a mechanism of immunologic memory and defense against foreign DNA6. Using CRISPR, bacteria incorporate short sequences of exogenous DNA from invading pathogens, for example from bacteriophages or viruses that infect bacteria, into their own genome. When transcribed from the bacterial host genome, these sequences are processed into CRISPR RNAs (crRNAs) that complex with a trans-activating crRNA (tracrRNA). The crRNA/tracRNA duplex directs Cas9 to cleave target double-stranded DNA that is complementary to the 20-nucleotide guide sequence within the crRNA, creating a site-specific double strand break (DSB). In the laboratory, a single 80 to 100-nucleotide RNA transcript synthesized in the form of a single guide RNA (sgRNA) can mimic the structure and function of the crRNA/tracrRNA duplex (Figure 1)2. The simplicity and multiplexing capacity of CRISPR-Cas9 nuclease activity is based on the easy-to-design sgRNA or crRNA, whose RNA sequences can be modified to direct the Cas nuclease to target different sequences in the dsDNA genome.\n\n(A) Mechanism of CRISPR-Cas9 genome editing. Cas9 nuclease complexes with a single guide RNA (sgRNA) to form a ribonucleoprotein (RNP). The sgRNA guides Cas9 to create a double strand break (DSB) three to four base pairs proximal to an “NGG” PAM sequence. After creating a DSB, dsDNA can be repaired by either non-homologous end joining (NHEJ) or, when a homologous dsDNA donor template is available, homology-directed repair (HDR). (B) Strategies for gene modification therapies in humans. Ex vivo gene editing strategies involve the extraction and manipulation of patient-derived cells in vitro in cell culture. Gene-corrected cells are expanded in culture and are subsequently re-infused or grafted onto the patient. In vivo gene editing involves the direct delivery of CRISPR-Cas DNA, RNA, and/or protein via viral or nonviral means. (C) Traditional gene therapy versus genome editing with CRISPR-Cas technology. Traditional gene therapy involves the addition of a functioning gene to replace a mutant allele. The replacement gene is usually inserted randomly into the host genome via a viral vector. In contrast, genome editing with CRISPR-Cas involves the direct, site-specific editing of the host genome.\n\nIn eukaryotic cells, following the formation of a site-specific DSB by Cas9, one of two cellular repair processes can occur: non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Figure 1)7. NHEJ is an error-prone process that can result in mutations or nucleotide insertions and deletions (indels), interrupting the sequence of a target gene. In contrast, HDR is a high-fidelity DNA repair strategy whereby the DSB is repaired using homologous DNA as a template. HDR can be facilitated by co-administration of homologous donor DNA with the Cas nuclease. This donor sequence can be used as a synthetic template for the cell to copy when repairing the Cas-induced DSB. HDR can be used to direct the repair of a mutated gene, albeit with lower efficiency than NHEJ8.\n\nTo date, most genome engineering strategies for dermatological disease have involved the ex vivo editing of patient-derived primary cells (Figure 1)9. To perform ex vivo editing, patient cells are isolated and genetically modified in vitro, potentially for subsequent autologous transplantation. This strategy allows for the clonal selection, characterization, and expansion of genetically modified cells prior to use for engraftment in the affected organ or tissue. Ex vivo approaches facilitate targeting and delivery of the CRISPR-Cas therapeutic and, by allowing for enrichment of modified cells, reduce the requirement for highly efficient and specific CRISPR-Cas editing constructs10. However, cell expansion in culture can lead to unwanted cellular differentiation, particularly in induced pluripotent stem cells (iPSCs)11. In addition, cell-based transplantations can be technologically challenging, especially for non-hematopoietic cells. In contrast to ex vivo gene manipulation, in vivo gene editing involves the direct modification of somatic cells in situ (Figure 1). Using CRISPR-Cas constructs, in vivo gene editing is achieved through systemic or local administration of packaged CRISPR-Cas components (protein, DNA, and/or RNA) into the body to induce gene editing outcomes in specific organs or cells. In vivo editing requires the development of effective targeting strategies to generate cell-specific changes with minimal off-target effects and precludes comprehensive characterization of all edited cells. Safe in vivo gene editing techniques could have utility for a wide range of systemic and localized diseases, but many hurdles and concerns remain to be addressed.\n\nMost genodermatoses are monogenic in nature and therefore serve as an attractive disease model for gene therapy12. Because there are no widely available effective treatments for these disorders, current therapies are focused primarily on symptom management. Early success in the use of gene therapy for the treatment of the monogenic inherited epidermolysis bullosa (EB) disorders provided particular promise for the development of curative gene therapies for genodermatoses. In 2006, a patient suffering from nonlethal junctional EB (JEB) underwent successful long-term skin transplantation with epidermal sheets made from gene-corrected autologous keratinocytes13,14. These keratinocytes were corrected using a retroviral vector encoding the beta 3 chain of laminin-332, compensating for the mutated version of the gene in this patient. Such successes laid the groundwork for further research in not only gene replacement therapies for cutaneous disorders, but also for gene editing therapies. Unlike classically defined gene therapy, which involves the random insertion of one or more exogenous genes into cells to replace the function of a missing or mutated gene, gene editing involves the direct, site-specific manipulation of the genome using targeted nucleases such as ZFNs, TALENs, and CRISPR-Cas enzymes (Figure 1)15. The therapeutic potential of direct genome manipulation is immense. Researchers have leveraged CRISPR-Cas constructs to develop diverse treatment strategies for genodermatoses including the targeted addition of genes to specific genomic sites, the correction of disease-causing point mutations, and the removal of disease-causing genes or genomic sequences. Such strategies expand the scope of gene therapy beyond additive approaches, allowing for corrective gene editing and the targeted ‘knockout’ of mutant alleles in dominant negative disorders. It is also hoped that targeted gene editing strategies will reduce the risks associated with random insertion of exogenous transgenes.\n\nEB. Of the genodermatoses, the inherited EB disorders have been the most extensively studied as potential candidates for gene editing therapy3. The EB disorders are a diverse group of inherited blistering diseases that affect the skin and, in some subtypes, mucous membranes and other organs16. They are caused by mutations in over 20 different genes that code for different proteins expressed at the cutaneous basement membrane zone17.\n\nTwo EB subtypes—EB Simplex (EBS) and dominant dystrophic EB (DDEB)—are caused by dominant negative mutations that cannot be corrected with traditional additive gene therapies. Thus, these disorders are particularly well-suited for treatment with CRISPR-Cas genome editing, which allows for the direct modification of the dominant, disease-causing allele. EBS is caused by dominant negative missense mutations in either the keratin 14 (KRT14) or keratin 5 (KRT5) genes that code for intermediate filaments (IFs) expressed in the basal layer of the epidermis18,19. Kocher et al.20 used CRISPR-Cas9-induced HDR to correct the disease-causing mutated KRT14 allele in EBS patient keratinocytes in vitro. Gene-corrected clones showed normal phenotype without characteristic mutant cytoplasmic aggregates in cell culture. DDEB is caused by dominant negative mutations in COL7A1, which codes for collagen 7 (C7)21,22. Shinkuma et al.23 successfully used CRISPR-Cas9 to induce site-directed mutagenesis of the mutated COL7A1 allele in iPSCs derived from DDEB patient keratinocytes. Edited cells expressed a truncated version of C7 that was incapable of forming deleterious trimers with WT C7 and would hypothetically allow for normal anchoring fibril formation at the DEJ23.\n\nUnlike EBS and DDEB, JEB and RDEB are inherited in an autosomal recessive manner. Therefore, CRISPR-Cas therapeutics for these disorders must achieve gene correction to allow for production of new, functional proteins. This can be accomplished through precise correction of the disruptive mutation (i.e., via CRISPR-Cas9 induced HDR with a gene-corrected donor template) or through methods that produce a functional protein without fully correcting the disease-causing mutation. For example, in specific cases, targeted deletions can be used to either remove a premature stop codon or causative mutation directly or to disrupt splicing signals, allowing for therapeutic exon skipping. JEB is caused by autosomal recessive mutations in genes encoding subunits of the heterotrimeric laminin-5 (laminin-332) protein (e.g., LAMA3, LAMB3, and LAMC2)24,25, and RDEB is caused by autosomal recessive mutations in COL7A126,27. For both of these conditions, CRISPR-Cas9-mediated HDR has been used to successfully correct disease-causing mutations ex vivo in patient-derived primary keratinocytes28–31 and iPSCs32. When grafted onto immunodeficient mice, gene-corrected keratinocytes displayed restored WT functionality and adhesion at the DEJ29–31. However, the efficiency of HDR during ex vivo gene modification remains low, particularly without antibiotic section protocols or other methods of enriching for modified cells. Exon skipping approaches have been explored for the treatment of RDEB. Exon 80 contains a common disease-causing point mutation in the COL7A1 gene33. By inducing targeted Cas9-mediated DSBs on either side of the mutation-bearing exon 80 in COL7A1, it is possible to mediate complete excision of the disease-causing mutation. Bonafant et al. delivered paired Cas9/sgRNA RNPs to patient keratinocytes ex vivo through electroporation. They were able to achieve efficient rates of deletion of exon 80, generating cells with restored C7 expression that, when grafted to immunodeficient mice, showed long-term dermal-epidermal adhesion34.\n\nNotably, Cas9-mediated therapies for RDEB have recently expanded to include in vivo approaches, with Wu and colleagues35 restoring C7 function in an RDEB mouse model using the exon skipping approach. Wu et al. designed two sgRNAs targeting the 5’ and 3’ side of exon 80 and delivered them as an sgRNA/Cas9 ribonucleoprotein complex (RNP) via intradermal injection into mouse tail skin. They then directly electroporated the mouse tail to facilitate penetration of RNPs into epidermal stem cells. By causing DSBs on either side of the mutation-bearing exon 80 in COL7A1, they were able to achieve complete excision of the disease-causing mutation in epidermal stem cells. Electroporated mice displayed restored C7 function, and their dermal-epidermal adhesion area improved from 30% to 60% after one treatment35. The success of this approach demonstrated the potential for CRISPR-Cas9-induced gene correction of epidermal stem cells in vivo without the cost and technical challenge of ex vivo cell modification. Still, however, several limitations of this method are apparent. Only 2% of epidermal cells were capable of being targeted with this novel in vivo delivery method, and no long-term follow-up to assess sustainability of the treatment could be performed. Moreover, potential off-target effects of the Cas9/sgRNA RNPs at sites other than exon 80 were not analyzed.\n\nEpidermolytic palmoplantar keratoderma (EPPK). EPPK is an autosomal dominant keratin disease of the hereditary palmoplantar keratoderma (PPK) group characterized by an abnormal thickening of skin on the palms and soles36. The disease is caused by dominant-negative missense mutations in the KRT9 gene, leading to the formation of a mutant keratin 9 (K9) protein that interferes with the function of the wild-type K937. EPPK has relatively localized disease manifestations that are well suited for targeted delivery of CRISPR therapeutics.\n\nLuan and colleagues showed that through local delivery of a lentiviral (LV) vector carrying an sgRNA and Cas9 directed to the mutant KRT9 allele, they could disrupt the formation of the mutant K9 protein in vivo in an EPPK mouse model and induce phenotypic correction of the disease37. Mice who received nine subcutaneous injections of the Cas9/sgRNA-containing LV particles into their right forepaw over the course of 24 days displayed restored epidermal proliferation of the forepaw and a reduction in disease-associated K9 expression. Off-target effects of the genome editing system were reportedly minimal, but analyses were limited to 10 predicted off-target sites in the mouse genome37. In addition, long-term effects of the treatment on the mice were not recorded, and there was no analysis of potential immune response to the LV Cas9 vector—an occurrence which has been reported for other LV vector systems38. Still, this study demonstrates the potential for robust in vivo effects of CRISPR-Cas9-mediated gene editing, particularly for diseases that have localized manifestations.\n\nCRISPR-Cas systems, which evolved in bacteria to fight invading bacteriophages, have also been repurposed to serve a similar function in virally infected human cells. In human cells, CRISPR-Cas enzymes have the potential to target latent viruses that are able to escape eradication by immune surveillance and standard antiviral therapies. As such, there has been extensive research into the ability of CRISPR-Cas systems to target specific viral genomic sequences, enabling targeted disruption and even complete excision of components of the viral genome39. In addition, researchers have leveraged the high sensitivity of certain Cas enzymes for the purposes of viral pathogen detection in human tissue samples40. Cas12 and Cas13—two Cas enzymes that demonstrate indiscriminate trans-cleavage of ssDNA when activated by their guide-complementary target nucleotide sequence—enable ultrasensitive nucleic acid detection in viral biosensing systems such as DETECTR41, SHERLOCK/SHERLOCKv242 and HOLMES/HOLMESv243,44\n\nThough most CRISPR-mediated antiviral research to date has focused on systemic viruses that lack primary cutaneous involvement, the unique accessibility of the skin suggests that CRISPR therapeutics and diagnostics might be more effectively applied to cutaneous viruses. Current research suggests CRISPR-Cas technology may be effective in detecting and modifying human papillomavirus (HPV), herpes simplex virus (HSV), and Kaposi sarcoma-associated herpesvirus (KSHV).\n\nHPV. HPV is a dsDNA virus that infects the basal cells of stratified epithelium. Here, the virus has the potential to integrate its viral DNA into the host genome. The HPV E6 and E7 proteins of certain high-risk strains, including 16, 18, 31, 33, cause malignant transformation of epithelial cells by inactivating p53 and Rb, respectively45,46, and can lead to anogenital squamous cancers and other neoplasms. E7 expression from lower risk strains of HPV, particularly strains 6 and 11, is associated with the uncontrolled proliferation of epithelial cells that cause genital warts47,48.\n\nSeveral researchers have effectively used CRISPR-Cas9 to disrupt of E6 and E7 genes in cervical cancer cells, both in vitro and in vivo in animal models44–46,49–51. One of the first antiviral CRISPR-Cas9 clinical trial in humans will involve in vivo targeting of E6/E7 in HPV-infected neoplastic cervical cells52.\n\nThough fewer studies have been performed with the aim of using CRISPR-Cas to cure the dermatological manifestations of HPV, researchers have begun to develop CRISPR-Cas constructs targeting the virus in HPV-associated anal cancer and genital warts. Hsu et al.53 were able to successfully decrease tumor burden in a mouse model of HPV-16-associated anal cancer. Using an adeno-associated virus (AAV) vector, they delivered Cas9 nuclease in conjunction with two gRNAs: one gRNA specific for HPV-16 E6 and the other specific for HPV-16 E7. When delivered via three intratumoral injections over one week to mice with patient-derived xenografts of HPV-16 anal cancer cells, the CRISPR-Cas9 and dual-gRNA caused a twofold decrease in tumor volume, providing proof of concept for a novel in vivo gene editing strategy for HPV-16-associated anal cancer. For HPV-associated genital warts, CRISPR-Cas9 has been used to successfully target the E7 gene in vitro, promoting apoptosis of HPV-6 and -11-infected keratinocyte cell lines54. Notably, however, the Cas9 and gRNAs were transfected transiently into the keratinocyte cell line and achieved only incomplete E7 deactivation. In addition, efficacy of the genome editing construct was not confirmed in vivo.\n\nCRISPR-Cas systems hold potential for not only HPV therapeutics, but also HPV diagnostics. DETECTR is a nucleic acid biosensing system that uses the enzyme Cas12a and a ssDNA fluorescent reporting scheme to identify specific sequences in amplified dsDNA from human samples41. Chen et al.41 showed that by using their DETECTR platform, they could detect human papillomavirus (HPV) DNA in patient anal swab samples with attomolar sensitivity, even reliably distinguishing between different genotypes of the virus. Moreover, their assay was performed in just one hour and involved only isothermal amplification of DNA, suggesting that DETECTR could serve as a rapid, low-cost, point-of-care detection assay for HPV with similar sensitivity and specificity to conventional diagnostic PCR.\n\nHerpesviruses. The herpesviruses are large dsDNA viruses that establish lifelong infection in human hosts55,56. HSV-1 and HSV-2 classically infect the oral and genital mucosal epithelium, respectively, leading to the local production of ulcers. After undergoing partial clearance by the host immune system, HSV establishes latent infection in the form of episomal DNA in sensory ganglia. KSHV, or human herpesvirus 8, infects human endothelial cells and is the causative agent of Kaposi’s sarcoma57. Like HSV and other herpesviruses, KSHV remains latent in most infected cells. Latent herpes infections avoid immunological surveillance by limiting viral gene transcription58 and are extremely difficult to treat.\n\nAs conventional therapies targeting viral replication are ineffective against latent virus, CRISPR-mediated targeting of viral DNA has emerged as an alternative approach for HSV, KSHV, and other herpesviruses. Roehm and colleagues59 were able to completely abrogate HSV-1 replication in human fibroblast cells in vitro by disrupting two essential viral genes using CRISPR-Cas9. Other researchers have achieved similar success against HSV-1, inhibiting viral replication in vitro in oligodendroglioma cells and epithelial cells using Cas9/gRNA editing complexes without evident off-target effects59,60. For KSHV, researchers have demonstrated an ability to reduce the burden of KSHV in latently infected epithelial and endothelial cell lines by AAV-CRISPR-Cas9-mediated disruption of the KSHV latency-associated nuclear antigen (LANA)61.\n\nStill, challenges remain in the use of CRISPR-Cas for herpesviruses. First, despite demonstrated ability to abrogate active viral replication of HSV-1, it remains to be seen whether CRISPR-Cas can be effectively used to eradicate latent HSV-1 in neurons62. Oh et al. were able to disrupt quiescent HSV-1 genomes, but at a much lower rate compared to HSV-1 virions in the lytic cycle63. Because other targeted nucleases, such as MNs, have been shown to successfully target latent HSV-1 infection64, it is suspected that current failure to eliminate latent HSV-1 using CRISPR-Cas9 may be due to epigenetic modifications of the latent HSV-1 genome that block Cas9 activity62,65. Moreover, further demonstration of the effectiveness of anti-HSV and anti-KSHV CRISPR-Cas systems in vivo will also be important. For HSV-1 and -2, in vivo delivery may be simpler than for other systems because latent HSV-1 and 2 have very specific tropism for the trigeminal and sacral ganglia, respectively, and possible delivery strategies could be designed to specifically home to these ganglia.\n\nIn addition to viral infections, CRISPR-Cas technology also shows promise against resistant bacterial infections66,67. Bacterial infections that are resistant to common antibiotics represent a growing public health concern68,69. Yet, despite this risk, antibiotics continue to be overprescribed70 while the development of novel antimicrobials for new pathogens lags behind71. Recently, CRISPR-Cas antimicrobials have emerged as a novel treatment strategy against bacterial infections66,67. Specifically, CRISPR-Cas9 has been leveraged to selectively remove antimicrobial resistance genes from populations of bacteria, re-sensitizing populations of bacteria to common antimicrobials72,73. Though systemic delivery of CRISPR-based antimicrobials remains a challenge, the accessibility of the skin enables the delivery of CRISPR constructs via convenient topical formulations66 and places cutaneous bacterial infections at the forefront of CRISPR antimicrobial research.\n\nStaphylococcus aureus. Staphylococcus aureus, a common cutaneous bacterial pathogen known for its antimicrobial resistance, is responsible for 76% of all skin and soft tissue infections74 and is associated with high morbidity and mortality75. Antimicrobial resistance to S. aureus continues to emerge as the pathogen gains plasmids and other mobile genetic elements that confer antibiotic resistance and virulence genes76. Moreover, outbreaks remain common as the pathogen maintains a high prevalence in the population, asymptomatically colonizing the nostrils of 20–30% of healthy adults77.\n\nBikard and colleagues72 developed a novel approach to target virulent strains of S. Aureus using CRISPR-Cas9. They developed gRNAs targeted to specific S. Aureus antimicrobial resistance genes, including the methicillin resistance gene, mecA. When delivered with Cas9 via a phage capsid to mixed populations of bacteria in vitro, these gRNA/Cas9 constructs were able to eradicate resistant S. Aureus strains and completely remove specific plasmids carrying antimicrobial resistance genes. Moreover, when delivered topically to a mouse model of S. Aureus skin colonization in vivo, these gRNA/Cas9 constructs were able to significantly decrease colonization by resistant bacteria. These results demonstrated promise for the topical application of CRISPR antimicrobials in vivo and laid the foundation for future multiplexed CRISPR antimicrobials designed to simultaneously target either several bacterial species or multiple gene sequences within the same bacterium.\n\nSome of the first CRISPR-Cas clinical trials in humans have involved the use of CRISPR-Cas technology in immunotherapy for cancers, including melanoma and non-small-cell lung cancer (NSCLC)4. Much of this research has centered on the use of gene editing to inactivate key immune checkpoint inhibitors such as programmed cell death protein-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4)—two proteins that normally inhibit the anti-tumor cytotoxic effect of endogenous and exogenous T cells78.\n\nMelanoma is well-known to have exceptional immunogenic potential, owing largely to the high mutational burden that drives the formation of immune-stimulating neoantigens79. Thus, under optimal conditions, melanoma cells are particularly susceptible to destruction by the human immune system80. Yet, clinically, the tumor microenvironment in melanoma is highly immunosuppressive81, and advanced disease has exceptionally poor treatment response82. Consequently, melanoma serves as an ideal target for immunotherapies that are designed to relieve tumor immunosuppression.\n\nThe first human trial designed to test the use of CRISPR-Cas for melanoma builds on the demonstrated success of previous immunotherapies, including PD-1 inhibitors83 and T cells transduced with the NY-ESO-1 T-cell receptor (TCR)84. Specifically, researchers aim to amplify the therapeutic effects of these existing approaches by using CRISPR-Cas9 to knock out PD-1 gene loci in autologous NY-ESO-1 TCR-transduced T cells. Autologous T cells are first taken from a patient and transduced with a LV vector that expresses the NY-ESO-1 TCR, priming them to recognize a highly immunogenic NY-ESO-1 antigen expressed on melanoma cells85. They are then electroporated with RNA-guided CRISPR-Cas9 nucleases designed to disrupt the expression of both PD-1 and the endogenous TCR subunits, TCRα and TCRβ4. Disrupting PD-1 prevents immune suppressive signaling, and blocking the endogenous TCR subunits inhibits aberrant immune responses that may result from TCR-mediated targeting of unknown antigens. With re-introduction of these melanoma-targeted, immune-avid T cells, researchers hope to obtain a more robust tumor-specific immune response—a response that has been difficult to achieve with previous T-cell therapies for solid tumors86. Moreover, beyond having an enhanced anti-tumor effect, such an approach is also expected to minimize unwanted treatment side effects. Using this treatment model, PD-1 blockade will be limited to the specific TCR-transduced T-cells that are designed to home to and attack NY-ESO-1-expressing melanoma cells. This stands in contrast to traditional anti-PD1 receptor antibodies which, when administered systemically, have the potential to affect T-cells more broadly and cause widespread immunogenic side effects87.\n\n\nPerspectives and future directions\n\nTaken together, a wide variety of studies underscore the potential for CRISPR-based therapeutics for genodermatoses, cutaneous infections, and melanoma. Future research will likely continue to expand on this success, with the aim of increasing the translatability of CRISPR therapeutics as well as developing expanded strategies to target other dermatologic diseases.\n\nThere are many other monogenic skin disorders and cutaneous infections that could be targeted with CRISPR-Cas therapeutics. Pachyonychia congenita and xeroderma pigmentosum have been targeted with RNAi-based therapies88 and designer nucleases89, respectively, and could likely also be targeted with CRIPSR-based genome engineering. Moreover, with the advent of hypoimmunogenic universal donor iPSCs—iPSCs that are genetically modified by CRISPR-Cas9 to avoid inciting a host immune response90,91—existing ex vivo gene modification strategies for genodermatoses could be applied to patients more broadly.\n\nThere are many cutaneous viruses that hold promise for targeting with gene editing technologies, including the oncoviruses, Merkel-cell polyomavirus (MCPyV), and human T-cell leukemia virus type 1 (HTLV-1). MCPyV causes up to 80% of Merkel cell carcinomas (MCC) and is randomly integrated into different sites in the MCC tumor genome92. Excision of MCV from MCC tumor cells could represent a potential therapeutic strategy for aggressive MCCs that are often refractory to many current therapies92–94. Preliminary studies in Merkel cell cancer cell lines demonstrate that CRISPR-Cas9-mediated disruption of the MCPyV tumor antigens leads to diminished cell growth95. HTLV-1, the retrovirus that causes HTLV-1-associated/tropical spastic myelopathy and adult T cell leukemia/lymphoma, has yet to be explored as a target for CRISPR-Cas therapeutics. Studies focusing on HIV96–99—a similarly structured retrovirus—would suggest that HTLV-1 could likely be completely excised from infected cells using CRISPR-Cas9. The relative genetic conservation of HTLV-1 relative to HIV-1100 would make it an even more appropriate candidate for targeting by RNA-guided endonucleases.\n\nLastly, CRISPR-Cas technology will likely develop clinical utility in the diagnosis of dermatological disease. CRISPR-Cas diagnostic platforms using Cas9101, Cas1241,43,102, Cas1342, and Cas14103 have the potential to revolutionize the detection of nucleic acid sequences, allowing for the ultrasensitive, low-cost, and portable detection of cutaneous viruses and single point mutations in cutaneous tumors103,104.\n\n\nChallenges to therapeutic application\n\nThere remain several challenges to the widespread application of CRISPR-based therapeutics. The studies reported here largely demonstrate the ability of CRISPR-Cas systems to treat human disease both in cell culture models and through ex vivo modification of primary patient cell lines. While such methods currently represent the safest approach to gene editing in humans, such a technique is technologically challenging and of limited use for routine clinical practice. In vivo treatment options would be ideal, but fewer studies have explored these possibilities. Studies on the in vivo treatment of non-dermatologic disorders, including Duchenne muscular dystrophy105–107, hereditary liver disease108–110, congenital eye disease111, and Huntington Disease112 have shown tremendous promise in animal models. However, of the studies in dermatology focused on in vivo delivery of CRISPR-therapeutics, all have been limited to localized effects in mouse models, and none have demonstrated high efficiency or success in long-term follow up35,37,53.\n\nFor in vivo genome editing via CRISPR-Cas technology to be clinically translatable not only in dermatology but also in other fields, there are several major challenges that are yet to be effectively addressed. CRISPR-Cas guide RNAs and nucleases must 1) be optimized for specific on-target effects with minimal off-target effects, 2) be delivered efficiently to specific human cells, and 3) have minimal antigenic properties so that they are accepted by human immune systems113. Novel CRISPR-Cas enzymes and delivery systems are being developed to tackle these challenges. To improve specificity of CRISPR-Cas9, researchers have modified Cas9 construction114, optimized sgRNA design115, and developed a CRISPR-Cas9 double nickase approach116 that introduces only single-strand nicks at target sites. In addition, researchers have developed sensitive methods to scan the entire genome for unintended off-target genome editing effects, including GUIDE-Seq117, Digenome-seq118, SITE-seq119, CIRCLE-Seq120, and, most recently, VIVO121. Such methods would allow for screening of off-target effects of a given Cas/gRNA construct prior to therapeutic use.\n\nNovel delivery methods are also being explored that expand upon traditional AAV- and LV-associated delivery systems. Gene delivery methods with viral vectors have the potential to cause integrational mutagenesis and lifelong Cas9 expression in cells. For this reason, delivery of Cas9 nuclease by way of a transiently expressed Cas9/sgRNA RNP may be preferred. Scientists have developed lipid nanoparticles that can carry CRISPR-Cas nucleotide sequences or RNP complexes that are targeted to specific organs122–124. Still, it will be challenging to achieve systemic delivery of such therapies, particularly for bloodborne illnesses that would require the delivery of CRISPR-Cas constructs to every circulating B or T cell39.\n\nLastly, to limit the possible immunologic response to Cas9 by preformed antibodies in human serum125, researchers have discovered novel Cas enzymes such as the structurally distinct Cas12e and Cas12d from ground bacteria126. CRISPR therapeutics employing nucleases from bacteria to which humans are not exposed may not be subject to pre-existing immunity, allowing for a more robust genome editing effect.\n\n\nConclusion\n\nThough significant work remains to be done prior to its widespread therapeutic use in humans, preliminary research suggests great potential for CRISPR-Cas technology in the treatment of dermatological pathologies. As researchers continue to optimize delivery methods and off-target screening approaches, more human trials for the treatment of dermatologic diseases with CRISPR-based gene editing therapeutics will likely be initiated.\n\n\nData availability\n\nNo data are associated with this study.", "appendix": "References\n\nFinn JD, Smith AR, Patel MC, et al.: A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. Cell Rep. 2018; 22(9): 2227–2235. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoench JG, Fusi N, Sullender M, et al.: Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34(2): 184–191. 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): 1380–1389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai SQ, Zheng Z, Nguyen NT, et al.: GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2014; 33(2): 187–197. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim D, Bae S, Park J, et al.: Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015; 12(3): 237–43. PubMed Abstract \| Publisher Full Text\n\nCameron P, Settle AH, Fuller CK, et al.: SITE-Seq: A Genome-wide Method to Measure Cas9 Cleavage. 2017. Publisher Full Text\n\nTsai SQ, Nguyen NT, Malagon-Lopez J, et al.: CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods. 2017; 14(6): 607–614. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkcakaya P, Bobbin ML, Guo JA, et al.: In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature. 2018; 561(7723): 416–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZuris JA, Thompson DB, Shu Y, et al.: Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015; 33(1): 73–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiang C, Mei M, Li B, et al.: A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell Res. 2017; 27(3): 440–443. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMout R, Ray M, Yesilbag Tonga G, et al.: Direct Cytosolic Delivery of CRISPR/Cas9–Ribonucleoprotein for Efficient Gene Editing. ACS Nano. 2017; 11(3): 2452–2458. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCharlesworth CT, Deshpande PS, Dever DP, et al.: Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019; 25(2): 249–254. PubMed Abstract \| Publisher Full Text\n\nBurstein D, Harrington LB, Strutt SC, et al.: New CRISPR-Cas systems from uncultivated microbes. Nature. 2016; 542(7640): 237–241. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "62746", "date": "18 May 2020", "name": "Bindu Hegde", "expertise": [ "Reviewer Expertise Drug discovery", "Immunology", "Cancer immunotherapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 review article, Baker and Hayden discuss the role of CRISPR technology in genome editing for dermatological diseases. Overall, the manuscript is well-written with clear and unambiguous language. The article highlights the studies that use CRISPR-Cas 9 approach for gene editing in various cutaneous diseases and provides a balanced assessment by noting both advantages and limitations of using this technology. However, it could improved with the following suggestions:\n\nPage 4 - Since EB can be caused by mutation in several genes and multiple mutations can be observed within the same patient, strategies correcting a single mutation may not reverse the disease completely. How would some of these studies, which correct a single mutation, translate clinically? Are there any efforts to correct multiple mutations at once using CRISPR?\n\nPage 7 - iPSCs are an attractive tool for cell therapy in some ways because of their low immunogenicity. However, there are several safety concerns with these cells. They could acquire mutations while being expanded in culture and develop into teratomas (Dinella 20141). What methods are used to overcome these concerns?\n\nPage 7 - Challenges of therapeutic application should be discussed in more detail. Low efficiency of gene editing is also one of the drawbacks. What are the new techniques being applied to overcome this? (For example, one of the ways to improve the efficiency of HDR is the use of NHEJ inhibitors)\n\nPage 7 - In your discussion of strategies to improve the specificity of Cas9, include the possibility of using several modifications of Cas9 like high-fidelity Cas9 (eSpCas9), evolved Cas9 (evoCas9) in addition to the ones you have mentioned.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "5976", "date": "07 Oct 2020", "name": "Catherine Baker", "role": "Author Response", "response": "Dear Dr. Hegde, Thank you for your thoughtful review of our manuscript. We have carefully reviewed the comments and have revised the manuscript accordingly. Our responses are given in a point-by-point manner below. Since EB can be caused by mutation in several genes and multiple mutations can be observed within the same patient, strategies correcting a single mutation may not reverse the disease completely. How would some of these studies, which correct a single mutation, translate clinically? Are there any efforts to correct multiple mutations at once using CRISPR? A sentence regarding the potential use of CRISPR for targeting rare cases of EB caused by >1 mutation was added to the “Perspectives and future directions” section. iPSCs are an attractive tool for cell therapy in some ways because of their low immunogenicity. However, there are several safety concerns with these cells. They could acquire mutations while being expanded in culture and develop into teratomas (Dinella 2014). What methods are used to overcome these concerns? The challenges of gene editing with iPSCs are alluded to in the last paragraph of the “Mechanisms of genome engineering with CRISPR-Cas” section. Challenges of therapeutic application should be discussed in more detail. Low efficiency of gene editing is also one of the drawbacks. What are the new techniques being applied to overcome this? (For example, one of the ways to improve the efficiency of HDR is the use of NHEJ inhibitors) Expanded second paragraph of “Challenges to therapeutic application” to include mention of NHEJ inhibitors and brief mention of other strategies to improve efficiency of Cas9-mediated HDR. In your discussion of strategies to improve the specificity of Cas9, include the possibility of using several modifications of Cas9 like high-fidelity Cas9 (eSpCas9), evolved Cas9 (evoCas9) in addition to the ones you have mentioned. Cas9 variants with enhanced specificity have been listed as strategies to improve the specificity of Cas9 (second paragraph of “Challenges to therapeutic application” section)." } ] }, { "id": "63774", "date": "01 Jun 2020", "name": "Jillian Richmond", "expertise": [ "Reviewer Expertise dermatology research", "autoimmunity", "skin diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 review, Baker & Hayden describe the field of gene editing for the treatment of dermatologic conditions. This is a very well-written and interesting review. I only have minor suggestions prior to publication.\nMinor:\nIn your discussion of NHEJ and HDR, could you provide specifics about the efficiency of HDR (as a freq, etc)?\n\nUnder \"Genodermatoses\" you have a few sentences that seem redundant, please consider streamlining your intro paragraph before going into specific disease areas.\n\nLast paragraph under EB: any comment/concern about electroporating a mouse tail? Feasibility in humans? Side effects?\n\nIn the melanoma section, any thoughts about \"kill switch\" T cells?\n\nMaybe include a brief discussion about the skin microbiome, either in the bacteria section or future directions section? Could focus on the impact of gene editing on microbiome dynamics, or therapeutic potential for correcting dysbioses.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "5977", "date": "07 Oct 2020", "name": "Catherine Baker", "role": "Author Response", "response": "Dear Dr. Richmond, Thank you for your thoughtful review of our manuscript. We have carefully reviewed the comments and have revised the manuscript accordingly. Our responses are given in a point-by-point manner below. In your discussion of NHEJ and HDR, could you provide specifics about the efficiency of HDR (as a freq, etc)? This is a difficult question because relative efficiencies of NHEJ and HDR vary quite widely depending on cell type and conditions. A sentence addressing this issue, as well as two references to articles addressing this topic, were added to the HDR/NHEJ discussion in “Mechanisms of genome engineering with CRISPR-Cas.” Under \"Genodermatoses\" you have a few sentences that seem redundant, please consider streamlining your intro paragraph before going into specific disease areas. Repetitive words/sentences were eliminated in this paragraph. Last paragraph under EB: any comment/concern about electroporating a mouse tail? Feasibility in humans? Side effects? Two sentences on the feasibility of electroporation for human patients with EB were added. In the melanoma section, any thoughts about \"kill switch\" T cells? A paragraph on engineered T-cell therapies, including “kill switch” T cells, was added to the “Perspectives and future directions” section. Maybe include a brief discussion about the skin microbiome, either in the bacteria section or future directions section? Could focus on the impact of gene editing on microbiome dynamics, or therapeutic potential for correcting dysbioses. In “Cutaneous bacterial infections,” a section on the potential role for CRISPR in modulating the cutaneous microbiome was added, particularly as it pertains to atopic dermatitis and S. aureus colonization." } ] } ]	1	https://f1000research.com/articles/9-281
https://f1000research.com/articles/9-781/v1	28 Jul 20	{ "type": "Brief Report", "title": "Soluble nitrogen forms in sand soil of Pallag: a quantitative report", "authors": [ "Ida Kincses", "Jesus R. Melendez", "Lenin Ramírez-Cando", "Diego Burbano-Salas", "Daniel A. Lowy", "Gerardo Cuenca Nevarez", "Viviana Talledo Solórzano", "Juan Morales Arteaga", "Benito Mendoza", "Zsolt Sándor", "Jesus R. Melendez", "Lenin Ramírez-Cando", "Diego Burbano-Salas", "Daniel A. Lowy", "Gerardo Cuenca Nevarez", "Viviana Talledo Solórzano", "Juan Morales Arteaga", "Benito Mendoza" ], "abstract": "Nitrogen (N) is a crop macronutrient of major importance, which affects both plant growth and yield. In this paper we discuss the humus content (%) and various soluble N forms (NO3-, total N, nitrate-N, ammonium-N, and organic nitrogen) available in humus sand soil samples originating from the Pallag Experimental Station of Horticulture at the University of Debrecen, Hungary. We found 45.4% nitrate-N and 13.8% nitrite-N of total N content present in the soil. Considering the percentage distribution of soluble N forms present at the Pallag Experimental Station, we recommend using this soil in further pot experiments, given that this has optimal nutrient supply capacity. In addition, we examined possible statistical correlations between humus% and N forms.", "keywords": [ "pot experiment", "total N", "nitrate", "nitrite", "ammonium", "nutrient supply capacity" ], "content": "Introduction\n\nNitrogen (N) is an essential element for plants, takes various forms in soil1, and is one of the most limiting nutrients for various crops2–4. Nitrogen is present in soils as inorganic nitrogen (nitrate, ammonium, and dinitrogen) and organic nitrogen (urea and amino acids)5.\n\nConversions between organic and mineral N forms are primarily affected by soil microorganisms, which enable conversion to forms that plants can uptake6. In addition, some pollutant N forms are present in soil, so nitrogen conversions may indirectly affect human health and load the environment1. For this reason, quantifying studies aim to better understand nitrogen form ratios in soil and contribute to reaching sustainable agriculture practice. Recently, Jakab published a similar study7 quantifying another vital element for plants, phosphorus, in soil from the same region.\n\nHumus is a main fertility component of the soil, 65–75% of its structure being made up of organic matter8,9. We decided to include humus content in this N-related study as it represents a known indicator of soil quality10.\n\nCalcium chloride (CaCl2)-soluble nitrogen forms are the most available N ions for assimilation by plants11,12. Therefore, we quantified all N forms in CaCl2 solution. Additionally, we determined nitrate-N via a potassium chloride (KCl)-based method.\n\nThe main objectives of our study were: (a) to map out all easily absorbed nitrogen forms for better understanding of available nutrient composition and (b) to determine the ratio of pollutant (nitrite) and absorbable (ammonium ion, nitrate) N forms in humus sand soil in pot experiments.\n\n\nMethods\n\nRandomized soil sampling (approx. 60kg; 15 cores from control parcel) was done at the Pallag Experimental Station of Horticulture at the University of Debrecen, Hungary on May 20, 2020, according to Hungarian national standard MSZ 08020213 from the -20 cm of topsoil using a vane. Next, samples were transferred for measurements to a greenhouse located at Department of Agriculture, at the University of Debrecen. To measure chemical parameters, samples were sieved through a 2mm mesh.\n\nWe determined soil moisture content gravimetrically, according to Klimes-Szmik14, drying the soil samples at 105°C for 24 hours and weighing the mass loss. To evaluate texture, Arany-type plasticity index was measured, using the methodology recommended by Hungarian national standard MSZ-08 0206/1-7815; briefly from a burette distilled water is added to 100g sample until soil reaches the upper limit of its water holding capacity. Soil pH was determined in 1 mol L-1 KCl solution (soil/water = 1.0/2.5 wt./wt.), according to Buzás16 using a glass electrode (Model Seven2Go Advanced Single-Channel Portable pH Meter, Mettler, Toledo. We quantified humus content with potassium dichromate, according to Székely17: to calculate humus% we first determined organic-C%. The latter was measured following Hungarian standard MSZ-08 0210-7718; briefly, 10 mL K2SO4 was added to 1.0 g of soil sample in a 300 mL Erlenmeyer flask, then 0.10 g Ag2SO4 aqueous solution was added, boiled for 5 minutes, cooled to room temperature, and titrated with 0.2 N Mohr's salt solution. Ferroin was used was the indicator, given that its color change is reversible, pronounced, and fast. We calculated C% according to Equation (E1):\n\n\n\nwhere a is the difference between blank and soil sample titration (mL), multiplied by the Mohr-salt factor and b represents the weight of the soil sample (g).\n\nNext, humus% was calculated according to Equation (E2):\n\n\n\nAll measurements of soluble N forms in soil were conducted according to Hungarian standard MSZ 20135:199919; briefly, to determine KCl-NO3 (mg/kg) content, the sample was prepared with 1 mol L-1 KCl of soil solution (74.5g KCl were added to 1 L distilled water20. Spectra were recorded with a Model PU 8610 UV/VIS kinetics spectrophotometer by Pye Unicam (Cambridge, GB). For assessing total soluble nitrogen (UV degraded) content, soil samples were extracted with 0.01 mol L-1 CaCl2 solution. Soil samples were mixed with sodium tetraborate buffer, then oxidized with excess K2S2O8 and passed into an ultraviolet digester. Nitrate was reduced on a cadmium-copper column and then converted to a colored azo compound via the Griess-Ilosvay reaction; dissolving 0.5 g of sulfanilic acid and 0.05 g of 1-naphthylamine in 150 mL of dilute acetic acid. A colored solution was obtained, which was analyzed by visible photometry at 540 nm (SKALAR photometry (San Plus Analyser, S.F.A.S).\n\nWe determined CaCl2-ammonium-N (mg/kg) using a modified Berthelot reaction-based method in which ammonia was initially converted to monochloramine and then to 5-aminosalicylate, according to Buzás16 using 1% EDTA solution and 1 ml 0.06 N NaOH. After oxidation, a green color complex was obtained, with a maximum light absorption at 660 nm. Nitrate-N was determined as described above for the measurement of N content (after reduction, conversion to azo dye, and photometric measurement at 540 nm). All soluble forms were detected by SKALAR photometry (San Plus Analyser, S.F.A.S) in a segmented continuous flow (SCF) system. Finally, we calculated nitrite-N (mg/kg) using Equation (E3), proposed by Buzás16:\n\n\n\nTo better understand the correlation between humus% (Y) and N-forms (X), we carried out two different tests (i) the Pearson test to establish the relation between the variance (Y) and covariance (XY); and (ii) the Kendall test to verify whether the two variables may be regarded as statistically dependent. All statistical analysis was performed with R Statistical Software 3.5.1 (Foundation for Statistical Computing, Vienna, Austria).\n\n\nResults\n\nSoil pH was slightly acidic: pH (KCl) 5.5. As KA = 30, according to Arany the soil is considered humus sand17. For these samples, 1.4 % humus content was found, which represents a reasonable value for a sand soil17. Nitrate content was determined using two methods: KCl-NO3 (12.66 mg/kg) and CaCl2-NO3 (13.53 mg/kg), and the average value of nitrate content (12.48 mg/kg) served for the calculation of percentages in Figure 1.\n\nAverage ammonium-N was 6.8 mg/kg, which represents 24.7% of the total-N content. In addition, 3.8 mg/kg of nitrite-N (pollutant nitrogen form) was assessed and found to be one order of magnitude less than the nitrate-N content, as expected for well-ventilated sand soil. Organic-N of 4.4 mg/kg contributed by 16.1% to the total nitrogen content of soil (Table 1).\n\nConsidering all forms of N under study, the results of correlation analysis evidenced that only the ammonium-N form was in strong correlation with humus%, with r ≈ 0.97 and T ≈ 0.99 (Figure 2).\n\nThese promising initial results call for additional experiments to confirm the strong correlation between these two variables. Total-N also showed a good correlation with humus%; however, it was not as strong as that found between humus% and ammonium-N.\n\n\nConclusions\n\nOur results reveal that the ratio of nitrate-N and nitrite-N (which is determined mostly by soil oxidation-reduction conditions) is optimal in sand soil samples originating from the Pallag Experimental Station of Horticulture at the University of Debrecen, Hungary. Based on the percentage distribution of soluble N forms present at Pallag Experimental Station, authors recommend using this soil in further pot experiments, given the soil’s optimal nutrient supply capacity20.\n\n\nData availability\n\nFigshare: Supporting data - N forms and Humus% in Sand soil, Nyírség. https://doi.org/10.6084/m9.figshare.12581303.v321\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nRobertson GP, Groffman PM: 13 - NITROGEN TRANSFORMATIONS (E. A. B. T.-S. M. PAUL Ecology and Biochemistry (Third Edition) (ed.);2007; 341–364. Publisher Full Text\n\nNascente AS, Kluthcouski J, Crusciol CAC, et al.: Fertilization of common bean cultivars in tropical lowlands. Pesquisa Agropecuária Tropical. 2012; 42(4): 407–415. Publisher Full Text\n\nNascente AS, Carvalho, MDS, Melo LC, et al.: Nitrogen management effects on soil mineral nitrogen, plant nutrition and yield of super early cycle common bean genotypes. Acta Scientiarum Agronomy. 2017; 39(3): 369–378. Publisher Full Text\n\nBloom AJ, Jackson LE, Smart DR: Root growth as a function of ammonium and nitrate in the root zone. Plant Cell Environ. 1993; 16(2): 199–206. Publisher Full Text\n\nCrawford NM, Glass ADM: Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci. 1998; 3(10): 389–395. Publisher Full Text\n\nSchimel JP, Bennett J: Nitrogen mineralization: challenges of a changing paradigm. Ecology. 2004; 85(3): 591–602. Publisher Full Text\n\nJakab A: The ammonium lactate soluble potassium and phosphorus content of the soils of north-east Hungary region: a quantifying study. DRC Sustainable Future. 2020; 1: 7–13. Publisher Full Text\n\nPettit RE: Organic Matter, Humus, Humate, Humic Acid, Fulvic Acid and Humin: their Importance in Soil Fertility and Plant Health.2008; 17. Corpus ID: 15995040 (Accessed: July 1, 2020.). Reference Source\n\nWinkelbauer J, Völkel J, Leopold M, et al.: Methods of surveying the thickness of humous horizons using ground penetrating radar (GPR): an example from the Garmisch-Partenkirchen area of the Northern Alps. Eur J For Res. 2011; 130(5): 799–812. Publisher Full Text\n\nHoffmann S, Csitári G, Hegedüs L: The humus content and soil biological properties as a function of organic and mineral fertilization. Arch. Agron. Soil Sci. 2002; 48(2): 141–146. Publisher Full Text\n\nHouba YJG, Novozamsky I, Temminghoff E: Soil anlysis procedures. Extraction with 0.01 M CaCl2 syllabus. Dept. of Soil Sci. and Plant Nutrition Wageningen Agricultural University-The Netherlands, 1994.\n\nJászberényi I, Loch J, Sarkadi J: Experiences of 0.01 M calcium chloride extraction as soil testing procedure in Hungary Commun. Soil Sci Plant Anal. 1994; 25(9–10): 1771–1777. Publisher Full Text\n\nStandard: MSZ 080202: Hungarian Standards Institution. Budapest, Hungary. 2002.\n\nKlimes-Szmik A: A talajok fizikai tulajdonságainak vizsgálata.Talaj és trágyvizsgálati módszerek.1970; (48): 83–161.\n\nStandard: MSZ-08 0206/1-78: Hungarian Standards Institution. Budapest, Hungary. 1978.\n\nBuzás I: A talajok fizikai-kémiai és kémiai vizsgálati módszerei (Physico-chemical and chemical analysis methods of soils). Mezőgazdasági Kiadó, Budapest.1988. Reference Source\n\nSzékely Á: Talaj-és agrokémiai vizsgálati módszerek 2. Mezogazdasági kiadó, 1988; 115. Reference Source\n\nStandard: MSZ-08-0210-77: Determination of soil organic carbon. In orginal language: “A talaj szerves szén tartalmának meghatározása.” Magyar Szabványügyi Hivatal, Budapest MSZH- Nyomda, 1977; 6.\n\nStandard: MSZ 20135: Hungarian Standards Institution. Budapest, Hungary Magyar Szabványügyi Hivatal, Budapest MSZH- Nyomda.1999.\n\nBerényi S, Bertáné Szabó E, Pepó P, et al.: Effect of fertilization and irrigation on N fractions determined in 0.01 M calcium chloride on lowland pseudomyceliar chernozem. Agrokémia és Talajtan. 2009 ; 58(2): 251–264 . Publisher Full Text\n\nDama Research Center limited: Supporting data - N forms and Humus% in Sand soil, Nyírség. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12581303.v3" }	[ { "id": "69180", "date": "24 Aug 2020", "name": "Katalin Juhos", "expertise": [ "Reviewer Expertise soil quality", "nutrient management" ], "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 manuscript is not entirely clear:\nExactly what land-use type the samples come from (what do control and greenhouse mean? I don't see the comparison in the results section.)\n\nWhat do you mean by \"pollutant nitrogen\" (= nitrite?)?\n\nOn the basis of the soluble N-forms measured once in the upper 20 cm layer, how can it be concluded that the test soil is suitable for use? Soluble N-forms change very rapidly in soil.\nN-test methods are up-to-date, but it is not clear what soil-liquid ratio was used for CaCl2 and KCl extracts? Nor is it clear how many soil samples were tested?\n\nThe detailed determination of soluble N-forms is very important. It would have been interesting to see this in the deeper layers of the soil as well. Furthermore, based on the total (Kjeldahl) N content of the soil, a picture of the source of soluble components could be obtained. Did the total soluble N content not correlate with SOM?\n\"Soil organic matter (SOM)\" is proposed instead of \"Humus\" and \"soil organic carbon (SOC)\" is proposed instead of \"C%\".\nIn the description of the SOM method, K2SO4 is erroneously used instead of K-dichromate. When giving the averages, the number of items and the standard deviation must also be included (Table 1).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "70069", "date": "18 Sep 2020", "name": "Yuhua Kong", "expertise": [ "Reviewer Expertise Carbon and nitrogen cycles in terrestrial ecosystems" ], "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 and significance of the study are not very clear and lack of novelty.\n\nThe basic information of sampling site is not mentioned, like land-use type, the mean annual precipitation and air temperature, etc.\n\nHow many replicates were carried out in the study? In table 1, the standard deviation should be included.\n\nIt is suggested that the Pearson correlation analysis between soil humus and all nitrogen forms be shown in the results part.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "71958", "date": "29 Sep 2020", "name": "Paul S. Agachi", "expertise": [ "Reviewer Expertise Chemical engineering", "clean coal technology", "process control", "modeling", "optimization." ], "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 deals with the assessment of the appropriation of the sandy Pallag soil for raising crops, due to the humus and Nitrogen content. The ratio of nitrate-N and nitrite-N recommends the soil for crops.\n\nThe importance of this study is given mainly by the fact it is done on experimental plots, in real conditions.\nMy objections are related to the transparency of the Equations 1 and 2. Additional explanations should be added. Why the authors calculated this way the organic and the humus %? The values of the constants should be explained. Maybe they are known for the category of the soil scientists, but for larger dissemination and understanding, additional explanations are needed.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-781
https://f1000research.com/articles/9-674/v1	03 Jul 20	{ "type": "Research Article", "title": "In vitro efficacy of a copper iodine complex PPE disinfectant for SARS-CoV-2 inactivation", "authors": [ "Emily Mantlo", "Tanya Rhodes", "Jenny Boutros", "Laura Patterson-Fortin", "Alex Evans", "Slobodan Paessler", "Emily Mantlo", "Tanya Rhodes", "Jenny Boutros", "Laura Patterson-Fortin", "Alex Evans" ], "abstract": "Background: The ability to protect workers and healthcare professionals from infection by SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is of great concern. Hospitals, nursing homes and employers are adopting infection control strategies based on guidance from leading public health organizations such as the CDC, OSHA, FDA, and other government bodies. Certain hard surface disinfectants are effective against SARS-CoV-2 but are not suitable for use on skin or personal protective equipment (PPE) that comes into contact with skin. Furthermore, near-ubiquitous alcohol-based hand sanitizers are acceptable for use on skin, but they are not suitable for use on PPE. PPE, especially masks, are also commonly being used for longer durations than normal. There is a need for new products and techniques that can effectively disinfect PPE during wear time without having detrimental effects on surrounding skin. Clyraguard spray is a novel copper iodine complex designed to be used on non-critical PPE. Methods: In this study, the Clyraguard copper iodine complex was tested for its ability to inactivate SARS-CoV-2 in solution. Results: These data indicate the product to be effective in reducing SARS-CoV-2 titers in a time-dependent manner, with the virus being reduced below the detection limits within 30 minutes. Conclusions: These results suggest that Clyraguard may be an effective tool for mitigating cross-contamination of non-critical PPE that may come into contact with SARS-CoV-2.", "keywords": [ "COVID", "PPE", "iodine", "disinfection", "decontamination", "virus" ], "content": "Introduction\n\nIn late 2019, the world saw the spread of a novel coronavirus (SARS-CoV-2) that caused a global pandemic (ongoing as of this writing) that has claimed the lives of more than 260,000 people and infected 3.8 million as of May 2020 (Johns Hopkins Coronavirus Resource Center, 2020). The spread of this virus has led governments worldwide to implement unprecedented and far-reaching public health guidelines and lockdowns in an effort to flatten the infection curve and reduce the burden on their respective healthcare systems. Healthcare workers currently have the highest risk of exposure and can easily become vectors of viral transmission. On April 16th 2020, the United Kingdom cabinet reported that 16.2% of positive cases were critical workers in NHS (Centre for Evidence-Based Medicine, 2020), and the United States-based CDC reported that up to 11% of positive cases were healthcare workers in some states (CDC, 2020a).\n\nSymptomatic COVID-19 patients display symptoms including a dry cough, fever, shortness of breath and sore throat (CDC, 2020b), and in severe cases can experience severe pneumonia, pulmonary edema, organ failure, and death (Chen et al., 2020). The virus incubation period varies from 3 to 20 days, during which time a patient can be infectious while asymptomatic, as some evidence suggests (Bai et al., 2020). While all modes of transmission for SARS-CoV-2 are not completely understood, it is generally believed that the primary mechanism of transmission is through micron-sized droplets and aerosols produced when an infected person, sneezes, coughs, talks or breathes (WHO, 2020a). Accordingly, public health organizations around the world are urging citizens to practice physical distancing (i.e., “social distancing”), use facemasks when in public, frequently wash their hands, and disinfect commonly touched areas in their surroundings (CDC, 2020c; WHO, 2020b).\n\nIn most countries, many front-line healthcare professionals are required to wear approved N95 masks as well as face shields to protect themselves against infectious droplets and cross-contamination (CDC, 2020d). However, due to shortages of N95 masks and other protective PPE, health organizations including the CDC are now recommending extended use and reuse of PPE including N95 masks. These new recommended practices may put healthcare workers at additional risk if the PPE cannot be effectively disinfected between uses. Therefore, effective disinfection of protective masks and other PPE before reuse is critical (CDC, 2020e). One recent study (Fischer et al., 2020) validated several CDC-recommended PPE decontamination techniques by demonstrating effective disinfection of N95 masks using UV-radiation, dry heat, and hydrogen peroxide vapor application. However, authors showed that while ethanol was effective in disinfecting N95 masks, it also led to reduced filtration capacity in the mask. Another study suggested that ethanol- and chlorine-based disinfectants are not suitable for decontamination due to their capacity to adsorb on fabric material and interfere with their electrostatic properties, thereby reducing filtration capacity (Liao et al., 2020). In keeping with these findings, bleach, sanitizing wipes, and ethyl oxides are not recommended for use to decontaminate masks (SAGES, 2020). Recommended PPE decontamination methods (UV, dry heat, hydrogen peroxide vapors) cannot, however, be easily applied during times of extended daily use of PPE, warranting the need for new or additional disinfection methods that can be applied “on the go” in these settings without causing harm to adjacent skin.\n\nIodine-containing solutions such as povidone iodine (PVP-I) have been proven effective as surface disinfectants against a wide variety of viruses including influenza A, poliovirus, adenovirus type 3, mumps, SARS, MERS, and HIV (Eggers et al., 2015; Kawana et al., 1997; Wada et al., 2016), with some indication of greater viricidal spectrum of activity compared to other commercially available disinfectants. Furthermore, iodine-based formulations have also been shown to be an effective preventative method to reduce upper respiratory tract infections and oral tract pathogens (Eggers et al., 2018; Sakai et al., 2008).\n\nClyraguard copper iodine complex, developed by Clyra Medical Technologies, Inc., is a novel FDA-registered product intended to be used for decontaminating non-critical PPE. The formula has proven antimicrobial activity (FDA submission data, see here) and is cleared for use on skin and wounds. In contrast, other iodine-based products, such as Lugol’s Iodine and PVP-I, may cause staining and skin sensitivity.\n\nIn this study, Clyraguard copper iodine complex was assessed for its efficacy in inactivating SARS-CoV-2 using a Vero cell monolayer infection model to provide a basis for whether or not Clyraguard may represent a potentially effective tool to disinfect and prolong the useful protective life of PPE, as well as to provide additional antiviral protection to PPE such as masks.\n\n\nMethods\n\nVero cells were obtained from ATCC and grown in DMEM (Corning) containing 1x L-glutamine and 1% MEM vitamins, and supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37°C in 5% CO2. Cells were used at 85–95% confluent growth.\n\nThe SARS-CoV-2 (USA-WA1/2020) virus was obtained from The World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), University of Texas Medical Branch, Galveston, TX. All experiments involving infectious virus were conducted by S.P.’s laboratory at the University of Texas Medical Branch (Galveston, TX) in approved biosafety level 3 laboratories in accordance with institutional health and safety guidelines and federal regulations.\n\nFrom the original stock, SARS-CoV-2 was propagated for one passage in infection medium (DMEM containing 1x L-Glutamine and 1% MEM vitamins, supplemented with 1% penicillin/streptomycin (Gibco) and 2% FBS) at 37°C in 5% CO2. Briefly, SARS-CoV-2 was added at a MOI of 0.01 to Vero cells and incubated 1 hour at 37°C. Viral inoculum was removed, and fresh infection medium added. Cells were incubated for an additional 48 hours before supernatant was collected. Supernatant was centrifuged for 5 minutes at 3000 rpm to remove cell debris. Virus stocks were stored at -80°C at a concentration of 1x106 median tissue culture infectious dose (TCID50) per mL.\n\nViral titers were measured by TCID50 on Vero Cells in 96-well plates. Each log10 dilution (10-1 through 10-6) of the virus was inoculated in quadruplicates. On day 4 post-infection, the cells were fixed with 10% formalin for 45 minutes and subsequently stained with crystal violet. Cleared wells were quantified to calculate titer.\n\nAliquots of stock virus (10 μl) were mixed with 90 μl of Clyra, diluted Clyra, or control solutions. Clyra was mixed either 1:10 or 1:100 with sterile saline for dilution. Room temperature water was used as a negative control for virus inactivation, while boiling water (water pre-heated to 100°C) was used as a positive control. All mixtures were incubated for 30 seconds, 10 minutes, 30 minutes, or 60 minutes at room temperature, at which time 900 µl infection medium was added to neutralize antiviral activity. Subsequently, the SARS-CoV-2 viral titer (TCID50/mL) for each test substance was determined. The experiment was conducted in triplicate.\n\nStatistical significance was determined using Student’s t-test, conducted using GraphPad Prism (software version 8.3.0). A p-value of 0.05 or below was considered statistically significant.\n\n\nResults\n\nIn this study, the ability of Clyraguard to inactivate SARS-CoV-2 at various timepoints and at various concentrations was assessed (Figure 1; Table 1). While diluted Clyraguard was unable to inactivate SARS-CoV-2 after any length of time, undiluted Clyraguard was effective at significantly reducing viral titers after just 10 minutes of incubation. After incubation with undiluted Clyraguard for 10 minutes, viral titers dropped by 2 logs (one-tailed t-test p-value = 0.0140). Furthermore, after incubation with undiluted Clyraguard for either 30 minutes or 60 minutes, viral titers dropped below the limit of detection (<75 TCID50 per ml).\n\nOne part SARS-CoV-2 by volume was added to nine parts Clyraguard, diluted Clyraguard, or control saline solutions. Solutions were incubated for the indicated times before titration via TCID50.\n\n\nDiscussion\n\nIn this study, Clyraguard copper iodine complex was assessed for its ability to inactivate SARS-CoV-2. These data demonstrate that the product has significant viricidal activity against SARS-CoV-2 within 10 minutes and yields complete SARS-CoV-2 deactivation by 30 minutes.\n\nAntiviral activity against human CoV viruses including SARS-CoV and MERS-CoV has been previously been demonstrated using iodine (Eggers et al., 2015; Eggers et al., 2018). The mechanism of action is not widely understood but researchers have hypothesized that iodine attacks viruses in multiple ways, attacking the protein structure and interfering with hydrogen bonding associated with cysteine, histidine, and tyrosine, thus altering virus membrane structure and causing inhibition of viral release and spread from infected cells (Qin, 2009).\n\nIodine-based materials that have shown effective antiviral activity such as PVP-I and Lugol’s solution also exhibit toxicity concerns and can only be tolerated on skin for short periods of time. The copper iodine complex investigated in this study has also been tested according to ISO 10993 standards (FDA submission data, see here) and found to be safe for skin and wounds, suggesting that it may represent a potential alternative to current iodine-based regimens.\n\nIn addition, long-term antimicrobial performance studies with organisms including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Psuedomonas aeruginosa, Enterobacter aerogenes, Bacillus fragilis (FDA submission data, see here) have been conducted with the Clyraguard formula, wherein it was found to be efficacious for up to 72 hours. It is therefore recommended that further studies be carried out under good laboratory practices to directly determine any extended activity against the SARS-CoV-2 virus. This would verify the potential extended use for PPE against SARS-CoV-2, potentially providing additional protection for healthcare professionals working during the COVID-19 pandemic.\n\nThis study demonstrates clear evidence to the potential suitability of the copper iodine complex (Clyraguard) for decontaminating non-critical PPE to help mitigate cross-contamination of SARS-CoV-2.\n\n\nData availability\n\nFigshare: In vitro efficacy of a copper iodine complex PPE disinfectant for SARS-CoV-2 inactivation - Raw Data in CSV. https://doi.org/10.6084/m9.figshare.12493682.v1 (Mantlo & Paessler, 2020).\n\nThis project contains the raw viral titers for each repeat produced in this experiment.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe thank Drs. Kenneth Plante (The World Reference Center for Emerging Viruses and Arboviruses, UTMB) and Natalie Thornburg from the CDC for providing the SARS-CoV-2 stock virus. E.K.M was supported by NIH T32 training grant AI060549.\n\n\nReferences\n\nBai Y, Yao L, Wei T, et al.: Presumed Asymptomatic Carrier Transmission of COVID-19. JAMA. 2020; 323(14): 1406–1407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCDC COVID-19 Response Team: Characteristics of Health Care Personnel with COVID-19 — United States, February 12–April 9, 2020a. MMWR Morb Mortal Wkly Rep. 2020a; 69(15): 477–481. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention: Symptoms of Coronavirus. Accessed 7 May, 2020b. Reference Source\n\nCenters for Disease Control and Prevention: How to Protect Yourself & Others. Accessed 7 May, 2020c. Reference Source\n\nCenters for Disease Control and Prevention: Strategies to Optimize the Supply of PPE and Equipment. Accessed 7 May, 2020d. Reference Source\n\nCenters for Disease Control and Prevention: Decontamination and Reuse of Filtering Facepiece Respirators. Access 7 May, 2020e. Reference Source\n\nChen N, Zhou M, Dong X, et al.: Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020; 395(10223): 507–513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEggers M, Eickmann M, Zorn J: Rapid and Effective Virucidal Activity of Povidone-Iodine Products Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Modified Vaccinia Virus Ankara (MVA). Infect Dis Ther. 2015; 4(4): 491–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEggers M, Koburger-Janssen T, Eickmann M: In Vitro Bactericidal and Virucidal Efficacy of Povidone-Iodine Gargle/Mouthwash Against Respiratory and Oral Tract Pathogens. Infect Dis Ther. 2018; 7(2): 249–259. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFischer RJ, Morris DH, van Doremalen N, et al.: Assessment of N95 respirator decontamination and re-use for SARS-CoV-2. MedRxiv. preprint, 2020. Publisher Full Text\n\nJohns Hopkins University: COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE). Accessed 7 May, 2020. Reference Source\n\nKawana R, Kitamura T, Nakagomi O, et al.: Inactivation of Human Viruses by Povidone-Iodine in Comparison with Other Antiseptics. Dermatology. 1997; 195(Suppl 2): 29–35. PubMed Abstract \| Publisher Full Text\n\nLiao L, Xiao W, Zhao M, et al.: Can N95 Respirators be Reused After Disinfection? How Many Times? ACS Nano. 2020; 14(5): 6348–6356. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMantlo EK, Paessler S: In vitro efficacy of a copper iodine complex PPE disinfectant for SARS-CoV-2 inactivation - Raw Data in CSV. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12493682.v1\n\nQin Y: Antimicrobial textile dressings in managing wound infection. In Advanced Textiles for Wound Care. edited by S. Rajendran (Woodhead Publishing Series in Textiles) 2009; 179–197. Publisher Full Text\n\nSakai M, Shimbo T, Omata K, et al.: Cost-effectiveness of gargling for the prevention of upper respiratory tract infections. BMC Health Serv Res. 2008; 8: 258. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSociety of American Gastrointestinal and Endoscopic Surgeons: N95 Mask Re-use Strategies. Accessed 7 May, 2020. Reference Source\n\nThe Centre for Evidence-Based Medicine: COVID-19 How many Healthcare workers are infected?. Accessed 7 May, 2020. Reference Source\n\nWada H, Nojima Y, Ogawa S, et al.: Relationship between Viricidal Efficacy and Free Iodine Concentration of Povidone-Iodine in Buffer Solution. Biocontrol Sci. 2016; 21(1): 21–27. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations. Accessed 7 May, 2020a. Reference Source\n\nWorld Health Organization: Coronavirus disease (COVID-19) advice for the public. Accessed 7 May, 2020b. Reference Source" }	[ { "id": "66377", "date": "06 Jul 2020", "name": "Ilya Frolov", "expertise": [ "Reviewer Expertise Virology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents new data, which are important to the field and public health. However, to make the results more reproducible by other groups, the authors need to provide the source of Vero cell and catalog number, number of cells seeded into the wells of 96-well plates and to clarify \"infection medium.\"\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? Partly\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? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "68324", "date": "18 Aug 2020", "name": "Paul Dabisch", "expertise": [ "Reviewer Expertise My research focuses on the examination of factors influencing transmission of infectious diseases", "including the influence of environmental conditions on the persistence of infectious material on surfaces and in aerosols." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study describes novel data on the efficacy of a copper iodine complex for inactivation of SARS-CoV-2 in liquid suspension. The results demonstrate that the undiluted copper iodine complex causes a significant decrease in viral infectivity in a cell-based microtitration assay after 10 minutes, with greater than a 5-log reduction in 30 minutes. The methodologies and conclusions are generally appropriate. However, there are several limitations to the study:\nThe study only investigates the ability of the copper iodine complex to inactivate the virus in a liquid suspension. Thus, while the present study provides useful preliminary data, additional testing with virus deposited as droplets or aerosol particles on relevant surfaces needs to be conducted.\n\nAs a justification for the use of copper iodine complex, the authors discuss the incompatibility of certain disinfectants for N95 masks, as well as the difficulty of using other decontamination methods while PPE is being worn. However, while the authors cite some evidence that copper iodine complex is safe on skin, the present study does not perform testing to demonstrate the benefit of copper iodine complex in this regard, specifically its compatibility with N95 masks or ability to be used while PPE is being worn.\n\nThe manuscript would benefit from additional discussion/comparison of the results obtained in the study to those published for other decontamination methods for SARS-CoV-2, SARS-CoV-1, and MERS.\n\nThe methods state that a t-test was used for statistical comparisons. However, it is unclear from the text what comparisons were made. In looking at the data, it appears that either a one- or two-way ANOVA with a multiple comparisons post-test would be more appropriate.\n\nRegarding data presentation, while Table 1 provides mean values, the standard deviation would also be helpful.\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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5948", "date": "17 Sep 2020", "name": "Alex Evans", "role": "Author Response", "response": "Thank you for your review. Your comments have provided valuable insights, and we are presently working to edit the paper to address each of the issues raised in your comments. We hope to re-submit this new version in the coming weeks." } ] } ]	1	https://f1000research.com/articles/9-674
https://f1000research.com/articles/9-201/v1	23 Mar 20	{ "type": "Study Protocol", "title": "Controlled administration of cannabis to mitigate cannabis-attributable harm among recreational users: a quasi-experimental study in Germany", "authors": [ "Jakob Manthey", "Jens Kalke", "Jürgen Rehm", "Moritz Rosenkranz", "Uwe Verthein", "Jens Kalke", "Jürgen Rehm", "Moritz Rosenkranz", "Uwe Verthein" ], "abstract": "Background: New approaches are required to slow down or reverse increasing trends of levels of delta-9-tetrahydrocannabinol (THC) and cannabis-attributable hospitalizations in Germany. Legal access to cannabis may constitute one viable effective policy response; however, available evidence does not suffice to inform a regulation model for Germany. The proposed study aims to reduce harm for cannabis users through legal access to herbal cannabis through pharmacies. Protocol: A quasi-experimental study comparing cannabis users with legal access to herbal cannabis (Berlin, intervention group) to those without legal access (Hamburg, control group) (total N=698). As the primary outcome, we hypothesize that: 1) illegal THC consumption will reduce by at least 50% in the intervention group and 2) total THC exposure in the intervention group will be reduced by at least 10% lower than that of the control group, taking into account baseline values. Secondary outcomes comprise measures of frequency of use, THC-impaired driving, and mode of administration. Paired t-tests and multilevel regression models will be performed for statistical analyses. Discussion: This study proposal is currently being reviewed by the ‘Federal Institute for Drugs and Medical Devices’ – the body responsible for approving research studies on classified substances, including cannabis. Upon approval and prior to the start of the study, a full ethical review will be undertaken. Results may inform a regulation model for Germany and other jurisdictions and are expected to deepen the understanding of the effects of legal access to cannabis. Pre-registration: German Clinical Trials Register (DRKS), DRKS00020829", "keywords": [ "Cannabis", "Marihuana", "THC", "legal", "prohibition", "Germany", "model study", "pharmacy" ], "content": "Introduction\n\nCannabis remains the most prevalent illegal drug in Europe and other regions worldwide1. In the EU, 14.4% of people aged 15–34 years indicated past-year use of cannabis2. In the past decade, several trends adverse to public health have been observed, including rising prevalence of cannabis use, growing treatment demand for cannabis problems3, and increases in potency levels in both herbal cannabis and resin4. These trends are also mirrored in Germany, where, since 2006, prevalence of past-year use increased by 75%5, treatment demand increased by 113%, and potency of resin increased by 119% (data for herbal cannabis not available6). Moreover, the number of cannabis-related offences has risen by 38% in the same period7.\n\nTo date, effective policy responses to the above-outlined trends could not be observed. In fact, these trends demonstrate that current cannabis policies in Germany have not resulted in reductions in drug demand. Furthermore, the possibilities for reducing or slowing down increases in potency are very limited in an illegal or unregulated drug market. However, potency levels constitute a crucial determinant for public health as the main ingredient, delta-9-tetrahydrocannabinol (THC), has been linked to severity of cannabis dependence8, cognitive impairment9, and incidence of psychotic disorders10. Consequently, THC concentrations in legally available products are deemed to be increasingly important by policymakers11,12. With the legalization of recreational cannabis in Canada and several US American states, it has become mandatory to label all commercially produced cannabis products with their respective THC level (for Canada, see 13,14).\n\nIn addition to the lack of control over potency levels, illegal markets also make it impossible to establish minimum criteria for safety and purity of available products. Analyses of herbal cannabis acquired on the Swiss black market showed that only one in three samples passed the microbiological test for human consumption15. According to one systematic review, microbes, heavy metals, and pesticides constitute the most prevalent contaminants in cannabis products16. While the risks from contaminants to human health have not yet been quantified, case reports highlight that the use of contaminated products may be potentially life-threatening17.\n\nConsequently, legalizing the cannabis market would allow for the control over the rising THC exposure and cannabis-attributable sequelae, and to ensure safety standards of available products are being met. However, there is a multitude of options to realize legal access to cannabis. One option is the creation of so-called ‘cannabis social clubs’, which are established by consumers with the aim of growing and distributing herbal cannabis on a non-for profit basis (e.g., in Uruguay, Spain, Belgium18,19). More prominently, legal markets in North America have been established by private retailers or government monopolies, while some jurisdictions allow users to grow their own cannabis themselves.\n\nA combination of different modes of access to legal cannabis can be observed in Uruguay, where users may grow their own cannabis, acquire cannabis through membership in a social club or through licensed pharmacies, which get a limited supply of herbal cannabis from government-licensed suppliers20,21. In fact, a pilot project was proposed in 1997 for a regulated sale of cannabis in pharmacies in a German state22. While this proposal was never implemented due to lack of political support, the ‘pharmacy model’ has several advantages over other private models as pharmacists are trained to test substances with regard to purity and are also more familiar with recognizing substance misuse than are commercial vendors, especially those who are selling product over the Internet. Further, pharmacists are already familiar with dispensing medical marijuana in Germany, and already meet the requirements for dealing with classified substances, such as cannabis.\n\nTo inform a cannabis regulation model for Germany, evidence gathered from evaluations of jurisdictions legalizing cannabis in the Americas may serve as a useful base. However, North American cannabis users differ from European users in regard to use modes (e.g. lower co-use of tobacco23, higher use of concentrates24,25) and patterns (e.g., the stark increase of daily use in the USA26), demanding different requirements for a regulation model in Germany. Further, evidence collected from large-scale natural experiments has its limitations, which may be overcome in small-scale, controlled experiments27. Thus, evaluating tightly regulated cannabis administration models in European jurisdictions is required.\n\nIn this study protocol, we outline the proposal for the regulated sale of cannabis to a limited number of users in Berlin, Germany. Responding to calls to make cannabis safer28, this study sets out to reduce harm to recreational users through: a) use of legal cannabis products free from contaminants and pollutants, and b) capping maximum THC-levels and incentivizing the purchase of low-potency cannabis products by aligning retail prices with potency levels and thereby reducing THC exposure among users.\n\n\nProtocol\n\nVersion: 1 (26 February 2020).\n\nThis protocol has been written in accordance with the SPIRIT guidelines61.\n\nThe aims of the study will be evaluated using a quasi-experimental two-group study design, as summarized in Figure 1. The intervention will constitute an individualized licence to purchase herbal cannabis in selected pharmacies for a duration of 12 months in Berlin, Germany. Control group participants will be recruited in Hamburg, a city comparable to Berlin in terms of sociodemographic indicators (population, deprivation) and user characteristics29.\n\nPrior to any data collection, study team members will take consent of each participant. A model consent form is available as Extended data60. In both study groups, participants will be asked to complete a questionnaire and provide their usual consumption unit (e.g., joints, mix of tobacco and cannabis to smoke in water pipe, etc.) at both baseline and again after one year (T12). In predetermined intervals, additional questionnaires will be completed by all participants. Further, individual sales data will be collected for intervention group participants, which will be part of the primary outcome analyses.\n\nAll participants will be recruited via posters, or through consumer groups and local stakeholders in each city. To incentivize study participation and minimize loss to follow-up, participants will be reimbursed with 25€ upon completion of the questionnaires at each wave. Further, providing details regarding their usual consumption unit will be reimbursed with 15€ (at baseline and at one-year follow-up).\n\nEvery resident in the respective control or intervention municipality aged 18 years or above reporting to have used cannabis at least monthly in the past 6 months may participate in the study. Following international recommendations to minimize cannabis use risks30, persons with a familial predisposition to psychotic or substance use disorders, pregnant women, persons in current psychiatric treatment or with a preceding psychotic disorder will be excluded from this study. Further, persons with a prescription to use cannabis for medical purposes will not be eligible to participate as the study focuses on recreational use only.\n\nThe term dispensary model describes how intervention group participants can acquire cannabis in pharmacies and all measures undertaken to achieve the postulated aim of mitigating use risks. Registered users will be eligible to purchase up to 5 grams of herbal cannabis per transaction and up to 15 grams of herbal cannabis per week. All herbal cannabis will be sold in 1-gram packages, which will be labelled with the concentration of THC and Cannabidiol (CBD,31,32), and contain further information (e.g., on other constituents, producer, date of production, safety warnings).\n\nTwo core measures will deter intervention group participants from using high-potency cannabis varieties. First, we will restrict the sale to varieties containing at most 12% THC, which is just below the median value of 13% identified in herbal cannabis batches seized by the federal police in 201733. Second, the retail price will be bound to the THC level, with lower potencies being more affordable. Specifically, the variety lowest in THC will be sold at the current black-market price, as determined by the federal police. More potent varieties will be sold at higher prices, with the most potent variety to be sold at 50% above the current black-market price. In this way, purchasing varieties with high THC levels will be disincentivized.\n\nTo further mitigate the risks of THC, an upper threshold of the THC/CBD ratio will be established as CBD has been found to attenuate some of the adverse effects of THC (see e.g.9,34). In the absence of any systematic evidence on the specific risk profile of THC/CBD ratios, an upper threshold of 50:1 was defined based on US data, indicating a worrying ratio increase from 23:1 to 104:1 between 2008 and 201725.\n\nAll herbal cannabis to be sold in the pharmacies will be acquired through the medical supply system35. As of 2019, six out of the 20 varieties available for medical use in Germany36 met the outlined criteria (cap of potency and cap of THC/CBD ratio) to be sold to the intervention group participants in this study. As prescribed for medical cannabis, each batch of herbal cannabis will undergo routine pharmaceutical tests. Further, producers of medical marijuana are legally required to perform systematic tests to detect variations in potency levels and contaminants. In accordance with the requirements of the narcotics law, cannabis stored in the pharmacies will not be accessible to anyone but trained staff. All cannabis products will be stored in a steel cabinet protected by a safety lock, in accordance with the standards prescribed by the German ‘Federal Institute for Drugs and Medical Devices’ (Bundesinstitut für Arzneimittel und Medizinprodukte, BfArM).\n\nIn all participating pharmacies, users will find printed information material on mitigating use risks and will be offered the option of renting devices for vaporizing herbal cannabis. The use of vaporizers can reduce exposure to some hazardous compounds and is hence considered a harm-reducing method37. Further, the vending staff in all participating pharmacies will be trained in the following: legal framework, safer use guidelines (original:30, for a German translation:38), identification of vulnerable users, cooperation with drug counselling and prevention services. If required, study participants will be able to hold conversations with staff in private on the pharmacy premises.\n\nTwo important aspects will determine the study’s success and will be combined in the primary outcome, measured at baseline and at 12-month follow-up: 1) dominance of legal over illegal consumption, and 2) reduction of THC exposure. The hypothesis for the primary outcome is:\n\n“As compared to baseline, intervention group participants will reduce their use of illegally acquired THC within the past 30 days by at least 50% at 12-month follow-up. Taking into account baseline exposure levels, the total amount of THC consumed among intervention group participants within the past 30 days will fall below the total amount of THC consumed among control group participants within the past 30 days by at least 10% at 12-month follow-up.”\n\nAs first aspect, we expect that illicit cannabis use will be largely replaced by consumption of legally acquired products. A shift from an illegal to a legal consumption environment implies that exposure to contaminants and pollutants but also legal consequences (revocation of driver’s licence, imprisonment) will be largely diminished or even completely prevented. As an evaluation of changes in these specific outcomes would require very large sample sizes, and go beyond the scope of this study, we will restrict the first part of the primary outcome to a proxy, i.e., to the percentage of legal cannabis consumption. This will be operationalized as the percentage of an individual’s THC exposure within the past 30 days acquired through purchases in pharmacies.\n\nFor the second aspect, we expect to halt the increasing THC exposure among intervention group users. While THC levels in seized and legally available cannabis products are increasing, intervention group participants are expected to reduce their overall THC consumption as compared to control group participants. While THC exposure is the core determinant for cannabis-attributable harm, specific effects on adverse consequences (e.g. on incidence of psychotic disorders) require larger sample sizes which, again, are beyond the scope of this study.\n\nWhile the first aspect will only be evaluated within the intervention group, the second aspect will require between-group comparisons across a 12-month period. Only if both aspects can be positively evaluated, can an overall success of the study be inferred.\n\nFor secondary outcomes, we will adhere to the criteria proposed to evaluate the legalization of cannabis in Canada39, encompassing all cannabis use behaviours linked to adverse consequences, for which substantial evidence exists30. As intervention group participants will receive information on risky cannabis use30 and enclosed within each cannabis package sold by the pharmacies, we hypothesize that risky cannabis use practices will not increase among these users – despite the presumably increased availability of cannabis products. Specifically, we hypothesize that intervention group participants will: A) not increase their use frequency, B) not increase frequency of THC-impaired driving, and C) will not smoke cannabis products more frequently (for operationalization, see Table 1). The secondary outcomes will be evaluated at one-year follow-up, which involves testing for differences between control and intervention group while taking baseline values into account.\n\nAs further secondary outcomes, we will examine acceptance and satisfaction of the dispensary model among intervention group participants, which may be important for explaining a possible dominance of illegal over legal consumption (see first aspect of primary outcome).\n\nFor both aspects of the primary outcome, THC exposure within the past 30 days needs to be determined for each respondent by combining the following data sources. First, THC levels per average use occasion will be determined through analyses of usual consumption samples provided at both baseline and after one year (T12). Second, questionnaire data will be used to determine the number of use occasions within the past 30 days. Third, and only for the intervention group, sales data will be used to determine THC exposure levels of legally acquired cannabis over a 12-month period.\n\nIn summary, for the control group, THC exposure levels within the past 30 days will be determined multiplying THC levels in a usual consumption unit with the number of use occasions reported in the questionnaire. For the intervention group, THC exposure levels will be determined analogously while correcting for the proportion of legally acquired and consumed THC using sales and questionnaire data.\n\nAt all waves — T0, T3, T6, and T12 — a set of questionnaires will be administered (see Table 2). For the primary and secondary outcomes, use characteristics will be assessed using items from the ‘Daily Sessions, Frequency, Age of Onset, and Quantity of Cannabis Use Inventory’ (DFAQ-CU), which will be translated and adapted for this study. All remaining questionnaire data will serve to explain unexpected findings, to control for potential confounders in the statistical analyses, or for the economic evaluation of this study.\n\nAt T18, we will conduct a post-intervention assessment of intervention group participants to examine how their cannabis use has developed after being denied further legal purchases of cannabis products. This assessment will include all instruments outlined in Table 2, in addition to several free-text items.\n\nThere will be three main types of study data: (1) chemical analyses of standard consumption units, (2) sales data, (3) survey responses of participants. For (1), we will adhere to the standard operating procedures issued by the BfArM40. For (3), we will aim to carry out digital survey assessments to minimize human error in data entry. Further, consistency checks will be performed before data analyses.\n\nThe study outcomes will be analysed according to ‘Intention-to-treat’ (ITT) principles. Specifically, the sample to be analysed is defined as all users who have provided a usual consumption unit within 4 weeks after completing the baseline questionnaire. Over-recruitment will compensate for participants failing to provide a consumption unit within this period. According to ITT principles, only those participants who provided their baseline data, including their usual consumption unit, will be included in the analyses.\n\nTo examine between-group differences with a t-test, the required sample size of this study was calculated assuming a power of 80%, a 5% alpha error, and a THC standard deviation equal to one-third of its mean (approximated using Canadian data,41; adding 25% to account for uncertainty). The required sample size was estimated to sum up to n=698 participants (control: n=349; intervention: n=349) for detecting group differences in THC exposure.\n\nNot relevant for sample size considerations, we expect that 20% (control: 30%; intervention: 10%) of all participants of the ITT sample will be lost to T12 follow-up. Subjects dropping out of the study will not be replaced and missing values will be imputed using the ‘Last Observation Carried Forward‘ (LOCF) technique. The critique regarding LOCF imputation methods42 does not apply to our study, as this method will bias the data towards the null hypothesis (assuming no change over time) and therefore representing a conservative imputation approach. The assumptions of LOCF will be examined in sensitivity analyses using advanced multiple imputation techniques.\n\nTo evaluate both aspects of the primary outcome, two analyses need to be conducted. First, the THC exposure levels in the past 30 days ascribed to illegal cannabis acquisition within the intervention group needs to be compared between baseline and intervention, with a 50% reduction denoting the minimum threshold for a positive evaluation. Second, a between-group comparison of THC exposure in the past 30 days at T12 adjusting for baseline data will be conducted using a t-test. The dependent variable will be calculated as follows:\n\n\n\nThe actual between-group comparison will be evaluated against the following condition:\n\n\n\nMultilevel regression analyses will be performed additionally for evaluating the primary outcome, which serve to rule out the impact of possibly confounding variables. For evaluating the secondary outcomes, the proposed analyses (correction for baseline values, comparison via t-test, confirmation via multilevel regression analyses) will be performed analogously.\n\nIf more than 60% of the intervention group participants drop out within the first three months, the study will be stopped immediately, because it will be taken as an indicator for subjects not accepting the administration model.\n\nIn addition, the Data Safety Monitoring Board (DSMB) will be formed by pharmacists and social workers working in the addiction and youth protection field. As stipulated in Good Clinical Practice guidelines, the DSMB, as an independent and multidisciplinary group will be established to review, at intervals, accumulating trial data, in order to monitor the progress of the trial and to make recommendations on whether to continue, modify or stop the trial for safety or ethical reasons.\n\nAdverse events will be closely monitored and documented. Criteria have been pre-specified to define a preterm stop to the study. Further, the study team will be in close contact with the participating pharmacies in order to capture all events not foreseen at study inception. In regular meetings, the DSMB will evaluate the progress of the study and may decide to stop the study.\n\nIn a ‘cost-benefit-analysis’ (CBA), the proposed dispensary model will undergo an additional economic evaluation, in which the economic benefits from attenuated THC exposure will be contrasted to the programme costs50, with estimates applied to Germany as a whole. Building on the approach of a previous CBAs for Australia51 and using the extended framework of generalized cost-effectiveness analyses52, we will compare the following scenarios:\n\nNull scenario: no implementation of cannabis-specific measures (i.e., no police enforcement, no treatment of cannabis use disorders)\n\nStatus quo: implementation of random traffic controls to reduce THC-impaired driving and psychosocial interventions for cannabis use disorders\n\nDispensary model: as ‘status quo’ but with legal sale of herbal cannabis in pharmacies\n\nFor the CBA, the so-called net social benefit will be calculated as the difference between projected costs and benefits discounted over the study period. On the cost side, we will consider all economic costs that can be ascribed to cannabis-related law enforcement, treatment of cannabis use disorders and cannabis-attributable diseases (e.g. psychoses), loss of productivity, and all costs pertaining to establishing and maintaining the dispensary model. On the benefit side, we will consider all economic benefits arising in the following domains: reductions in law enforcement and health-care costs, as well as increases in productivity. Based on the CBA results, a cost-utility-analyses will additionally be conducted by estimating the costs required to avoid one ‘disability adjusted life year’ (DALY53) for the scenario of a nationwide implementation of the dispensary model.\n\nData collection will be completed using electronic means and all required measures will be implemented to protect the data and anonymity of all study participants at all times. In particular, the study will adhere to the European ‘General Data Protection Regulation’ (GDPR) according to which, health care data is particularly sensitive and should be handled with the greatest care. Further, cannabis possession will remain a federal crime, further emphasizing the sensitive nature of the study data. Thus, all data will be stored on hard drives encrypted using state-of-the-art encryption techniques (AES-256). Upon completion of the study, the study data will be kept on encrypted hard drives in physically locked cabinets.\n\nConditioned on the approval from the public study sponsor, all study data shall be made available to other researchers. All efforts will be undertaken to anonymize the study data (in the sense of GDPR) in order to publish the data in public repositories making the data findable, openly accessible, interoperable, and re-usable (FAIR principles issued by the European Union). Upon publication of the primary outcome analyses, study data shall be published alongside the respective code of the statistical programme. Through these means, we hope that our analyses will be reproducible and that the data will be used for other purposes than those described, increasing the merit of this study. Lastly, study results will be published under an open access licence to allow for a widespread recognition of our findings.\n\nThis study has not undergone ethical review yet. A full-length study proposal is currently being evaluated by the BfArM. According to the German narcotics law, studies on cannabis may, by an exception, be allowed for scientific purposes and only if approved by the BfArM. Only once a positive decision is received from the BfArM will a complete study outline be reviewed by the responsible ethics board. The study has been pre-registered with the German Clinical Trials Register (a primary registry within the WHO registry network) and will be formally registered upon ethical approval (registry number: DRKS00020829). If any amendments of the outlined study design or protocol will be requested by the BfArM or ethics board, they will be reflected in this publication, as well.\n\nThere are several limitations of this study. First, given a lack of randomization, causal inferences cannot be drawn from the study findings without controlling for relevant confounders. We have sought to include a broad variety of questionnaires to capture information on all possible determinants; however, we cannot rule out that some important confounding variables will not be assessed or be biased through self-report. Further, resin will not be part of the dispensary model for it is not available from medical suppliers. As far as we know, resin cannot be prescribed for medical purposes despite favourable THC/CBD ratios (for Dutch data, see 54). For cannabis users preferring resin over herbal cannabis, study participation may therefore be unattractive, and this may bias the study sample towards predominant herbal cannabis users.\n\n\nStudy status\n\nThe full-length study proposal was submitted in December 2019 to the BfArM and a response is due in March 2020.\n\n\nConclusion\n\nAfter decades of prohibition and the prospective rescheduling of cannabis in international treaties55, opportunities to reform the regulation of cannabis will continue to emerge in many countries. As with medical marijuana, there is considerable economic pressure for a liberal market of recreational cannabis56, however, public health concerns should be considered in the decisions to change the regulation for the better57. So far, jurisdictions legalizing cannabis for recreational purposes have done so before studying the effects of these policy changes in a closed environment. While these large-scale natural experiments provide valuable insights, small-scale experiments have the advantage of allowing for the study of the effects on an individual level with more control over confounding variables.\n\nThe proposed study covers a comprehensive evaluation of a tightly regulated dispensary model of cannabis for recreational users in Germany. To the knowledge of the authors, it will be the first controlled study to investigate the effects of legal access to cannabis in a spatially and temporarily limited framework. Study findings are expected to shape the discussion on the best regulation model for Germany, for Europe, and globally. The evaluation focusing on the primary psychoactive constituent THC acknowledges the causal pathways of cannabis-attributable harm. Findings are expected to inform policy responses to counteract an increase of THC exposure as observed in many European and North American jurisdictions. For Germany specifically, studies evaluating different modes of access to illicit substances have had considerable impact on legislation in the past58,59. Thus, this study may accelerate the process of legalizing cannabis in Germany and elsewhere. Lastly, results from the economic evaluation will be of interest to policymakers and will serve as essential argument for regulated models of cannabis legalization.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: Model consent form, https://doi.org/10.6084/m9.figshare.11903301.v160\n\nFigshare: SPIRIT checklist, https://doi.org/10.6084/m9.figshare.11903322.v161\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nUnited Nations Office on Drugs and CrimeWorld Drug Report 2018: United Nations publication, 2018. Reference Source\n\nEuropean Monitoring Centre for Drugs and Drug Addiction.European Drug Report 2019: Trends and Developments. Luxembourg: Publications Office of the European Union, 2019. Reference Source\n\nManthey J: Cannabis use in Europe: Current trends and public health concerns. Int J Drug Policy. 2019; 68: 93–96. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "61804", "date": "02 Apr 2020", "name": "Alfred Uhl", "expertise": [ "Reviewer Expertise Research methodology", "epidemiology", "substance use" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 motivation for the treatment group is to get \"legal\" cannabis. This is very different from the control situation, where subjects only are awarded 25 Euro for filling in a questionnaire. The protocol does not explain exactly which control variables are used to compensate for this possible bias.\nOne idea of the study is to sell cannabis with a limited THC/CBD ratio to the experimental group and to see if the total THC consumption can be reduced that way. This requires knowing the amount and purity of illegally consumed cannabis in both groups though. The amount of cannabis can be measured (provided the subjects give correct answers) - but there is no plan to systematically assess the THC content of the illegally acquired cannabis consumed by both groups, as far as the protocol states.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5848", "date": "27 Aug 2020", "name": "Jakob Manthey", "role": "Author Response", "response": "Regarding the different incentives for the two groups of study participants: We acknowledge that differing incentives in the two groups may have an impact on their motivation. However, we belief that the vast majority of study participants agreeing to participate in the study, will do so because of their internal motivation in supporting this research. The 25€ compensation will only serve to ensure high rates of follow-up. For the control group, we decided not to provide any compensation, as this could be perceived as incentive to buy (and use) cannabis legally. Prior to conducting primary and secondary outcome analyses, we will compare user characteristics (socio-economic variables and cannabis use indicators) of both groups and will take them into account in all analyses, if applicable. Regarding the assessment of THC exposure: We have added some details on the assessment of the primary outcome to the text. In both groups, we will collect usual consumption units to determine the THC level per use occasion for baseline and 12 months follow-up. For the intervention group, the THC exposure at follow-up will be determined by triangulating baseline data (i.e. from illegally acquired cannabis), sales data, as well as questionnaire data. This will allow to calculate the proportion of total THC consumption attributable to illegally acquired cannabis and thus lay the foundation for the primary outcome analyses." } ] }, { "id": "67190", "date": "03 Aug 2020", "name": "Josefine Atzendorf", "expertise": [ "Reviewer Expertise lifestyle risk factors", "mental health", "substance use", "epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 protocol describes a very interesting study in order to investigate the effects of legal access to cannabis using a quasi-experimental design. The authors describe the study design, sample, data assessment statistical analyses, data protection issues and the importance of their research object very clearly and detailed.\nMinor remark:\nThe study protocol states: “THC levels per average use occasion will be determined through analyses of usual consumption samples provided at both baseline and after one year (T12). Second, questionnaire data will be used to determine the number of use occasions within the past 30 days. Third, and only for the intervention group, sales data will be used to determine THC exposure levels of legally acquired cannabis over a 12-month period.” (S. 5f). The study protocol does not mention if study participants in the intervention group are being asked about their occasions buying cannabis legally and illegally in the questionnaire. I think it would be interesting to have the number of both occasions since the THC exposure of THC bought illegally might be different.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [ { "c_id": "5849", "date": "27 Aug 2020", "name": "Jakob Manthey", "role": "Author Response", "response": "In order to facilitate understanding of the assessment of the primary outcome (THC exposure), we have added the following half-sentence in the respective section:Second, questionnaire data will be used to determine the number of use occasions within the past 30 days, separately for legally and illegally acquired cannabis.THC exposure will thus be quantified for illegally and legally acquired cannabis for the intervention group as this is a prerequisite for analysing the primary outcome." } ] } ]	1	https://f1000research.com/articles/9-201
https://f1000research.com/articles/9-1205/v1	06 Oct 20	{ "type": "Systematic Review", "title": "Could malaria explain the global distribution of the angiotensin converting enzyme I/D polymorphism? A systematic review and ecological study", "authors": [ "Chris Kenyon", "Anna Rosanas", "Anna Rosanas" ], "abstract": "Background: The D-allele of the angiotensin converting enzyme (ACE1) has been linked to an increased risk of certain diseases including hypertension and COVID-19 but a decreased risk of cerebral malaria. We hypothesized that malaria played a role in determining variations in the global distribution of ACE1 I/D polymorphism. Methods: A systematic review was conducted to summarize the frequency of ID/DD genotypes in all countries with available data. Results: The ID/DD genotype frequency was found to be highest in Africa (86.4%, IQR 83.6-94.7%) and Eastern Mediterranean (median 84.5%, IQR 78.3-89.8%) and lowest in South East Asia (55%, 49.5-67.8%) and Western Pacific (61.1%, IQR 55.0-67.2%). Linear regression revealed positive associations between ID/DD genotype frequency and the incidence of malaria, malaria mortality as well as hemoglobin S allele frequency (all P<0.05). Conclusions: Our findings are compatible with the hypothesis that malaria played a role in establishing the differential frequency of the D-allele.", "keywords": [ "Malaria", "ACE", "polymorphism" ], "content": "Summary\n\nThe D-allele of the angiotensin converting enzyme (ACE1) has been linked to a number of diseases including hypertension, obesity and most recently COVID-19. Previous research has suggested that the prevalence of this allele may vary between world regions. Because the D-allele has been shown to be protective against cerebral malaria, we hypothesized that malaria played a role in determining variations in the global distribution of ACE1 I/D polymorphism. We conducted a systematic review of published literature to estimate the prevalence of the D-allele by country and World Health Organization region. The D-allele was found to be most prevalent in Africa and least prevalent in the Western Pacific. A positive association was found between the prevalence of the D-allele and three markers of malaria burden of disease. Our findings are thus compatible with the hypothesis that malaria played a role in establishing the differential frequency of the D-allele.\n\n\nIntroduction\n\nThe renin-angiotensin system plays an important role in the regulation of cardiovascular, renal and immune physiology1–3. The angiotensin converting enzyme-1 (ACE1) converts angiotensin I to angiotensin II which is a key effector molecular in this system2. The ACE1 gene insertion (I) and a 287 bp Alu repeat deletion (D) polymorphism explain a large proportion of the individual variation in ACE1 levels both in serum and tissue2. ACE1 DD homozygotes and ID heterozygotes have approximately 65% and 30%, respectively, higher ACE1 levels in serum ACE1 than II homozygote individuals3,4. It is unknown why the frequency of this polymorphism varies between different populations around the world5,6.\n\nThis increased ACE1 expression is thought to partly explain the association between the D-allele and an increased risk for diseases such as hypertension, heart failure, cerebrovascular disease, diabetic nephropathy, arthritis, various cancers, asthma, acute respiratory distress syndrome (ARDS) and possibly COVID-19 incidence and mortality3,5,7,8. Increased ACE1 in D-allele carriers generates higher angiotensin II levels, whose stimulation of the AT21 receptor then activates vasoconstriction as well as a number of pro-inflammatory and fibrotic pathways which increase the risk of these conditions3,7–10. The D-allele has been found to be associated in case-control studies with protection against progression to severe malaria11–13. In vitro and in vivo studies have demonstrated that angiotensin II can reduce the entry of plasmodia into erythrocytes and protect the blood-brain barrier against invasion by plasmodia12–15. These findings have led a number of authors to propose that differential intensity in malaria exposure may have driven variations in the prevalence of the D-allele13,14 in a similar way to how malaria influenced the prevalence of haemoglobin S (HbS), and other polymorphisms associated with different susceptibility to malaria16. In this study, we aimed to assess the association between D allele frequency and malaria using three indicators of malaria exposure: malaria incidence, malaria mortality and the prevalence of the HbS allele12.\n\nTo do this we first conducted a systemic review of the frequency of the ID/DD genotypes in different countries around the world. A prior systematic review measured D allele frequencies by ethnicity, sex and age and reported D allele frequencies of 56.2% in “whites’, 60.3% in ‘Blacks’ (60.3%) and the lowest (39.1%) in ‘Asians’6. Results were similar for when DD genotype was measured. This study did not evaluate the geographical variation of the I/D polymorphism.\n\n\nMethods\n\nIn April 2020, we used PubMed and Google Scholar to conduct a systematic review with the objective of obtaining country-level ACE1 estimates of DD and ID+DD frequency. We used the following MeSH terms: 'ACE' OR 'angiotensin-converting enzyme' AND ('polymorphism' OR 'deletion' OR 'insertion'). The following inclusion criteria were used: sample size of at least 50; published in English and the population could be considered representative of the general population (the studies were typically the control groups of case control studies evaluating the associations between I/D polymorphisms and specific diseases). Studies from previous systematic reviews of associations between D-allele frequency and various diseases could be included if they met the entry criteria. No date restrictions were used. Duplicate studies were removed (Figure 1). The data was extracted by a single author (CK), and manually checked for accuracy. Risk of bias was not assessed. The principle summary measure used was the prevalence of the genotype of interest expressed as a percentage. The PRISMA guidelines were followed for the manuscript (see Extended data, STable 117).\n\nHbS: The HbS allele frequency (expressed as a percentage) was obtained from a study that estimated national HbS allele frequencies for 190 countries18.\n\nMalaria incidence: The national incidence of malaria per 1,000 population at risk for the year 2000 (the earliest year with available data), was obtained from the World Health Organization (WHO), Global Health Observatory Data Repository. A value of zero cases per 1,000 was used for countries classified as having eliminated malaria in 2000.\n\nMalaria mortality. The age-standardized national number of deaths from malaria per 100,000 individuals. The data was obtained from the International Health Metrics and Evaluation Global Burden of Diseases study for the year 1990 – the first year with available data.\n\nGeographical grouping. For the global analyses, countries were classified into 6 regions according to the WHO system.\n\nA previous study limited to European countries found an ecological association between COVID-19 incidence and D-allele frequency, with both having higher values in Southern European countries5. This provided the motivation to compare the ID/DD prevalence between European regions. We used the four European Regions as defined by the United Nations Geoscheme for Europe for this purpose.\n\n#\n\nStatistical analysis. Linear regression was used to evaluate the country-level association between the prevalence of ID/DD genotypes and the three measures of intensity of malaria exposure. Differences in ID/DD genotype prevalence between world regions were assessed via the Wilcoxon rank-sum test. The analyses were performed in STATA version 16 (Stata Corp, College Station, Tx). The maps were produced with Data Wrapper v2.\n\n\nResults\n\nSearches of the literature identified 5099 references. The application of the selection criteria restricted the total to 248 informative references published between 1993 and 2020, providing 253 prevalence estimates from 71 countries (Figure 1; Extended data, STable 217). Of these, 7 were located in the Americas, 15 in Eastern Mediterranean, 26 in Europe, 8 in sub Saharan Africa, 5 in South East Asia and 10 in Western Pacific (Table 2).\n\nThe ID/DD frequency was found to be highest in Africa (86.4%, IQR 83.6-94.7%) and Eastern Mediterranean (84.5%, IQR 78.3-89.8%) and lowest in South East Asia (55%, 49.5-67.8%) and Western Pacific (61.1%, IQR 55.0-67.2%; Figure 2 & Figure 5; Table 1). Within Europe, the prevalence was highest in Southern Europe (85.3%, IQR 84.3-88.2%) and lowest in Northern Europe (75.7%, IQR 73.9-76.7%; Table 2).\n\na Europe was used as the reference category as it had the largest sample size.\n\nb The number of studies used to generate the median prevalence estimates\n\na Northern Europe was used as the reference category as it had the largest sample size.\n\nb The number of studies used to generate the median prevalence estimates\n\nLinear regression revealed positive associations between ID/DD frequency and the incidence of malaria (coef. 4.96, 95% confidence interval [CI] 1.73- 8.20; N=63; Table 3 and Figure 3), malaria mortality (coef. 0.69, 95% CI 0.20- 1.19; N=70) as well as HbS allele frequency (coef. 0.12, 95% CI 0.04- 0.19; N=68). Results were similar when these regressions were run with DD frequency as the outcome variable (Extended data, STable 417).\n\nScatter plots of the associations between prevalence of the ACE1 ID/DD genotypes and malaria incidence per 1,000 people (a), malaria deaths per 100,000 people and allele frequency of the hemoglobin S allele (c).\n\n\nDiscussion\n\nDifferential exposure to malaria has been found to play a role in driving the large geographical variations in the prevalence of various haemoglobinopathies, thalassemias, glucose 6-phosphate dehydrogenase deficiencies, erythrocyte membranopathies and various mediators of inflammation and immunity19,20. We found country- and regional-level differences in the prevalence of the ACE1 D-allele that were associated with malaria incidence and mortality and HbS allele. These findings add into previous individual-level studies showing protection against severe malaria by the D allele, suggesting that malaria may have played a role in the global variations in D-allele frequency.\n\nAn important weakness of the study is our use of malaria incidence and mortality data from 1990 and 2000. Although this was the earliest data we had available, it underestimates the historical burden of malaria for much of the world’s population up to the beginning/mid-20th century as shown in Figure 420. This figure of malaria prevalence from the preintervention period (around 1900) was produced by Lysenko in 1968. Unfortunately it has not been translated into estimated numerical values of national prevalence that could be used to test numerical associations21. Although we did not test this statistically, it does appear that a number of the countries with high D-allele prevalence but low malaria incidence in 2000 in our analysis had a high malaria prevalence in 1900 (Figure 3 and Figure 4)20. For example, all 10 countries with ID/DD prevalence of above 81% and malaria incidence of 0 in 2000 were classified as being at least hypoendemic for malaria transmission in 1900 according to Lysenko21. Our finding of higher prevalence of the D-allele in Southern than Northern Europe is also commensurate with the higher historical prevalence of malaria in this part of Europe20,21.\n\nEndemicity as defined by Lysenko is according to the parasite rate in the 2–10-year age cohort (hypoendemic <0.1; mesoendemic 0.11–0.5; hyperendemic 0.51–0.75) except the holoendemic class (0.75) where the PR refers to the 1-year age group. The black line was added by Hay et al. to delineate the 2002 limit of malaria risk20.\n\nAnother weakness of our study is the small number of countries with data for D-allele frequency and malaria burden of disease. We also acknowledge the fact that some of the differences in ID/DD frequency between studies and countries may be attributed to differences in how control groups were selected, differences in age distribution and in the methodology used to genotype individuals6. It is also important to note that recent genome wide association studies have not found the ACE1 I/D polymorphism to be associated with severe malaria22,23. The lack of an association in these studies may however be related to the high prevalence of the D-allele in malaria endemic areas, as well as the high level of genetic diversity and weak linkage disequilibrium in Africans compared to elsewhere22. The association between malaria and the D-allele could be explained by confounding. It is possible that the actual driver of the D-allele is an environmental factor such as another infection that is associated with malaria. Finally, not all studies we drew our data from reported if the I- and D-alleles were in Hardy-Weinberg equilibrium. Including data from studies where these alleles were not in Hardy-Weinberg equilibrium would, however, be expected to introduce a non-differential misclassification bias into the analysis24. This would be expected to dilute the strength of association between the prevalence of the D-allele and malaria severity.\n\nGiven these concerns, we can only conclude that our data is compatible with the hypothesis that malaria played a role in establishing frequency distribution of the D-allele. This association could provide a potential evolutionary explanation for the commonly observed higher prevalence of hypertension in individuals with African genetic background25,26. If the association between the D-allele and COVID-19 incidence/morbidity is confirmed, our mapping of D-allele prevalence could assist with estimating morbidity and mortality in different populations.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Could malaria explain the global distribution of the angiotensin converting enzyme I/D polymorphism? A systematic review and ecological study. https://doi.org/10.6084/m9.figshare.12982112.v217.\n\nThis project contains the following extended data:\n\nSTable 1. PRISMA checklist.\n\nSTable 2. Study characteristics of studies included in systematic review, including frequency of II, ID and DD genotypes of ACE1, arranged according to country and year of study.\n\nSTable 3. Study references for STable 2.\n\nSTable 4. Linear regression assessing the country-level association between the prevalence of the DD genotypes of the ACE1 gene and malaria incidence per 1,000, malaria mortality per 100,000 and the allele frequency of hemoglobin S.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAuthors contributions\n\nCK conceptualized the study, was responsible for the acquisition, analysis and interpretation of data including the systematic review and wrote the analysis up as a manuscript. AR assisted with the study design and analysis. Both authors contributed to and approved the final draft.", "appendix": "References\n\nSriram K, Insel PA: A hypothesis for pathobiology and treatment of COVID-19: The centrality of ACE1/ACE2 imbalance. Br J Pharmacol. 2020. PubMed Abstract \| Publisher Full Text\n\nSayed-Tabatabaei FA, Oostra BA, Isaacs A, et al.: ACE polymorphisms. Circ Res. 2006; 98(9): 1123–33. PubMed Abstract \| Publisher Full Text\n\nGard PR: Implications of the angiotensin converting enzyme gene insertion/deletion polymorphism in health and disease: a snapshot review. Int J Mol Epidemiol Genet. 2010; 1(2): 145–57. PubMed Abstract \| Free Full Text\n\nRigat B, Hubert C, Alhenc-Gelas F, et al.: An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990; 86(4): 1343–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelanghe JR, Speeckaert MM, De Buyzere ML: The host's angiotensin-converting enzyme polymorphism may explain epidemiological findings in COVID-19 infections. Clin Chim Acta. 2020; 505: 192–193. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStaessen JA, Ginocchio G, Wang JG, et al.: Genetic variability in the renin-angiotensin system: prevalence of alleles and genotypes. J Cardiovasc Risk. 1997; 4(5–6): 401–22. PubMed Abstract \| Publisher Full Text\n\nDeng X, Zhang S, Jin K, et al.: Angiotensin-converting enzyme I/D polymorphism and acute respiratory distress syndrome. J Renin Angiotensin Aldosterone Syst. 2015; 16(4): 780–6. PubMed Abstract \| Publisher Full Text\n\nZmorzynski S, Szudy-Szczyrek A, Popek-Marciniec S, et al.: ACE Insertion/Deletion Polymorphism (rs4646994) Is Associated With the Increased Risk of Multiple Myeloma. Front Oncol. 2019; 9: 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nItoyama S, Keicho N, Quy T, et al.: ACE1 polymorphism and progression of SARS. Biochem Biophys Res Commun. 2004; 323(3): 1124–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang D, Chai XQ, Magnussen CG, et al.: Renin-angiotensin-system, a potential pharmacological candidate, in acute respiratory distress syndrome during mechanical ventilation. Pulm Pharmacol Ther. 2019; 58: 101833. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDhangadamajhi G, Mohapatra BN, Kar SK, et al.: Gene polymorphisms in angiotensin I converting enzyme (ACE I/D) and angiotensin II converting enzyme (ACE2 C-->T) protect against cerebral malaria in Indian adults. Infect Genet Evol. 2010; 10(2): 337–41. PubMed Abstract \| Publisher Full Text\n\nGallego-Delgado J, Rodriguez A: Malaria and hypertension. Another co-evolutionary adaptation? Front Cell Infect Microbiol. 2014; 4: 121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilva LS, Silva JL, Caruso-Neves C, et al.: New Concepts in Malaria Pathogenesis: The Role of the Renin-Angiotensin System. Front Cell Infect Microbiol. 2016; 5: 103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGallego-Delgado J, Walther T, Rodriguez A: The High Blood Pressure-Malaria Protection Hypothesis. Circ Res. 2016; 119(10): 1071–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGallego-Delgado J, Baravian C, Edagha I, et al.: Angiotensin II Moderately Decreases Plasmodium Infection and Experimental Cerebral Malaria in Mice. PLoS One. 2015; 10(9): e0138191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiel FB, Patil AP, Howes RE, et al.: Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis. Nat Commun. 2010; 1: 104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenyon C: Could malaria explain the global distribution of the angiotensin converting enzyme I/D polymorphism? A systematic review and ecological study. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12982112.v2\n\nPiel FB, Patil AP, Howes RE, et al.: Global epidemiology of sickle haemoglobin in neonates: a contemporary geostatistical model-based map and population estimates. Lancet. 2013; 381(9861): 142–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMarquet S: Overview of human genetic susceptibility to malaria: From parasitemia control to severe disease. Infect Genet Evol. 2018; 66: 399–409. PubMed Abstract \| Publisher Full Text\n\nHay SI, Guerra CA, Tatem AJ, et al.: The global distribution and population at risk of malaria: past, present, and future. Lancet Infect Dis. 2004; 4(6): 327–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLysenko A, Semashko I: Medical geography: a medical-geographical study of an ancient disease. In: Lebedew AW, editor. Medical Geography. Academy of Sciences, USSR, Moscow. 1968; 25–146. Reference Source\n\nDamena D, Denis A, Golassa L, et al.: Genome-wide association studies of severe P. falciparum malaria susceptibility: progress, pitfalls and prospects. BMC Med Genomics. 2019; 12(1): 120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRavenhall M, Campino S, Sepulveda N, et al.: Novel genetic polymorphisms associated with severe malaria and under selective pressure in North-eastern Tanzania. PLoS Genet. 2018; 14(1): e1007172. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChyou PH: Patterns of bias due to differential misclassification by case-control status in a case-control study. Eur J Epidemiol. 2007; 22(1): 7–17. PubMed Abstract \| Publisher Full Text\n\nCappuccio FP: Ethnicity and cardiovascular risk: variations in people of African ancestry and South Asian origin. J Hum Hypertens. 1997; 11(9): 571–6. PubMed Abstract \| Publisher Full Text\n\nSampson UK, Edwards TL, Jahangir E, et al.: Factors associated with the prevalence of hypertension in the southeastern United States: insights from 69,211 blacks and whites in the Southern Community Cohort Study. Circ Cardiovasc Qual Outcomes. 2014; 7(1): 33–54. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "74480", "date": "24 Nov 2020", "name": "Bin Pei", "expertise": [ "Reviewer Expertise Evidence-based medicine", "clinical epidemiology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors performed a systematic review to analyze the association between ACE1 I/D polymorphism and malaria. The topic is novel, but there are some serious defects that cannot be ignored.\nThis study included previous systematic reviews regarding ACE1 I/D, which is inconsistent with the scientificity of a systematic review. Besides, the search strategy and databases could not ensure the comprehensiveness of the researches included in this study, and the included 248 literatures were neither listed in this paper nor in the supplement. The data was extracted by only a single author, this is inconsistent with the basic requirement of data extracting method in systematic reviews. Moreover, the author did not evaluate the publication bias and method quality of the included studies, which may influence the reliability of the conclusion.\n\nFor the systematic review of gene polymorphism, Hardy-Weinberg test was not performed in this study. Whether the included researches meets Hardy-Weinberg test is important to reflect the representativeness of the population.\n\nThere are defects in statistical analysis. Linear regression was used to evaluate the country-level association between the prevalence of ID/DD genotypes and the three measures of intensity of malaria. However, the scatter plots (Figure 3) could not reflect the feasibility of linear regression, and the authors did not explain whether the data meet the assumption of linear regression. Besides, the prevalence of the ACE1 ID/DD genotypes should be obtained only from the healthy population, but the authors did not display the data resource in the figures or tables. Additionally, in Table 1 and Table 2, there is a methodological error to analyze the association between gene polymorphisms and malaria in other regions based on the region with the largest sample size, which may obtain fundamentally wrong results and conclusions.\n\nThe results of ID/DD genotype were analyzed in the study. Although it was mentioned in the last sentence of the results section that “Results were similar when these regressions were run with DD frequency as the outcome variable”, it could not be concluded that the D allele is associated with malaria. It is suggested that on the basis of overcoming the above problems, the authors should separately study the correlation between I allele and malaria and the correlation between D allele and malaria.\n\nThe research on gene polymorphism is always divided by race rather than region.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Partly\n\nIs the statistical analysis and its interpretation appropriate? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "76910", "date": "26 Jan 2021", "name": "Alfred Amambua-Ngwa", "expertise": [ "Reviewer Expertise parasitology", "genomics", "genetics", "epidemiology", "transmission", "drug resistance" ], "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 somewhat systematic review of publications to determine the relationship between ACE1 ID/DD alleles and malaria. The authors used malaria incidence rather than prevalence information as well as the distribution of Hbs and malaria mortality. The review has a strong rationale given the suspected role of ACE1 in COVID-19 and associations with high blood pressure in populations of African descent. However, there are several limitations with the data gathering and analysis process that does not render full credibility to the conclusions of malaria being a contributing factor to the prevalence of the ID/DD alleles:\nData curation by a single author is not in line with standards for systematic reviews.\n\nThe authors have not clearly stated the number of different publication types that were included, short communications, letters, posters, reviews, and conference abstracts. The method seemed limited by the number of databases or sources used for the search and the numbers in the flow chat need further clarification on how some publications were eliminated. For example, why were only 312 texts assessed for eligibility out of the 402 texts obtained after screening? Why were the final 248 texts included after excluding 90 out of the 312 assessed?\n\nBoth first (Title/abstract) and second (full text) screening were done by one author which might have biased the screening strategy. Moreover, the author CK seems to have done everything in the manuscript, and this questions the contribution(s) of the second author.\n\nIt is not clear how the authors assessed the criteria of “the population being a representative of the general population” in the selected 248 articles. This is important because allele frequencies depend on census sizes from the population. Yet the authors did not provide country-level information on the number of samples. This reflects a general flaw in the statistics as results are presented per sub-region. Country-level malaria incidence for example can be widely different within sub-regions and so country-level data would have been more informative. Given that the frequency of the D allele is almost fixed in most of Africa and equally high in malaria-free regions, the regression in my opinion cannot be robust. No explanation for example was given for the lower frequency in South East Asia where there is historically a higher prevalence of malaria than in Europe.\n\nFrom figure 3 there is a higher prevalence of the D allele in higher malaria prevalence regions, but the relationship is far from linear. Hence the coefficients presented cannot be accurate.\n\nThe authors should consider adding a reference for the WHO system mentioned in the geographical grouping section of the methods.\n\nIn the first paragraph of the results, the authors are referring to table 2 instead of table 1 and in the second paragraph, they refer the reader to table 1 instead of table 2.\n\nThe author should consider rearrangement of figures for easy flow. Changing figure 5 to figure 3 will enable a better understanding of the second paragraph of the results section by the reader.\n\nThe authors might consider capturing the P-values in figure 2.\n\nThe limitation of drawing data from studies without a report that I and D alleles are in Hardy-Weinberg equilibrium can and should be corrected. Withdrawing data from such studies in a review on polymorphism can create big bias in the analysis and conclusions drawn. I herein suggest the authors go back to all included 248 papers, sort out those without reports that the I and D alleles are in Hardy-Weinberg equilibrium, and then do additional analysis on data from only studies with reports on Hardy-Weinberg equilibrium. This will enable the authors to present results of data drawn from studies with and without reports from Hardy-Weinberg equilibrium as well as results with data drawn from only studies with reports on I and D alleles being in Hardy-Weinberg equilibrium, hence reducing bias in the analysis and final conclusion(s).\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "86865", "date": "21 Jun 2021", "name": "Rasheedunnisa Begum", "expertise": [ "Reviewer Expertise Immunogenetics", "disease biology", "vitiligo", "autoimmunity", "case-control studies" ], "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 tried to present a systematic review to study the role of Malaria in ACE I/D genotype distribution. The review is novel and interesting as ACE I/D polymorphism might be associated with malaria and COVID-19 susceptibility as suggested by the authors. However, there seems to be a few reservations for the article and concluding the association of Malaria with ACE I/D genotype distribution.\nComments:\n\nHow do the authors justify the effect of Malaria on distribution of ACE I/D genotypes in the studied population? In fact ACE I/D polymorphism might be a susceptibility factor for malaria in these populations. To confirm this, respective analysis should be carried out and if the ACE I/D polymorphism is found to be a susceptibility factor to malaria then, the conclusion “malaria played a role in establishing frequency distribution of the D-allele” must be revised as “ACE I/D polymorphism may have a role in malaria susceptibility”.\n\nIn the introduction the authors have mentioned that previous case-control studies suggest that the D-allele is associated with protection against progression to severe malaria but in the current review they have found positive association for malaria incidence, malaria mortality and D allele which is contradictory and they must justify this. Moreover, the authors should carry out a meta-analysis for the association of ACE I/D polymorphism with Malaria susceptibility and severity.\n\nIt is important to study the association of ACE protein levels with malaria incidence, malaria mortality.\n\nAdditionally, the article lacks the following crucial points in the systematic review, which could be included:\n\nRisk of publication bias and sensitivity analyses. The authors must screen the studies before inclusion of studies in the systematic review to avoid any bias. The methodology part lacks the exclusion criteria for the studies. Table 2 (Supplementary) lacks the crucial data like HWE, age, gender, ACE protein levels. Quality assessment for the included studies should be done. The accession number (rs number) of the ACE I/D polymorphism has not been mentioned anywhere in the article.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] }, { "id": "359145", "date": "03 Feb 2025", "name": "Abhinav Sinha", "expertise": [ "Reviewer Expertise Molecular epidemiology", "malaria", "molecular parasitology", "epidemiology", "public health" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors take up a very intriguing but complex hypothesis for the systematic review. The rationale behind the systematic review is very good but it appears that there are multiple confusions in the understanding of this complex evolutionary relationship between malaria and hypertension. There are two hypotheses that are put forth to explain the above relation that malaria is associated with hypertension - direct and indirect. Although the authors hint towards the indirect association but it is not explicitly stated anywhere in the manuscript. Further, if we presume that the authors take up the indirect hypothesis, it must be made very clear that exposure pressure to severe malaria (and not malaria) predisposes them to the genetic risk of hypertension (and not directly increased blood pressure). Therefore, correlation with malaria incidence, mortality, blood pressure and HbS may not be the most appropriate way to address this hypothesis..\nOther major negative issues relate to the lack of clarity on multiple aspects such as reasons for choosing the WHO regions, why the DD and ID genotypes were not separately analyzed, errors in citing references, issues about representativeness of population, choice of regression analysis, incomplete PRISMA flow chart, not registering the systematic review on PROSPERO, misinterpretation of p-values, inadequate information on selected allele of HbS, and many more minor mistakes.\nOverall, there is no doubt that authors selected a very pertinent rationale but were unable to do justice with the intricate hypothesis.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Partly\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? No\n\nAre the conclusions drawn adequately supported by the results presented in the review? No\n\nIf this is a Living Systematic Review, is the ‘living’ method appropriate and is the search schedule clearly defined and justified? (‘Living Systematic Review’ or a variation of this term should be included in the title.) Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/9-1205
https://f1000research.com/articles/9-1203/v1	06 Oct 20	{ "type": "Research Article", "title": "Bioinformatic study to discover natural molecules with activity against COVID-19", "authors": [ "Sweta Singh", "Hector Florez", "Sweta Singh" ], "abstract": "Background: In 2020, the world has struggled to deal with coronavirus disease 2019 (COVID-19), which started in 2019 in China and has spread throughout the globe, affecting at least 31,175,835 humans globally and claiming 962,634 lives reported till 22nd September, 2020 by the World Health Organization. The main causative agent for this disease is known as severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). So far, there is no cure or proven therapeutics available till date. Therefore, we undertook this study to ﬁnd the most probable drug candidate through a bioinformatics study. Methods: Thus, we virtually screened the Zinc natural database using HTVS tool through molecular docking studies to analyze molecules recommended for the treatment of COVID-19. Results: Ramipril benzyl ester, propafenone dimer and Lariciresinol are three important drugs found from the present study due to their medicinal application which could be helpful in treating the disease. Stylopine, quillaic acid, cinobufagin, vitisinol C, segetalin A, scopolamine, 3-oxo glycyrrhetinic acid, conchinone B, lactimidomycin and cardinalins 4 are the other lead molecules that could be used as therapeutics against COVID-19 disease. Conclusions: The studied molecules could act as an effective inhibitory drug against COVID-19.", "keywords": [ "COVID-19", "SARS-CoV-2", "Molecular Docking", "High throughput virtual screening" ], "content": "Introduction\n\nA novel virus was first found infecting human in China on 30th December, 2019. The causative agent for this disease was found to be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) was declared as a pandemic by the World Health Organization on 11th March, 2020. The virus has been responsible for ’locking down’ much of the human race in most countries of the world for several months. The infectivity of this virus is extremely high and the mortality of this disease is around 5–7 percentage of the total infected cases1. However, the infection fatality rate was reported to be 0.5–3.6% and the case fatality rate 2.3–7.2%, as per other reports since it differs from region to region over time2\n\nCoronaviruses consist of an enveloped RNA genome, and these viruses are zoonotic in nature. They cause respiratory, hepatic neurological, and enteric diseases in birds and mammals. As per the recent reports, this disease has claimed 962,634 lives worldwide, infecting 31,175,835 people globally till 22nd September, 20203. SARS-CoV-2 consists of 30 kb nucleotides as its genome4. Two polyproteins essential for replication and transcription, pp1a and pp1ab, are encoded by the SARS CoV-2 replicase gene. The main protease (3C-like protease) or Mpro (a 33.8 kDa protein) is responsible for releasing the functional viral polypeptides from polyproteins after cleaving pp1a and pp1ab4. Therefore, Mpro is identified as an important drug target owing to its functional significance in the life cycle of the virus and also due to the absence of its homologues in the human race4.\n\nA few drugs such as hydroxychloroquine and remdesivir that were found via bioinformatics approach have been reported. Hydroxychloroquine is a drug that is presently used against malaria. But when it was tested on SARS-CoV-2 virus, it showed activity against the virus in vitro. However, it was not found to be effective against the virus in COVID 19 patients during clinical trials5,6. Later, this drug was prohibited from use in certain countries due to it side effects. Remdesivir is an antiviral drug that was found to show activity against the virus and it did inhibit the SARS-CoV-2 virus to certain extent. Remdesivir is presently used against COVID-19 in certain countries7.\n\nIn the present work, we carried out high throughput virtual screening (HTVS). It is a rapid way to search for the potential drug against the target. HTVS of Zinc natural database was performed through Python prescription software known as PyRx. This software has in built dock, Vina and Autodock tools along with openbabel. Autodock software utilizes MGLTools that has computer aided drug discovery pipeline. CADD is required for virtual high throughput screening of large databases for probable hits as therapeutic agents. This type of virtual screening helps to dock several ligands on one protein. PyRx is publicly obtainable HTVS software. Analysis and identification of distinct poses visibly and using a scoring algorithm quantitatively forms two basis of docking technique8.\n\nThe free binding energy (ΔG) between the protein and the ligands is calculated in docking. The free binding energy, thus calculated, forms the foundations of complex systems of subjects such as molecular biology and biochemistry8.\n\n\nMethods\n\nAs per our earlier published article8, we found that fexofenadine acetate (Allegra), atovaquone, ethamidindole, baicalin,justicidin D, euphol,curine and glycyrrhetic acid could be used as COVID-19 inhibitory drugs8. In that article, we selected 129 known drugs for drug repurposing, from which we found atovaquone, presently in use for treating malaria and Pneumocystis carinii pneumonia. We had considered another aspect in which we had taken 992 small molecules from Zinc natural database from which fexofenadine acetate (Allegra), an already in use drug for anti-allergic medicine8. Notably, the small molecules from natural database had least binding energy. Therefore, we decided to dock 1100 small molecules (see Extended data9) from the ZINC Natural Products database to identify additional lead molecules against the disease.\n\nIn this study, we utilized a bioinformatic approach that uses predictions for the arrangement of a molecule bounded to another molecule, known as molecular docking10,11. Molecular docking can be performed by two main approaches. The former describes complementation surfaces of the ligand and the protein12. The latter simulates the docking process that calculates the free binding energy ΔG while protein-ligand interaction13–15. We have used PyRx (Python prescription) version 0.8 that performs HTVS. PyRx uses Autodock, ock and Vina as docking tools. Further, MGLTools, providing a computer aided drug discovery or rational drug design pipeline to search large databases to obtain potential leads as therapeutic drugs, is present as inbuilt software in the Autodock tool of PyRx.\n\nThe X-ray crystal structure of Mpro (PDB ID: 6M03 (resolution: 2 Å) was considered for our study Å). The database considered for docking was the ZINC Natural Products database. For processing of ligands and macromolecules, the energy of 3D molecules were minimized. The Universal Force Field or UFF was used for the energy minimization of small molecules before docking. Removal of water molecules and insertion of polar hydrogen into the protein crystal structure was carried out in pre-processing of the macromolecule. Later, PDBQT files of ligands and macromolecule were generated using AutoDock version 4.\n\nThe grid box was as per our previous article, with dimension coordinates of X:29.9242, Y:64.1097, and Z:48.1126 and center coordinates of X:12.2632, Y:12.3998, and Z:5.4737)8. Maximum grid box space was chosen for docking in order to maximize the possibility of finding a good lead molecule owing to the fact that there are still no known molecule to inhibit the virus completely. Therefore, it was thought to be better to maximize the active site space. Docking was performed with the Vina software present in PyRx tool, in which the protein-ligand interaction was studied. Analysis of separate poses were done in the AutoDock and the docked pose with the least binding affinity in kcal/mol was chosen for further analysis.\n\nConversion of these final docked ligands into PDB format was done using Pymol software version 1.7.4; Discovery studio 4.1 was used to study their interaction. Pymol could also be used for studying the interactions.\n\nIn addition, remdesivir, an antiviral agent that was found to act to some extent against this disease, was used as the reference molecule. Hence, it was initially docked with 6M03 in order to know (ΔG) value as the reference range for acquiring the hits.\n\n\nResults\n\nMpro has six stranded antiparallel beta barrels which provides the substrate binding site between domain I (10–99 residues) and domain II (100–182 residues)16. Dimerization of Mpro is regulated by domain III(198–303 residues) which is a globular cluster of five helices. It is primarily done by forming a salt-bridge interaction between Glu290 of one protomer and Arg4 of the other16. The two molecules domain II of molecule A and the NH2-terminal residues(“N-finger”) of molecule B are arranged perpendicularly to one another to provide a tight dimer for Mpro16. N-finger of each of the two protomers is found to be interacting with Glu166 of the other protomer, therefore, the N-finger is firmly pressed in between domains II and III of the parent monomer and domain II of the other monomer. Dimerization is essential for the catalytic activity of Mpro. Hence, these residues (10 to 99, 100 to 182 and 198 to 303) are essential for increasing the catalytic activity of Mpro16. Docking results for each molecule are given in the Extended data9.\n\nThe free binding energy for our reference molecule, which is remdesivir, was found to be -6.3 kcal/mol and it interacted with Leu272, Leu287, Tyr237, and Asn238 forming six hydrogen bonds with Lys137, Thr199, and Tyr239 residues of the main protease, Mpro (6M03). Figure 1 presents the interaction of remdesivir with Mpro.\n\nThe best 25 molecules, with binding energies of -8.8 kcal/mol to -8.7 kcal/mol, were shortlisted and are depicted in Table 1.\n\nThe results obtained through the molecular docking analysis of some of the presented molecules exhibit different insights that allows asserting whether it is possible to use them for medical treatment of COVID-19.\n\nRamipril benzyl ester forms two hydrogen bonds with Asp153 as well as Thr304 residues apart from interacting with Asn151, Phe294, Asp295, and Val303 residues of Mpro as presented in Figure 2.\n\nEscholtzine / (S)-stylopine forms two hydrogen bonds with Thr111 and Ile152 apart from interacting with Asp153 residues of Mpro. Its interaction is presented in Figure 3.\n\n3-oxoglycyrrhetinic acid forms two hydrogen bonds with Leu287 as well as Asp289, whereas Glu288, Leu272, and Tyr239 are the other interacting residues of Mpro. Figure 4 presents 3-oxoglycyrrhetinic acid interaction with Mpro.\n\nLariciresinol forms a hydrogen bond with Arg298 as well as interacts with Phe294, Ile106, and Val104 residues of Mpro. Figure 5 presents the interaction of this molecule with 6M03.\n\nCochinchinone B forms a hydrogen bond with Arg298 along with interacting with Ile249, Pro252, Leu253, Pro293, Phe294, Val297, and Val303 residues of Mpro. The interaction of this molecule with Mpro is presented in Figure 6\n\nLactimidomycin interacts with Phe8, Phe305, Val303, Arg298, Phe294, Val297, and Val104. In addition, it forms two hydrogen bonds with Gln110 and Asp295 residues of Mpro as presented in Figure 7.\n\nCardinalin 4 forms five hydrogen bonds, one with Asp197 and four with Lys5 apart from interacting with other residues of Mpro such as Lys137, Met276, Leu286, Leu287, and Asp289. Its interaction with Mpro is presented in Figure 8\n\nCinobufagin forms two hydrogen bonds with Thr111 residue and interacts with Val297, Phe294, Arg298, Val303, Phe305, Phe8, and Pro252 residues of Mpro. Figure 9 presents Ccnobufagin interaction with Mpro.\n\nQuillaic acid interacts with Val104, Phe294, Arg298, Val303, and Phe305 residues of 6M03. The quillaic acid interaction with Mpro is presented in Figure 10.\n\nVitisinol C forms two hydrogen bonds with Asn151 and Arg298 apart from interacting with Gln110, Phe294, Val297, Val303, and Phe8. Figure 11 presents vitisinol C interaction with Mpro.\n\nCitrusinol forms four hydrogen bonds with two residues, Asn151 and Gln110, as well as interacts with Val297, Arg298, and Val303 residues of Mpro. Figure 12 presents its interaction with Mpro.\n\nSegetalin A forms three hydrogen bonds with Gln110, Ser158, and Asp153 and interacting with residues such as Phe294, Arg298, Val303 of Mpro. Figure 13 presents segetalin A interaction with 6M03.\n\nPropafenone dimer interacts with Gln110, Phe294, Arg298, Val303, and Phe305 and forms two hydrogen bonds with Thr111 and Asn151 of Mpro residues as presented in Figure 14\n\nScopolamine 4-hydroxybenzoate forms hydrogen bonds with Phe294 and Thr304 apart from interacting with Asp295 and Val303 residues of Mpro. Figure 15 presents the scopolamine 4-hydroxybenzoate interaction.\n\n\nDiscussion\n\nNatural molecules are known derivatives of most of the present drugs in use against many diseases. In this study, we have also found some good leads and most of the 25 selected molecules show medicinal properties. Ramipril benzyl ester (protected form of ramipril), which is an inhibitor of angiotensin-converting enzyme ACE is the host receptor protein where the SARS-CoV-2 spike protein binds. Ramiprilat, which is an active compound of ramipril benzyl ester, is formed on the hydrolysis of the ester group, which later inhibits ACE. This leads to the prevention of the catalytic conversion of angiotensin I to angiotensin II that causes vasodilation17. Stylopine18 and quillaic acid exhibits anti-inflammatory activity19. Cinobufagin, CBF, has antitumor activity as it is known to inhibit the growth of PC3 cells both in vitro and in vivo along with inducing apoptosis of tumor cells as per reports20. It was found that intake of the plant ligands such as lariciresinol reduced endothelial dysfunction and vascular inflammation in post-menopausal women and middle aged/elderly men21. Vitisinol C as per earlier reports was found to exhibit an essential activity against amyloid aggregation, which is present in Vitis vinifera grapevine shoots22.\n\nSegetalins are known to have vasorelaxant activity23. Scopolamine is known as antimuscarinics that blocks the effects of certain natural substance (acetylcholine) on the central nervous system, it is also used to prevent nausea and vomiting caused by motion sickness or medications used during surgery24.\n\n3-oxo-glycyrrhetinic acid has antitumor activity and expectorant (antitussive) properties25. Lactimidomycin has antifungal, antiviral, and anti-cancer properties apart from acting as a direct inhibitor of protein translation inribosomes which is a glutarimide antibiotic derived from the bacterium Streptomyces amphibiosporus26. Cardinalins dinalins 4 exhibit anti-cancer properties. Conchinone B has an antioxidant activity. Rotenone used to treat head lice on humans, scabies, and parasitic mites on chickens, livestock, and pet animals. Propafenone is used for the treatment of irregular heartbeat like atrial fibrillation, paroxysmal supraventricular and tachycardia in order to restore normal heart rhythm for maintaining a regular, steady heartbeat27. These small molecules or potential drugs could be used as therapeutics for COVID-19 disease due to their binding affinity with the viral protein as well as interaction with Mpro. These small molecules were found to interact with the residues involved in enhancing the catalytic activity of Mpro or SARS CoV-2 main protease. Therefore, these could prove to play important role in inhibiting the viral activity. However, there are three small molecules ramipril benzyl ester, propafenone dimer and lariciresinol found from this study that might prove to be useful against this virus especially due to their present medical application, docking score and interaction with Mpro, as illustrated in Figure 16, Figure 17 and Figure 18.\n\n\nConclusions\n\nRamipril benzyl ester, stylopine, quillaic acid, cinobufagin, vitisinol C, segetalin A, scopolamine, 3-oxo glycyrrhetinic acid, lariciresinol, conchinone B, lactimidomycin, cardinalins 4, and propafenone dimer are 13 molecules that are predicted to form good interaction with Mpro. These molecules showed better binding affinity for Mpro than the reference molecule. Ramipril benzyl ester, apart from binding to the main protease of the virus, was also found to inhibit the virus receptor ACE present in humans. ACE2 is the receptor to which virus spike protein is known to bind to the host. The spike protein and Mpro of the SARS-CoV-2 virus are responsible for the virulence of the virus. Ramipril benzyl ester drug is reported to inhibit ACE and also found to inhibit main protease of SARS-CoV-2 virus from this study. Ramipril benzyl ester is presently used against heart stroke. Ramipril benzyl ester and propafenone are two important small molecules found from our study due to their present medicinal use as drugs for other diseases in human along with lariciresinol due to its medical application as stated in the discussion. As a result, these molecules have medicinal properties which could be used for the treatment of COVID-19 disease after further evaluation. This study presents a significant possibility in finding the cure for the COVID-19 disease from the drugs repurposing point of view. The drug targets mentioned in our study could bring relief to all those who are infected with the disease and these may also act as a prophylactic treatment when tested by the frontline healthcare providers. This study could provide relief to everyone if the drugs mentioned in our study proved to inhibit the virus in COVID19 positive patients since they are known natural derivatives with minimal side effects in individuals. Thus, our study could help in flattening the curve with speedy recovery in COVID -19 positive patients.\n\n\nData availability\n\nThe COVID-19 main protease structure was downloaded from the Protein Data Bank, ID 6M03: https://www.rcsb.org/structure/6M03.\n\nLigands were obtained from the Zinc Natural Products database (http://zinc.docking.org/).\n\nZenodo: Molecular Docking ZINC DB. https://doi.org/10.5281/zenodo.40505769.\n\nThis project contains the following extended data:\n\nDocking results for thee 100 best molecules (XLSX).\n\nInteraction_images_viral_protein_molecules (subfolder). (Interaction images of the best 42 molecules.)\n\nZinc_natural_database_pdbqt_minimized (subfolder). (Molecular docking results for 1100 small molecules from the ZINC Natural Products database.)\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CCBY 4.0).", "appendix": "Acknowledgements\n\nThe authors would like to acknowledge the ITI research group members for their valuable feedback.\n\n\nReferences\n\nChowdhury MS, Rathod J, Gernsheimer J: A Rapid Systematic Review of Clinical Trials Utilizing Chloroquine and Hydroxychloroquine as a Treatment for COVID-19. Acad Emerg Med. 2020; 27(6): 493–504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVermund SH, Pitzer VE: Asymptomatic transmission and the infection fatality risk for COVID-19: Implications for school reopening. Clin Infect Dis. 2020; ciaa855. 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PubMed Abstract \| Publisher Full Text\n\nNiu T, Zhao L, Lin X, et al.: Cinobufagin, a bufadienolide from traditional Chinese medicine Bufo bufo gargarizans CANTOR, inhibits PC3 cell growth in vitro and in vivo. Journal of Traditional Chinese Medical Sciences. 2019; 6(2): 175–183. Publisher Full Text\n\nPellegrini N, Valtueña S, Ardigò D, et al.: Intake of the plant lignans matairesinol, secoisolariciresinol, pinoresinol, and lariciresinol in relation to vascular inflammation and endothelial dysfunction in middle age-elderly men and post-menopausal women living in Northern Italy. Nutr Metab Cardiovasc Dis. 2010; 20(1): 64–71. PubMed Abstract \| Publisher Full Text\n\nChaher N, Arraki K, Dillinseger E, et al.: Bioactive stilbenes from Vitis vinifera grapevine shoots extracts. J Sci Food Agric. 2014; 94(5): 951–954. 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Publisher Full Text" }	[ { "id": "72545", "date": "12 Oct 2020", "name": "Pritha Ghosh", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Singh and Florez presents the discovery of drugs against COVID19 using molecular docking methods.\nMajor:\nIt might be worthwhile to re-check the binding affinity values for the top hits because all of the top 15 hits have a value of -8.8 kcal/mol. Does this mean that these drugs are equally effective and can be used interchangeably?\n\nThe authors have also not discussed the importance of the residues Tyr237, Leu272, Leu287 etc. Are these known to be critical residues in the functioning of the main protease?\nMinor:\nPlease use a uniform nomenclature (COVID 19 or COVID-19).\n\nThe manuscript needs to be proofread by a proficient English speaker.\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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "72544", "date": "23 Oct 2020", "name": "Maria Florencia Pollo-Cattaneo", "expertise": [ "Reviewer Expertise Artificial IntelligenceIntelligent Systems" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 proposes a study for the search of drugs to combat COVID-19. This search will be carried out by virtually screened the Zinc natural database using HTVS tool through molecular docking studies to analyze molecules recommended for the treatment of COVID-19.\nThe methodology used is consistent with the objective and the results are promising.\nResearchers are encouraged to continue with the proposed line and advance their research.\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/9-1203
https://f1000research.com/articles/9-379/v1	19 May 20	{ "type": "Research Article", "title": "Evaluation of antimicrobial, antioxidant and cytotoxic properties of bioactive compounds produced from endophytic fungi of Himalayan yew (Taxus wallichiana) in Nepal", "authors": [ "Dhurva Prasad Gauchan", "Pratistha Kandel", "Astha Tuladhar", "Ashesh Acharya", "Upendra Kadel", "Aayush Baral", "Arjan Bir Shahi", "María Rosario García-Gil", "Pratistha Kandel", "Astha Tuladhar", "Ashesh Acharya", "Upendra Kadel", "Aayush Baral", "Arjan Bir Shahi", "María Rosario García-Gil" ], "abstract": "Background: Endophytic fungi are largely underexplored in the discovery of natural bioactive products though being rich sources of novel compounds with promising pharmaceutical potential. In this study, Taxus wallichiana, which has huge medicinal value, was investigated for its endophytic diversity and capability to produce bioactive secondary metabolites by analyzing antioxidant, antimicrobial and cytotoxic properties. Methods: The endophytes were identified by ITS-PCR using genomic DNA samples. The secondary metabolites were extracted by solvent extraction method using ethyl acetate. The antioxidant activity was analyzed by Thin Layer Chromatography, Total Phenol Content (TPC), Total Flavonoid Content (TFC) and DPPH assay, and the antimicrobial activity was analyzed by agar-well diffusion method. Brine shrimp lethality assay was used to analyze the cytotoxicity of the fungal extracts. Results: Out of 16 different Taxus trees sampled from different locations of Dhorpatan, 13 distinctive endophytic fungi were isolated and grouped into 9 different genera: Bjerkandera, Trichoderma, Preussia, Botrytis, Arthrinium, Alternaria, Cladosporium, Sporormiella and Daldinia. The ethyl acetate extracts isolated from three endophytic fungi: Alternaria alternata, Cladosporium cladosporioides and Alternaria brassicae showed significant TPC values of 204±6.144, 312.3±2.147 and 152.7±4.958µg GAE/mg of dry extract, respectively, and TFC values of 177.9±2.911, 644.1±4.202 and 96.38±3.851µg RE/mg of dry extract, respectively. Furthermore, these three extracts showed a dose dependent radical scavenging activity with IC50 concentration of 22.85, 22.15 and 23.001 µg/ml, respectively. The extracts of C. cladosporioides and A. brassicae also showed promising antimicrobial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis with a minimum inhibitory concentration of 250μg/ml for all bacteria. Both the samples showed cytotoxic property against shrimp nauplii with LC50 of 104.2 and 125.9µg/ml, respectively. Conclusions: The crude fungal extracts obtained from endophytes: A. alternata, C. cladosporioides and A. brassicae upon purification and further identification of the bioactive compounds can be a fascinating source for novel pharmaceutical agents.", "keywords": [ "Taxus wallichiana", "Endophytic", "DPPH", "GAE", "RE", "cytotoxic" ], "content": "Introduction\n\nTaxus wallichiana Zucc. (Himalayan yew) is an evergreen coniferous tree (10 to 28 meters in height), native to the Himalaya and parts of southeast Asia that grows on steep, moist mountain slopes at altitudes of about 2000m to 3500m above sea level. It has flat and dark green leaves, which are arranged spirally on the stem1. The leaves and bark of Taxus species are the primary source of the chemotherapeutic drug Paclitaxel used in the treatment of breast and ovarian cancer, resulting in it receiving huge attention worldwide2. The Himalayan yew also has an exceptional history of its application in the traditional system of medicine for the treatment of fever and painful inflammatory conditions. It is also consumed as decoctions, herbal tea, and juice for treating cold, cough, respiratory infections, indigestion, and epilepsy and also on infected wounds and burns as poultice1,3. Some research indicates various medicinal properties of T. wallichiana, such as analgesic, antipyretic, anti-inflammatory, immunomodulatory, antibacterial, antifungal, antiplatelet, antispasmodic activities antiallergic, anticonvulsant, antiosteoporotic, and vasorelaxing effect3,4,5,6. Due to the persistent over-exploitation of Himalayan yew for its leaves and bark, it is now on the verge of extinction and classified as endangered by the International Union for Conservation of Nature (IUCN). Alternative measures to utilize the medicinal components of Taxus while also addressing the vexing problem of its mass exploitation are needed.\n\nPlants growing in unique environmental conditions or strange locations and having ethnobotanical uses generally harbour novel endophytic microorganisms7. Endophytes are microbial species that colonize the inner tissues of higher plants at some time in their life cycles without causing apparent damage or producing external structures8,9. There is a symbiotic interaction between endophytes and their host plants that may result in mutualistic benefits for both partners, such that these endophytes may provide protection and survival conditions for the plants by producing a myriad of bioactive substances and in return obtain carbon for energy. These bioactive substances exhibit important properties, such as antioxidant, anticancer, antimicrobial, cytotoxic, immunomodulatory, antiviral, anti-parasitic and insecticidal properties, which when isolated and characterized, may also have the potential for use in industry, agriculture, and medicine10–12. The majority of endophytic fungi possess biosynthetic capabilities similar to the associated host because of their long history of co-evolution and genetic recombination.\n\nThe development of resistance by pathogenic microorganisms to available drugs is a growing problem, which leads to an immediate need for an intensive search for newer and effective antimicrobial agents. Many endophytic fungi have the ability to produce antimicrobial substances13. Our body continuously produces reactive oxygen species (ROS) as by-products of aerobic metabolism14. Excessive production of these ROS can damage our cells, tissues and organs and can inhibit the normal function of DNA. These damages can in turn cause various diseases such as cancer, male factor infertility, heart disease and Alzheimer’s disease14–16. Fungi can potentially produce a wide variety of antioxidants such as alkaloids, flavonoids, phenols, steroids, etc. that can scavenge the excessive ROS produced in our body17.\n\nThough sufficient research has shown that endophytic fungi harboring T. wallichiana have the capability to produce Taxol, to our knowledge, no reports are available on antioxidant, antimicrobial and cytotoxic properties of these endophytes. This study focuses on investigating T. wallichiana for its endophytic fungal diversity and capability of these fungal endophytes to produce bioactive secondary metabolites having antimicrobial, antioxidant and cytotoxic properties. This study aims to establish endophytic fungi as sustainable natural reservoirs for novel drug discovery useful in medicine, food and agriculture industries.\n\n\nMethods\n\nBark and twigs with leaf needles of 16 different T. wallichiana trees were collected from the periphery of Dhorpatan Hunting Reserve using the simple random sampling method in the month of May, 2018. Dhorpatan is a village in Nepal's Baglung District at 2900m elevation. The collected samples were placed in sterile polyethylene bags. The samples were first verified with consultation of the local people under the vernacular name 'Lauth Salla' and then from the District Forest Office, Baglung. Bark and leaf samples collected are shown in Figure 1.\n\n(A) Twigs with leaf needles of T. wallichiana. (B) Bark of T. wallichiana.\n\nBark and needles were cut and washed thoroughly under running tap water to remove the dirt and debris. The pieces of bark and leaves were treated with 70% ethanol (v/v) for 1 minute and 0.1% mercuric chloride (v/v) for 2 minutes and rinsed twice with sterile distilled water. For bark, the outer layer was removed by a sharp, sterilized blade to expose the inner layer. The inner pinkish-white layer was placed on the surface of potato dextrose agar (PDA) media. For needles, the individual needle was cut from the midrib and also from edges by a sterile sharp blade and placed on the surface of PDA media exposing the inner tissues. In another method, both bark and needles were ground into a paste with 2 ml of sterilized water using mortar and pestle separately under aseptic conditions. 1ml of bark paste and 1 ml of paste was poured on Petri plates and molten PDA media was poured into each plate. PDA plates were then incubated at 25±2°C for 10 days to allow the growth of endophytic fungi. Pure culture of fungal endophytic isolates was obtained by hyphal-tip transfer to fresh PDA media.\n\nMorphological screening. Microscopic characterization of the isolated endophytic fungi was carried out using the tape lift mount method using lactophenol cotton blue as dye and observed under 40X, 100X and 400X magnification in a compound microscope and then photographs were captured by a digital camera. Shape, color, margin, elevation, spore structure and other morphological characteristics of the endophytic isolates were observed.\n\nExtraction of secondary metabolites. Pieces of agar blocks (10 mm) of the mycelial mats from an actively growing fungal culture was inoculated into 250 ml Erlenmeyer flasks containing 100 ml potato dextrose broth media using a sterile borer. Flasks were then incubated at 150 rpm at 25±2°C for 10 days in a shaking incubator. The entire fermented broth was then filtered through filter paper. An equal volume of ethyl acetate was added to the filtrate broth and vigorously shaken for 5 minutes. The mixture was transferred to a separating funnel and the organic layer of ethyl acetate was allowed to separate from the aqueous layer. The ethyl acetate fraction, which comprised of the upper organic layer in the separating funnel, was then transferred to a new flask and allowed to dry at room temperature using an evaporator. The dried extract obtained from each fungal sample was weighed and stored at 4°C for future use.\n\nMolecular screening and phylogenetic analysis. Genomic DNA of endophytic fungi was isolated using Quick-DNA™ Fungal/Bacterial Miniprep Kit from Zymo Research, as per the manufacturer’s instructions. The obtained pure DNA was run on 0.8% agarose gel electrophoresis and the bands were observed. The isolated DNA was used as a template to amplify the ITS regions together with the 5.8s rRNA gene (ITS 1-5.8S-ITS2 rDNA) through PCR using universal primers ITS1 (5'TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). PCR was performed using all reagents from Thermofisher, in a 25µl reaction mixture using 5 µl template DNA, 0.25 µl of 1.25 U Taq DNA polymerase, 2.5 µl of 10X Amplification buffer, 0.8µl of 10mM dNTP, 0.75 µl of 50mM MgCl2, 13.2 µl PCR grade water, 1.25µl of 10μM each of forward and reverse primer. PCR amplification was carried out in BioRad300 thermocycler. The thermo-cycling conditions involved an initial denaturation at 94°C for 3mins, 37 cycles of 30 secs at 94°C, 30 secs at 55°C, 1 min at 72°C and a final extension at 72°C for 5 mins. The PCR amplification products were analyzed by gel electrophoresis on 2% agarose (w/v). 100 bp DNA ladder from Thermo-fisher was used. The remaining volume was sent for sequencing to Macrogen, Korea.\n\nThe ITS sequences of fungal isolates were subjected to BLASTn sequence homology search and compared with data in the National Centre for Biotechnology Information (NCBI). Multiple sequence alignment of related species was performed using CLUSTAL W. Phylogenetic analysis was carried out by Maximum Likelihood (ML) method based on the Tamura-Nei model18 using MEGA X software. Initial tree(s) for the heuristic search were obtained by Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value19. One thousand bootstrap replicates were analyzed to assess reliable nodal support values.\n\nBenzene: Chloroform: Acetone: Methanol (20:92.5:15:7.5) was used as the solvent system. The sample was prepared by dissolving the fungal extracts in ethyl acetate. TLC was carried out on 20×20 alumina TLC plate. Ascorbic acid, Rutin, Quercetin, and Gallic acid were used as standards. The samples were loaded equidistantly onto the TLC plate and allowed to air dry. The TLC plate was allowed to run in the TLC chamber containing the mobile phase. The TLC plate was then air-dried and sprayed with 10% AlCl3 solution followed by 0.04% DPPH solution.\n\nDPPH radical scavenging assay. DPPH radical scavenging assay was performed as proposed by Makris et al. with minor modifications20. DPPH reagent (0.04 mg/ml) was prepared in methanol. Ascorbic acid (Vitamin C) was used as a positive control. Different concentration of ascorbic acid as well as fungal extracts were made (2, 4, 6, 8, 10, 12, 14, 16, and 18µg/ml) from 1mg/ml stock solution. These samples were mixed with DPPH reagent in the ratio 1:1 and incubated at 27°C in the dark for 30 minutes. The radical scavenging activity of the samples against DPPH free radical was determined by measuring the UV absorbance at 517 nm against the blank. The mixture of 1 ml DPPH solution and 1 ml blank methanol was used as control. The radical scavenging activity of the standard (Ascorbic acid), as well as the samples (fungal extracts), were calculated using the formula:\n\nDPPH radical scavenging activity (%) = (Abs control – Abs sample)/Abs control x 100\n\nWhere Abs control is the absorbance of control and Abs sample is absorbance of test samples. From calibration curves obtained from different concentrations of the extracts and ascorbic acid, the concentration of sample required to scavenge 50% of DPPH free radicals (50% inhibition concentration, IC50) was determined.\n\nTotal phenol content (TPC). The TPC of the fungal endophytes was determined using the Folin-Ciocalteu method described by Lin and Tang with minor modifications21. Each extract solution (0.5 mg/ml, 1 ml) was diluted to 5 ml deionized water in a 25 ml test tube. 0.25 ml volume of Folin-Ciocalteu reagent and 5 ml of 7.5% Sodium carbonate (Na2CO3, w/v) were added. This mixture was incubated at 40°C for 60 mins, diluted with deionized water up to 12.5 ml total volume and mixed. The absorbance was measured at 733 nm against the blank on a spectrophotometer. The TPC was calculated on the basis of the Gallic acid calibration curve. The results were expressed as μg of Gallic acid equivalents (GAEs) per mg of dry extract.\n\nTotal flavonoid content (TFC). The TFC was determined using NaNO2-Al(NO3)3-NaOH colorimetric assay described by Zhu et al. with minor modifications22. Each extract solution (0.5 mg/ml, 1 ml) and 2 ml of 60% alcoholic solution (v/v) were accurately added to a 25 ml test tube and 0.5 ml of NaNO2 solution (5% w/v) was added. The mixture was shaken and left to stand for 6 mins. 0.5 ml of the Al(NO3)3 (10% w/v) solution was added. After 6 mins, 5 ml of NaOH solution (10% w/v) was added, followed by dilution with deionized water up to 12.5 ml volume. The solution was mixed and incubated at room temperature for 15 mins. The reaction mixture absorbance was measured at 507nm against the blank in the spectrophotometer. The TFC was calculated on the basis of the calibration curve of Rutin. The results were expressed as μg of Rutin Equivalents (RAEs) per mg of dry extract.\n\nAgar-well diffusion method. The antimicrobial activity of the fungal extracts was determined using the agar-well diffusion method. A gram-negative bacterial strain: Escherichia coli (ATCC 25922), and two gram-positive bacterial strains: Staphylococcus aureus (ATCC 12600) and Bacillus subtilis (ATCC6633) were used as indicator organisms. The dried extracts of each fungal isolate were dissolved in dimethyl-sulphoxide (DMSO). The microbial cultures were spread evenly using L-shaped spreader. Wells of 6 mm diameter were made on the plates using a sterile borer. 30µl of 7mg/ml of fungal extract dissolved in DMSO was added to the well. DMSO was used as negative control and Gentamycin (10μg disc) was used as a positive control. The plates were incubated at 37±2°C overnight. The zones of inhibition were observed and measured.\n\nDetermination of minimum inhibitory concentration (MIC). 2mg/ml stock solution of fungal extract was diluted into concentrations of 1, 500, 250, 125, 62.5, 31.25, 15.625, 31.25, 15.625, 7.81, 3.9 and 1.95μg/ml. Each concentration of extract was mixed with Muller Hinton Broth (MHB) in equal volume to make a total volume of 500µl. 500μl of E. coli culture was added to the test tubes to make a final volume of 1 ml. Kanamycin sulphate was also diluted into concentrations of 1, 500, 250, 125, 62.5, 31.25, 15.625, 31.25, 15.625, 7.81, 3.9 and 1.95 μg/ml and used as positive control. Each concentration of kanamycin sulphate was mixed with MHB in equal volume to make a total volume of 500µl. 500μl of E. coli culture was added to the test tubes to make a final volume of 1 ml. Control 11, which consisted of media, DMSO and E. coli culture, was taken as negative control and Control 12, which consisted of media and DMSO only, was taken to test the sterility of the process. All test tubes were incubated at 37°C overnight. The MIC of fungal extracts were determined by observing the turbidity and clarity of the test tubes.\n\nCytotoxicity assay by using brine shrimp. Brine shrimps (Artemia salina) were hatched using brine shrimp eggs in a vessel filled with simulated sterile artificial seawater (brine solution). 38 g of sea salt, sodium chloride was dissolved in 1000 mL of distilled water and the pH was adjusted to 8.5 using 1N NaOH under constant aeration for 48 hours. The active shrimps were collected and used for the assay23. Samples were prepared by dissolving crude fungal extracts in 1% aqueous DMSO to give stock solutions. From the stock, five different concentrations: 1,000, 500, 250, 125, and 62.5 ppm were prepared in seawater. An aliquot of each concentration (0.5ml) was transferred in triplicate into sterile vials, and aerated seawater (4.5 ml) was added. Ten shrimp nauplii were transferred to each vial. Doxorubicin 1% aqueous solution and 1% DMSO in seawater were used as positive and negative controls, respectively. After 24 hrs, the numbers of live nauplii were counted and the percentage of death was calculated. The concentration that killed 50% of the nauplii (LC50 in μg/mL) was determined. Criterion of toxicity for fractions was established i.e. LC50 values > 1000 μg/ml (non-toxic), ≥ 500 and ≤ 1000 μg/ml (low toxicity), and < 500 μg/ml (toxic)24.\n\n\nResults and Discussion\n\nBark and leaf samples from 16 different trees of T. wallichiana were collected. The information regarding the names of plant samples, geographical features of the location and the girth of the tree from which samples were collected are given in Table 1.\n\nOut of the 16 bark and leaf samples collected, 13 endophytic fungal isolates with different morphological features were obtained. The name codes given to the individual plant samples and their respective fungal isolates are shown in Table 2. The pure fugal isolates obtained are shown in Figure 2 and the microscopic view of all the fungal isolates are shown in Figure 3.\n\nGB: Ground Bark; IB: Intact Bark; GL: Ground Leaves\n\nPure culture of 13 fungal endophytic isolates (DOR-1 to DOR-13) obtained from bark and leaves of Taxus wallichiana by hyphal-tip transfer to fresh potato dextrose agar media.\n\nMicroscopic characterization of the isolated endophytic fungi was carried out using tape lift mount method using lactophenol cotton blue and viewed under 400x magnification in a compound microscope.\n\nPCR amplification products of ITS regions together with the 5.8s rRNA gene (ITS 1-5.8S-ITS2 rDNA) were analyzed by gel electrophoresis on 2% agarose (w/v) as shown in Figure 4.\n\n500-600bp sized bands observed after PCR amplification of the ITS gene of fungal samples D0R-1 to DOR-13.The PCR amplification products were analyzed by gel electrophoresis on 2% agarose (w/v) using 100bp ladder from Thermo-fisher. -C is negative control.\n\nThe fungal isolates DOR-1 to DOR-13 were subjected to molecular identification based on the ITS rDNA sequence analysis. The sequences are deposited in GenBank of NCBI with accession numbers MN696546 to MN696558. The details of putative taxonomic affinities are listed in Table 3. The phylogenetic tree of fungal isolates is shown in Figure 5. The 13 identified isolates were grouped into 9 genera: Bjerkandera, Trichoderma, Preussia, Botrytis, Arthrinium, Alternaria, Cladosporium, Sporormiella and Daldinia. Bjerkandera adusta belonged to phylum Basidiomycota while all others belonged to phylum Ascomycota. Sporormiella showed the highest colonization frequency accounting for 23% followed by Alternaria and Trichoderma with 15.4% each and the rest showed 7.7% each of colonization frequencies.\n\nThe tree with the highest log likelihood (-4362.92) is shown. Multiple sequence alignment of related species was performed using ClustalW. Phylogenetic analysis was carried out by Maximum Likelihood method using MEGA X software. Initial tree(s) for the heuristic search were obtained by Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach.\n\nAscorbic acid, Rutin, Quercetin, and Gallic acid were used as standards. These standards produced yellowish bands on the TLC plate. 10µl with 10mg/ml concentration of fungal extracts were loaded to the plate. The extracts producing yellow bands on the purple background were considered as antioxidants. The purple background color was visualized after spraying the plate with 0.4%2,2-diphenyl-1-picrylhydrazy (DPPH) reagent. Of all the 13 extracts, five of them: DOR-4, DOR-6, DOR-7, DOR-9, and DOR-13 showed clearer yellow bands compared to other samples, hence indicating good DPPH radical scavenging activity (Figure 6). The purple background was due to the presence of DPPH radical while the yellowish bands were formed due to the reduction of the DPPH radical. The yellowish bands indicate the presence of antioxidant compounds that can scavenge DPPH radical by donating an electron/hydrogen atom25. The difference in the band intensities of developed yellow color could be due to the varied differences in the amount as well as the chemical characteristics of the compound present in of each extract.\n\n20×20 alumina TLC plate showing yellow bands of all fungal extracts and standards: Ascorbic acid (A), Rutin (R), Quercitin (Q) and Gallic acid (G) after spraying with 10% AlCl3 solution followed by 0.04% DPPH solution.\n\nPhenols are basically secondary metabolites of plants or fungi formed against predation and protection. Nearly 8000 phenolics are known to exist, encompassing a wide range of molecules which can give up hydrogen atoms from their hydroxyl groups to radicals and form stable phenoxy radicals, hence playing an important role in antioxidant activity by reducing oxidative stress and the risks of cardiovascular diseases, osteoporosis as well as neurodegenerative diseases26. Several methods for the determination of TPC have been introduced because of the heterogeneous nature of natural phenols. In this study, TPC was estimated using a modified Folin-Ciocalteu method.\n\nA blue-colored solution was observed due to the presence of phospomolybdic phosphotungstic-phenol complex when the fungal extracts reacted with the Folin-Ciocalteu reagent in alkaline medium. The phenolic content was calculated from the regression equation of the calibration curve (y=0.007702x+0.1682, R2=0.9933), expressed in μg Gallic Acid Equivalent (GAE) per mg of dried extract which is shown in Table 4. The total TPC of the samples showed large variations, with significant values of three samples: DOR-6, DOR-7 and DOR-13 with 204±6.144, 312.3±2.147 and 152.7±4.958µg GAE/mg of dry extract, respectively. The calibration curve of Gallic acid and the GAE/mg value of fungal extracts showing high phenolic contents are shown in Figure 7 and Figure 8, respectively.\n\nValues are means of three biological replicates (n =3); GAE: Gallic Acid Equivalent\n\nRegression equation of Gallic acid y=0.007702x+0.1682, R2=0.9933 was obtained using the calibration curve. Error bars represent standard deviation.\n\nThe phenolic content was calculated from the regression equation of the calibration curve (y=0.007702x+0.1682, R2=0.9933), expressed in μg GAE per mg of dried extract. Error bars in the graph represents standard deviation.\n\nFlavonoids include flavones, flavonols and condensed tannins that are secondary metabolites, whose antioxidant activity depends on the presence of free OH groups, especially 3-OH27. Flavonoids are known to inhibit formation of reactive oxygen species, chelate trace elements involved in free-radical production, scavenge such reactive species and protect in antioxidant defenses28. The flavonoids are reported to exhibit antioxidative, anticarcinogenic, anti-inflammatory, anti-aggregatory property and vasodilating effects29. The TFC of the endophytic fungal extracts was estimated by NaNO2-Al(NO3)3-NaOH colorimetric method.\n\nThe flavonoid content was calculated from the regression equation of the calibration curve (y=0.004294x+0.09578, R2=0.999), expressed in μg Rutin Equivalent (RE) per mg of dried extract, which is shown in Table 5. The TFC of the samples also showed large variations with significant values of three samples: DOR-6, DOR-7 and DOR-13 with values of177.9±2.911, 644.1±4.202 and 96.38±3.851µg RE/mg of dry extract, respectively. The calibration curve of Rutin and the RE/mg value of fungal extracts showing high flavonoid contents are shown in Figure 9 and Figure 10, respectively.\n\nValues are means of three biological replicates (n =3); RE: Rutin Equivalent\n\nRegression equation y=0.004294x+0.09578, R2=0.9989 of Rutin was obtained using the above calibration curve. Error bars represent standard deviation.\n\nThe flavonoid content was calculated from the regression equation of the calibration curve (y=0.004294x+0.09578, R2=0.9989), expressed in μg RE per mg of dried extract. Error bars in the graph represents standard deviation.\n\nDPPH assay is a widely used method to evaluate the antioxidant ability of extracts. It measures the ability of compounds to act as hydrogen donors or free radical scavengers30,31. Various types of antioxidant compounds may be produced by fungal endophytes including multiple flavonoid compounds, saponin, phenols, sterols, terpenoids, etc., which may remove DPPH free radicals25. These antioxidant compounds have important health effects such as reducing the risk of cancer, heart disease, and neuro-degenerative disorders that are found in abundant amounts in plants, vegetables, fruits, and natural products32. DPPH is a stable nitrogen-centered free radical that can readily undergo scavenging by antioxidants33. DPPH gives rise to the deep violet color, with maximum absorption at a wavelength of 517 nm. DPPH reduces to diphenyl picrylhydrazine after the odd electron of a nitrogen atom in DPPH receives an electron or hydrogen atom from antioxidants and changes its color from the deep violet to pale yellow34. As antioxidant donates a proton to this radical, the absorbance decreases due to the disappearance of DPPH radical.\n\nAmong the 13 fungal extracts, three of the extracts, DOR-6, DOR-7, and DOR-13, showed the highest radical scavenging activity. The radical scavenging activity of Ascorbic acid as well as the fungal extracts DOR-6, DOR-7 and DOR-13 were found to be dose dependent (Table 6 and Figure 11). IC50 values of these fungal extracts and ascorbic acid was calculated from the equation obtained from linear fitting curve as shown in Figure 12. The fungal extracts DOR-6, DOR-7 and DOR-13 showed a significant radical scavenging activity with a 50% inhibition (IC50) at a concentration of 22.85, 22.15 and 23.001 µg/ml as compared with standard ascorbic acid that showed 50% inhibition at a concentration of 8.86µg/ml.\n\nValues are means of three biological replicates (n =3).\n\nComparison of % DPPH radical scavenging activity of selected fungal extracts DOR-6, DOR-7, and DOR-13 with the standard antioxidant Ascorbic acid at different concentrations (2, 4, 6, 8, 10, 12, 14, 16, and 18µg/ml). The radical scavenging activity of the standard (Ascorbic acid), as well as the EA fungal extracts, were calculated using the formula: DPPH radical scavenging activity (%) = (Abs. control – Abs. sample)/Abs. control x 100, where Abs. control is the absorbance of DPPH radical solution without samples.\n\n(A) Comparison of 50% inhibition concentration of fungal extract DOR-6 with the standard antioxidant Ascorbic acid. The IC50 value of standard ascorbic acid, fungal extract DOR-6 was determined from equations y=5.0908x+4.8973, y=2.3311x–3.2573 respectively. (B) Comparison of 50% inhibition concentration of fungal extract DOR-7 with the standard antioxidant Ascorbic acid. The IC50 value of standard ascorbic acid, fungal extract DOR-7 was determined from the equations y=5.0908x+4.8973, y=2.1271x+2.8799 respectively. (C) Comparison of 50% inhibition concentration of fungal extract DOR-7 with the standard antioxidant Ascorbic acid. The IC50 value of standard ascorbic acid, fungal extract DOR-7 was determined from the equations y=5.0908x+4.8973, y=2.29035x–2.6812 respectively.\n\nEndophytic fungi have the ability to produce a large variety of bioactive compounds that can protect the plants against various pathogens7,35. The screening of all the fungal extracts was conducted using agar well diffusion method. 20 mg/ml concentration of the sample was used. Gentamycin was used as positive control and 10% DMSO was used as a negative control. Among the 13 fungal extracts three of the extracts: DOR-6, DOR-7, and DOR-13 showed the highest antimicrobial activity during primary screening against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 12600) and were selected for secondary screening.\n\nDuring the secondary screening, the antimicrobial activity of these three fungal extracts was appraised on three bacterial strains: Gram-positive bacteria- Staphylococcus aureus (ATCC 12600), and Bacillus subtilis (ATCC 6633), and a Gram-negative bacterium: Escherichia coli (ATCC 25922) (Figure 13). DOR-6 showed inhibition diameter of 9.333±0.577, 7.33±0.557 and 6.5±.866mm, DOR-7 showed inhibition diameter of 14.33±0.577, 10.33±0.577 and 10±0.5mm, and DOR-13 showed inhibition diameter of 10.167±0.764, 8±0 and 8±0.5mm against E. coli, S. aureus and B. subtilis, respectively (Table 7). MIC value of these three extracts was further assessed. Positive control Gentamycin showed 19.333±0.577, 21.167±0.764 and 17.667±0.557mm inhibition diameter against E. coli, S. aureus and B. subtilis. DOR-7 and DOR-13 showed good antimicrobial activity, hence were selected for determination of MIC. The figure exhibiting the comparison of zone of inhibition of fungal extracts DOR-6, DOR-7 and DOR-13 with negative and positive control is shown in Figure 14 and Figure 15, respectively.\n\nValues are expressed as Mean±SD.\n\nAntimicrobial activity of fungal extracts DOR-6, DOR-7 and DOR-13 was determined using a gram-negative bacterial strain: Escherichia coli (ATCC 25922), and two gram-positive bacterial strains: Staphylococcus aureus (ATCC 12600) and Bacillus subtilis (ATCC6633) with 10 µg Gentamycin disc, as a positive control and DMSO as negative control.\n\nComparison of mean zones of inhibition measurements (mm) of fungal crude extracts (DOR-6, DOR-7, and DOR-13) with DMSO as a negative control (NC) for bacterial test pathogens. Error bars represent standard deviation. An asterisk (*) indicates a significant difference in mean zones of inhibition between fungal crude extracts and negative control observed for each test pathogen (compared using Dunnett’s test, P < 0.05). If no inhibition was seen, a value of 6 was assigned, which was the diameter of the agar-well used.\n\nComparison of mean zones of inhibition measurements (mm) of fungal crude extracts (DOR-6, DOR-7 and DOR-13) with antibiotic - 10 µg Gentamycin disc, as a positive control (PC) for bacterial test pathogens. Error bars represent standard deviation. An asterisk indicates significant difference in mean zones of inhibition between positive control and fungal crude extracts observed for each test pathogen (compared using Dunnett’s test, P < 0.05). The less potency of the fungal extract in comparison to antibiotic disc of Gentamycin can be attributed to the crude nature of the extracts used. If no inhibition was seen, a value of 6 was assigned, which was the diameter of the agar-well used.\n\nCrude extracts showing potent antimicrobial activity was further examined for MIC by tube dilution technique. The maximum inhibition diameter was shown by fungal extracts DOR-7 and DOR- 13 in E. coli. So, the minimum inhibitory concentration of these two extracts was assessed using E. coli as an indicator organism. Kanamycin sulphate was used as positive control.\n\nThe microorganisms were subjected to a range of antibiotic concentrations: 1, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, and 1.9531µg/ml. Serial dilution of the fungal extracts was performed in the same concentration range between each successive test tube. The macroscopic inhibition of growth was measured by observing the absence of turbidity in the medium (Figure 16). The MIC of samples DOR-7, as well as DOR-13, was determined to be 250 µg/ml. It means the concentration of a sample less than 250 µg/ml showed growth of bacteria. The MIC of kanamycin sulphate was 15.625µg/ml, i.e. bacterial growth was observed only in those test tubes containing antibiotic concentration less than 15.625µg/ml. The results of the present study suggest that 250 µg/ml of fungal extracts DOR-7 and DOR-13 consists of bioactive compounds equivalent to 15.625µg/ml kanamycin sulphate. These bioactive compounds with antibacterial property might be the potential source for the development of antimicrobial compounds.\n\nMIC of kanamycin sulfate was observed at K-7 i.e. at 15.625µg/ml concentration. MIC of extract DOR-7 and DOR-13 observed at DOR-7-3 (250µg/ml) and DOR-13-3(250µg/ml) respectively. 11 and 12 are negative controls and 1 and 10 represent the highest and lowest concentration, i.e. 1mg/ml and 1.9531ug/ml, respectively.\n\nBrine shrimp (Artemia salina Leach) is an invertebrate inhabiting saline aquatic and marine ecosystem. It can be used in a laboratory bioassay in order to determine the toxicity of plants by the estimation of the medium lethality concentration LC5036. In this study, cytotoxicity test was done by brine shrimp bioassay method. We calculated the cytotoxicity of our fungal extracts by their determining their ability to kill the shrimp nauplii in 24 hours. The lethal concentration required to kill half the population (LC50) was calculated. 1% doxorubicin and 1% DMSO were used as positive and negative control respectively. Death percentage of nauplii in 24 hours was calculated and is shown in Table 8. A graph of a log of extract concentration in ppm versus death percentage was plotted (Figure 17).\n\nThe cytotoxicity of the fungal extracts at different concentrations: 1,000, 500, 250, 125, and 62.5 ppm was determined by their ability to kill the shrimp nauplii in 24 hours. The lethal concentration required to kill half the population (LC50) was calculated. 1% doxorubicin and 1% DMSO were used as positive and negative control respectively.\n\nLC50 of extracts DOR-7 and DOR-13 were 104.2 ppm and 125.9 ppm, respectively. In our study, both extracts showed a low value of LC50 (<200ug/ml), which is an indication of the presence of the potent cytotoxic compound. Throughout our experiment, the shrimp nauplii did not receive food; therefore the death of the nauplii can be considered either due to the effect of our bioactive fungal extract or because of starvation. However, any hatched nauplii still feed on their yolk-sac, they can survive for up to 48 hours without food37. In addition, we had also used a control sample containing only nauplii for ensuring the mortality effect of our fungal extract. Since brine shrimp lethality assay is a primary assay to detect cytotoxic property, further experiments are required to establish the cytotoxicity of the fungal extracts against cell lines.\n\n\nConclusions\n\nEndophytic fungi are largely underexplored in the discovery of natural products. To date, only a few plants are investigated for their endophytic biodiversity and their potential to produce bioactive secondary metabolites. Nepal has an abundance of medicinal plants, so this research helps shed a light in the possibilities of discovering novel compounds with pharmaceutical potential from a variety of endophytic fungi residing within them. Our research primarily concludes Himalayan yew to be a promising reservoir for large endophytic diversity with the capability to produce natural compounds showing significant antioxidant, antimicrobial and cytotoxic properties. A total of 9 different genera of fungi were isolated from 16 Taxus samples, which further suggests that this diversity could offer an abundant source of varied novel metabolites. The fungal extracts isolated from three endophytic fungi, Alternaria alternata, Cladosporium cladosporioides and Alternaria brassicae, showed significant bioactivity. These crude fungal extracts upon purification and further identification of the bioactive compounds can be a fascinating source for novel pharmaceutical agents. Although these compounds are produced in small quantities in nature, these promising endophytes can be further employed to fast growth and amplification through isolation, genetic manipulation, and industrial scale-up on an industrial level. Moreover, the utilization of endophytes as alternative sources of bioactive compounds also eliminates the exploitation of the host plant, hence conserving biodiversity.\n\n\nData availability\n\nSequences of fungal isolates (DOR-1 to DOR-13) are deposited in GenBank of NCBI with accession numbers MN696546 to MN696558. See Table 3 for a full list of accession numbers.\n\nOpen Science Framework: Evaluation of antimicrobial, antioxidant and cytotoxic properties of bioactive compounds produced from endophytic fungi of Himalayan Yew (Taxus wallichiana) in Nepal, https://doi.org/10.17605/OSF.IO/73PAX38.\n\nThis project contains the following underlying data:\n\n- Uncropped, unedited images for Figure 4 and Figure 6.\n\n- DPPH, TPC, TFC results\n\n- Agar well diffusion (Primary and secondary screening results)\n\n- Brine shrimp assay results\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nWe would like to acknowledge Department of Biotechnology, Kathmandu University for providing us laboratory facilities to carry out this research. 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Reference Source\n\nGauchan D: Evaluation of antimicrobial, antioxidant and cytotoxic properties of bioactive compounds produced from endophytic fungi of Himalayan yew (Taxus wallichiana) in Nepal. 2020. https://www.doi.org/10.17605/OSF.IO/73PAX" }	[ { "id": "63720", "date": "02 Jul 2020", "name": "Anaya Pokhrel", "expertise": [ "Reviewer Expertise Microbiology", "molecular biology", "and natural products." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “Evaluation of antimicrobial, antioxidant and cytotoxic properties of bioactive compounds produced from endophytic fungi of Himalayan yew (Taxus wallichiana) in Nepal” discusses isolation of endophytic fungi. Antimicrobial, antioxidant, and cytotoxic properties of bioactive compounds isolated from endophytic fungi is studied. The manuscript is clear, concise, and flows smoothly. However, there are minor corrections suggested before consideration of the manuscript for indexing.\n\nSuggestions\nIntroduction section\nSentence: Some research indicates various medicinal properties of T. wallichiana, such as analgesic, antipyretic, anti-inflammatory, immunomodulatory, antibacterial, antifungal, antiplatelet, antispasmodic activities antiallergic, anticonvulsant, antiosteoporotic, , and vasorelaxing effect3–6.\n\nAntispasmodic activities: remove activities in this sentence antiosteoporotic, ,: Remove one comma\nMethod section\n\nUnder the title “Molecular screening and phylogenetic analysis” in this sentence:\nThe obtained pure DNA was run on 0.8% agarose gel electrophoresis and the bands were observed\nSuggestions:\n“Pure DNA” is a claim not supported by evidence, please replace it with genomic DNA instead.\n\nReplace the word “run” with “analyzed using” and remove the part that says the bands were observed. This description belongs to the result section.\n\nIn the PCR amplification condition, please add the number of cycles to make the data reproducible.\n\nUnits for volume and time are not consistent. There is space right after the number in some cases while there is no space in some other. Please make it consistent.\nFor example in this section:\nPCR was performed using all reagents from Thermofisher, in a 25μl reaction mixture using 5 μl template DNA, 0.25 μl of 1.25 U Taq DNA polymerase, 2.5 μl of 10X Amplification buffer, 0.8μl of 10mM dNTP, 0.75 μl of 50mM MgCl2, 13.2 μl PCR grade water, 1.25μl of 10μM each of forward and reverse primer. PCR amplification was carried out in BioRad300 thermocycler. The thermo-cycling conditions involved an initial denaturation at 94°C for 3 mins, 37 cycles of 30 secs at 94°C, 30 secs at 55°C, 1 min at 72°C and a final extension at 72°C for 5 mins. The PCR amplification products were analyzed by gel electrophoresis on 2% agarose (w/v).\n\nInitial tree(s) for the heuristic search were obtained by Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value19.\n\nResult section\n\nIn the following sentence: Sporormiella showed the highest colonization frequency accounting for 23% followed by Alternaria and Trichoderma with 15.4% each and the rest showed 7.7% each of colonization frequencies.\n\nThe colonization frequencies are discussed. However, the colonization frequency is not discussed in the method section. Please describe the method used for the calculation of colonization frequency somewhere in the methods section.\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? Partly\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": [ { "c_id": "5968", "date": "05 Oct 2020", "name": "Dhurva P. Gauchan", "role": "Author Response", "response": "Dear Dr. Anaya Pokhrel, Thank you for your meticulous observation especially in the materials and methods section which will definitely improve the quality of the manuscript. We addressed all the issues in version 2 of the manuscript as suggested. All typographical corrections suggested in the introduction and method sections have been corrected accordingly. The number of PCR cycles was included in the original version. (The thermo-cycling conditions involved an initial denaturation at 94°C for 3 mins, 37 cycles of 30 secs at 94°C, 30 secs at 55°C, 1 min at 72°C and a final extension at 72°C for 5 mins.) The units and spacing in between are made consistent throughout the paper as suggested. We have also added the method for calculation of colonization frequency in the revised paper. Regards Dhurva Prasad Gauchan" } ] }, { "id": "70764", "date": "07 Sep 2020", "name": "Dipesh Dhakal", "expertise": [ "Reviewer Expertise Natural Product Biosynthesis", "Bioactivity of natural products as antibacterial", "anticancer", "anti-oxidant etc", "Engineering of natural products." ], "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 provides showcase about the diversified fungal consortium existing in Taxus wallichiana. Further they have isolated secondary metabolites and analyzed their antioxidant, antimicrobial and anticancer activities.The findings are very interesting to cater the biological importance of such potent fungus. However, the study has room for improvement.\nThe author can analyze the secondary metabolite profile by HPLC and GC, to determine whether they are already known molecule or entirely new molecules.\n\nThe author can perform MTT assay against few cancer cell lines and normal cell line, which will provide better insight on practical application of such metabolites.\n\nThe author showed that the extract has better activity against Gram negative strains, hence it would be interesting if author would add additional Gram negative bacterias as Pseudomonas or ESKAPE pathogens for their testing.\n\nDoes the author isolate entirely new species of fungus or they recovered already known species of fungus. In case the fungus are known, then the author can include some information about potential compounds from such fungal species.\n\nIn section \"Determination of MIC\", please correct 1000 instead of 1.\n\nIn section, Extraction of secondary metabolites, the author should mentioned that they weighed the extract hence the author should provide information about the total yield.\n\nI find the author have reiterated the datas on figures as well as table, hence they can just summarize in Figure and include the data in figure legend for e.g. Table 4 and Figure 8 can be integrated.\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? Partly\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?\nPartly\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": [ { "c_id": "5969", "date": "05 Oct 2020", "name": "Dhurva P. Gauchan", "role": "Author Response", "response": "Dear Dr. Dipesh Dhakal, Thank you for your comments. Your comments helped us to add a few points to make the manuscript clearer. We addressed all the issues in version 2 of the manuscript as suggested. The suggestion for the employment of HPLC/GC for identification of isolated bioactive compounds, MTT assay, and antimicrobial assay on other gram-negative bacteria on our research has been acknowledged and research being currently investigated adopting various parameters. Matter of fact, our next phase of the research focuses on the identification of bioactive compounds using HPLC/LCMS and its effect on various cancer cell lines doing MTT assay. At the advance level, we have planned to evaluate the effectiveness of bioactive compounds extending our experiments on the animal models which will help to develop anticancer drugs in near future. We recovered already identified species of fungi. Therefore, some information about bioactive compounds previously isolated and studied from the identified fungal species is added to the conclusion section as suggested. In the determination of the MIC section, the initial diluted concentration is changed from 1 to 1000 μg/ml. Thank you for pointing out the error. The percent yield of fungal extracts is added in Table 3. The formula used for calculation of the yield is also added in the method section. Table 4 and Figure 8; Table 5 and Figure 10 are integrated as advised. Regards Dhurva P. Gauchan" } ] } ]	1	https://f1000research.com/articles/9-379

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